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In coming years, the development in genetics is going to have a big impact on medicine, especially in the areas of diagnosis, prevention, and treatment of diseases. The following link explores some of those applications.

http://web.ornl.gov/sci/techresources/Human_Genome/publicat/primer2001/primer11

Read about Genetics and DNA technology in chapter 8. Based on what you learned in chapter 8 and from the article on the website, post two paragraphs on

Prompts:

Choose a technology and the applications of DNA technology in medicine.
Discuss the pros and cons of the technology you chose

Please don’t copy sentences out of the book or websites. Please specify the technology you are discussing.

8
Virtually all the microbial traits you have read about in earlier chapters are controlled or influenced by heredity. The
inherited characteristics of microbes include shape,
structural features, metabolism, ability to move, and
interactions with other organisms. Individual organisms
transmit these characteristics to their offspring through
genes.

The development of antibiotic resistance in microorganisms
is often carried on plasmids such as those in the photo,
which are readily transferred between bacterial cells. They
are responsible for the emergence of methicillin-resistant
Staphylococcus aureus and the recent emergence of carbapenem-
resistant Klebsiella pneumoniae. The emergence of vancomycin-
resistant S. aureus (VRSA) poses a serious threat to patient care.
In this chapter you will see how VRSA acquired this characteristic.

Emerging diseases provide another reason why it is important
to understand genetics. New diseases are the results of genetic
changes in some existing organism; for example, E. coli O157:H7
acquired the genes for Shiga toxin from Shigella.

Currently, microbiologists are using genetics to study
unculturable microbes and the relationship between hosts and
microbes.

The Big Picture on pages 206–207 highlights key
principles of genetics that are explained in greater detail
throughout the chapter.

In the Clinic
As a nurse at a U.S. military hospital, you treat service members injured in the recent Middle
East conflicts. You notice that wounds infected by Acinetobacter baumannii are not responding
to antibiotics. The Centers for Disease Control and Prevention reports that the antibiotic-
resistance genes found in A. baumannii are the same as those in Pseudomonas, Salmonella, and
Escherichia. Cephalosporin-resistance genes are on the chromosome, tetracycline resistance is
encoded by a plasmid, and streptomycin resistance is associated with a transposon. Can you
suggest mechanisms by which Acinetobacter acquired this
resistance?

Hint: Read about genetic recombination on pages 229–235.

Microbial Genetic

Play In the Clinic Video
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▶ Plasmids exist in cells separate from
chromosomes.

204

CHAPTER 8 Microbial Genetics 20

Structure and Function
of the Genetic Material
LEARNING OBJECTIVES

8-1 Define genetics, genome, chromosome, gene, genetic code,
genotype, phenotype, and genomics.

8-2 Describe how DNA serves as genetic information.

8-3 Describe the process of DNA replication.

8-4 Describe protein synthesis, including transcription, RNA
processing, and translation.

8-5 Compare protein synthesis in prokaryotes and eukaryotes.

Genetics is the science of heredity. It includes the study of genes:
how they carry information, how they replicate and pass to sub-
sequent generations of cells or between organisms, and how the
expression of their information within an organism determines
its characteristics. The genetic information in a cell is called the
genome. A cell’s genome includes its chromosomes and plas-
mids. Chromosomes are structures containing DNA that physi-
cally carry hereditary information; the chromosomes contain
the genes. Genes are segments of DNA (except in some viruses,
in which they are made of RNA) that code for functional prod-
ucts. Usually these products are proteins, but they can also be
RNAs (ribosomal RNA, transfer RNA, or microRNA).

We saw in Chapter 2 that DNA is a macromolecule composed
of repeating units called nucleotides. Each nucleotide consists of
a nucleobase (adenine, thymine, cytosine, or guanine), deoxyri-
bose (a pentose sugar), and a phosphate group (see Figure 2.16,
page 45). The DNA within a cell exists as long strands of nucle-
otides twisted together in pairs to form a double helix. Each
strand has a string of alternating sugar and phosphate groups
(its sugar-phosphate backbone), and a nitrogenous base is attached
to each sugar in the backbone. The two strands are held together
by hydrogen bonds between their nitrogenous bases. The base
pairs always occur in a specific way: adenine always pairs with
thymine, and cytosine always pairs with guanine. Because of
this specific base pairing, the base sequence of one DNA strand
determines the base sequence of the other strand. The two
strands of DNA are thus complementary.

The structure of DNA helps explain two primary features
of biological information storage. First, the linear sequence of
bases provides the actual information. Genetic information
is encoded by the sequence of bases along a strand of DNA,
in much the same way as our written language uses a linear
sequence of letters to form words and sentences. The genetic
language, however, uses an alphabet with only four letters—the
four kinds of nucleobases in DNA (or RNA). But 1000 of these
four bases, the number contained in an average-sized gene, can
be arranged in 41000 different ways. This astronomically large
number explains how genes can be varied enough to provide
all the information a cell needs to grow and perform its func-
tions. The genetic code, the set of rules that determines how a

nucleotide sequence is converted into the amino acid sequence
of a protein, is discussed in more detail later in this chapter.

Second, the complementary structure allows for the pre-
cise duplication of DNA during cell division. Each offspring
cell receives one of the original strands from the parent, thus
ensuring one strand that functions correctly.

Much of cellular metabolism is concerned with translating
the genetic message of genes into specific proteins. A gene is
usually copied to make a messenger RNA (mRNA) molecule,
which ultimately results in the formation of a protein. When
the ultimate molecule for which a gene codes (a protein, for
example) has been produced, we say that the gene has been
expressed. The flow of genetic information can be shown as
flowing from DNA to RNA to proteins, as follows:

RNA Protein

This theory was called
the central dogma by Francis
Crick in 1956, when he first
proposed that the sequence
of nucleotides in DNA deter-
mines the sequence of amino
acids in a protein.

Genotype and Phenotyp

The genotype of an organism is its genetic makeup—all its
DNA—the information that codes for all the particular char-
acteristics of the organism. The genotype represents potential

ASM: Although the central dogma is
universal in all cells, the processes differ

in prokaryotes and eukaryotes, as we shall
see in this chapter.

205 224 227 23

CLINICAL CASE Where There’s Smoke

Marcel DuBois, a 70-year-old grandfather of 12, quietly hangs up the phone. His doctor has just called him with
the results of his stool DNA test that he undertook at the
Mayo Clinic last week. Marcel’s doctor suggested this new,
noninvasive screening tool for colorectal cancer because
Marcel is not comfortable with the colonoscopy procedure
and usually tries to postpone getting one. The stool DNA test,
however, uses stool samples, which contain cells that have
been shed from the colon lining. The DNA from these cells
is tested for DNA markers that may indicate the presence of
precancerous polyps or cancerous tumors. Marcel makes an
appointment to come in to see his doctor the next afternoon.

Once in the office, the doctor explains to Marcel and his
wife, Janice, that the stool DNA test detected the presence
of serrated colorectal polyps. This type of polyp is usually
difficult to see with a colonoscopy because it is not raised and
can be the same color as the colon wall.

How can DNA show whether a person has cancer?
Read on to find out.

206

GeneticsBIG PICTURE
Genetics is the science of heredity. It
includes the study of genes: how they
are replicated, expressed, and passed
on from one generation to another.
The central dogma of molecular biology describes how, typically,
DNA is transcribed to messenger RNA, which, in turn, is translated
into proteins that carry out vital cellular functions. Mutations
introduce change into this process—ultimately leading to new or
lost functions.

How mutations
alter a genome

DNA

RNA

Protei

Function

Mutated DNA

Altered

mRNA

Altered protein

Altered function

Typical chain of events
described by

central dogma

Mutations can be caused by base substitutions or frameshift
mutations.

In base substitution mutations, a single DNA base
pair is altered.

T A C T T

C A

A U G A A G

T A A T T C A

A U A A G TT

In frameshift mutations, DNA base pairs are added or removed
from the sequence, causing a shift in the sequence reading.

T A C T T C A
A U G A A G T

T A T T C A

A U A A G T

Groups of genes in operons can be inducible or repressible.

Active
repressor

DNA
DNA

Inactive
repressor

Inducer

“OFF” (gene
not expressed)

“ON” (gene
expressed)

An inducible operon includes genes that are in the “off”
mode, with the repressor bound to the DNA, and is turned
“on” by the environmental inducer.

“ON” (gene
expressed)

“OFF” (gene
not expressed)

A repressible operon includes genes that are in the “on”
mode, without the repressor bound to the DNA, and is turned
“off” by the environmental corepressor and repressor.

DNA
DNA
Inactive
repressor
Active
repressor

Corepressor

AFM
7 nmAtomic force micrograph showing DNA molecules.

207

Alteration of bacterial genes and/or gene expression may cause disease, prevent
disease treatment, or be manipulated for human benefit.

TEM 0.4 mm

Diseases: Many bacterial diseases are caused by t

presence of toxic proteins that damage human tissue. These
toxic proteins are coded for by genes. Vibrio cholerae, shown
above, produces an enterotoxin that causes diarrhea and
severe dehydration, which can be fatal if left untreated.

Antibiotic resistance: Mutations in the bacterial genome
are one of the first steps toward the development of antibiotic
resistance. This process has occurred with Staphylococcus
aureus, which is currently resistant to beta-lactam antibiotics
such as penicillin. Methicillin was introduced to treat
penicillin-resistant S. aureus. Methicillin-resistant
S. aureus (MRSA), shown in purple above, is now a
leading cause of healthcare-associated infections.

SEM 0.3 mm

Biofilms: Biofilms, such as the one seen here growing on a
toothbrush bristle, are produced by altered bacterial gene
expression when populations are large enough. Various
Streptococcus species, including S. mutans, form biofilms on
teeth and gums, contributing to the development of dental
plaque and dental caries.

SEM 5 mm

Biotechnology: Scientists can alter a microorganism’s
genome, adding genes that will produce human proteins used
in treating
disease. Insulin,
used for treatmen

of diabetes, is
produced in this
manner.

DNA expression leads to cell function
via the production of proteins.

Genes in operons are turned on or off
together.

Mutations alter DNA sequences.

DNA mutations can change bacterial
function.

●

●
●
●

KEY CONCEPTS

Play MicroFlix 3D Animation
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208 PART ONE Fundamentals of Microbiology

properties, but not the properties themselves. Phenotype refers
to actual, expressed properties, such as the organism’s ability to
perform a particular chemical reaction. Phenotype, then, is
the manifestation of genotype. For example, E. coli with the stx
gene can produce the stx (Shiga toxin) protein.*

In a sense, an organism’s phenotype is its collection of pro-
teins, because most of a cell’s properties derive from the struc-
tures and functions of proteins. In microbes, most proteins
are either enzymatic (catalyze particular reactions) or structural
(participate in large functional complexes such as membranes
or flagella). Even phenotypes that depend on structural mac-
romolecules such as lipids or polysaccharides rely indirectly
on proteins. For instance, the structure of a complex lipid or
polysaccharide molecule results from catalytic activities of
enzymes that synthesize, process, and degrade those molecules.
Thus, saying that phenotypes are due to proteins is a useful
simplification.

DNA and Chromosomes
Bacteria typically have a single circular chromosome consist-
ing of a single circular molecule of DNA with associated pro-
teins. The chromosome is looped and folded and attached at
one or several points to the plasma membrane. The DNA of
E. coli has about 4.6 million base pairs and is about 1 mm
long—1000 times longer than the entire cell (Figure 8.1). How-
ever, the chromosome takes up only about 10% of the cell’s
volume because the DNA is twisted, or supercoiled.

The entire genome does not consist of back-to-back genes.
Noncoding regions called short tandem repeats (STRs) occur
in most genomes, including that of E. coli. STRs are repeating
sequences of two- to five-base sequences. These are used in
DNA fingerprinting (discussed on page 258).

Now, the complete base sequences of chromosomes can
be determined. Computers are used to search for open reading
frames, that is, regions of DNA that are likely to encode a
protein. As you will see later, these are base sequences between
start and stop codons. The sequencing and molecular charac-
terization of genomes is called genomics. The use of genomics
to track Zika virus is described in the Clinical Focus box on
page 218.

The Flow of Genetic Information
DNA replication makes possible the flow of genetic informa-
tion from one generation to the next. This is called vertical
gene transfer. As shown in Figure 8.2, the DNA of a cell repli-
cates before cell division so that each offspring cell receives a
chromosome identical to the parent’s. Within each metaboliz-
ing cell, the genetic information contained in DNA also flows
in another way: it is transcribed into mRNA and then trans-
lated into protein. We describe the processes of transcription
and translation later in this chapter.

Chromosome

1 mm
TEM

Figure 8.1 A prokaryotic chromosome.

How many times longer than the 2-mm cell is the chromosome?

CHECK YOUR UNDERSTANDIN

✓ 8-1 Give a clinical application of genomics.

✓ 8-2 Why is the base pairing in DNA important?

DNA

Replication

In DNA replication, one “parental” double-stranded DNA mol-
ecule is converted to two identical offspring molecules. The
complementary structure of the nitrogenous base sequences in
the DNA molecule is the key to understanding DNA replica-
tion. Because the bases along the two strands of double-helical
DNA are complementary, one strand can act as a template for
the production of the other strand (Figure 8.3a).

DNA replication requires the presence of several cellular
proteins that direct a particular sequence of events. Enzymes
involved in DNA replication and other processes are listed
in Table 8.1. When replication begins, the supercoiling is
relaxed by topoisomerase or gyrase. The two strands of parental
DNA are unwound by helicase and separated from each other
in one small DNA segment after another. Free nucleotides
present in the cell cytoplasm are matched up to the exposed
bases of the single-stranded parental DNA. Where thymine
is present on the original strand, only adenine can fit into
place on the new strand; where guanine is present on the
original strand, only cytosine can fit into place, and so on.
Any bases that are improperly base-paired are removed and *Gene names are italicized, but the protein name is not italicized.

CHAPTER 8 Microbial Genetics

209

●
●
●

Genetic information can be
transferred horizontally between
cells of the same generation.

Genetic information can be
transferred vertically to the
next generation of cells.

Genetic information is used
within a cell to produce the
proteins needed for the cell
to function.

New combinations
of genes

Offspring cells

Transcription

DNA

Parent cell

Cell metabolizes and grows Recombinant cell

Translation

expression recombination replication

DNA is the blueprint for a cell’s proteins, including
enzymes.

DNA is obtained either from another cell in the same
generation or from a parent cell during cell division.

DNA can be expressed within a cell or transferred to
another cell through recombination and replication.

KEY CONCEPTS

The Flow of Genetic Information
FOUNDATION

FIGURE
8.2

replaced by replication enzymes. Once aligned, the newly
added nucleotide is joined to the growing DNA strand by
an enzyme called DNA polymerase. Then the parental DNA
is unwound a bit further to allow the addition of the next
nucleotides. The point at which replication occurs is called
the replication fork.

As the replication fork moves along the parental DNA, each
of the unwound single strands combines with new nucleotides.
The original strand and this newly synthesized daughter strand
then rewind. Because each new double-stranded DNA molecule
contains one original (conserved) strand and one new strand,
the process of replication is referred to as semiconservative
replication.

Before looking at DNA replication in more detail, let’s dis-
cuss the structure of DNA (see Figure 2.16 on page 45 for an
overview). It is important to understand that the paired DNA
strands are oriented in opposite directions (antiparallel) rela-
tive to each other. The carbon atoms of the sugar component
of each nucleotide are numbered 1′ (pronounced “one prime”)

to 5′. For the paired bases to be next to each other, the sugar
components in one strand are upside down relative to the
other. The end with the hydroxyl attached to the 3′ carbon is
called the 3′ end of the DNA strand; the end having a phos-
phate attached to the 5′ carbon is called the 5′ end. The way
in which the two strands fit together dictates that the 5′ S 3′
direction of one strand runs counter to the 5′ S 3′ direction
of the other strand (Figure 8.3b). This structure of DNA affects
the replication process because DNA polymerases can add new
nucleotides to the 3′ end only. Therefore, as the replication fork
moves along the parental DNA, the two new strands must grow
in different directions.

One new strand, called the leading strand, is synthesized con-
tinuously in the 5′ S 3′ direction (from a template parental
strand running 3′ S 5′). In contrast, the lagging strand of the
new DNA is synthesized discontinuously in fragments of about
1000 nucleotides, called Okazaki fragments. These must be
joined later to make the continuous strand.

209

210 PART ONE Fundamentals of Microbiology

A
A
A
A

Parental
strand

3¿ end

Daughter

strand
forming

5¿ end

Daughter

strand Parental
strand

Parental
strand
G

Replication
fork

T
C
3¿ end
3¿ end
5¿ end
5¿ end

Deoxyribose sugar

Phosphate

(a) The replication fork

(b) The two strands of DNA are antiparallel.
The sugar-phosphate backbone of one strand is
upside down relative to the backbone of the
other strand. Turn the book upside down to
demonstrate this.

A
A
C

T A

KEY

T
T
C

1 The double helix of the
parental DNA separates
as weak hydrogen
bonds between the
nucleotides on opposite
strands break in
response to the action
of replication enzymes.

Hydrogen bonds form
between new
complementary
nucleotides and each
strand of the parental
template to form new
base pairs.

Enzymes catalyze
the formation of
sugar-phosphate
bonds between
sequential nucleotides
on each resulting
daughter strand.

O
O

OH
O

–O

–O
–O
–O
P

H2C

H2C
H2C
H2C
5¿ end
3¿ end
5¿ end
3¿ end
O
O
O
P
O
O
O

O–

O–
O–
O–
P

CH2

CH2
CH2
CH2
O
O
O
P
O
O
O
P
O
O
O
O
O
O
O
O
O
O
O
P
O

HO
O

C G

G G

G C

T AT

T T

Adenine ThymineA T

Guanine CytosineG C

Figure 8.3 DNA replication.

What is the advantage of semiconservative replication?

TABLE 8.1 Important Enzymes in DNA Replication, Expression, and Repair

DNA Gyrase Relaxes supercoiling ahead of the replication fork

DNA Ligase Makes covalent bonds to join DNA strands; Okazaki fragments, and new segments in excision repair

DNA Polymerases Synthesize DNA; proofread and facilitate repair of DNA

Endonucleases Cut DNA backbone in a strand of DNA; facilitate repair and insertions

Exonucleases Cut DNA from an exposed end of DNA; facilitate repair

Helicase Unwinds double-stranded DNA

Methylase Adds methyl group to selected bases in newly made DNA

Photolyase Uses visible light energy to separate UV-induced pyrimidine dimers

Primase An RNA polymerase that makes RNA primers from a DNA template

Ribozyme RNA enzyme that removes introns and splices exons together

RNA Polymerase Copies RNA from a DNA template

snRNP RNA-protein complex that removes introns and splices exons together

Topoisomerase or Gyrase Relaxes supercoiling ahead of the replication fork; separates DNA circles at the end of DNA replication

Transposase Cuts DNA backbone, leaving single-stranded “sticky ends”

CHAPTER 8 Microbial Genetics 211

G C

C
OH

Sugar

Phosphate
OH

OHOH

C G C G

P
P P

A T A T

A T A

G C
C

New
strand

Template
strand

When a nucleoside
triphosphate bonds
to the sugar, it loses
two phosphates.

Hydrolysis of the
phosphate bonds
provides the energy
for the reaction.

P P i

Figure 8.4 Adding a nucleotide to DNA.

Why is one strand “upside down” relative to the other strand?
Why can’t both strands “face” the same way?

Energy Needs
DNA replication requires a great deal of energy. The energy is
supplied from the nucleotides, which are actually nucleoside
triphosphates. You already know about ATP; the only differ-
ence between ATP and the adenine nucleotide in DNA is the
sugar component. Deoxyribose is the sugar in the nucleosides
used to synthesize DNA, and nucleoside triphosphates with
ribose are used to synthesize RNA. Two phosphate groups are
removed to add the nucleotide to a growing strand of DNA;
hydrolysis of the nucleoside is exergonic and provides energy
to make the new bonds in the DNA strand (Figure 8.4).

Figure 8.5 provides more detail about the many steps that
go into this complex process.

DNA replication by some bacteria, such as E. coli, goes
bidirectionally around the chromosome (Figure 8.6). Two repli-
cation forks move in opposite directions away from the origin
of replication. Because the bacterial chromosome is a closed
loop, the replication forks eventually meet when replication is
completed. The two loops must be separated by a topoisomer-
ase. Much evidence shows an association between the bacterial
plasma membrane and the origin of replication. After dupli-
cation, if each copy of the origin binds to the membrane at

Enzymes unwind the
parental double
helix.

Proteins stabilize the
unwound parental DNA.

DNA polymerase

The leading strand is synthesized
continuously from the primer by
DNA polymerase.

3
Replication
fork

The lagging strand is
synthesized discontinuously.
Primase, an RNA polymerase,
synthesizes a short RNA primer,
which is then extended by
DNA polymerase.

4 DNA polymerase
digests RNA primer
and replaces it with DNA.

DNA
polymerase

Primase
RNA primer

DNA ligase joins
the discontinuous
fragments of the
lagging strand.

DNA polymerase

DNA ligaseOkazaki fragment

Parental
strand

5¿

3¿

5¿
3¿

REPLICATIO

Figure 8.5 A summary of events at the DNA replication fork.

Why is one strand of DNA synthesized discontinuously? Q

212 PART ONE Fundamentals of Microbiology

20 nmSEM(a) An E. coli chromosome in the process of replicating

(b) Bidirectional replication of a circular bacterial DNA molecule

Origin of
replication

Replication
fork

Daughter
strands

Parental
strand

Termination
of replication

Replication
fork
REPLICATION
Replication
fork

Replication fork
Figure 8.6 Replication of bacterial DNA.

What is the origin of replication? Q

opposite poles, then each offspring cell receives one copy of the
DNA molecule—that is, one complete chromosome.

DNA replication is an amazingly accurate process. Typi-
cally, mistakes are made at a rate of only 1 in every 10 billion
bases incorporated. Such accuracy is largely due to the proof-
reading capability of DNA polymerase. As each new base is
added, the enzyme evaluates whether it forms the proper com-
plementary base-pairing structure. If not, the enzyme excises
the improper base and replaces it with the correct one. In this
way, DNA can be replicated
very accurately, allowing
each daughter chromosome
to be virtually identical to
the parental DNA.

Play DNA Replication:
Overview, Forming the
Replication Fork,

Replication Proteins, Synthesis
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CHECK YOUR UNDERSTANDIN

✓ 8-3 Describe DNA replication, including the functions of DNA
gyrase, DNA ligase, and DNA polymerase.

RNA and Protein Synthesis
How is the information in DNA used to make the proteins that
control cell activities? In the process of transcription, genetic
information in DNA is copied, or transcribed, into a comple-
mentary base sequence of RNA. The cell then uses the infor-
mation encoded in this RNA to synthesize specific proteins
through the process of translation. We now take a closer look at
these two processes as they occur in a bacterial cell.

Transcription in Prokaryotes
Transcription is the synthesis of a complementary strand of
RNA from a DNA template. We will discuss transcription in
prokaryotic cells here. Transcription in eukaryotes is discussed
on page 215.

Ribosomal RNA (rRNA) forms an integral part of ribo-
somes, the cellular machinery for protein synthesis. Transfer
RNA is also involved in protein synthesis, as we will see.
Messenger RNA (mRNA) carries the coded information for
making specific proteins from DNA to ribosomes, where pro-
teins are synthesized.

During transcription, a strand of mRNA is synthesized
using a specific portion of the cell’s DNA as a template. In
other words, the genetic information stored in the sequence of
nucleobases of DNA is rewritten so that the same information
appears in the base sequence of mRNA.

As in DNA replication, a guanine (G) in the DNA template
dictates a cytosine (C) in the mRNA being made, and a C in the
DNA template dictates a G in the mRNA. Likewise, a thymine
(T) in the DNA template dictates an adenine (A) in the mRNA.

CHAPTER 8 Microbial Genetics

213

However, an adenine in the DNA template dictates a uracil (U)
in the mRNA, because RNA contains uracil instead of thymine.
(Uracil has a chemical structure slightly different from thy-
mine, but it base-pairs in the same way.) If, for example, the
template portion of DNA has the base sequence 3’-ATGCAT,
the newly synthesized mRNA strand will have the complemen-
tary base sequence 5’-UACGUA.

The process of transcription requires both an enzyme called
RNA polymerase and a supply of RNA nucleotides (Figure 8.7).
Transcription begins when RNA polymerase binds to the DNA
at a site called the promoter. Only one of the two DNA strands
serves as the template for RNA synthesis for a given gene.
Like DNA, RNA is synthesized in the 5′ S 3′ direction. RNA
synthesis continues until RNA polymerase reaches a site on the
DNA called the terminator.

Transcription allows the cell to produce short-term copies
of genes that can be used as the direct source of information
for protein synthesis. Messenger RNA acts as an intermedi-
ate between the permanent
storage form, DNA, and the
process that uses the infor-
mation, translation.

Translation
We have seen how the genetic information in DNA transfers
to mRNA during transcription. Now we will see how mRNA
serves as the source of information for the synthesis of pro-
teins. Protein synthesis is called translation because it involves
decoding the “language” of nucleic acids and converting it into
the “language” of proteins.

UG
G

C
U
U
U
G

A
TT

T T
T

T T T T
A

A A

A
AA
A
T

C C

A
A

C C

G
C

G
G
G G
A
U
A
U
A
U
T A
C
G
A
G

RNA polymerase
binds to the
promoter, and
DNA unwinds at
the beginning of
a gene.

RNA is synthesized
by complementary
base pairing of free
nucleotides with the
nucleotide bases on
the template strand
of DNA.

The site of synthesis
moves along DNA;
DNA that has been
transcribed rewinds.

Transcription reaches
the terminator.

RNA and RNA
polymerase are
released, and the
DNA helix re-forms.

1
3
4
5
2
DNA
mRNA

Protein

TRANSCRIPTION

10 nm
AFMRNA polymerase bound to DNA

DNA

RNA
polymerase

RNA synthesis

Complete
RNA strand

Promoter

(gene begins)

RNA polymerase

RNA Terminator
(gene ends)

RNA

RNA nucleotides
RNA polymerase

Template strand of DNA

Promoter

Figure 8.7 The process of transcription. The orienting diagram indicates the relationship
of transcription to the overall flow of genetic information within a cell.

When does transcription stop? Q

Play Transcription:
Overview, Process
@MasteringMicrobiology

214 PART ONE Fundamentals of Microbiology

The language of mRNA is in the form of codons, groups of
three nucleotides, such as AUG, GGC, or AAA. The sequence
of codons on an mRNA molecule determines the sequence of
amino acids that will be in the protein being synthesized. Each
codon “codes” for a particular amino acid. This is the genetic
code (Figure 8.8).

Codons are written in terms of their base sequence in
mRNA. Notice in Figure 8.8 that there are 64 possible codons
but only 20 amino acids. This means that most amino acids
are signaled by several alternative codons, a situation referred
to as the degeneracy of the code. For example, leucine has
six codons, and alanine has four codons. Degeneracy allows for
a certain amount of misreading of, or mutation in, the DNA
without affecting the protein ultimately produced.

Of the 64 codons, 61 are sense codons, and 3 are nonsense
codons. Sense codons code for amino acids, and nonsense codons
(also called stop codons) do not. Rather, the nonsense codons—
UAA, UAG, and UGA—signal the end of the protein molecule’s
synthesis. The start codon that initiates the synthesis of the pro-
tein molecule is AUG, which is also the codon for methionine. In
bacteria, the start AUG codes for formylmethionine rather than
the methionine found in other parts of the protein. The initiat-
ing methionine is often removed later, so not all proteins contain
methionine.

During translation, codons of an mRNA are “read” sequen-
tially; and, in response to each codon, the appropriate amino
acid is assembled into a growing chain. The site of translation
is the ribosome, and transfer RNA (tRNA) molecules both rec-
ognize the specific codons and transport the required amino
acids.

Each tRNA molecule has an anticodon, a sequence of three
bases that is complementary to a codon. In this way, a tRNA
molecule can base-pair with its associated codon. Each tRNA
can also carry on its other end the amino acid encoded by the
codon that the tRNA recognizes. The functions of the ribo-
some are to direct the orderly binding of tRNAs to codons and
to assemble the amino acids brought there into a chain, ulti-
mately producing a protein.

Figure 8.9 shows the details of translation. The two ribo-
somal subunits, a tRNA with the anticodon UAC, and the
mRNA molecule to be translated, along with several addi-
tional protein factors, all assemble. This sets up the start
codon (AUG) in the proper position to allow translation to
begin. After the ribosome joins the first two amino acids with
a peptide bond, the first tRNA molecule leaves the ribosome.
The ribosome then moves along the mRNA to the next codon.
As the proper amino acids are brought into line one by one,
peptide bonds are formed between them, and a polypeptide
chain results. (Also see Figure 2.14, page 42.) Translation

Second position

Fi
rs

t p
os

iti
on

Th
ir

d
po

si
tio

C A G

C
A
G
U
U

UUU

UUC

UUA

UUG

Phe

Leu

UCU

UCA

UCG

Ser

Tyr Cys
U

C
A
G

CUU

CUC

CUA

CUG

Leu

CCU

CCC

CCA

CCG

Pro

CAU

CAA

CAG

His

Gln

CGU

CGC

CGG

Arg

U
C
A
G

Ile

ACU

ACC

ACA

ACG

Thr

AAU

AAC

AAA

AAG

Asn

Lys

AGU

AGC

AGA

AGG

Ser
Arg
U
C
A
G

GUU

GUC

GUA

GUG

Val

GCU

GCC

GCA

GCG

Ala

GAU

GAC

GAA

GAG

Asp

Glu

GGU

GGC

GGA

GGG

Gly

U
C
A
G

UAU

UAC

UAA

UAG

Stop

Stop Trp

UGU

UGC

UGA

UGG

Stop

AUU

AUC

AUA

AUG Met/start

Figure 8.8 The genetic code. The three nucleotides in an mRNA
codon are designated, respectively, as the first position, second
position, and third position of the codon on the mRNA. Each set of
three nucleotides specifies a particular amino acid, represented by
a three-letter abbreviation (see Table 2.5, page 41). The codon AUG,
which specifies the amino acid methionine, is also the start of protein
synthesis. The word Stop identifies the nonsense codons that signal the
termination of protein synthesis.

What is the advantage of the degeneracy of the genetic code? Q

ends when one of the three nonsense codons in the mRNA is
reached. The ribosome then comes apart into its two subunits,
and the mRNA and newly synthesized polypeptide chain are
released. The ribosome, the mRNA, and the tRNAs are then
available to be used again.

The ribosome moves along the mRNA in the 5′ S 3′ direc-
tion. As a ribosome moves along the mRNA, it will soon allow
the start codon to be exposed. Additional ribosomes can then
assemble and begin synthesizing protein. In this way, there
are usually a number of ribosomes attached to a single mRNA,
all at various stages of protein synthesis. In prokaryotic cells,
the translation of mRNA into protein can begin even before

CHAPTER 8 Microbial Genetics 2

transcription is complete (Figure 8.10). Because mRNA is pro-
duced in the cytoplasm in prokaryotes, the start codons of an
mRNA being transcribed are available to ribosomes before the
entire mRNA molecule is even made.

Transcription in Eukaryotes
In eukaryotic cells, transcription takes place in the nucleus. The
mRNA must be completely synthesized and moved through
the nuclear membrane to the cytoplasm before translation can
begin. In addition, the RNA undergoes processing before it leaves
the nucleus. In eukaryotic cells, the regions of genes that code for
proteins are often interrupted by noncoding DNA. Thus, eukary-
otic genes are composed of exons, the regions of DNA expressed,
and introns, the intervening regions of DNA that do not encode
protein. In the nucleus, RNA polymerase synthesizes a molecule
called an RNA transcript that contains copies of the introns.
Particles called small nuclear ribonucleoproteins, abbreviated
snRNPs and pronounced “snurps,” remove the introns and splice
the exons together. In some organisms, the introns act as ribo-
zymes to catalyze their own removal (Figure 8.11).

* * *

To summarize, genes are the units of biological information
encoded by the sequence of nucleotide bases in DNA. A gene is
expressed, or turned into a product within the cell, through the
processes of transcription and translation. The genetic informa-
tion carried in DNA is transferred to a temporary mRNA mol-
ecule by transcription. Then, during translation, the mRNA
directs the assembly of amino acids into a polypeptide chain: a
ribosome attaches to mRNA, tRNAs deliver the amino acids to
the ribosome as directed by the mRNA codon sequence, and
the ribosome assembles the
amino acids into the chain
that will be the newly synthe-
sized protein.

CHECK YOUR UNDERSTANDING

✓ 8-4 What is the role of the promoter, terminator, and mRNA
in transcription?

✓ 8-5 How does mRNA production in eukaryotes differ from
the process in prokaryotes?

A cell’s genetic and metabolic machineries are integrated and inter-
dependent. The bacterial cell carries out an enormous number of
metabolic reactions (see Chapter 5). The common feature of all
metabolic reactions is that they are catalyzed by enzymes that are
proteins synthesized via transcription and translation. Feedback
inhibition stops a cell from performing unneeded chemical reac-
tions (Chapter 5, page 116) by stopping enzymes that have already
been synthesized. We will now
look at mechanisms to prevent
synthesis of enzymes that are
not needed.

Because protein synthesis requires a huge amount of
energy, cells save energy by making only those proteins needed
at a particular time. Next we look at how chemical reactions
are regulated by controlling gene expression.

Many genes, perhaps 60–80%, are not regulated but are
instead constitutive, meaning that their products are con-
stantly produced at a fixed rate. Usually these genes, which
are effectively turned on all the time, code for enzymes
that the cell needs in fairly large amounts for its major life
processes. Glycolysis enzymes are examples. The produc-
tion of other enzymes is regulated so that they are present
only when needed. Trypanosoma, the protozoan parasite that
causes African sleeping sickness, has hundreds of genes cod-
ing for surface glycoproteins. Each protozoan cell turns on
only one glycoprotein gene at a time. As the host’s immune
system kills parasites with one type of surface molecule,
parasites expressing a different surface glycoprotein can con-
tinue to grow.

Pre-transcriptional Control
Two genetic control mechanisms known as repression and
induction regulate the transcription of mRNA and, conse-
quently, the synthesis of enzymes from them. These mecha-
nisms control the formation and amounts of enzymes in the
cell, not the activities of the enzymes.

The Operon Model of Gene Expression
Details of the control of gene expression by induction and
repression are described by the operon theory formulated in
the 1960s by François Jacob and Jacques Monod. An operon is
a group of genes that are transcribed together and controlled
by one promoter. We’ll look first at an inducible operon, in
which transcription must be turned on. In E. coli, the enzymes
of the lac operon are needed to metabolize lactose. In addition
to b-galactosidase, these enzymes include lac permease, which
is involved in the transport of lactose into the cell, and trans-
acetylase, which metabolizes certain disaccharides other than
lactose.

The genes for the three enzymes involved in lactose
uptake and utilization are next to each other on the bacterial

Play Translation: Overview,
Genetic Code, Process
@MasteringMicrobiology

ASM: The regulation of gene expression
is influenced by external and internal

molecular cues and/or signals.

The Regulation of Bacterial
Gene Expression
LEARNING OBJECTIVES

8-6 Define operon.

8-7 Explain pre-transcriptional
regulation of gene
expression in bacteria.

8-8 Explain post-transcriptional regulation of gene expression.

Play Interactive Microbiology
@MasteringMicrobiology See
how operons affect a patient’s
health

216 PART ONE Fundamentals of Microbiology

Met

U ACA

A U

A U G G G

U A

mRNA

The second codon of the mRNA pairs with a tRNA carrying
the second amino acid at the A site. The first amino acid
joins to the second by a peptide bond. This attaches the
polypeptide to the tRNA in the P site.

Peptide bond forms

A site

E site

3
Leu

Met Phe

The ribosome moves along the mRNA until the second tRNA is
in the P site. The next codon to be translated is brought into the
A site. The first tRNA now occupies the E site.

U U UAA
AA

U
U C

C A
G G G

A C

mRNA

Ribosome moves
along mRNA

AA
A
4
Leu

When the ribosome reaches a stop codon,
the polypeptide is released.

Phe
Phe
Met

U
C U

A AG U AG

Polypeptide
released

mRNA

Stop codon

Met
Met

7 Finally, the last tRNA is released, and the ribosome comes
apart. The released polypeptide forms a new protein.

Gly

GlyGly
Leu

Leu

Arg
Leu P

he
Phe
Met
Met
Met

Gl
y

Gly
Gly
Leu
Leu
Arg

LeuU C

U
mRNA

New protein

Gly

chromosome and are regulated together (Figure 8.12). These
genes, which determine the structures of proteins, are called
structural genes to distinguish them from an adjoining control
region on the DNA. When lactose is introduced into the cul-
ture medium, the lac structural genes are all transcribed and
translated rapidly and simultaneously. We will now see how
this regulation occurs.

In the control region of the lac operon are two relatively
short segments of DNA. One, the promoter, is the segment
where RNA polymerase initiates transcription. The other is
the operator, which is like a traffic light that acts as a go or

Met
Leu

On the assembled ribosome, a tRNA carrying the first
amino acid is paired with the start codon on the mRNA.
The place where this first tRNA sits is called the P site.
A tRNA carrying the second amino acid approaches.

U
A C

U G

C
A
A U

U U A

Start
codon

Second
codon

mRNA

2Components needed to begin
translation come together.

U
A C
A U G

tRNA

mRNA

Anticodon

Ribosomal
subunit

C A
U A
C

Ribosome

P Site

The second amino acid joins to the third by another peptide
bond, and the first tRNA is released from the E site.

U U A U U U

A AG G U

tRNA released

5
Gly

A A U

Leu
Met

The ribosome continues to move along the mRNA,
and new amino acids are added to the polypeptide.

CC
A
A

U
G

G G U U A U GU U
U U A

Growing
polypeptide
chain

mRNA
mRNA

AA
A

CA
U

6
Met
Met
Leu
Gly
Phe

Phe
Met

DNA
mRNA
Protein

TRANSLATION

Figure 8.9 The process of translation.
The overall goal of translation is to produce
proteins using mRNAs as the source of
biological information. The complex cycle of
events illustrated here shows the primary
role of tRNA and ribosomes in the decoding

of this information. The ribosome acts as the
site where the mRNA-encoded information is
decoded, as well as the site where individual
amino acids are connected into polypeptide
chains. The tRNA molecules act as the
actual “translators”—one end of each tRNA

recognizes a specific mRNA codon, while the
other end carries the amino acid encoded by
that codon.

Q When does translation stop?

stop signal for transcription of the structural genes. A set of
operator and promoter sites and the structural genes they con-
trol define an operon; thus, the combination of the three lac
structural genes and the adjoining control regions is called the
lac operon.

A regulatory gene called the I gene encodes a repressor pro-
tein that switches inducible and repressible operons on or off.
The lac operon is an inducible operon (see Figure 8.12). In the
absence of lactose, the repressor binds to the operator site, thus
preventing transcription. If lactose is present, the repressor binds

CHAPTER 8 Microbial Genetics 217

Met

U ACA A U
A U G G GU U A

mRNA
The second codon of the mRNA pairs with a tRNA carrying
the second amino acid at the A site. The first amino acid
joins to the second by a peptide bond. This attaches the
polypeptide to the tRNA in the P site.
U
Peptide bond forms
A site
E site
3
Leu
Met Phe
The ribosome moves along the mRNA until the second tRNA is
in the P site. The next codon to be translated is brought into the
A site. The first tRNA now occupies the E site.
U U UAA
AA
U
U C
C A
G G G

U A C

mRNA
Ribosome moves
along mRNA
AA
A
4
Leu
When the ribosome reaches a stop codon,
the polypeptide is released.
Phe
Phe
Met
U
C U
A AG U AG
Polypeptide
released
mRNA
Stop codon
Met
Met
7 Finally, the last tRNA is released, and the ribosome comes
apart. The released polypeptide forms a new protein.
8
Gly
GlyGly
Leu
Leu
Arg
Leu P
he
Phe
Met
Met
Met
Gl
y
Gly
Gly
Leu
Leu
Arg
LeuU C
U
mRNA
New protein
Gly

Figure 8.9 The process of translation. (continued)

DNA
mRNA
Protein
TRANSLATION

Direction of transcription

Polyribosome

Peptide

RNA
polymerase

Direction of translation

Ribosome
DNA
mRNA
5¿

60 nm
TEM

Figure 8.10 Simultaneous transcription and translation
in bacteria. Many molecules of mRNA are being synthesized
simultaneously. The longest mRNA molecules were the first to be
transcribed at the promoter. Note the ribosomes attached to the newly
forming mRNA. The micrograph shows a polyribosome (many ribosomes)
in a single bacterial gene.

Why can translation begin before transcription is complete in
prokaryotes but not in eukaryotes?

Q
Met
Leu
On the assembled ribosome, a tRNA carrying the first
amino acid is paired with the start codon on the mRNA.
The place where this first tRNA sits is called the P site.
A tRNA carrying the second amino acid approaches.
U
A C
A U G
C
A
A U
U U A
Start
codon
Second
codon
mRNA
2Components needed to begin
translation come together.
U
A C
A U G
tRNA
mRNA
Anticodon
1
Ribosomal
subunit
Ribosomal
subunit
C A
U A
C
Ribosome
P Site
The second amino acid joins to the third by another peptide
bond, and the first tRNA is released from the E site.
U U A U U U
A
A AG G U
tRNA released
5
Gly
A A U
Leu
Met
The ribosome continues to move along the mRNA,
and new amino acids are added to the polypeptide.
CC
A
A
U
G
G G U U A U GU U
U U A
Growing
polypeptide
chain
mRNA
mRNA
AA
A
CA
U
6
Met
Met
Leu
Gly
Phe
Phe
Met
DNA
mRNA
Protein
TRANSLATION

218 PART ONE Fundamentals of Microbiology
CLINICAL FOCUS Tracking Zika Virus

to a metabolite of lactose instead of to the operator, and lactose-
digesting enzymes are transcribed.

In repressible operons, the structural genes are tran-
scribed until they are turned off (Figure 8.13). The genes for
the enzymes involved in the synthesis of tryptophan are reg-
ulated in this manner. The structural genes are transcribed
and translated, leading to tryptophan synthesis. When excess

In 2014, Brazilian physicians reported clusters of patients with fever and rash. Reverse transcription polymerase chain
reaction (RT-PCR) was used to detect
dengue, chikungunya, West Nile, and Zika
viruses. Public health
officials were relieved
when the cause was
identified as Zika
virus (ZIKV) because
ZIKV had never made
anyone sick enough
to go to the hospital.
ZIKV is an arbovirus
(arthropod-borne
virus) that is spread
between susceptible
vertebrate hosts
by blood-feeding
arthropods, such
as mosquitoes. At
the same time, local health officials saw
a fourfold increase in microcephaly—fetal
brains were not developing at the same rate
as the body. Some mothers reported having
rashes and achy joints, but these symptoms
weren’t long-lasting, and Zika virus disease
was common.

Adriana Melo, an obstetrician, sent
samples of amniontic fluid from two patients
to be tested. RT-PCR confirmed the presence
of ZIKV. By 2016, nearly 5000 cases of
microcephaly had been reported in Brazil.

This Old World
flavivirus was first
identified in 1947 in
monkeys in the Zika
Forest of Uganda.
Prior to 2000,
only 14 human
cases had been
documented in the
world. In 2007, an
outbreak occurred
on the island of
Yap in Micronesia.
Over 70% of Yap
residents were
infected with

ZIKV. However, no deaths or neurological
complications were reported.

The ZIKV genome consists of a positive,
single-stranded RNA consisting of 10,794
base pairs. (Positive RNA can act as mRNA
and be translated.) The polyprotein encoded
by the genome is cut to produce the proteins

that make up the virus. The virus has
acquired several mutations, and researchers
are looking for clues in these mutations to
determine the virus’s journey around the
world.

1. Using the portions of the genomes
(shown below) that encode viral proteins,
can you determine how similar these
viruses are? Can you figure out its
movement around the world?
Determine the amino acids encoded, and
group the viruses based on percentage of
similarity to the Uganda strain.

2. Based on amino acids, there are two
groups called clades.
Can you identify the two groups?

3. The two clades are the African and
Asian.
Calculate the percentage of difference
between nucleotides to see how the viruses
are related within their clade.

4. The virus in the Americas is most
closely related to the Asian strain that
circulated in French Polynesia.

Source: GenBank genome sequences.

TEM
40 mmZika virus.

Brazil K K R R S A E T S G L L L T A M A V S K

Colombia K K R R S A E T S G L L L T A M A V N K

French Polynesia K K R R G A D T S L L L L T A M A V S K

Haiti K K R R G A D T S L L L L T A M A I S K

Mexico K K R R S A E T S L L L L T A M A V N E

Micronesia K K R R G A D T S L L L L T A M A I S K

Nigeria R K R R G A D T S L L L L T V M A I S K

Uganda 1947 R K R R G A D A S L L L L T V M A I S K

United States K K R R G A E T S L L L L T A M A V S K

tryptophan is present, the tryptophan acts as a corepressor
binding to the repressor protein. The repressor protein can now
bind to the operator, stop-
ping further tryptophan
synthesis.

218

Play Operons: Overview,
Induction, Repression
@MasteringMicrobiology

CHAPTER 8 Microbial Genetics 219

CHECK YOUR UNDERSTANDING

✓ 8-6 Use the following metabolic pathway to answer the
questions that follow it.

enzyme a enzyme b
Substrate A Intermediate B End-product C

a. If enzyme a is inducible and is not being synthesized at
present, a (1)            protein must be bound tightly to the
(2)            site. When the inducer is present, it will bind to the
(3)            so that (4)            can occur.

b. If enzyme a is repressible, end-product C, called a (1) ,
causes the (2) to bind to the (3) . What causes
derepression?

Regulatory
gene

Promoter

Operator

I P O Z Y A

Control region

Operon

Structural genes

I P Z Y A

RNA polymerase

2 Repressor active, operon off. The repressor protein binds with the
operator, preventing transcription from the operon.

3 Repressor inactive, operon on. When the inducer allolactose binds
to the repressor protein, the inactivated repressor can no longer block
transcription. The structural genes are transcribed, ultimately resulting
in the production of the enzymes needed for lactose catabolism.

Repressor
mRNA

Transcription
Translation

Active
repressor
protein

I P O Z Y A
Transcription
Translation

Operon
mRNA

Allolactose
(inducer)

b-Galactosidase

Inactive
repressor
protein

Permease
Transacetylase

Structure of the operon. The operon consists of the promoter (P) and
operator (O ) sites and structural genes that code for the protein. The
operon is regulated by the product of the regulatory gene (I ).

DNA

What causes transcription of an inducible enzyme? Q

Figure 8.12 An inducible operon. Lactose-digesting enzymes are
produced in the presence of lactose. In E. coli, the genes for the three
enzymes are in the lac operon. b-galactosidase is encoded by lacZ. The
lacY gene encodes the lac permease, and lacA encodes transacetylase,
whose function in lactose metabolism is still unclear.

Positive Regulation
Regulation of the lactose operon also depends on the level of
glucose in the medium, which in turn controls the intracel-
lular level of the small molecule cyclic AMP (cAMP), a sub-
stance derived from ATP that serves as a cellular alarm signal.
Enzymes that metabolize glucose are constitutive, and cells
grow at their maximal rate with glucose as their carbon source
because they can use it most efficiently (Figure 8.14). When
glucose is no longer available, cAMP accumulates in the cell.
The cAMP binds to the allosteric site of catabolic activator
protein (CAP). CAP then binds to the lac promoter, which ini-
tiates transcription by making it easier for RNA polymerase
to bind to the promoter. Thus transcription of the lac operon
requires both the presence of lactose and the absence of glu-
cose (Figure 8.15).

Cyclic AMP is an example of an alarmone, a chemical alarm
signal that promotes a cell’s response to environmental or
nutritional stress. (In this case, the stress is the lack of glucose.)

Exon Intron Exon Intron Exon

mRNA

RNA
trans-
cript

DNA

Nucleus

Cytoplasm

1 In the nucleus, a gene composed of exons and
introns is transcribed to RNA by RNA polymerase.

2 Processing involves snRNPs in the nucleus to
remove the intron-derived RNA and splice
together the exon-derived RNA into mRNA.

3 After further modification, the mature
mRNA travels to the cytoplasm,
where it directs protein synthesis.

Figure 8.11 RNA processing in eukaryotic cells.

Why can’t the RNA transcript be used for translation? Q

220 PART ONE Fundamentals of Microbiology

P E D C B A

2 Repressor inactive, operon on. The repressor is inactive, and
transcription and translation proceed, leading to the synthesis
of tryptophan.

3 Repressor active, operon off. When the corepressor tryptophan binds
to the repressor protein, the activated repressor binds with the
operator, preventing transcription from the operon.

RNA polymerase
Active
repressor
protein
Inactive
repressor
protein

Polypeptides
comprising the
enzymes for
tryptophan
synthesis

Repressor
mRNA
Transcription
Translation
Operon
mRNA

I P E D C B A

Tryptophan
(corepressor)

11 Structure of the operon. The operon consists of the promoter (P ) and
operator (O ) sites and structural genes that code for the protein. The
operon is regulated by the product of the regulatory gene (I ).

Regulatory
gene
Promoter Operator
O
Control region
Operon
Structural genes
DNA

Figure 8.13 A repressible operon. Tryptophan, an amino acid,
is produced by anabolic enzymes encoded by five structural genes.
Accumulation of tryptophan represses transcription of these genes,
preventing further synthesis of tryptophan. The E. coli trp operon is
shown here.

What causes transcription of a repressible enzyme? Q

The same mechanism involving cAMP allows the cell to use
other sugars. Inhibition of the metabolism of alternative car-
bon sources by glucose is termed catabolite repression (or the
glucose effect). When glucose is available, the level of cAMP in
the cell is low, and consequently CAP is not bound.

Epigenetic Control
Eukaryotic and bacterial cells can turn genes off by methyl-
ating certain nucleotides—that is, by adding a methyl group
1¬ CH32. The methylated (off) genes are passed to offspring
cells. Unlike mutations, this isn’t permanent, and the genes
can be turned on in a later generation. This is called epigene-
tic inheritance (epigenetic = on genes). Epigenetics may explain
why bacteria behave differently in a biofilm.

Post-transcriptional Control
Some regulatory mechanisms stop protein synthesis after tran-
scription has occurred. A part of an mRNA molecule, called a
riboswitch, that binds to a substrate can change the mRNA
structure. Depending on the type of change, translation can
be initiated or stopped. Both eukaryotes and prokaryotes use
riboswitches to control expression of some genes.

Lo
g 1

0
o

f n
um

be
r

of
c

el
ls

Lo
g 1
0
o
f n
um
be
r
of
c
el
ls

(a) Bacteria growing on
glucose as the sole carbon
source grow faster than on
lactose.

(b) Bacteria growing in a
medium containing glucose
and lactose first consume
the glucose and then, after a
short lag time, the lactose.
During the lag time, intra-
cellular cAMP increases, the
lac operon is transcribed,
lactose is transported into
the cell, and d-galacto-
sidase is synthesized to
break down lactose.

Time

Glucose

Lactose

All glucose
consumed

Glucose
used Lag

time

Lactose used

Figure 8.14 The growth rate of E. coli on glucose and lactose.

When both glucose and lactose are present, why will cells use
glucose first?

CHAPTER 8 Microbial Genetics 221

lacI

lacZ

RNA
polymerase
can bind
and transcribe

CAP-binding site

CAP-binding site RNA
polymerase
can’t bind

Active
CAP

Inactive
CAP

cAMP

DNA
DNA
Promoter

Inactive lac
repressor

Operator

(a) Lactose present, glucose scarce (cAMP level high). If glucose is
scarce, the high level of cAMP activates CAP, and the lac operon produces
large amounts of mRNA for lactose digestion.

lacI lacZ
Inactive
CAP
Promoter
Inactive lac
repressor
Operator

(b) Lactose present, glucose present (cAMP level low). When glucose is
present, cAMP is scarce, and CAP is unable to stimulate transcription.

Figure 8.15 Positive regulation of the lac operon.

Will transcription of the lac operon occur in the presence
of lactose and glucose? In the presence of lactose and the
absence of glucose? In the presence of glucose and the
absence of lactose?

Single-stranded RNA molecules of approximately
22 nucleotides, called microRNAs (miRNAs), inhibit protein
production in eukaryotic cells. In humans, miRNAs pro-
duced during development allow different cells to produce
different proteins. Heart cells and skin cells have the same
genes, but the cells in each organ produce different proteins
because of miRNAs produced in each cell type during devel-
opment. Similar short RNAs in bacteria enable the cell to
cope with environmental stresses, such as low temperature
or oxidative damage. An miRNA base-pairs with a comple-
mentary mRNA, forming a double-stranded RNA. This
double-stranded RNA is enzymatically destroyed so that the
mRNA-encoded protein is not made (Figure 8.16). The action
of another type of RNA, siRNA, is similar and is discussed on
page 256.

1 Transcription of
miRNA occurs.

2 miRNA binds to target
mRNA that has at least six
complementary bases.

3 mRNA is degraded.

miRNA

DNA
mRNA

Figure 8.16 MicroRNAs control a wide range of activities in cells.

In mammals, some miRNAs hybridize with viral RNA. What
would happen if a mutation occurred in the miRNA gene?

CHECK YOUR UNDERSTANDING

✓ 8-7 What is the role of cAMP in regulating gene expression?

✓ 8-8 How does miRNA stop protein synthesis?

Changes in Genetic Material
LEARNING OBJECTIVES

8-9 Classify mutations by type.

8-10 Describe two ways mutations can be repaired.

8-11 Describe the effect of mutagens on the mutation rate.

8-12 Outline the methods of direct and indirect selection of mutants.

8-13 Identify the purpose of and outline the procedure for the
Ames test.

A cell’s DNA can be changed by mutations and horizontal gene
transfer. Changes in DNA result in genetic variations that can
impact microbial function (e.g., biofilm formation, pathoge-
nicity, and antibiotic resistance). Survival and reproduction of

222 PART ONE Fundamentals of Microbiology

encoded by the gene. Silent mutations commonly occur when
one nucleotide is substituted for another in the DNA, espe-
cially at a location corresponding to the third position of the
mRNA codon. Because of the degeneracy of the genetic code,
the resulting new codon might still code for the same amino
acid. Even if the amino acid is changed, the function of the
protein may not change if the amino acid is in a nonvital por-
tion of the protein, or is chemically very similar to the original
amino acid.

The most common type of mutation involving single base
pairs is base substitution (or point mutation), in which a single
base at one point in the DNA sequence is replaced with a dif-
ferent base. When the DNA replicates, the result is a substituted
base pair (Figure 8.17). For example, AT might be substituted for
GC, or CG for GC. If a base substitution occurs within a gene
that codes for a protein, the mRNA transcribed from the gene
will carry an incorrect base at that position. When the mRNA is
translated into protein, the incorrect base may cause the inser-
tion of an incorrect amino acid in the protein. If the base substi-
tution results in an amino acid substitution in the synthesized

the bacteria with a new genotype can be favored by natural and
human-influenced environments and result in a huge diversity
of microorganisms. The survival of new genotypes is called
natural selection.

Mutation
A mutation is a permanent change in the base sequence of DNA.
Such a change will sometimes cause a change in the product
encoded by that gene. For example, when the gene for an enzyme
mutates, the enzyme encoded by the gene may become inactive
or less active because its amino acid sequence has changed. Such
a change in genotype may be disadvantageous, or even lethal,
if the cell loses a phenotypic trait it needs. However, a mutation
can be beneficial if, for instance, the altered enzyme encoded by
the mutant gene has a new or enhanced activity that benefits the
cell. See the Clinical Focus box in Chapter 26, page 771.

Types of Mutations
Many simple mutations are silent (neutral); the change in DNA
base sequence causes no change in the activity of the product

During DNA replication,
a thymine is incorporated
opposite guanine by
mistake.

T
TA

A
T

A
G C
G
C
G
T
T
A
T
TA
A
T
A
G
A
T
T
A

U UG U UA U UG GU U

C
T
TA
A
T
A

G
T

A
C

Parental DNA

Daughter DNA

Granddaughter DNA

2 If not corrected, in the next round
of replication, adenine pairs with
the new thymine, yielding an AT pair
in place of the original GC pair.

3 When mRNA is transcribed from
the DNA containing this substitution,
a codon is produced that, during
translation, encodes a different
amino acid: tyrosine instead
of cysteine.

Daughter DNA
mRNA

Amino acids

Transcription
Translation
Replication
Replication

Cysteine Tyrosine Cysteine Cysteine

Figure 8.17 Base substitutions. This mutation leads to an altered protein in a grandchild cell.

Does a base substitution always result in a different amino acid? Q

CHAPTER 8 Microbial Genetics 223

results in the change from glutamic acid to valine in the protein.
This causes the shape of the hemoglobin molecule to change
under conditions of low oxygen, which, in turn, alters the shape
of the red blood cells.

By creating a nonsense (stop) codon in the middle of an
mRNA molecule, some base substitutions effectively prevent
the synthesis of a complete functional protein; only a fragment
is synthesized. A base substitution resulting in a nonsense
codon is thus called a nonsense mutation (Figure 8.18c).

Besides base-pair mutations, there are also changes in DNA
called frameshift mutations, in which one or a few nucleotide
pairs are deleted or inserted in the DNA (Figure 8.18d). This
mutation can shift the “translational reading frame”—that
is, the three-by-three grouping of nucleotides recognized as
codons by the tRNAs during translation. For example, delet-
ing one nucleotide pair in the middle of a gene causes changes
in many amino acids downstream from the site of the original
mutation. Frameshift mutations almost always result in a long
stretch of altered amino acids and the production of an inac-
tive protein from the mutated gene. In most cases, a nonsense
codon will eventually be encountered and thereby terminate
translation.

Base substitutions and frameshift mutations may occur
spontaneously because of occasional mistakes made during
DNA replication. These spontaneous mutations apparently
occur in the absence of any mutation-causing agents.

protein, this change in the DNA is known as a missense mutation
(Figure 8.18a and Figure 8.18b).

The effects of such mutations can be dramatic. For example,
sickle cell disease is caused by a single change in the gene for
globin, the protein component of hemoglobin. Hemoglobin is
primarily responsible for transporting oxygen from the lungs to
the tissues. A single change from an A to a T at a specific site

T A C T T C A A A C C G A T T

G AA U G A A G U U U G C U A

T A C T T C A A A C G A T T

G AA U G A A G U U U C U A

T
A

T A C T C A A A C G A T T

G AA U G A G U U U C U A

A
U G
C

T A C T C A A C G A T T

Met Lys Phe Gly

DNA (template strand)

mRNA

Amino acid sequence

(a) Normal DNA molecule

Met Lys Phe Ser

DNA (template strand)
mRNA
Amino acid sequence

(b) Missense mutation

Met

G AA U G A G U U U A

(d) Frameshift mutation

A
GA

Lys Leu Ala

Transcription
Translation
Stop
Stop
Stop
Met

Figure 8.18 Types of mutations and their effects on the
amino acid sequences of proteins.

Q What happens if base 9 in (a) is changed to a C?

CHECK YOUR UNDERSTANDING

✓ 8-9 How can a mutation be beneficial?

Mutagens

Chemical Mutagens
Agents in the environment, such as certain chemicals and
radiation, that directly or indirectly bring about mutations are
called mutagens.

One of the many chemicals known to be a mutagen is
nitrous acid. Figure 8.19 shows how exposing DNA to nitrous
acid can convert the base adenine to a form that pairs with
cytosine instead of the usual thymine. When DNA contain-
ing such modified adenines replicates, one daughter DNA
molecule will have a base-pair sequence different from
that of the parent DNA. Eventually, some AT base pairs of
the parent will have been changed to GC base pairs in a
granddaughter cell. Nitrous acid makes a specific base-pair
change in DNA. Like all mutagens, it alters DNA at random
locations.

Another type of chemical mutagen is the nucleoside
analog. These molecules are structurally similar to normal
nitrogenous bases, but they have slightly altered base-pairing

224 PART ONE Fundamentals of Microbiology

CH2OH

H
H

O
OH
H
H
H
H
N
N
N
N
H
H

N H H

N
N

N H

O
H

N
CH2OH O

H
H H

H
H
OH

AC
C
G
T
A
G

C T

A T

HNO2

Normal parent DNA

Replication

Normal daughter DNA

TG
AC
C
G
G
C T
A

Altered parent DNA

TG
AC
C
G
T
A
G
C T
A

Altered daughter DNA

TG
AC
C
G
G
C T
A

Mutated granddaughter DNA

TG
AC
C
G
G
C T
A
G

Altered granddaughter DNA

TG
AC
C
G
G
C T

A
Replication

C
C
C
A
A
A

(a) Adenosine nucleoside normally base-pairs by
hydrogen bonds with an oxygen and a hydrogen
of a thymine or uracil nucleotide.

(b) The altered adenine pairs with cytosine instead of
thymine.

Altered adenine will hydrogen bond with a
hydrogen and a nitrogen of a cytosine nucleotide.

Figure 8.19 Oxidation of nucleotides makes a mutagen. The nitrous acid emitted into
the air by burning fossil fuels oxidizes adenine.

Q What is a mutagen?

205 224 227 234

CLINICAL CASE

A person’s DNA can undergo mutations. One improper nucleotide in DNA creates a mutation, which could alter
the function of the gene. Cancer is abnormal cell growth
caused by mutations. These mutations can be inherited.

As Marcel and his wife, Janice, drive home from the
doctor’s office, they review Marcel’s family history. Marcel’s
brother, Robert, passed away from colon cancer 10 years
ago, but Marcel has always been the picture of health. Even
at 70, he hasn’t given a thought to retiring from his Memphis
barbeque restaurant that he once co-owned with his brother
until Robert’s death.

What factors may have contributed to Marcel’s colon cancer?

properties. Examples, 2-aminopurine and 5-bromouracil,
are shown in Figure 8.20. When nucleoside analogs are given
to growing cells, the analogs are randomly incorporated into
cellular DNA in place of the normal bases. Then, during DNA
replication, the analogs cause mistakes in base pairing. The

(a) The 2-aminopurine is incorporated into DNA in place of adenine but can
pair with cytosine, so an AT pair becomes a CG pair.

(b) The 5-bromouracil is used as an anticancer drug because it is mistaken
for thymine by cellular enzymes but pairs with cytosine. In the next DNA
replication, an AT pair becomes a GC pair.

Adenine nucleoside

Normal nitrogenous base

2-Aminopurine nucleoside

Analog

Thymine nucleoside 5-Bromouracil nucleoside

CH2OH
H
H
O
OH
H
H
H
H
N
N
N
N
H
H
N H H
N

N
N

NH2

NCH2OH
O

H
H H
H
H
OH
CH2OH
H
H
O
OH
H
H
H
O
O

H
H

CH3

CH2OH
H
H
O
OH
H
H
H
O
O
NN
H
H

Figure 8.20 Nucleoside analogs and the nitrogenous bases
they replace. A nucleoside is phosphorylated, and the resulting
nucleotide used to synthesize DNA.

Q Why do these drugs kill cells?

Play MicroFlix 3D Animation
@MasteringMicrobiology

CHAPTER 8 Microbial Genetics 2

DNA that is complementary to the correct strand. For many
years biologists questioned how the incorrect base could be
distinguished from the correct base if it was not physically dis-
torted like a thymine dimer. In 1970, Hamilton Smith provided

incorrectly paired bases will be copied during subsequent rep-
lication of the DNA, resulting in base-pair substitutions in the
progeny cells. Some antiviral and antitumor drugs are nucleo-
side analogs, including AZT (azidothymidine), used to treat
HIV infection.

Still other chemical mutagens cause small deletions or
insertions, which can result in frameshifts. For instance,
under certain conditions, benzopyrene, which is present
in smoke and soot, is an effective frameshift mutagen.
Aflatoxin— produced by Aspergillus flavus (a-sper-JIL-lus
FLĀ-vus), a mold that grows on peanuts and grain—is a
frameshift mutagen. Frameshift mutagens usually have
the right size and chemical properties to slip between the
stacked base pairs of the DNA double helix. They may work
by slightly offsetting the two strands of DNA, leaving a gap
or bulge in one strand or the other. When the staggered DNA
strands are copied during DNA synthesis, one or more base
pairs can be inserted or deleted in the new double-stranded
DNA. Interestingly, frameshift mutagens are often potent
carcinogens.

Radiation
X rays and gamma rays are forms of radiation that are potent
mutagens because of their ability to ionize atoms and mole-
cules. The penetrating rays of ionizing radiation cause electrons
to pop out of their usual shells (see Chapter 2). These electrons
bombard other molecules and cause more damage, and many
of the resulting ions and free radicals (molecular fragments
with unpaired electrons) are very reactive. Some of these ions
oxidize bases in DNA, resulting in errors in DNA replication
and repair that produce mutations (see Figure 8.19). An even
more serious outcome is the breakage of covalent bonds in
the sugar-phosphate backbone of DNA, which causes physical
breaks in chromosomes.

Another form of mutagenic radiation is ultraviolet (UV)
light, a nonionizing component of ordinary sunlight. However,
the most mutagenic component of UV light (wavelength
260 nm) is screened out by the ozone layer of the atmo-
sphere. The most important effect of direct UV light on DNA is
the formation of harmful covalent bonds between pyrimidine
bases. Adjacent thymines in a DNA strand can cross-link to
form thymine dimers. Such dimers, unless repaired, may cause
serious damage or death to the cell because it cannot properly
transcribe or replicate such DNA.

Bacteria and other organisms have enzymes that can repair
UV-induced damage. Photolyases, also known as light-repair
enzymes, use visible light energy to separate the dimer back to
the original two thymines. Nucleotide excision repair, shown
in Figure 8.21, is not restricted to UV-induced damage; it can
repair mutations from other causes as well. Enzymes cut out
the incorrect base and fill in the gap with newly synthesized

T
T T
T T
T

1 Exposure to ultraviolet light
causes adjacent thymines to
become cross-linked, forming
a thymine dimer and disrupting
their normal base pairing.

2 An endonuclease cuts the
DNA, and an exonuclease
removes the damaged DNA.

3 DNA polymerase fills the gap
by synthesizing new DNA,
using the intact strand as
a template.

4 DNA ligase seals the
remaining gap by joining the
old and new DNA.

Thymine dimer

Ultraviolet light

T T

New DNA

Figure 8.21 The creation and repair of a thymine dimer
caused by ultraviolet light. After exposure to UV light, adjacent
thymines can become cross-linked, forming a thymine dimer. In the
absence of visible light, the nucleotide excision repair mechanism is
used in a cell to repair the damage.

Q How do excision repair enzymes “know” which strand is
incorrect?

226 PART ONE Fundamentals of Microbiology

the answer with the discovery of methylases. These enzymes
add a methyl group to selected bases soon after a DNA strand
is made. A repair endonuclease then cuts the nonmethylated
strand.

The Frequency of Mutation
The mutation rate is the probability that a gene will mutate
when a cell divides. The rate is usually stated as a power of 10,
and because mutations are very rare, the exponent is always a
negative number. For example, if there is one chance in a mil-
lion that a gene will mutate when the cell divides, the mutation
rate is 1/1,000,000, which is expressed as 10-6. Spontaneous
mistakes in DNA replication occur at a very low rate, perhaps
only once in 109 replicated base pairs (a mutation rate of one in
a billion). Because the average gene has about 103 base pairs,
the spontaneous rate of mutation is about one in 106 (a mil-
lion) replicated genes.

Mutations usually occur more or less randomly along a
chromosome. The occurrence of random mutations at low fre-
quency is an essential aspect of the adaptation of species to
their environment, for evolution requires that genetic diver-
sity be generated randomly and at a low rate. For example, in
a bacterial population of significant size—say, greater than 107
cells—a few new mutant cells will always be produced in every
generation. Most mutations either are harmful and likely to be
removed from the gene pool when the individual cell dies or
are neutral. However, a few mutations may be beneficial. For
example, a mutation that confers antibiotic resistance is ben-
eficial to a population of bacteria that is regularly exposed to
antibiotics. Once such a trait has appeared through mutation,
cells carrying the mutated gene are more likely than other cells
to survive and reproduce as long as the environment stays the
same. Soon most of the cells in the population will have the
gene; an evolutionary change will have occurred, although on
a small scale.

A mutagen usually increases the spontaneous rate of muta-
tion, which is about one in 106 replicated genes, by a factor of
10 to 1000 times. In other words, in the presence of a muta-
gen, the normal rate of 10-6 mutations per replicated gene
becomes a rate of 10-5 to 10-3 per replicated gene. Mutagens
are used experimentally to enhance the production of mutant
cells for research on the
genetic properties of
microorganisms and for
commercial purposes.

CHECK YOUR UNDERSTANDING

✓ 8-10 How can mutations be repaired?
✓ 8-11 How do mutagens affect the mutation rate?

Play Mutations: Types, Repair
@MasteringMicrobiology

Identifying Mutants
Mutants can be detected by selecting or testing for an altered
phenotype. Whether or not a mutagen is used, mutant cells
with specific mutations are always rare compared with other
cells in the population. The problem is detecting such a rare
event.

Experiments are usually performed with bacteria because
they reproduce rapidly, so large numbers of organisms (more
than 109 per milliliter of nutrient broth) can easily be used.
Furthermore, because bacteria generally have only one copy of
each gene per cell, the effects of a mutated gene are not masked
by the presence of a normal version of the gene, as in many
eukaryotic organisms.

Positive (direct) selection involves the detection of mutant
cells by rejection of the unmutated parent cells. For exam-
ple, suppose we were trying to find mutant bacteria that are
resistant to penicillin. When the bacterial cells are plated on
a medium containing penicillin, the mutant can be identi-
fied directly. The few cells in the population that are resistant
(mutants) will grow and form colonies, whereas the normal,
penicillin-sensitive parental cells cannot grow.

To identify mutations in other kinds of genes, negative
(indirect) selection can be used. This process selects a cell
that cannot perform a certain function, using the technique of
replica plating. For example, suppose we wanted to use replica
plating to identify a bacterial cell that has lost the ability to
synthesize the amino acid histidine (Figure 8.22). First, about
100 bacterial cells are inoculated onto an agar plate. This plate,
called the master plate, contains a medium with histidine on
which all cells will grow. After 18 to 24 hours of incubation,
each cell reproduces to form a colony. Then a pad of sterile
material, such as latex, filter paper, or velvet, is pressed over
the master plate, and some of the cells from each colony
adhere to the velvet. Next, the velvet is pressed down onto two
(or more) sterile plates. One plate contains a medium with-
out histidine, and one contains a medium with histidine on
which the original, nonmutant bacteria can grow. Any colony
that grows on the medium with histidine on the master plate
but that cannot synthesize its own histidine will not be able
to grow on the medium without histidine. The mutant colony
can then be identified on the master plate. Of course, because
mutants are so rare (even those induced by mutagens), many
plates must be screened with this technique to isolate a spe-
cific mutant.

Replica plating is a very effective means of isolating
mutants that require one or more new growth factors. Any
mutant microorganism having a nutritional requirement that
is absent in the parent is known as an auxotroph. For example,
an auxotroph may lack an enzyme needed to synthesize a par-
ticular amino acid and will therefore require that amino acid
as a growth factor in its nutrient medium.

CHAPTER 8 Microbial Genetics 227

Identifying Chemical Carcinogens
Many known mutagens have been found to be carcinogens,
substances that cause cancer in animals, including humans. In
recent years, chemicals in the environment, the workplace, and
the diet have been implicated as causes of cancer in humans.
Animal testing procedures are time-consuming and expensive,
so some faster and less expensive procedures for preliminary
screening of potential carcinogens that do not use animals
have been developed. One of these, called the Ames test, uses
bacteria as carcinogen indicators.

The Ames test is based on the observation that exposure
of mutant bacteria to mutagenic substances may cause new
mutations that reverse the effect (the change in phenotype)
of the original mutation. These are called reversions. Specifi-
cally, the test measures the reversion of histidine auxotrophs
of Salmonella (so-called his- cells, mutants that have lost the
ability to synthesize histidine) to histidine-synthesizing cells
(his+) after treatment with a mutagen (Figure 8.23). Bacteria are
incubated in both the presence and absence of the substance
being tested. Because animal enzymes must activate many
chemicals into forms that are chemically reactive for mutagenic
or carcinogenic activity to appear, the chemical to be tested

1 Sterile velvet is
pressed on the grown
colonies on the
master plate.

Handle

Velvet
surface
(sterilized)

2 Cells from each colony
are transferred from the
velvet to new plates.

3 Plates are incubated.

4 Growth on plates is compared.
A colony that grows on the
medium with histidine but could
not grow on the medium without
histidine is auxotrophic
(histidine-requiring mutant).

Petri plate with
medium lacking
histidine

Colony missing

Petri plate with
medium containing
histidine

Master plate
with medium
containing
histidine

Auxotrophic mutant

Figure 8.22 Replica
plating. In this example, the
auxotrophic mutant cannot
synthesize histidine. The plates
must be carefully marked
(with an X here) to maintain
orientation so that colony
positions are known in relation
to the original master plate.

Q What is an auxotroph?

205 224 227 234
CLINICAL CASE

Not all mutations are inherited; some are induced by genotoxins, that is, chemicals that damage a cell’s genetic
material. Marcel is not overweight and has never smoked.
Researchers have known since the 1970s that people who
consume cooked meat and meat products are more likely to
develop colon cancer. The suspect cancer-causing chemicals
are aromatic amines that form during high-heat cooking.

Marcel has owned his Memphis barbeque restaurant
for over 50 years. He is a hands-on type of employer and is
always in the kitchen overseeing the cooking process. All of
his barbequed meat is seared over high heat and then slow-
cooked for hours. Marcel is considered the expert in this
technique, but now it seems as if his profession could be a
factor in his disease.

What test can be used to determine whether a chemical is
genotoxic?

228 PART ONE Fundamentals of Microbiology

color change of the pH indicator. The Ames test is routinely
used to evaluate new chemicals and air and water pollutants.

About 90% of the substances found by the Ames test to be
mutagenic have also been shown to be carcinogenic in ani-
mals. By the same token, the more mutagenic substances have
generally been found to be more carcinogenic.

and the mutant bacteria are incubated together with rat liver
extract, a rich source of activation enzymes. If the substance
being tested is mutagenic, it will cause the reversion of his-
bacteria to his+ bacteria at a rate higher than the spontaneous
reversion rate. The number of observed revertants indicates the
degree to which a substance is mutagenic and therefore pos-
sibly carcinogenic.

The Ames test can be performed in liquid media with a
pH indicator in a 96-well plate. Several potential mutagens
or different concentations of mutagens can be qualitatively
tested in different wells. Bacterial growth is determined by a

Suspected
mutagen

Rat liver
extract

Experimental
sample

Incubation

Experimental plate

Colonies of
revertant
bacteria

Rat liver
extract

Control
(no suspected
mutagen added)

Control plate

Cultures of
histidine-dependent
Salmonella Media lacking histidine

1 2 3 4Two cultures are pre-
pared of Salmonella
bacteria that have lost
the ability to synthesize
histidine (histidine-
dependent).

The suspected
mutagen is added to
the experimental
sample only; rat liver
extract (an activator)
is added to both
samples.

Each sample is poured onto
a plate of medium lacking
histidine. The plates are
then incubated at 378C for two
days. Only bacteria whose
histidine-dependent
phenotype has mutated back
(reverted) to histidine-
synthesizing will grow into
colonies.

The numbers of colonies on the experimental
and control plates are compared. The control
plate may show a few spontaneous
histidine-synthesizing revertants. The test
plates will show an increase in the number of
histidine-synthesizing revertants if the test
chemical is indeed a mutagen and potential
carcinogen. The higher the concentration of
mutagen used, the more revertant colonies
will result.

Incubation

Figure 8.23 The Ames reverse gene mutation test.

Q Do all mutagens cause cancer?

CHECK YOUR UNDERSTANDING

✓ 8-12 How would you isolate an antibiotic-resistant
bacterium? An antibiotic-sensitive bacterium?

✓ 8-13 What is the principle behind the Ames test?

CHAPTER 8 Microbial Genetics 229

Like mutation, genetic
recombination contributes to a
population’s genetic diversity,
which is the source of variation
in evolution. In highly evolved
organisms such as present-day microbes, recombination is
more likely to be beneficial than mutation because recombi-
nation will less likely destroy a gene’s function and may bring
together combinations of genes that enable the organism to
carry out a valuable new function.

The major protein that constitutes the flagella of Salmonella
is also one of the primary proteins that causes our immune sys-
tems to respond. However, these bacteria have the capability of
producing two different flagellar proteins. As our immune system
mounts a response against those cells containing one form of the
flagellar protein, those organisms producing the second are not
affected. Which flagellar protein is produced is determined by a
recombination event that apparently occurs somewhat randomly
within the chromosomal DNA. Thus, by altering the flagellar pro-
tein produced, Salmonella can better avoid the defenses of the host.

Vertical gene transfer occurs when genes are passed from
an organism to its offspring. Plants and animals transmit their
genes by vertical transmission. Bacteria can pass their genes
not only to their offspring, but also laterally, to other microbes
of the same generation. This is known as horizontal gene
transfer (see Figure 8.2). Horizontal gene transfer between nor-
mal microbiota and pathogens may be important in the spread
of antibiotic resistance. Horizontal gene transfer between
bacteria occurs in several ways. In all of the mechanisms, the
transfer involves a donor cell that gives a portion of its total
DNA to a recipient cell. Once transferred, part of the donor’s
DNA can be incorporated into the recipient’s DNA; the remain-
der is degraded by cellular
enzymes. The recipient cell
that incorporates donor DNA
into its own DNA is called a
recombinant. The transfer of
genetic material between bacteria is by no means a frequent
event; it may occur in only 1% or less of an entire population.
Let’s examine in detail the specific types of genetic transfer.

Plasmids and Transposons
Plasmids and transposons are genetic elements that exist out-
side chromosomes. They occur in both prokaryotic and eukary-
otic organisms, but this discussion focuses on their role in
genetic change in prokaryotes. Plasmids and transposons are
called mobile genetic elements because they can move from
one chromosome to another or from one cell to another.

Plasmids

Recall from Chapter 4 (page 90) that plasmids are self- replicating,
gene-containing, circular pieces of DNA about 1–5% the size of

Genetic Transfer and Recombination
LEARNING OBJECTIVES

8-14 Describe the functions of plasmids and transposons.

8-15 Differentiate horizontal and vertical gene transfer.

8-16 Compare the mechanisms of genetic recombination in
bacteria.

Genetic recombination refers to the exchange of genes between
two DNA molecules to form new combinations of genes on a
chromosome. Figure 8.24 shows one mechanism for genetic
recombination. If a cell picks up foreign DNA (called donor
DNA in the figure), some of it could insert into the cell’s
chromosome—a process called crossing over—and some of the
genes carried by the chromosomes are shuffled. The DNA has
recombined, so that the chromosome now carries a portion of
the donor’s DNA.

If A and B represent DNA from different individuals, how are
they brought close enough together to recombine? In eukary-
otes, genetic recombination is an ordered process that usually
occurs as part of the sexual cycle of the organism. Crossing over
generally takes place during the formation of reproductive cells,
such that these cells contain recombinant DNA. In bacteria,
genetic recombination can happen in a number of ways, which
we will discuss in the following sections.

1 DNA from one cell
aligns with DNA in the
recipient cell. Notice that
there is a nick in the
donor DNA.

Recipient
chromosome

RecA protein

Donor DNA

2 DNA from the donor aligns with
complementary base pairs in the
recipient’s chromosome. This can
involve thousands of base pairs.

3 RecA protein catalyzes
the joining of the two
strands.

4 The result is that the recipient’s
chromosome contains new DNA.
Complementary base pairs between
the two strands will be resolved by
DNA polymerase and ligase. The
donor DNA will be destroyed. The
recipient may now have one or more
new genes.

Figure 8.24 Genetic recombination by crossing over. Foreign
DNA can be inserted into a chromosome by breaking and rejoining
the chromosome. This can insert one or more new genes into the
chromosome. A photograph of RecA protein is shown in Figure 3.11a,
page 60.

Q What type of enzyme breaks the DNA?

ASM: Genetic variations can
impact microbial functions (e.g., in

biofilm formation, pathogenicity, and
drug resistance).

Play Horizontal Gene
Transfer: Overview
@MasteringMicrobiology

The number of antibiotic-resistant bacteria in our intestinal microbiome increases with age. The reason:
exposure to antibiotics. In the presence
of a bacteria-killing drug, a resistant

mutant will grow while the nonresistant,
or susceptible, bacteria die off. So over
the human life span, which includes many
episodes of illnesses and treatments, we
end up populated with more and more
antibiotic-resistant microbes.

At first, this seems like a desirable
effect. For instance, if beneficial intestinal
microbes survive a course of drugs meant
to treat your pneumonia, then you may not
experience medication side effects such
as GI discomfort or diarrhea. Unfortunately,
recent evidence shows that a drug-resistant
microbiome may actually threaten us in
ways we previously didn’t understand.

Scientists suspect that drug resistance
in pathogenic bacteria often originates from
drug-resistant normal microbiota. Nearly
half of the resistance genes identified
in intestinal bacteria are identical to
resistance genes found in pathogens.
Swapping of genes between species that
come in contact with each other (horizontal
gene transfer) happens easily in the

intestines, where large numbers of different
microbes mingle. In one study, Escherichia
coli bacteria that were resistant to the
drugs sulfonamide and ampicillin were
found residing in volunteers who ingested
E. coli bacteria that were susceptible to
these antibiotics. How could this be? The
researchers traced the drug-resistance
genes to a plasmid found in E. coli that had
resided in the volunteers before the study—
the resistant bacteria had transferred the
plasmid to the drug-susceptible bacteria
once the different strains met up in the
intestine. Likewise, resistance to the
drug vancomycin is believed to have
transferred from the commensal bacterium
Enterococcus faecalis to pathogenic strains
of Staphylococcus aureus. The resistance
gene was found on a conjugative plasmid in
both species.

Antibiotics remain an essential part of
modern health care. However, these days,
weighing whether an antibiotic is truly
needed is all the more important.

EXPLORING THE MICROBIOME Horizontal Gene Transfer and the
Unintended Consequences of Antibiotic Usage

Plasmids can be transferred between unrelated bacteria through
cytoplasmic bridges between cells.

the bacterial chromosome (Figure 8.25). They are found mainly
in bacteria but also in some eukaryotic microorganisms, such
as Saccharomyces cerevisiae. The F factor is a conjugative plasmid
that carries genes for sex pili and for the transfer of the plasmid
to another cell. Although plasmids are usually dispensable, under
certain conditions genes carried by plasmids can be crucial to
the survival and growth of the cell. For example, dissimilation
plasmids code for enzymes that trigger the catabolism of certain
unusual sugars and hydrocarbons. Some species of Pseudomonas
can actually use such exotic substances as toluene, camphor, and
petroleum as primary carbon and energy sources because they
have catabolic enzymes encoded by genes carried on plasmids.
Such specialized capabilities permit the survival of those micro-
organisms in very diverse and challenging environments. Because
of their ability to degrade and detoxify a variety of unusual com-
pounds, many of them are being investigated for possible use in
the cleanup of environmental wastes.

Other plasmids code for proteins that enhance the patho-
genicity of a bacterium. The strain of E. coli that causes infant
diarrhea and traveler’s diarrhea carries plasmids that code for

toxin production and for bacterial attachment to intestinal cells.
Without these plasmids, E. coli is a harmless resident of the large
intestine; with them, it is pathogenic. Other plasmid-encoded
toxins include the exfoliative toxin of Staphylococcus aureus,
Clostridium tetani neurotoxin, and toxins of Bacillus anthracis. Still
other plasmids contain genes for the synthesis of bacteriocins,
toxic proteins that kill other bacteria. These plasmids have been
found in many bacterial genera, and they are useful markers for
the identification of certain bacteria in clinical laboratories.

Resistance factors (R factors) are plasmids that have signif-
icant medical importance. They were first discovered in Japan
in the late 1950s after several dysentery epidemics. In some of
these epidemics, the infectious agent was resistant to the usual
antibiotic. Following isolation, the pathogen was also found to
be resistant to a number of different antibiotics. In addition,
other normal bacteria from the patients (such as E. coli) proved
to be resistant as well. Researchers soon discovered that these
bacteria acquired resistance through the spread of genes from
one organism to another. The plasmids that mediated this
transfer are R factors.

230

CHAPTER 8 Microbial Genetics 231

In some cases, the accumulation of resistance genes on a
single plasmid is quite remarkable. For example, Figure 8.25a
shows a genetic map of resistance plasmid R100. This particular
plasmid can be transferred between a number of enteric genera,
including Escherichia, Klebsiella, and Salmonella.

R factors present very serious problems for treating infec-
tious diseases with antibiotics. The widespread use of anti-
biotics in medicine and agriculture (see the box in Chapter
20 on page 583) has led to the preferential survival (selec-
tion) of bacteria that have R factors, so populations of resis-
tant bacteria grow larger and larger. The transfer of resistance
between bacterial cells of a population, and even between
bacteria of different genera, also contributes to the problem.
The ability to reproduce sexually with members of its own
species defines a eukaryotic species. However, a bacterial
species can conjugate and transfer plasmids to other species.
Neisseria may have acquired its penicillinase-producing
plasmid from Streptococcus, and Agrobacterium can transfer
plasmids to plant cells (see Figure 9.20, page 262). Noncon-
jugative plasmids may be transferred from one cell to another
by inserting themselves into a conjugative plasmid or a chro-
mosome or by transformation when released from a dead cell.
Insertion is made possible by an insertion sequence, which
will be discussed shortly.

Plasmids are an important tool for genetic engineering, dis-
cussed in Chapter 9 (pages 243–247).

Transposons
Transposons are small segments of DNA that can move (be
“transposed”) from one region of a DNA molecule to another.
These pieces of DNA are 700 to 40,000 base pairs long.

In the 1950s, American geneticist Barbara McClintock dis-
covered transposons in corn, but they occur in all organisms
and have been studied most thoroughly in microorganisms.
They may move from one site to another site on the same chro-
mosome or to another chromosome or plasmid. As you might
imagine, the frequent movement of transposons could wreak
havoc inside a cell. For example, as transposons move about on
chromosomes, they may insert themselves within genes, inacti-
vating them. Fortunately, transposition occurs relatively rarely.
The frequency of transposition is comparable to the spontane-
ous mutation rate that occurs in bacteria—that is, from 10-5 to
10-7 per generation.

All transposons contain the information for their own
transposition. As shown in Figure 8.26a, the simplest transpo-
sons, also called insertion sequences (IS), contain only a gene
that codes for an enzyme (transposase, which catalyzes the cut-
ting and resealing of DNA that occurs in transposition) and
recognition sites. Recognition sites are short inverted repeat
sequences of DNA that the enzyme recognizes as recombina-
tion sites between the transposon and the chromosome.

R factors carry genes that confer upon their host cell resis-
tance to antibiotics, heavy metals, or cellular toxins. Many
R factors contain two groups of genes. One group is called the
resistance transfer factor (RTF) and Includes genes for plasmid
replication and conjugation. The other group, the r-determinant,
has the resistance genes; it codes for the production of enzymes
that inactivate certain drugs or toxic substances (Figure 8.25a).
Different R factors, when present in the same cell, can recombine
to produce R factors with new combinations of genes in their
r-determinants.

R
T

r-determ
inant

Origin of
replication

Mercury
resistance

Sulfonamide
resistance

Streptomycin
resistance

Pilus and
conjugation

proteins

Origin of
transfer

Chloram-
phenicol
resistance

Tetracycline
resistance

(a)

(b) SEM 20 nm

Figure 8.25 R factor, a type of plasmid. (a) A diagram of an
R factor, which has two parts: the RTF contains genes needed for
plasmid replication and transfer of the plasmid by conjugation, and the
r-determinant carries genes for resistance to four different antibiotics
and mercury (sul = sulfonamide resistance, str = streptomycin
resistance, cml = chloramphenicol resistance, tet = tetracycline
resistance, mer = mercury resistance); numbers are base pairs * 1000.
(b) Plasmids from E. coli bacteria.

Q Why are R factors important in the treatment of infectious
diseases?

232 PART ONE Fundamentals of Microbiology

Transposons with antibiotic resistance genes are of practi-
cal interest, but there is no limitation on the kinds of genes
that transposons can have. Thus, transposons provide a natu-
ral mechanism for the movement of genes from one chromo-
some to another. Furthermore, because they may be carried
between cells on plasmids or viruses, they can also spread from
one organism—or even species—to another. For example, van-
comycin resistance was transferred from Enterococcus faecalis to
Staphylococcus aureus via a transposon called Tn1546. Trans-
posons are thus a
potentially powerful
mediator of evolu-
tion in organisms.

Kanamycin resistance

Tn5

IS1

Transposase gene

Transposase gene
IS1

Inverted repeat

A
T
C
G
T
A
T
A
A
T
C
G
T
A
G
C
A
T
T
A
A
T
T
A
C
G
A
T
G
C
T
A
A
T
A
T
G
C
T
A
Inverted repeat
IS1

IS1
IS1
A
T
C
G
T
A
T
A
A
T
C
G
T
A
G
C
A
T
A
T

G
A

CTA
A

T
G
T
T
A
A
T
T
A
C
G
A
T
G
C
T
A
A
T
A
T
G
C
T

A
1 Transposase cuts DNA, leaving sticky ends.

2 Sticky ends of transposon and target DNA anneal.

T
C

Kanam
ycin resistance

(a) An insertion sequence (IS), the simplest transposon, contains a gene for
transposase, the enzyme that catalyzes transposition. The tranposase gene
is bounded at each end by inverted repeat sequences that function as
recognition sites for the transposon. IS1 is one example of an insertion
sequence, shown here with simplified IR sequences.

(b) Complex transposons carry other genetic material in addition to
transposase genes. The example shown here, Tn5, carries the gene for
kanamycin resistance and has complete copies of the insertion sequence
IS1 at each end.

Figure 8.26 Transposons and insertion.

Q Why are transposons sometimes referred to as “jumping genes”?

Complex transposons also carry other genes not connected
with the transposition process. For example, bacterial transpo-
sons may contain genes for enterotoxin or for antibiotic resis-
tance (Figure 8.26b). Plasmids such as R factors are frequently
made up of a collection of transposons (Figure 8.26c).

Play Transposons: Overview, Insertion
Sequences, Complex Transposons
@MasteringMicrobiology

CHECK YOUR UNDERSTANDING

✓ 8-14 What types of genes do plasmids carry?

Transformation in Bacteria
During the process of transformation, genes are transferred
from one bacterium to another as “naked” DNA in solution.
This process was first demonstrated over 70 years ago, although
it was not understood at the time. Not only did transforma-
tion show that genetic material could be transferred from one
bacterial cell to another, but study of this phenomenon even-
tually led to the conclusion that DNA is the genetic material.
The initial experiment on transformation was performed by
Frederick Griffith in England in 1928 while he was working
with two strains of Streptococcus pneumoniae. One, a virulent
strain, has a polysaccharide capsule that prevents phagocy-
tosis. The bacteria grow and cause pneumonia. The other, an
avirulent strain, lacks the capsule and does not cause disease.

Griffith was interested in determining whether injections of
heat-killed bacteria of the encapsulated strain could be used to
vaccinate mice against pneumonia. As he expected, injections
of living encapsulated bacteria killed the mouse (Figure 8.27a);
injections of live nonencapsulated bacteria (Figure 8.27b) or
dead encapsulated bacteria (Figure 8.27c) did not kill the mouse.
However, when the dead encapsulated bacteria were mixed with
live nonencapsulated bacteria and injected into the mice, many
of the mice died. In the blood of the dead mice, Griffith found
living, encapsulated bacteria. Hereditary material (genes) from
the dead bacteria had entered the live cells and changed them
genetically so that their progeny were encapsulated and there-
fore virulent (Figure 8.27d).

Subsequent investigations based on Griffith’s research
revealed that bacterial transformation could be carried out
without mice. A broth was inoculated with live nonencapsu-
lated bacteria. Dead encapsulated bacteria were then added to
the broth. After incubation, the culture was found to contain
living bacteria that were encapsulated and virulent. The nonen-
capsulated bacteria had been transformed; they had acquired

CHAPTER 8 Microbial Genetics 233

1 Living encapsulated
bacteria injected into
mouse.

2 Mouse died.

1 Living nonencapsulated
bacteria injected into
mouse.

1 Heat-killed encapsulated
bacteria injected into
mouse.

1 Living nonencapsulated and
heat-killed encapsulated
bacteria injected into mouse.

2 Mouse remained healthy. 2 Mouse died.

3 Colonies of encapsulated
bacteria were isolated from
dead mouse.

3 A few colonies of nonencap-
sulated bacteria were isolated
from mouse; phagocytes
destroyed nonencapsulated
bacteria.

3 No colonies were isolated
from mouse.

3 Colonies of encapsulated
bacteria were isolated from
dead mouse.

2 Mouse remained healthy.

(a) (b) (c) (d)

RECOMBINATION

Figure 8.27 Griffith’s experiment
demonstrating genetic transformation.
(a) Living encapsulated bacteria caused
disease and death when injected into a mouse.
(b) Living nonencapsulated bacteria are
readily destroyed by the phagocytic defenses
of the host, so the mouse remained healthy
after injection. (c) After being killed by heat,

encapsulated bacteria lost the ability to cause
disease. (d) However, the combination of
living nonencapsulated bacteria and heat-
killed encapsulated bacteria (neither of which
alone causes disease) did cause disease.
Somehow, the live nonencapsulated bacteria
were transformed by the dead encapsulated
bacteria so that they acquired the ability

to form capsules and therefore cause
disease. Subsequent experiments proved the
transforming factor to be DNA.

Q Why did encapsulated bacteria kill the
mouse while nonencapsulated bacteria
did not? What killed the mouse in (d)?

a new hereditary trait by incorporating genes from the killed
encapsulated bacteria.

The next step was to extract various chemical components
from the killed cells to determine which component caused the
transformation. These crucial experiments were performed in
the United States by Oswald T. Avery and his associates Colin
M. MacLeod and Maclyn McCarty. After years of research,
they announced in 1944 that the component responsible for
transforming harmless S. pneumoniae into virulent strains was
DNA. Their results provided one of the conclusive indications
that DNA was indeed the carrier of genetic information.

Since the time of Griffith’s experiment, considerable infor-
mation has been gathered about transformation. In nature,
some bacteria, perhaps after death and cell lysis, release their

DNA into the environment. Other bacteria can then encounter
the DNA and, depending on the particular species and growth
conditions, take up fragments of DNA and integrate them
into their own chromosomes by recombination. A protein
called RecA binds to the cell’s DNA and then to donor DNA
causing the exchange of strands. A recipient cell with this
new combination of genes is a kind of hybrid, or recombi-
nant cell (Figure 8.28). All the descendants of such a recom-
binant cell will be identical to it. Transformation occurs
naturally among very few genera of bacteria, including Bacillus,
Haemophilus (hē-MAH-fil-us), Neisseria, Acinetobacter (a-sin-E-
tō-bak9ter), and certain strains of the genera Streptococcus and
Staphylococcus.

234 PART ONE Fundamentals of Microbiology

Conjugation differs from transformation in two major
ways. First, conjugation requires direct cell-to-cell contact.
Second, the conjugating cells must generally be of opposite
mating type; donor cells must carry the plasmid, and recipi-
ent cells usually do not. In gram-negative bacteria, the plasmid
carries genes that code for the synthesis of sex pili, projec-
tions from the donor’s cell surface that contact the recipient
and help bring the two cells into direct contact (Figure 8.29a).
Gram- positive bacterial cells produce sticky surface molecules
that cause cells to come into direct contact with each other.
In the process of conjugation, the plasmid is replicated during
the transfer of a single-stranded copy of the plasmid DNA to
the recipient, where the complementary strand is synthesized
(Figure 8.29b).

Because most experimental work on conjugation has been
done with E. coli, we will describe the process in this organ-
ism. In E. coli, the F factor (fertility factor) was the first
plasmid observed to be transferred between cells during con-
jugation. Donors carrying F factors (F+ cells) transfer the plas-
mid to recipients (F- cells), which become F+ cells as a result
(Figure 8.30a). In some cells carrying F factors, the factor

Even though only a small portion of a cell’s DNA is trans-
ferred to the recipient, the molecule that must pass through
the recipient cell wall and membrane is still very large. When a
recipient cell is in a physiological state in which it can take up
the donor DNA, it is said to be competent. Competence results
from alterations in the cell
wall that make it permeable
to large DNA molecules.

Conjugation in Bacteria
Another mechanism by which genetic material is transferred
from one bacterium to another is known as conjugation.
Conjugation is mediated by a conjugative plasmid, (discussed on
page 229).

1 Recipient cell takes
up donor DNA.

2 Donor DNA aligns
with complementary
bases.

b
c
d

Recipient cell

Chromosomal DNADNA fragments
from donor cells

3 Recombination occurs
between donor DNA
and recipient DNA.

Genetically transformed cell

Degraded
unrecombined DNA

a
b
c
d

3¿5¿

A D

C
B
A D
C
b
c
d
B
A D
C

a D

B C

5¿ 3¿

Figure 8.28 The mechanism of genetic transformation in
bacteria. Some similarity is needed for the donor and recipient to
align. Genes a, b, c, and d may be mutations of genes A, B, C, and D.

Q What type of enzyme cuts the donor DNA?

Play Transformation
@MasteringMicrobiology

205 224 227 234

CLINICAL CASE Resolved

The Ames test allows rapid screening of chemicals for genotoxicity. The his- mutant Salmonella bacteria used in
the Ames test are spread over glucose–minimal salts agar
plates. A paper disk saturated with 2-aminofluorene (2-AF),
an aromatic amine, is placed on the culture. The figure, for
example, shows that reversion of the his- mutation allowed
the Salmonella to grow. This indicates that the chemical is
mutagenic and is therefore potentially carcinogenic.

There are studies indicating
that 2-AF activated by enzymes
is more damaging than 2-AF
alone, suggesting that the
interaction between diet and
intestinal microbiota is more
likely to cause cancer than just
diet. Variations in diet produce
little change in the kinds of
bacteria in the intestine, but they
produce dramatic changes in the
metabolic activity of the bacteria.

The detection of serrated colorectal polyps from Marcel’s
stool DNA test led to a diagnosis of an early, rather than late,
stage of colorectal cancer. The offending polyps are found
and removed, and Marcel undergoes chemotherapy to kill any
missed cancer cells in his colon.

CHAPTER 8 Microbial Genetics 2

Sex pilus

F+ cell

F– cell

Mating
bridge

(a) Sex pilus (b) Mating bridge TEMSEM
0.3 mm1.5 mm

Figure 8.29 Bacterial conjugation.

Q What is an F+ cell?

integrates into the chromosome, converting the F+ cell to an
Hfr cell (high frequency of recombination) (Figure 8.30b).
When conjugation occurs between an Hfr cell and an F- cell,
the Hfr cell’s chromosome (with its integrated F factor) repli-
cates, and a parental strand of the chromosome is transferred
to the recipient cell (Figure 8.30c). Replication of the Hfr chro-
mosome begins in the middle of the integrated F factor, and a
small piece of the F factor leads the chromosomal genes into
the F- cell. Usually, the chromosome breaks before it is com-
pletely transferred. Once within the recipient cell, donor DNA
can recombine with the recipient’s DNA. (Donor DNA that is
not integrated is degraded.) Therefore, by conjugation with
an Hfr cell, an F- cell may acquire new versions of chromo-
somal genes (just as in transformation). However, it remains
an F- cell because it did not receive a complete F factor during
conjugation.

Conjugation is used to map the location of genes on a bac-
terial chromosome (Figure 8.31). The genes for the synthesis of
threonine (thr) and leucine (leu) are first, reading clockwise from
0. Their locations were determined by conjugation experiments.
Assume that conjugation is allowed for only 1 minute between an
Hfr strain that is his+, pro+, thr+, and leu+, and an F- strain that
is his-, pro-, thr-, and leu-. If the F- acquired the ability to syn-
thesize threonine, then the thr gene is located early in the chro-
mosome, between 0 and 1 minute. If after 2 minutes the F- cell
now becomes thr+ and leu+,
the order of these two genes
on the chromosome must be
thr, leu.

Transduction in Bacteria
A third mechanism of genetic transfer between bacteria is
transduction. In this process, bacterial DNA is transferred from
a donor cell to a recipient cell inside a virus that infects bacte-
ria, called a bacteriophage, or phage. (Phages will be discussed
further in Chapter 13.)

To understand how transduction works, we will consider
the life cycle of one type of transducing phage of E. coli; this
phage carries out generalized transduction (Figure 8.32).

During phage reproduction, phage DNA and proteins are
synthesized by the host bacterial cell. The phage DNA should
be packaged inside the phage protein coat. However, bacterial
DNA, plasmid DNA, or even
DNA of another virus may be
packaged inside a phage pro-
tein coat.

All genes contained within
a bacterium infected by a generalized transducing phage are
equally likely to be packaged in a phage coat and transferred.
In another type of transduction, called specialized transduction,
only certain bacterial genes are transferred (see page 375). In
one type of specialized transduction, the phage codes for certain
toxins produced by their bacterial hosts, such as diphtheria
toxin for Corynebacterium diphtheriae (kor9Ī-nē-bak-TI-rē-um dif-
THI-rē-Ī), erythrogenic toxin for Streptococcus pyogenes, and Shiga
toxin for E. coli O157:H7.

CHECK YOUR UNDERSTANDING

✓ 8-15 Differentiate horizontal and vertical gene transfer.
✓ 8-16 Compare conjugation between the following pairs: F + *

F -, Hfr * F -.

Play Conjugation: Overview,
F Factor, Hfr Conjugation,
Chromosome Mapping
@MasteringMicrobiology

Play Transduction:
Generalized Transduction
@MasteringMicrobiology

236 PART ONE Fundamentals of Microbiology

Recombination between
F factor and chromosome,
occurring at a specific site
on each

Insertion of

F factor

into chromosome

Hfr cellF+ cell

Integrated F factor

F+ cell F– cell

Replication
and transfer
of F factor

F+ cell F+ cell

(a) When an F factor (a plasmid) is transferred from a donor (F+) to a recipient (F–), the F– cell is converted to an F+ cell.

(b) When an F factor becomes integrated into the chromosome of an F+ cell, it makes the cell a high frequency of recombination (Hfr) cell.

Replication
and transfer
of part of the
chromosome

In the recipient,
recombination
between the
Hfr chromosome
fragment and the
F– chromosome

Hfr cell

Recombinant
F– cell

Hfr cell F– cell

Bacterial
chromosome

F factor

Mating bridge

RECOMBINATION

Figure 8.30 Conjugation in E. coli.

Q Do bacteria reproduce during conjugation?

CHAPTER 8 Microbial Genetics 237

Bacterial
DNA

Phage
DNA

Recipient
bacterial
DNA

Many cell
divisions

Donor
bacterial
DNA

1 A phage infects the
donor bacterial cell.

3 Occasionally during phage assembly,
pieces of bacterial DNA are pack-
aged in a phage capsid. Then the
donor cell lyses and releases phage
particles containing bacterial DNA.

Phage protein coat

Bacterial
chromosome

Donor
cell

Phage DNA

Recipient
cell

Recombinant
cell reproduces
normally

2 Phage DNA and proteins are made,
and the bacterial chromosome is
broken into pieces.

4 A phage carrying
bacterial DNA infects
a new host cell, the
recipient cell.

5 Recombination can
occur, producing a
recombinant cell with
a genotype different
from both the donor
and recipient cells.

RECOMBINATION

Figure 8.32 Transduction by a bacteriophage. Shown here is
generalized transduction, in which any bacterial DNA can be transferred
from one cell to another.

Q How could E. coli acquire the Shiga toxin gene?

Genes and Evolution
LEARNING OBJECTIVE

8-17 Discuss how genetic
mutation and
recombination provide
material for natural
selection to act upon.

We have now seen how gene activity can be controlled by the
cell’s internal regulatory mechanisms and how genes them-
selves can be altered or rearranged by mutation, transposition,
and recombination. All these processes provide diversity in the
descendants of cells. Diversity provides the raw material for evo-
lution, and natural selection provides its driving force. Natural
selection will act on diverse populations to ensure the survival of
those fit for that particular environment. The different kinds of
microorganisms that exist today are the result of a long history
of evolution. Microorganisms have continually changed by alter-
ations in their genetic properties and acquisition of adaptations

Play Interactive Microbiology
@MasteringMicrobiology
See how the selection of
antibiotic-resistant microbes
affects a patient’s health

CHECK YOUR UNDERSTANDING

✓ 8-17 Natural selection means that the environment favors
survival of some genotypes. From where does diversity in
genotypes come?

Amino acid metabolism

DNA replication and repair

Lipid metabolism

KEY

Carbohydrate metabolism

Membrane synthesis

50
60

3480 kbp

bp1

1160 kbp

2320 kbp

Figure 8.31 A genetic map of the chromosome of E. coli.
This map is made by observing recombinant cells after conjugation.
The numbers inside the circle indicate the number of minutes it takes
to transfer the genes during mating between two cells; the numbers in
colored boxes indicate the number of base pairs. 1 kbp = 1000 base
pairs.

Q How many minutes of conjugation would be needed to transfer
genes for membrane synthesis on this chromosome?

to many different habitats. See Exploring the Microbiome on
page 230 and the box on antibiotic resistance in Chapter 26,
page 771, for examples of natural selection.

238 PART ONE Fundamentals of Microbiology

Structure and Function of the Genetic
Material (pp. 205–217)
1. Genetics is the study of what genes are, how they carry

information, how their information is expressed, and how they
are replicated and passed to subsequent generations or other
organisms.

2. DNA in cells exists as a double-stranded helix; the two strands are
held together by hydrogen bonds between specific nitrogenous
base pairs: AT and CG.

3. A gene is a sequence of nucleotides, that encodes a functional
product, usually a protein.

4. The DNA in a cell is duplicated before the cell divides, so each
offspring cell receives the same genetic information.

Genotype and Phenotype (pp. 205, 208)
5. Genotype is the genetic composition of an organism, its entire

complement of DNA.

6. Phenotype is the expression of the genes: the proteins of the cell
and the properties they confer on the organism.

DNA and Chromosomes (p. 208)
7. The DNA in a chromosome exists as one long double helix

associated with various proteins that regulate genetic activity.

8. Genomics is the molecular characterization of genomes.

The Flow of Genetic Information (p. 208)
9. Following cell division, each offspring cell receives a chromosome

that is virtually identical to the parent’s.

10. Information contained in the DNA is transcribed into RNA and
translated into proteins.

DNA Replication (pp. 208–212)
11. During DNA replication, the two strands of the double helix

separate at the replication fork, and each strand is used as a
template by DNA polymerases to synthesize two new strands of
DNA according to the rules of complementary base pairing.

12. The result of DNA replication is two new strands of DNA, each
having a base sequence complementary to one of the original strands.

13. Because each double-stranded DNA molecule contains one
original and one new strand, the replication process is called
semiconservative.

14. DNA is synthesized in one direction designated 5’ S 3’. At the
replication fork, the leading strand is synthesized continuously and
the lagging strand discontinuously.

15. DNA polymerase proofreads new molecules of DNA and removes
mismatched bases before continuing DNA synthesis.

RNA and Protein Synthesis (pp. 212–217)
16. During transcription, the enzyme RNA polymerase synthesizes a

strand of RNA from one strand of double-stranded DNA, which
serves as a template.

17. RNA is synthesized from nucleotides containing the bases A, C, G, and
U, which pair with the bases of the DNA strand being transcribed.

18. RNA polymerase binds the promoter; transcription begins at AUG;
the region of DNA that is the end point of transcription is the
terminator; RNA is synthesized in the 5’ S 3’ direction.

19. Translation is the process in which the information in the
nucleotide base sequence of mRNA is used to dictate the amino
acid sequence of a protein.

20. The mRNA associates with ribosomes, which consist of rRNA and
protein.

21. Three-base codons of mRNA specify amino acids.

22. The genetic code refers to the relationship among the nucleotide
base sequence of DNA, the corresponding codons of mRNA, and
the amino acids for which the codons code.

23. Specific amino acids are attached to molecules of tRNA. Another
portion of the tRNA has a base triplet called an anticodon.

24. The base pairing of codon and anticodon at the ribosome results in
specific amino acids being brought to the site of protein synthesis.

25. The ribosome moves along the mRNA strand as amino acids are joined
to form a growing polypeptide; mRNA is read in the 5’ S 3’ direction.

26. Translation ends when the ribosome reaches a stop codon on the
mRNA.

The Regulation of Bacterial Gene Expression
(pp. 217–221)

1. Regulating protein synthesis at the gene level is energy-efficient
because proteins are synthesized only as they are needed.

2. Constitutive genes are expressed at a fixed rate. Examples are genes
for the enzymes in glycolysis.

Pre-transcriptional Control (pp. 217–220)
3. In bacteria, a group of coordinately regulated structural genes with

related metabolic functions, plus the promoter and operator sites
that control their transcription, is called an operon.

4. In the operon model for an inducible system, a regulatory gene
codes for the repressor protein.

5. When the inducer is absent, the repressor binds to the operator,
and no mRNA is synthesized.

6. When the inducer is present, it binds to the repressor so that it
cannot bind to the operator; thus, mRNA is made, and enzyme
synthesis is induced.

7. In repressible systems, the repressor requires a corepressor in order
to bind to the operator site; thus, the corepressor controls enzyme
synthesis.

8. Transcription of structural genes for catabolic enzymes (such as
b-galactosidase) is induced by the absence of glucose. Cyclic AMP
and CRP must bind to a promoter in the presence of an alternative
carbohydrate.

9. Methylated nucleotides are not transcribed in epigenetic control.

Post-transcriptional Control (pp. 220–221)
10. mRNA as a riboswitch regulates translation.

11. MicroRNAs combine with mRNA; the resulting double-stranded
RNA is destroyed.

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CHAPTER 8 Microbial Genetics 239

2. In crossing over, genes from two chromosomes are recombined
into one chromosome containing some genes from each original
chromosome.

3. Vertical gene transfer occurs during reproduction when genes are
passed from an organism to its offspring.

4. Horizontal gene transfer in bacteria involves a portion of the cell’s
DNA being transferred from donor to recipient.

5. When some of the donor’s DNA has been integrated into the
recipient’s DNA, the resultant cell is called a recombinant.

Plasmids and Transposons (pp. 229–232)
6. Plasmids are self-replicating circular molecules of DNA carrying

genes that are not usually essential for the cell’s survival.

7. There are several types of plasmids, including conjugative plasmids,
dissimilation plasmids, plasmids carrying genes for toxins or
bacteriocins, and resistance factors.

8. Transposons are small segments of DNA that can move from one
region to another region of the same chromosome or to a different
chromosome or a plasmid.

9. Complex transposons can carry any type of gene, including
antibiotic-resistance genes, and are thus a natural mechanism for
moving genes from one chromosome to another.

Transformation in Bacteria (pp. 232–234)
10. During this process, genes are transferred from one bacterium to

another as “naked” DNA in solution.

Conjugation in Bacteria (pp. 234–235)
11. This process requires contact between living cells.

12. One type of genetic donor cell is an F+; recipient cells are F-. F cells
contain plasmids called F factors; these are transferred to the F-
cells during conjugation.

Transduction in Bacteria (pp. 235–237)
13. In this process, DNA is passed from one bacterium to another in a

bacteriophage and is then incorporated into the recipient’s DNA.

14. In generalized transduction, any bacterial genes can be
transferred.

Genes and Evolution (p. 237)
1. Diversity is the

precondition for
evolution.

2. Genetic mutation and
recombination provide
diversity of organisms,
and the process of natural selection allows the growth of those best
adapted to a given environment.

Changes in Genetic Material (pp. 221–228)
1. Mutations and horizontal gene transfer can change a bacterium’s

genotype.

Mutation (p. 222)
2. A mutation is a change in the nitrogenous base sequence of DNA;

that change causes a change in the product coded for by the
mutated gene.

3. Many mutations are neutral, some are disadvantageous, and others
are beneficial.

Types of Mutations (pp. 222–223)
4. A base substitution occurs when one base pair in DNA is replaced

with a different base pair.

5. Alterations in DNA can result in missense mutations, frameshift, or
nonsense mutations.

6. Spontaneous mutations occur without the presence of any
mutagen.

Mutagens (pp. 223–226)
7. Mutagens are agents in the environment that cause permanent

changes in DNA.

8. Ionizing radiation causes the formation of ions and free radicals
that react with DNA; base substitutions or breakage of the sugar-
phosphate backbone results.

9. Ultraviolet (UV) radiation is nonionizing; it causes bonding
between adjacent thymines.

The Frequency of Mutation (p. 226)
10. Mutation rate is the probability that a gene will mutate when a cell

divides; the rate is expressed as 10 to a negative power.

11. A low rate of spontaneous mutations is beneficial in providing the
genetic diversity needed for evolution.

Identifying Mutants (p. 226)
12. Mutants can be detected by selecting or testing for an altered

phenotype.

13. Positive selection involves the selection of mutant cells and the
rejection of nonmutated cells.

14. Replica plating is used for negative selection—to detect, for
example, auxotrophs that have nutritional requirements not
possessed by the parent (nonmutated) cell.

Identifying Chemical Carcinogens (pp. 227–228)
15. The Ames test is a relatively inexpensive and rapid test for

identifying possible chemical carcinogens.

16. The test assumes that a mutant cell can revert to a normal cell
in the presence of a mutagen and that many mutagens are
carcinogens.

Genetic Transfer and Recombination (pp. 229–237)
1. Genetic recombination, the rearrangement of genes from separate

groups of genes, usually involves DNA from different organisms; it
contributes to genetic diversity.

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240 PART ONE Fundamentals of Microbiology

c. Write the code for the complementary strand of DNA completed
in part (a).

d. What would be the effect if C were substituted for T at base 10?
e. What would be the effect if A were substituted for G at base 11?
f. What would be the effect if G were substituted for T at base 14?
g. What would be the effect if C were inserted between bases 9

and 10?
h. How would UV radiation affect this strand of DNA?
i. Identify a nonsense sequence in this strand of DNA.

5. When iron is not available, E. coli can stop synthesis of all proteins,
such as superoxide dismutase and succinate dehydrogenase, that
require iron. Describe a mechanism for this regulation.

6. Identify when (before transcription, after transcription but before
translation, after translation) each of the following regulatory
mechanisms functions.
a. ATP combines with an enzyme, altering its shape.
b. A short RNA is synthesized that is complementary to mRNA.
c. Methylation of DNA occurs.
d. An inducer combines with a repressor.

7. Which sequence is the best target for damage by UV radiation:
AGGCAA, CTTTGA, or GUAAAU? Why aren’t all bacteria killed
when they are exposed to sunlight?

8. You are provided with cultures with the following characteristics:

Culture 1: F+, genotype A+ B+ C+

Culture 2: F-, genotype A- B- C-

a. Indicate the possible genotypes of a recombinant cell resulting
from the conjugation of cultures 1 and 2.

b. Indicate the possible genotypes of a recombinant cell resulting
from conjugation of the two cultures after the F+ has become an
Hfr cell.

9. Why are mutation and recombination important in the process of
natural selection and the evolution of organisms?

10. NAME IT Normally a commensal in the human intestine, this
bacterium became pathogenic after acquiring a toxin gene from a
Shigella bacterium.

Multiple Choice
Match the following terms to the definitions in questions 1and 2.

a. conjugation
b. transcription
c. transduction
d. transformation
e. translation

1. Transfer of DNA from a donor to a recipient cell by a
bacteriophage.

2. Transfer of DNA from a donor to a recipient as naked DNA in
solution.

3. Feedback inhibition differs from repression because feedback
inhibition
a. is less precise.
b. is slower acting.
c. stops the action of preexisting enzymes.
d. stops the synthesis of new enzymes.
e. all of the above

3. Match the following examples of mutagens.

Column A

_______ a. A mutagen that is incorporated
into DNA in place of a normal
base

_______ b. A mutagen that causes the
formation of highly reactive
ions

_______ c. A mutagen that alters adenine
so that it base-pairs with
cytosine

_______ d. A mutagen that causes
insertions

_______ e. A mutagen that causes the
formation of pyrimidine
dimers

Column B

1. Frameshift mutagen

2. Nucleoside analog

3. Base-pair mutagen

4. Ionizing radiation

5. Nonionizing radiation

4. The following is a code for a strand of DNA.

DNA 3’ A T A T _ _ _ T T T _ _ _ _ _ _ _ _ _
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

mRNA C G U U G A

tRNA U G G

Amino Acid Met

ATAT = Promoter sequence

For answers to the Knowledge and Comprehension questions, turn to
the Answers tab at the back of the textbook.

Knowledge and Comprehension

Review
1. Briefly describe the components of DNA, and explain its functional

relationship to RNA and protein.

2. DRAW IT Identify and mark each of the following on the
portion of DNA undergoing replication: replication fork, DNA
polymerase, RNA primer, parent strands, leading strand, lagging
strand, the direction of replication on each strand, and the 5’ end
of each strand.

Study Questions

5¿
3¿

a. Using the genetic code provided in Figure 8.8, fill in the blanks
to complete the segment of DNA shown.

b. Fill in the blanks to complete the sequence of amino acids
coded for by this strand of DNA.

CHAPTER 8 Microbial Genetics 241

2. Replication of the E. coli chromosome takes 40 to 45 minutes,
but the organism has a generation time of 26 minutes. How
does the cell have time to make complete chromosomes for each
offspring cell?

3. Pseudomonas has a plasmid containing the mer operon, which
includes the gene for mercuric reductase. This enzyme catalyzes
the reduction of the mercuric ion Hg2+ to the uncharged form of
mercury, Hg0. Hg2+ is quite toxic to cells; Hg0 is not.
a. What do you suppose is the inducer for this operon?
b. The protein encoded by one of the mer genes binds Hg2+ in the

periplasm and brings it into the cell. Why would a cell bring in
a toxin?

Clinical Applications and Evaluation
1. Ciprofloxacin, erythromycin, and acyclovir are used to treat

microbial infections. Ciprofloxacin inhibits DNA gyrase.
Erythromycin binds in front of the A site on the 50S subunit of a
ribosome. Acyclovir is a guanine analog.
a. What steps in protein synthesis are inhibited by each drug?
b. Which drug is more effective against bacteria? Why?
c. Which drugs will have effects on the host’s cells? Why?
d. Use the index to identify the disease for which acyclovir is

primarily used. Why is it more effective than erythromycin for
treating this disease?

2. HIV, the virus that causes AIDS, was isolated from three
individuals, and the amino acid sequences for the viral coat were
determined. Of the amino acid sequences shown below, which two
of the viruses are most closely related? How can these amino acid
sequences be used to identify the source of a virus?

Patient Viral Amino Acid Sequence

A Asn Gln Thr Ala Ala Ser Lys Asn Ile Asp Ala Leu

B Asn Leu His Ser Asp Lys Ile Asn Ile Ile Leu Leu

C Asn Gln Thr Ala Asp Ser Ile Val Ile Asp Ala Leu

3. Human herpesvirus-8 (HHV-8) is common in parts of Africa, the
Middle East, and the Mediterranean, but is rare elsewhere except in
AIDS patients. Genetic analyses indicate that the African strain is
not changing, whereas the Western strain is accumulating changes.
Using the portions of the HHV-8 genomes (shown below) that
encode one of the viral proteins, how similar are these two viruses?
What mechanism can account for the changes? What disease does
HHV-8 cause?

Western 3’-ATGGAGTTCTTCTGGACAAGA
African 3’-AT A A AC TT T TTCT T GACAA CG

4. Bacteria can acquire antibiotic resistance by all of the following
except
a. mutation.
b. insertion of transposons.
c. conjugation.
d. snRNPs.
e. transformation.

5. Suppose you inoculate three flasks of minimal salts broth with
E. coli. Flask A contains glucose. Flask B contains glucose and
lactose. Flask C contains lactose. After a few hours of incubation,
you test the flasks for the presence of b-galactosidase. Which
flask(s) do you predict will have this enzyme?
a. A
b. B
c. C
d. A and B
e. B and C

6. Plasmids differ from transposons in that plasmids
a. become inserted into chromosomes.
b. are self-replicated outside the chromosome.
c. move from chromosome to chromosome.
d. carry genes for antibiotic resistance.
e. none of the above

Use the following choices to answer questions 7 and 8:

a. catabolite repression
b. DNA polymerase
c. induction
d. repression
e. translation

7. Mechanism by which the presence of glucose inhibits the lac
operon.

8. The mechanism by which lactose controls the lac operon.

9. Two offspring cells are most likely to inherit which one of the
following from the parent cell?
a. a change in a nucleotide in mRNA
b. a change in a nucleotide in tRNA
c. a change in a nucleotide in rRNA
d. a change in a nucleotide in DNA
e. a change in a protein

10. Which of the following is not a method of horizontal gene transfer?
a. binary fission
b. conjugation
c. integration of a transposon
d. transduction
e. transformation

Analysis
1. Nucleoside analogs and ionizing radiation are used in treating

cancer. These mutagens can cause cancer, so why do you suppose
they are used to treat the disease?

▶ Human immunodeficiency virus (HIV) (yellow)
budding from a host cell.

242

F or thousands of years, people have consumed foods produced by the action of microorganisms. Bread, chocolate, and soy
sauce are some of the best-known examples. But it
was only just over 100 years ago that scientists showed
that microorganisms are responsible for these products.
This knowledge opened the way for using microorganisms
to produce other important products. Since World War I,
microbes have been used to produce a variety of chemicals,
such as ethanol, acetone, and citric acid. Since World War II,
microorganisms have been grown to produce antibiotics. More
recently, microbes and their enzymes are replacing a variety of
chemical processes involved in manufacturing such products as
paper, textiles, and fructose. Using microbes or their enzymes
instead of chemical syntheses offers several advantages: microbes
may use inexpensive, abundant raw materials; microbes work at
normal temperatures and pressure, thereby avoiding the need for
expensive and dangerous systems; and microbes don’t produce
toxic, hard-to-treat wastes. In the past 30 years, DNA technology
has been added to the tools used to make products.

In this chapter you will learn the tools and techniques that
are used to research and develop a product. You will also learn
how DNA technology is used to track outbreaks of infectious
disease and to provide evidence for courts of law in forensic
microbiology. The Clinical Case illustrates the use of DNA
technology to track HIV (see the photo).

In the Clinic
A crime suspect claims he is innocent. His clothing became bloodstained, he says, when he
tried to resuscitate the victim. The pattern of blood on the suspect may have resulted from
striking the victim, but it is also consistent with spatter from the victim’s nose and mouth
during CPR. As a forensic nurse for a police department, you collect bloodstained fabric from
the suspect and a blood sample from the crime scene. You request PCR for streptococci on
both samples. The test is positive for the fabric, but negative for the blood at the scene.
How can a PCR test detect evidence of Streptococcus
bacteria in such small samples? Do these results help or
hurt the suspect?

Hint: Read about the polymerase chain reaction technique on page 247.

Biotechnology and
DNA Technology9

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CHAPTER 9 Biotechnology and DNA Technology 243

Introduction to Biotechnology
LEARNING OBJECTIVES

9-1 Compare and contrast biotechnology, genetic modification,
and recombinant DNA technology.

9-2 Identify the roles of a clone and a vector in making
recombinant DNA.

Biotechnology is the use of microorganisms, cells, or cell com-
ponents to make a product. Microbes have been used in the
commercial production of foods, vaccines, antibiotics, and
vitamins for years. Bacteria are also used in mining to extract
valuable elements from ore (see Figure 28.12, page 822). Addi-
tionally, animal cells have been used to produce viral vaccines
since the 1950s. Until the 1980s, products made by living cells
were all made by naturally occurring cells; the role of scientists
was to find the appropriate cell and develop a method for their
large-scale cultivation.

Now, microorganisms and plants are being used as “facto-
ries” to produce chemicals that the organisms don’t naturally
make. This is accomplished by inserting, deleting, or modify-
ing genes with recombinant
DNA (rDNA) technology,
which is sometimes called
genetic engineering. The devel-
opment of rDNA technology is expanding the practical applica-
tions of biotechnology almost beyond imagination.

Recombinant DNA Technology
Recombination of DNA occurs naturally in microbes (see
Chapter 8). In the 1970s and 1980s, scientists developed artifi-
cial techniques for making rDNA.

A gene from one organism can be inserted into the DNA of
a bacterium or a yeast. In many cases, the recipient can then be
made to express the gene, which may code for a commercially
useful product. Thus, bacteria with genes for human insulin are
now being used to produce insulin for treating diabetes, and a vac-
cine for hepatitis B is being made by yeast carrying a gene for part
of the hepatitis virus (the yeast produces a viral coat protein). Sci-
entists hope that such an approach may prove useful in produc-
ing vaccines against other infectious agents, thus eliminating the
need to use whole organisms, as in conventional vaccines.

The rDNA techniques can also be used to make thousands
of copies of the same DNA molecule—to amplify DNA—thus
generating sufficient DNA for various kinds of experimenta-
tion and analysis. This technique has practical application for
identifying microbes, such as viruses, that can’t be cultured.

An Overview of Recombinant DNA Procedures
An overview of some of the procedures typically used for mak-
ing rDNA, along with some promising applications, is shown
in Figure 9.1. A vector is a DNA molecule that transports

ASM: Cell genomes can be manipulated
to alter cell function.

foreign DNA into a cell. (See more on vectors on page 246.)
The gene of interest is inserted into the vector DNA in vitro. In
Figure 9.1, the vector is a plasmid. The DNA molecule chosen
as a vector must be self-replicating, such as a plasmid or a viral
genome. This recombinant vector DNA is taken up by a cell
such as a bacterium, where it can multiply. The cell contain-
ing the recombinant vector is then grown in culture to form a
clone of many genetically identical cells, each of which carries
copies of the vector, and therefore many copies of the gene of
interest. This is why DNA vectors are often called gene-cloning
vectors, or simply cloning vectors. (In addition to referring to a
culture of identical cells, the word clone is also routinely used
as a verb, to describe the entire process, as in “to clone a gene.”)

The final step varies according to whether the gene itself or
the product of the gene is of interest. From the cell clone, the
researcher may isolate (“harvest”) large quantities of the gene
of interest, which may then be used for a variety of purposes.
The gene may even be inserted into another vector for intro-
duction into another kind of cell (such as a plant or animal
cell). Alternatively, if the gene of interest is expressed (tran-
scribed and translated) in the cell clone, its protein product can
be harvested and used for a variety of purposes.

The advantages of using rDNA for obtaining such pro-
teins is illustrated by one of its early successes, the produc-
tion of human growth hormone (hGH) in E. coli bacteria.
Some individuals don’t produce adequate amounts of hGH, so
their growth is stunted. In the past, hGH had to be obtained
from human pituitary glands at autopsy. (Growth hormone

CLINICAL CASE No Ordinary Checkup

Dr. B. is closing his dental practice after 20 years. Four years ago, he went to his family doctor because of
debilitating exhaustion. He thought he had a flu virus that he
could not shake, and he was also having night sweats. His
doctor ordered a myriad of blood tests, but only one came
back positive. Dr. B. had HIV. Although he immediately began
an HIV treatment regimen, one year later he was diagnosed
with AIDS. Now, two years later, Dr. B. is very ill and can no
longer work.

Dr. B. lets his employees know the situation and suggests
that they all get tested for HIV. All of Dr. B.’s employees,
including the hygienists, test negative. Dr. B. also writes an
open letter to his patients informing them of his decision to
close his practice and why he is doing so. This letter prompts
400 former patients to be tested for HIV, seven of whom test
positive for antibodies against HIV.

What type of test can determine whether these patients
contracted HIV from Dr. B.? Read on to find out.

243 249 252 254 257

1 Vector, such as a
plasmid, is isolated.

2 DNA containing the gene of
interest from a different species is
cleaved by an enzyme into
fragments.

Bacterial
chromosome

Plasmid

DNA containing the
gene of intere

Plasmid
RNA
Protein product

Gene encoding protein for
pest resistance is inserted
into plant cells.

Gene encoding degradative
enzyme to clean up toxic
waste is inserted into
bacterial cells.

Amylase, cellulase, and other
enzymes prepare fabrics for
clothing manufacture.

Human growth hormone
treats stunted growth.

3 The desired gene is selected and inserted into a plasmid.

4 The plasmid is taken up by a cell,
such as a bacterium.

5 Cells with the gene of interest are cloned
with either of two goals in mind.

6a Create and harvest
copies of a gene.

6b Create and harvest
protein products of a gene.

bacterium

transformed
bacterium

recombinant
DNA (plasmid)

KEY CONCEPTS
●

Genes from one organism’s cells can be inserted and
expressed in another organism’s cells.

Genetically modified cells can be used to create a wide variety
of useful products and applications.

●

FOUNDATION
FIGURE

9.1 A Typical Genetic Modification Procedure

244

CHAPTER 9 Biotechnology and DNA Technology 2

cultures to radiation, the highest-yielding variant among the
survivors was selected for another exposure to a mutagen.
Using mutations, biologists increased the amount of penicillin
the fungus produced by over 1000 times.

Screening each mutant for penicillin production is a
tedious process. Site-directed mutagenesis is more targeted
and can be used to make a specific change in a gene. Suppose
you determine that changing one amino acid will make a laun-
dry enzyme work better in cold water. Using the genetic code
(see Figure 8.8, page 214), you could, using the techniques
described next, produce the sequence of DNA that encodes that
amino acid and insert it into that enzyme’s gene.

The science of molecular genetics has advanced to such a
degree that many routine cloning procedures are performed
using prepackaged materials and procedures that are very
much like cookbook recipes. Scientists have a grab bag of
methods from which to choose, depending on the ultimate
application of their experiments. Next we describe some of the
most important tools and techniques, and later we will con-
sider some applications.

Restriction Enzymes
Recombinant DNA technology has its technical roots in the
discovery of restriction enzymes, a special class of DNA-
cutting enzymes that exist in many bacteria. First isolated
in 1970, restriction enzymes in nature had actually been
observed earlier, when certain bacteriophages were found to
have a restricted host range. If these phages were used to infect
bacteria other than their usual hosts, restriction enzymes in
the new host destroyed almost all the phage DNA. Restriction
enzymes protect a bacterial cell by hydrolyzing phage DNA.
The bacterial DNA is protected from digestion because the cell
methylates (adds methyl groups to) some of the cytosines in its
DNA. The purified forms of these bacterial enzymes are used
in today’s laboratories.

What is important for rDNA techniques is that a restric-
tion enzyme recognizes and cuts, or digests, only one par-
ticular sequence of nucleotide bases in DNA, and it cuts this
sequence the same way each time. Typical restriction enzymes
used in cloning experiments recognize four-, six-, or eight-base
sequences. Hundreds of restriction enzymes are known, each
producing DNA fragments with characteristic ends. A few restric-
tion enzymes are listed in Table 9.1. You can see they are named
for their bacterial source. Some of these enzymes (e.g., HaeIII)
cut both strands of DNA in the same place, producing blunt
ends, and others make staggered cuts in the two strands—cuts
that are not directly opposite each other (Figure 9.2). These stag-
gered ends, or sticky ends, are most useful in rDNA because they
can be used to join two different pieces of DNA that were cut by
the same restriction enzyme. The sticky ends “stick” to stretches
of single-stranded DNA by complementary base pairing.

from other animals is not effective in humans.) This practice
was not only expensive but also dangerous because on several
occasions neurological diseases were transmitted with the hor-
mone. Human growth hormone produced by genetically modi-
fied E. coli is a pure and cost-effective product. Recombinant
DNA techniques also result in faster production of the hor-

mone than traditional methods might allow.

CHECK YOUR UNDERSTANDING

✓ 9-1 Differentiate biotechnology and rDNA technology.
✓ 9-2 In one sentence, describe how a vector and clone are

used.

Tools of Biotechnology
LEARNING OBJECTIVES

9-3 Compare selection and mutation.

9-4 Define restriction enzymes, and outline how they are used to
make rDNA.

9-5 List the four properties of vectors.

9-6 Describe the use of plasmid and viral vectors.

9-7 Outline the steps in PCR, and provide an example of its use.

Research scientists and technicians isolate bacteria and fungi
from natural environments such as soil and water to find,
or select, the organisms that produce a desired product. The
selected organism can be mutated to make more product or to
make a better product.

Selection
In nature, organisms with characteristics that enhance survival
are more likely to survive and reproduce than are variants that
lack the desirable traits. This is called natural selection. Humans
use artificial selection to select desirable breeds of animals or
strains of plants to cultivate. As microbiologists learned how
to isolate and grow microorganisms in pure culture, they were
able to select the ones that could accomplish a desired objec-
tive, such as brewing beer more efficiently or producing a new
antibiotic. Over 2000 strains of antibiotic-producing bacteria
have been discovered by testing soil bacteria and selecting the
strains that produce an antibiotic.

Mutation
Mutations are responsible for much of the diversity of life (see
Chapter 8). A bacterium with a mutation that confers resistance
to an antibiotic will survive and reproduce in the presence of
that antibiotic. Biologists working with antibiotic-producing
microbes discovered that they could create new strains by
exposing microbes to mutagens. After random mutations were
created in penicillin-producing Penicillium by exposing fungal

246 PART ONE Fundamentals of Microbiology

sources have been produced by the action of the same restric-
tion enzyme, the two pieces will have identical sets of sticky
ends and can be spliced (recombined) in vitro. The sticky
ends join spontaneously by hydrogen bonding (base pairing).
The enzyme DNA ligase is used to covalently link the backbones
of the DNA pieces,
producing an rDNA
molecule.

Vectors
Many different types of DNA molecules can serve as vectors,
provided they have certain properties. The most important
property is self-replication; once in a cell, a vector must be
capable of replicating. Any DNA that is inserted in the vector
will be replicated in the process. Thus, vectors serve as vehicles
for the replication of desired DNA sequences.

Vectors also need to be large enough to be manipulated out-
side the cell during rDNA procedures. Smaller vectors are more

TABLE 9.1 Selected Restriction Enzymes Used in rDNA
Technology

Enzyme Bacterial Source Recognition Sequence

BamHI Bacillus

amyloliquefaciens

G G A T C C
C C T A G G

EcoRI Escherichia

coli

A A T T C

C T T A A G

HaeIII Haemophilus

aegyptius

G G C C
C C G G

HindIII Haemophilus

influenzae

A A G C T T
T T C G A A

Recognition sites

Cut

G A A T T C

C T T A A G

Cut

Cut
G A A T T C
C T T A A G
A A T T C
G
G

C T T A A

Cut
G
C T T A A

G C T T A A

GA A T T C

G C T T A A
GA A T T C
G C T T A A
GA A T T C
A A T T C
G
G

Sticky end

G
A A T T C

A A T T C

G C T T A A

AATTC

CTT
AA G

G
G C T T A A

AATTC

CT
TAA G

G
1

2 These cuts produce a DNA fragment with
two sticky ends.

DNA from another source,
perhaps a plasmid, cut with
the same restriction enzyme

5
rDNA

DNA

A restriction enzyme cuts (red arrows)
double-stranded DNA at its particular
recognition sites, shown in blue.

When two such fragments of DNA cut by
the same restriction enzyme come
together, they can join by base pairing.

The enzyme DNA ligase is used to unite the
backbones of the two DNA fragments, producing
a molecule of rDNA.

4 The joined fragments will usually form either a linear
molecule or a circular one, as shown here for a plasmid.
Other combinations of fragments can also occur.

Figure 9.2 A restriction enzyme’s role in making rDNA.

Q Why are restriction enzymes used to make rDNA?

Play Recombinant DNA Technology
@MasteringMicrobiology

Notice in Figure 9.2 that the darker base sequences on the
two strands are the same but run in opposite directions. Stag-
gered cuts leave stretches of single-stranded DNA at the ends
of the DNA fragments. If two fragments of DNA from different

CHAPTER 9 Biotechnology and DNA Technology 247

lacZ

ori

amp
pUC19

HindIII

BamHI

EcoRI

being used to insert corrective genes into human cells that have
defective genes. Gene therapy is discussed on page 255.

CHECK YOUR UNDERSTANDING

✓ 9-3 How are selection and mutation used in biotechnology?
✓ 9-4 What is the value of restriction enzymes in rDNA technology?
✓ 9-5 What criteria must a vector meet?
✓ 9-6 Why is a vector used in rDNA technology?

Polymerase Chain Reaction
The polymerase chain reaction (PCR) is a technique by
which small samples of DNA can be quickly amplified, that is,
increased to quantities that are large enough for analysis.

Starting with just one gene-sized piece of DNA, PCR can be
used to make billions of copies in only a few hours. The PCR
process is shown in Figure 9.4.

Each strand of the target DNA will serve as a template for
DNA synthesis. Added to this DNA are a supply of the four
nucleotides (for assembly into new DNA) and the enzyme for
catalyzing the synthesis, DNA polymerase (see Chapter 8, page
209). Short pieces of nucleic acid called primers are also added
to help start the reaction. The primers are complementary to
the ends of the target DNA and will hybridize to the fragments
to be amplified. Then, the polymerase synthesizes new com-
plementary strands. After each cycle of synthesis, the DNA is
heated to convert all the new DNA into single strands. Each
newly synthesized DNA strand serves in turn as a template for
more new DNA.

As a result, the process proceeds exponentially. All of the nec-
essary reagents are added to a tube, which is placed in a thermal
cycler. The thermal cycler can be set for the desired temperatures,
times, and number of cycles. Use of an automated thermal cycler
is made possible by the use of DNA polymerase taken from a
thermophilic bacterium such as Thermus aquaticus; the enzyme
from such organisms can survive the heating phase without
being destroyed. Thirty cycles, completed in just a few hours, will
increase the amount of target DNA by more than a billion times.

The amplified DNA can be seen by gel electrophoresis. In
real-time PCR, or quantitative PCR (qPCR), the newly made DNA
is tagged with a fluorescent dye, so that the levels of fluores-
cence can be measured after every PCR cycle (that’s the real
time aspect). Another PCR procedure called reverse-transcription
(RT-PCR) uses viral RNA or a cell’s mRNA as the template. The
enzyme, reverse transcriptase, makes DNA from the RNA tem-
plate, and the DNA is then amplified.

Note that PCR can only be used to amplify relatively small,
specific sequences of DNA as determined by the choice of
primers. It cannot be used to amplify an entire genome.

PCR can be applied to any situation that requires the ampli-
fication of DNA. Especially noteworthy are diagnostic tests that
use PCR to detect the presence of infectious agents in situations in

Figure 9.3 A plasmid used for cloning. A plasmid vector used
for cloning in the bacterium E. coli is pUC19. An origin of replication
(ori) allows the plasmid to be self-replicating. Two genes, one encoding
resistance to the antibiotic ampicillin (amp) and one encoding the
enzyme β-galactosidase (lacZ), serve as marker genes. Foreign DNA can
be inserted at the restriction enzyme sites.

Q What is a vector in rDNA technology?

easily manipulated than larger DNA molecules, which tend to
be more fragile. Preservation is another important property of
vectors. The DNA molecule’s circular form protects the vector’s
DNA from being destroyed by its recipient. Notice in Figure 9.3
that the DNA of a plasmid is circular. Another preservation
mechanism occurs when a virus’s DNA inserts itself quickly
into the chromosome of the host.

When it is necessary to retrieve cells that contain the vector,
a marker gene in the vector often helps make selection easy.
Common selectable marker genes are for antibiotic resistance
or for an enzyme that carries out an easily identified reaction.

Plasmids are one of the primary vectors in use, particularly
variants of R factor plasmids. Plasmid DNA can be cut with the
same restriction enzymes as the DNA that will be cloned, so
that all pieces of the DNA will have the same sticky ends. When
the pieces are mixed, the DNA to be cloned will be inserted
into the plasmid (Figure 9.2). Note that other fragment com-
binations can occur as well, including the plasmid reforming a
circle with no DNA inserted.

Some plasmids are capable of existing in several different
species. They are called shuttle vectors and can be used to move
cloned DNA sequences among organisms, such as among bac-
terial, yeast, and mammalian cells, or among bacterial, fungal,
and plant cells. Shuttle vectors can be very useful in the process
of genetically modifying multicellular organisms—for example,
when herbicide resistance genes are inserted into plants.

A different kind of vector is viral DNA. This type of vec-
tor can usually accept much larger pieces of foreign DNA than
plasmids can. After the DNA has been inserted into the viral
vector, it can be cloned in the virus’s host cells. The choice of a
suitable vector depends on many factors, including the organ-
ism that will receive the new gene and the size of the DNA to
be cloned. Retroviruses, adenoviruses, and herpesviruses are

248 PART ONE Fundamentals of Microbiology

CHECK YOUR UNDERSTANDING

✓ 9-7 For what is each of the following used in PCR: primer,
DNA polymerase, 94°C?

Copy of target DNA

Copies of target DNA

2
3
4
5

Incubate at 94°C for 1 minute; this
temperature will separate the strands.

Incubate at 60°C for 1 minute; this allows primers
to attach to single-stranded DNA.

Incubate at 72°C for 1 minute; DNA polymerase
copies the target DNA at this temperature.

Repeat the cycle of heating and cooling
to make two more copies of target DNA.

FIRST CYCLE

SECOND CYCLE

5¿
3¿

3¿ 5¿5¿ 3¿

5¿ 3¿
5¿ 3¿

3¿ 5¿

Copies of target DNA

5¿ 3¿
3¿ 5¿

Copy of target DNA
3¿ 5¿

5¿
3¿ 5¿
3¿ 5¿
3¿
5¿ 3¿

3¿
5¿

Target DNA

Primer DNA
polymerase

Nucleotides

1 Add primers, nucleotides, and DNA polymerase.

PREPARATION
5¿
3¿

3¿
5¿

Figure 9.4 The polymerase chain reaction. Deoxynucleotides (dNTPs) base-pair with the
target DNA: adenine pairs with thymine, and cytosine pairs with guanine.

Q How does reverse-transcription PCR differ from this figure?

which they would otherwise be undetectable. A qPCR test provides
rapid identification of drug-resistant Mycobacterium tuberculosis.
Otherwise, this bacterium can
take up to 6 weeks to culture,
leaving patients untreated for
a significant period of time.

Play PCR: Overview,
Components, Process
@MasteringMicrobiology

Techniques of Genetic Modification
LEARNING OBJECTIVES

9-8 Describe five ways of getting DNA into a cell.

9-9 Describe how a genomic library is made.

9-10 Differentiate cDNA from synthetic DNA.

9-11 Explain how each of the following is used to locate a clone:
antibiotic-resistance genes, DNA probes, gene products.

9-12 List one advantage of modifying each of the following: E. coli,
Saccharomyces cerevisiae, mammalian cells, plant cells.

CHAPTER 9 Biotechnology and DNA Technology 249

glycol increases the frequency of fusion (Figure 9.5). In the
new hybrid cell, the DNA derived from the two “parent”
cells may undergo natural recombination. This method is
especially valuable in the genetic manipulation of plant and

algal cells.
A remarkable way of introducing foreign DNA into plant

cells is to literally shoot it directly through the thick cellu-
lose walls using a gene gun (Figure 9.6). Microscopic particles
of tungsten or gold are coated with DNA and propelled by a
burst of helium through the plant cell walls. Some of the cells
express the introduced DNA as though it were their own.

DNA can be introduced directly into an animal cell by
microinjection. This technique requires the use of a glass
micropipette with a diameter that is much smaller than the
cell. The micropipette punctures the plasma membrane, and
DNA can be injected through it (Figure 9.7).

Inserting Foreign DNA into Cells
Recombinant DNA procedures require that DNA molecules be
manipulated outside the cell and then returned to living cells.
There are several ways to introduce DNA into cells. The choice
of method is usually determined by the type of vector and host
cell being used.

In nature, plasmids are usually transferred between closely
related microbes by cell-to-cell contact, such as in conjuga-
tion. To modify a cell, a plasmid must be inserted into a cell
by transformation, a procedure during which cells can take
up DNA from the surrounding environment (see Chapter 8,
page 232). Many cell types, including E. coli, yeast, and mam-
malian cells, do not naturally transform; however, simple
chemical treatments can make all of these cell types compe-
tent, or able to take up external DNA. For E. coli, the procedure
for making cells competent is to soak them in a solution of
calcium chloride for a brief period. Following this treatment,
the now-competent cells are mixed with the cloned DNA and
given a mild heat shock. Some of these cells will then take up
the DNA.

There are other ways to transfer DNA to cells. A process
called electroporation uses an electrical current to form
microscopic pores in the membranes of cells; the DNA then
enters the cells through the pores. Electroporation is generally
applicable to all cells; those with cell walls often must be con-
verted to protoplasts first. Protoplasts are produced by enzy-
matically removing the cell wall, thereby allowing more direct
access to the plasma membrane.

The process of protoplast fusion also takes advantage
of the properties of protoplasts. Protoplasts in solution fuse
at a low but significant rate; the addition of polyethylene

CLINICAL CASE

Reverse-transcription PCR using a primer for an HIV gene can be used to amplify DNA for analysis. The Centers for
Disease Control and Prevention (CDC) interviews the seven
former patients to determine whether their histories show
any additional risk factors for contracting HIV. Five out of the
seven have no identified risk factors for HIV other than having
had invasive procedures performed on them by Dr. B. The

CDC then performs reverse-
transcription PCR on DNA from
white blood cells in Dr. B.’s
peripheral blood and the seven
HIV-positive patients (see the
figure).

What can be concluded from the PCR amplification in the
figure?

243 249 252 254 257

Patients

D
en

tis
t

B C D E F G

Bacterial cells

Protoplasts

Chromosome

Plasma membrane

Cell wall

1 Bacterial cell walls
are enzymatically
digested, producing
protoplasts.

2 In solution, protoplasts are
treated with polyethylene glycol.

3 Protoplasts fuse.

4 Segments of the two
chromosomes recombine.

5 Recombinant cell
grows new cell wall.

Recombinant
cell

Figure 9.5 Protoplast fusion. Removal of the cell wall leaves only
the delicate plasma membranes, which will fuse together, allowing the
exchange of DNA.

Q What is a protoplast?

250 PART ONE Fundamentals of Microbiology

Obtaining DNA
We have seen how genes can be cloned into vectors by using
restriction enzymes and how genes can be transformed or
transferred into a variety of cell types. But how do biologists
obtain the genes they are interested in? There are two main
sources of genes: (1) genomic libraries containing either natu-
ral copies of genes or cDNA copies of genes made from mRNA,
and (2) synthetic DNA.

Genomic Libraries
Isolating specific genes as individual pieces of DNA is seldom
practical. Therefore, researchers interested in genes from a
particular organism start by extracting the organism’s DNA,
which can be obtained from cells of any organism, whether
plant, animal, or microbe, by lysing the cells and precipitating
the DNA. This process results in a DNA mass that includes the
organism’s entire genome. After the DNA is digested by restric-
tion enzymes, the restriction fragments are then spliced into
plasmid or phage vectors, and the recombinant vectors are
introduced into bacterial cells. The goal is to make a collection
of clones large enough to ensure that at least one clone exists
for every gene in the organism. This collection of clones con-
taining different DNA fragments is called a genomic library;
each “book” is a bacterial or phage strain that contains a frag-
ment of the genome (Figure 9.8). Such libraries are essential for

Figure 9.6 A gene gun, which can be used to insert
DNA-coated “bullets” into a cell.

Q Name four other methods of inserting DNA into a cell.

80 mm
LM

Figure 9.7 The microinjection of foreign DNA into an egg. The
egg is first immobilized by applying mild suction to the large, blunt,
holding pipette (right). Several hundred copies of the gene of interest
are then injected into the nucleus of the cell through the tiny end of the
micropipette (left).

Q Why is microinjection impractical for bacterial and fungal cells?

Thus, there is a great variety of restriction enzymes, vec-
tors, and methods of inserting DNA into cells. But foreign
DNA will survive only if it’s either present on a self-replicating
vector or incorporated into one of the cell’s chromosomes by
recombination.

Plasmid Library

Genome to be stored
in library is cut up with
restriction enzyme

Recombinant
plasmid

Host
cell

Recombinant
phage DNA

Phage Library

Phage cloning
vector

Figure 9.8 Genomic libraries. Each fragment of DNA, containing
about one gene, is carried by a vector, either a plasmid within a bacterial
cell or a phage.

Q Differentiate a restriction fragment from a gene.

CHAPTER 9 Biotechnology and DNA Technology 251

(Figure 9.9). This synthesis is the reverse of the normal DNA-to-
RNA transcription process. A DNA copy of mRNA is produced
by reverse transcriptase. Following this, the mRNA is enzymati-
cally digested away. DNA polymerase then synthesizes a com-
plementary strand of DNA, creating a double-stranded piece of
DNA containing the information from the mRNA. Molecules of
cDNA produced from a mixture of all the mRNAs from a tissue
or cell type can then be cloned to form a cDNA library.

The cDNA method is the most common method of obtain-
ing eukaryotic genes. A difficulty with this method is that long
molecules of mRNA may not be completely reverse-transcribed
into DNA; the reverse transcription often aborts, forming only
parts of the desired gene.

Synthetic DNA
Under certain circumstances, genes can be made in vitro with
the help of DNA synthesis machines (Figure 9.10). A keyboard
on the machine is used to enter the desired sequence of nucleo-
tides, much as letters are entered into a word processor to com-
pose a sentence. A microprocessor controls the synthesis of the
DNA from stored supplies of nucleotides and the other neces-
sary reagents. A short chain of about 200 nucleotides, called an
oligonucleotide, can be synthesized by this method. Unless the
gene is very small, at least several chains must be synthesized
separately and linked together to form an entire gene.

The difficulty of this approach, of course, is that the
sequence of the gene must be known before it can be synthe-
sized. If the gene hasn’t already been isolated, then the only

Exon Intron Exon Intron Exon
DNA

1 A gene composed of exons and
introns is transcribed to RNA by
RNA polymerase.

RNA
transcript

2 Processing enzymes in the nucleus
remove the intron-derived RNA
and splice together the
exon-derived RNA into mRNA.

mRNA

Test tube

Cytoplasm
Nucleus

3 mRNA is isolated from the
cell, and reverse
transcriptase is added.

5 The mRNA is digested by
reverse transcriptase.

4 First strand
of DNA is
synthesized.

DNA strand
being synthesized

6 DNA polymerase is added to
synthesize second strand
of DNA.

cDNA of
gene without
introns

Figure 9.9 Making complementary DNA (cDNA) for a
eukaryotic gene. Reverse transcriptase catalyzes the synthesis of
double-stranded DNA from an RNA template.

Q How does reverse transcriptase differ from DNA polymerase?

maintaining and retrieving DNA clones; they can even be pur-
chased commercially.

Cloning genes from eukaryotic organisms presents a spe-
cific problem. Genes of eukaryotic cells generally contain both
exons, stretches of DNA that code for protein, and introns,
intervening stretches of DNA that do not code for protein.
When the RNA transcript of such a gene is converted to mRNA,
the introns are removed (see Figure 8.11 on page 219). To clone
genes from eukaryotic cells, it’s desirable to use a version of the
gene that lacks introns because a gene that includes introns may
be too large to work with easily. In addition, if such a gene is
put into a bacterial cell, the bacterium usually won’t be able
to remove the introns from the RNA transcript. Therefore, it
won’t be able to make the correct protein product. However,
an artificial gene that contains only exons can be produced
by using an enzyme called reverse transcriptase to synthe-
size complementary DNA (cDNA) from an mRNA template

Figure 9.10 A DNA synthesis machine. Short sequences of DNA
can be synthesized by instruments such as this one.

Q What four reagents (in the brown bottles) are necessary to
synthesize DNA?

252 PART ONE Fundamentals of Microbiology

The plasmid vector used contains a gene (amp) encoding
resistance to the antibiotic ampicillin. The host bacterium won’t
be able to grow on the test medium, which contains ampicillin,
unless the vector has transferred the ampicillin-resistance gene.
The plasmid vector also contains a second gene, this one for the
enzyme β-galactosidase (lacZ). Notice in Figure 9.3 that there
are several sites in lacZ that can be cut by restriction enzymes.

In the blue-white screening procedure shown in Figure 9.11,
a library of bacteria is cultured in a medium called X-gal. X-gal
contains two essential components other than those neces-
sary to support normal bacterial growth. One is the antibiotic
ampicillin, which prevents the growth of any bacterium that

CLINICAL CASE

The primer amplifies all eight samples and confirms that Dr. B. and seven of his former patients are all infected
with HIV. The CDC then sequences the amplified DNA and
compares the sequencing to an HIV isolate from Cleveland
(local control) and an isolate from Haiti (outlier). A portion of
the coding (5′ to 3′) is shown below.

Patient A GCTTG GGCTG GCGCT GAAGT GAGA

Patient B GCTAT TGCTG GCGCT GAATT GCAC

Patient C GCCAT AGCTG GCGCA GAAGT GCAC

Patient D GCTAT TGGCG TGGCT GACAG AGAA

Patient E GCACC TGCTG GCGCT GAAGT GAAA

Patient F CAGAT TGTGT TGATT GAACC TCAC

Patient G GCTAT TGCTG GCGCT GAAGT GAAA

Dentist GCTAT TGCTG GCGCT GAAGT GCAC

Local control CAGAC TACTG CTAGG AAAAA TATT

Outlier GAAGA CGAAA GGACT GCTAT TCAG

What is the percent similarity among the viruses?

243 249 252 254 257

way to predict the DNA sequence is by knowing the amino
acid sequence of the gene’s protein product. If this amino
acid sequence is known, in principle you can work backward
through the genetic code to obtain the DNA sequence. Unfor-
tunately, the degeneracy of the code prevents definitive deter-
mination; thus, if the protein contains a leucine, for example,
which of the six codons for leucine is the one in the gene?

For these reasons, it’s rare to clone a gene by synthesizing
it directly, although some commercial products such as insu-
lin, interferon, and somatostatin are produced from chemically
synthesized genes. Desired restriction sites are added to the
synthetic genes so the genes can be inserted into plasmid vec-
tors and cloned in E. coli. Synthetic DNA plays a much more
useful role in selection procedures, as we will see.

CHECK YOUR UNDERSTANDING

✓ 9-8 Contrast the five ways of putting DNA into a cell.
✓ 9-9 What is the purpose of a genomic library?
✓ 9-10 Why isn’t cDNA synthetic?

Selecting a Clone
In cloning, it’s necessary to select the particular cell that con-
tains the specific gene of interest. This is difficult to do because
out of millions of cells, only a very few might contain the
desired gene. Here we’ll examine a typical screening procedure
known as blue-white screening, from the color of the bacterial
colonies formed at the end of the screening process.

Bacterium

Colonies
with foreign
DNA

Plasmid

b-galactosidase gene (lacZ)

Ampicillin-resistance
gene (amp)

Restriction
site

Restriction
sites

Foreign DNA

1 Plasmid DNA and foreign DNA
are both cut with the same
restriction enzyme. The plasmid
has the genes for lactose hydrolysis
(the lacZ gene encodes the enzyme
b-galactosidase) and ampicillin
resistance.

2 Foreign DNA will insert into
the lacZ gene. The bacterium
receiving the plasmid vector
will not produce the enzyme
b-galactosidase if foreign
DNA has been inserted into
the plasmid.

3 The recombinant plasmid
is introduced into a
bacterium, which becomes
ampicillin resistant.

4 All treated bacteria are spread
on a nutrient agar plate
containing ampicillin and a
b-galactosidase substrate and
incubated. The b-galactosidase
substrate is called X-gal.

5 Only bacteria that picked up
the plasmid will grow in the
presence of ampicillin.
Bacteria that hydrolyze
X-gal produce galactose and
an indigo compound. The
indigo turns the colonies blue.
Bacteria that cannot hydrolyze
X-gal produce white colonies.

Recombinant
plasmid

Figure 9.11 Blue-white screening, one method of selecting
recombinant bacteria.

Q Why are some colonies blue and others white?

CHAPTER 9 Biotechnology and DNA Technology 253

has not successfully received the ampicillin-resistance gene
from the plasmid. The other, called X-gal, is a substrate for
β-galactosidase.

Only bacteria that picked up the plasmid will grow, because
they are now ampicillin resistant. Bacteria that picked up the
recombinant plasmid—in which the new gene was inserted
into the lacZ gene—will not hydrolyze lactose and will produce
white colonies. If a bacterium received the original plasmid
containing the intact lacZ gene, the cells will hydrolyze X-gal
to produce a blue-colored compound; the colony will be blue.

What remains to be done can still be difficult. The above
procedure has isolated white colonies known to contain for-
eign DNA, but it is still not known whether it’s the desired frag-
ment of foreign DNA. A second procedure is needed to identify
these bacteria. If the foreign DNA in the plasmid encodes the
production of an identifiable product, the bacterial isolate only
needs to be grown in culture and tested. However, in some
cases the gene itself must be identified in the host bacterium.

Colony hybridization is a common method of identifying
cells that carry a specific cloned gene. DNA probes, short seg-
ments of single-stranded DNA that are complementary to the
desired gene, are synthesized. If the DNA probe finds a match,
it will adhere to the target gene. The DNA probe is labeled with
an enzyme or fluorescent dye so its presence can be detected. A
typical colony hybridization experiment is shown in Figure 9.12.
An array of DNA probes arranged in a DNA chip can be used to
identify pathogens (see Figure 10.17, page 287).

Making a Gene Product
We have just seen how to identify cells carrying a particular
gene. Gene products are frequently the reason for genetic mod-
ification. Most of the earliest work in genetic modification used
E. coli to synthesize the gene products. E. coli is easily grown,
and researchers are very familiar with the bacterium and its
genetics. For example, some inducible promoters, such as that
of the lac operon, have been cloned, and cloned genes can be
attached to such promoters. The synthesis of great amounts
of the cloned gene product can then be directed by the addi-
tion of an inducer. Such a method has been used to produce
gamma interferon in E. coli (Figure 9.13). However, E. coli also
has several disadvantages. Like other gram-negative bacteria, it
produces endotoxins as part of the outer layer of its cell wall.
Because endotoxins cause fever and shock in mammals, their
accidental presence in products intended for human use would
be a serious problem.

Another disadvantage of E. coli is it doesn’t usually secrete
protein products. To obtain a product, cells must usually be
broken open and the product purified from the resulting
“soup” of cell components. Recovering the product from such
a mixture is expensive when done on an industrial scale. It’s
more economical to have an organism secrete the product so it

Master plate with
colonies of bacteria
containing cloned
segments of foreign
genes

Fluorescence-
labeled probes

Nitrocellulose
filter

Strands of
bacterial DNA

Bound
DNA
probe

Gene of
interest

Single-
stranded
DNA

Colonies containing
genes of interest

Replica plate

1 Make replica of master
plate on nitrocellulose
filter.

2 Treat filter with
detergent (SDS) to
lyse bacteria.

3 Treat filter with sodium
hydroxide (NaOH) to
separate DNA into
single strands.

4 Add labeled probes.

5 Probe will hybridize
with desired gene from
bacterial cells.

6 Wash filter to remove
unbound probe.

7 Compare filter with
replica of master plate
to identify colonies
containing gene of
interest.

Figure 9.12 Colony hybridization: using a DNA probe to
identify a cloned gene of interest.

Q What is a DNA probe?

254 PART ONE Fundamentals of Microbiology

CLINICAL CASE

The sequences from Dr. B. and patients A, B, C, E, and G share 87.5% of the nucleotide sequence, which
is comparable to reported similarities for known linked
infections.

Identify the amino acids encoded by the viral DNA. Did this
change the percent similarity? (Hint: Refer to Figure 8.8 on
page 214).

243 249 252 254 257

TEM 0.25 mm

against infection. To produce huge amounts of CSF industri-
ally, the gene is first inserted into a plasmid. Bacteria are used
to make multiple copies of the plasmid (see Figure 9.1), and the
resulting recombinant plasmids are then inserted into mam-
malian cells that are grown in bottles.

Plant cells can also be grown in culture, altered by rDNA
techniques, and then used to generate genetically modified
plants. Such plants may prove useful as sources of valuable
products, such as plant alkaloids (the painkiller codeine, for
example), the isoprenoids that are the basis of synthetic rubber,
and melanin (the animal skin pigment) for use in sunscreens.
Genetically modified plants have many advantages for the pro-
duction of human therapeutic agents, including vaccines and
antibodies. The advantages include large-scale, low-cost agri-
cultural production and a low risk of product contamination
by mammalian pathogens or cancer-causing genes. Geneti-
cally modifying plants often requires use of a bacterium. We’ll
return to the topic of genetically modified plants later in the
chapter (page 260).

CHECK YOUR UNDERSTANDING

✓ 9-11 How are recombinant clones identified?
✓ 9-12 What types of cells are used for cloning rDNA?

Applications of DNA Technology
LEARNING OBJECTIVES

9-13 List at least five applications of DNA technology.

9-14 Define RNAi.

9-15 Discuss the value of genome projects.

9-16 Define the following terms: random shotgun sequencing,
bioinformatics, proteomics.

9-17 Diagram the Southern blotting procedure, and provide an
example of its use.

9-18 Diagram DNA fingerprinting, and provide an example
of its use.

9-19 Outline genetic engineering with Agrobacterium.

Figure 9.13 E. coli genetically modified to produce gamma
interferon, a human protein that promotes an immune
response. The product, visible here as a red mass in a violet ring, can
be released by lysis of the cell.

Q What is one advantage of using E. coli for genetic engineering?
One disadvantage?

can be recovered continuously from the growth medium. One
approach has been to link the product to a natural E. coli pro-
tein that the bacterium does secrete. However, gram-positive
bacteria, such as Bacillus subtilis, are more likely to secrete their
products and are often preferred industrially for that reason.

Another microbe being used as a vehicle for expressing
rDNA is baker’s yeast, Saccharomyces cerevisiae. Its genome is
only about four times larger than that of E. coli and is prob-
ably the best understood eukaryotic genome. Yeasts may carry
plasmids, which are easily transferred into yeast cells whose
cell walls have been removed. As eukaryotic cells, yeasts may
be more successful in expressing foreign eukaryotic genes than
bacteria. Furthermore, yeasts are likely to continuously secrete
the product. Because of all these factors, yeasts have become
the eukaryotic workhorse of biotechnology.

Mammalian cells in culture, even human cells, can be
genetically modified much like bacteria to produce various
products. Scientists have developed effective methods of grow-
ing certain mammalian cells in culture as hosts for growing
viruses (see Chapter 13, page 371). Mammalian cells are often
the best suited to making protein products for medical use
because the cells secrete their products and there’s a low risk
of toxins or allergens. Using mammalian cells to make foreign
gene products on an industrial scale often requires a prelimi-
nary step of cloning the gene in bacteria. Consider the exam-
ple of colony-stimulating factor (CSF). A protein produced
naturally in tiny amounts by white blood cells, CSF is valuable
because it stimulates the growth of certain cells that protect

CHAPTER 9 Biotechnology and DNA Technology 255

DNA vaccines are usually circular plasmids that include a
gene encoding a viral protein that’s under the transcriptional
control of a promoter region active in human cells. The plas-
mids are then cloned in bacteria. A DNA vaccine to protect
against Zika virus disease is currently in clinical trials. Vac-
cines are discussed in further detail in Chapter 18 (page 503).
Table 9.2 lists some other important rDNA products used in
medical therapy.

The importance of rDNA technology to medical research
cannot be emphasized enough. Artificial blood for use in
transfusions can now be prepared with human hemoglobin
produced in genetically modified pigs. Sheep have also been
genetically modified to produce a number of drugs in their
milk. This procedure has no apparent effect upon the sheep,
and they provide a ready source of raw material for the product
that does not require sacrificing animals.

Gene therapy may eventually provide cures for some
genetic diseases. It is possible to imagine removing some cells
from a person and transforming them with a normal gene
to replace a defective or mutated gene. When these cells are
returned to the person, they should function normally. For
example, gene therapy has been used to treat hemophilia
B and severe combined immunodeficiency. Adenoviruses
and retroviruses are used most often to deliver genes; how-
ever, some researchers are working with plasmid vectors. An
attenuated retrovirus was used as the vector when the first
gene therapy to treat hemophilia in humans was performed
in 1990. Glybera® is a gene therapy drug licensed in Europe
to treat lipoprotein lipase deficiency. It uses an adenovirus
to deliver the lipase gene to cells. Antisense DNA (page 262)
introduced into cells is also being explored. Fomivirsen is an
antisense DNA drug used in the treatment of cytomegalovirus
retinitis.

Thus far, gene therapy results have not been impressive;
there have even been a few deaths attributed to the viral vec-
tors. A great deal of preliminary work remains to be done, but
cures may not be possible for all genetic diseases.

Gene editing is a promising new technology to correct
genetic mutations at precise locations. Gene editing uses
CRISPR (pronounced “crisper”), which stands for clustered
regularly interspaced short palindromic repeats. CRISPR
enzymes are found in archaea and bacteria, where they
destroy foreign DNA. A small RNA molecule, complementary
to the desired target, binds DNA, and then the Cas9 enzyme
cuts the DNA like molecular scissors. The cell’s DNA poly-
merase and DNA ligase reattach the ends. A researcher can
add template DNA for the correct gene, which can be attached
by the DNA ligase. If may be possible to correct mutations in
the human genome to treat genetic causes of disease. Gene
editing was used to repair a defective muscle protein gene in
mice with Duchenne muscular dystrophy. A parvovirus was
used to deliver the gene-editing system into mice. In 2016, the

We have now described the entire sequence of events in cloning
a gene. As indicated earlier, such cloned genes can be applied
in a variety of ways. One is to produce useful substances more
efficiently and less expensively. Another is to obtain infor-
mation from the cloned DNA that is useful for either basic
research, medicine, or forensics. A third is to use cloned genes
to alter the characteristics of cells or organisms.

Therapeutic Applications
An extremely valuable pharmaceutical product is the hormone
insulin, a small protein produced by the pancreas that controls
the body’s uptake of glucose from blood. For many years, peo-
ple with insulin-dependent diabetes controlled their disease by
injecting insulin obtained from the pancreases of slaughtered
animals. Obtaining this insulin is an expensive process, and
the insulin from animals is not as effective as human insulin.

Because of the value of human insulin and the protein’s
small size, producing human insulin by rDNA techniques was
an early goal for the pharmaceutical industry. To produce the
hormone, synthetic genes were first constructed for each of the
two short polypeptide chains that make up the insulin mol-
ecule. The small size of these chains—only 21 and 30 amino
acids long—made it possible to use synthetic genes. Follow-
ing the procedure described earlier (page 249), each of the
two synthetic genes was inserted into a plasmid vector and
linked to the end of a gene coding for the bacterial enzyme
β-galactosidase, so that the insulin polypeptide was copro-
duced with the enzyme. Two different E. coli bacterial cultures
were used, one to produce each of the insulin polypeptide
chains. The polypeptides were then recovered from the bacte-
ria, separated from the β-galactosidase, and chemically joined
to make human insulin. This accomplishment was one of the
early commercial successes of DNA technology, and it illus-
trates a number of the principles and procedures discussed in
this chapter.

Another human hormone that is now being produced com-
mercially by genetic modification of E. coli is somatostatin. At
one time 500,000 sheep brains were needed to produce 5 mg of
animal somatostatin for experimental purposes. By contrast,
only 8 liters of a genetically modified bacterial culture are
now required to obtain the equivalent amount of the human
hormone.

Subunit vaccines, consisting only of a protein portion of
a pathogen, are being made by genetically modifying yeasts.
Subunit vaccines have been produced for a number of diseases,
notably hepatitis B. One of the advantages of a subunit vac-
cine is that there is no chance that the vaccine will cause an
infection. The protein is harvested from genetically modified
cells and purified for use as a vaccine. Animal viruses such as
vaccinia virus can be genetically modified to carry a gene for
another microbe’s surface protein. When injected, the virus
acts as a vaccine against the other microbe.

256 PART ONE Fundamentals of Microbiology

TABLE 9.2 Some Pharmaceutical Products of rDNA

Product Comments

Cervical Cancer Vaccine Consists of viral proteins; produced by Saccharomyces cerevisiae or by insect cells

Epidermal Growth Factor (EGF) Heals wounds, burns, ulcers; produced by E. coli

Erythropoietin (EPO) Treatment of anemia; produced by mammalian cell culture

Interferon

IFN–a Therapy for leukemia, melanoma, and hepatitis; produced by E. coli and S. cerevisiae (yeast)

IFN–b Treatment for multiple sclerosis; produced by mammalian cell culture

IFN–g Treatment of chronic granulomatous disease; produced by E. coli

Hepatitis B Vaccine Produced by S. cerevisiae that carries hepatitis-virus gene on a plasmid

Human Growth Hormone (hGH) Corrects growth deficiencies in children; produced by E. coli

Human Insulin Therapy for diabetes; better tolerated than insulin extracted from animals; produced by E. coli

Influenza Vaccine Vaccine made from E. coli or S. cerevisiae carrying virus genes

Interleukins Regulate the immune system; possible treatment for cancer; produced by E. coli

Orthoclone OKT3
Muromonab-CD3

Monoclonal antibody used in transplant patients to help suppress the immune system, reducing the chance of
tissue rejection; produced by mouse cells

Pulmozyme (rhDNase) Enzyme used to break down mucous secretions in cystic fibrosis patients; produced by mammalian cell culture

Relaxin Used to ease childbirth; produced by E. coli

Superoxide Dismutase (SOD) Minimizes damage caused by oxygen free radicals when blood is resupplied to oxygen-deprived tissues;
produced by S. cerevisiae and Komagataella pastoris (yeast)

Taxol Plant product used for treating ovarian cancer; produced in E. coli

Tissue Plasminogen Activator Dissolves the fibrin of blood clots; therapy for heart attacks; produced by mammalian cell culture

Tumor Necrosis Factor (TNF) Causes disintegration of tumor cells; produced by E. coli

Veterinary Use

Canine Distemper Vaccine Canarypox virus carrying canine distemper virus genes

Feline Leukemia Vaccine Canarypox virus carrying feline leukemia virus genes

first clinical trials were approved to modify a patient’s T cells
(see page 476) to fight cancer.

Gene silencing is a natural process that occurs in a wide
variety of eukaryotes and is apparently a defense against viruses
and transposons. Gene silencing is similar to miRNA (page 219)
in that a gene encoding a small piece of RNA is transcribed.
Following transcription, RNAs called small interfering RNAs
(siRNAs) are formed after processing by an enzyme called Dicer.
The siRNA molecules bind to mRNA, which is then destroyed
by proteins called the RNA-induced silencing complex (RISC),
thus silencing the expression of a gene (Figure 9.14).

New technology called RNA interference (RNAi) holds
promise for gene therapy for treating genetic diseases. A small
DNA insert encoding siRNA against the gene of interest could
be cloned into a plasmid. When transferred into a cell, the cell
would produce the desired siRNA. Clinical trials are currently
being conducted to test RNAi to prevent Ebola and respiratory
syncytial virus infections.

CHECK YOUR UNDERSTANDING

✓ 9-13 Explain how DNA technology can be used to treat
disease and to prevent disease.

✓ 9-14 What is gene silencing?

Genome Projects
The first genome to be sequenced was from a bacteriophage in
1977. In 1995, the genome of a free-living cell—Haemophilus influ-
enzae—was sequenced. Since then, 1000 prokaryotic genomes
and over 400 eukaryotic genomes have been sequenced.

In shotgun sequencing, small pieces of a genome of a free-
living cell are sequenced, and the sequences are then assem-
bled using a computer. Any gaps between the pieces then
have to be found and sequenced (Figure 9.15). This technique
can be used on environmental samples to study the genomes
of microorganisms that haven’t been cultured. The study of

CHAPTER 9 Biotechnology and DNA Technology 257

genetic material taken directly from environmental samples is
called metagenomics.

The Human Genome Project was an international 13-year
effort, formally begun in October 1990 and completed in
2003. The goal of the project was to sequence the entire human
genome, approximately 3 billion nucleotide pairs, compris-
ing 20,000 to 25,000 genes. Thousands of people in 18 coun-
tries participated in this project. Researchers collected blood
(female) or sperm (male) samples from a large number of
donors. Only a few samples were processed as DNA resources,
and the source names are protected so that neither donors nor
scientists knew whose samples were used. Development of
shotgun sequencing greatly speeded the process, and 99% of
the genome has been sequenced.

One surprising finding was that less than 2% of the genome
encodes a functional product—the other 98% includes miRNA
genes, viral remnants, repetitive sequences (called short tandem
repeats), introns, the chromosome ends (called telomeres), and
transposons (page 231).

The next goal of researchers is the Human Proteome Proj-
ect, which will map all the proteins expressed in human cells.
Even before it is completed, however, it’s yielding data that are
of immense value to our understanding of biology. It will also

CLINICAL CASE Resolved

The amino acid sequence reflects the nucleotide sequence. Analysis of the amino acid signature pattern confirms
that the viruses from the dentist and patients are closely
related. HIV has a high mutation rate, so HIVs from different
individuals are genetically distinct. Dr. B.’s HIV is different
from the local control and from the outlier. Dr. B.’s amino acid
sequences and those of patients A, B, C, E, and G are distinct
from those in the control and in the outlier and from two
dental patients with known behavioral risks for HIV infection.

PCR and RFLP analyses have made it possible to track
transmission of disease between individuals, communities,
and countries. This tracking works best with pathogens that
have enough genetic variation to identify different strains.

* * *
Dr. B. died before the mode of transmission could be

established. But in the era when he practiced dentistry, it
was not always the norm to wear gloves when performing
procedures. Patient interviews indicated that Dr. B. didn’t like
to wear gloves. It is likely that HIV was transmitted when a cut
on the doctor’s bare hands allowed the virus to enter patients’
gums. Today the CDC and state health departments ask
dental care providers to use universal precautions, including
wearing gloves and masks and sterilizing equipment that is
to be reused. Had Dr. B. used standard precautions, it is
extremely unlikely he would have infected patients.

243 249 252 254 257

eventually be of great medical benefit, especially for the diag-
nosis and treatment of genetic diseases.

Scientific Applications
Recombinant DNA technology can be used to make products, but
this isn’t its only important application. Because of its ability to
produce many copies of DNA, it can serve as a sort of DNA “print-
ing press.” Once a large amount of a particular piece of DNA is
available, various analytic techniques, discussed in this section,
can be used to “read” the information contained in the DNA.

In 2010, researchers synthesized the smallest known cellu-
lar genome during the Minimal Genome Project. A copy of the
Mycoplasma mycoides genome was synthesized and transplanted
into an M. capricolum cell that had had its own DNA removed.
The modified cell produced M. mycoides proteins. This experi-
ment showed that large-scale changes to a genome can be made
and that an existing cell will accept this DNA.

DNA sequencing has produced an enormous amount of
information that has spawned the new field of bioinformatics,
the science of understanding the function of genes through
computer-assisted analysis. DNA sequences are stored in

1 An abnormal gene, cancer
gene, or virus gene is
transcribed in a host cell.

2 siRNA binds
mRNA.

3 RISC breaks
down the RNA
complex.

4 No protein
expression
occurs.

mRNA
DNA

RNA
transcript

Nucleus
Cytoplasm

siRNA

Figure 9.14 Gene silencing could provide treatments for a
wide range of diseases.

Q Does RNAi act during or after transcription?

258 PART ONE Fundamentals of Microbiology

web-based databases referred to as GenBank. Genomic infor-
mation can be searched with computer programs to find spe-
cific sequences or to look for similar patterns in the genomes of
different organisms. Microbial genes are now being searched to
identify molecules that are the virulence factors of pathogens.
By comparing genomes, researchers discovered that Chlamydia
trachomatis (tra-KŌ-ma-tis) produces a toxin similar to that of
Clostridium difficile (DIF-fi-sē-il).

The next goal is to identify the proteins encoded by these
genes. Proteomics is the science of determining all of the pro-
teins expressed in a cell.

Reverse genetics is an approach to discovering the function
of a gene from a genetic sequence. Reverse genetics attempts to
connect a given genetic sequence with specific effects on the
organism. For example, if you mutate or block a gene (see the
earlier discussions of gene editing on page 255 and gene silenc-
ing on page 256), you can then look for a characteristic the
organism lost.

An example of the use of human DNA sequencing is the
identification and cloning of the mutant gene that causes cystic
fibrosis (CF). CF is characterized by the oversecretion of mucus,
leading to blocked respiratory passageways. The sequence of the
mutated gene can be used as a diagnostic tool in a hybridization
technique called Southern blotting (Figure 9.16), named for Ed
Southern, who developed the technique in 1975.

In this technique, subject DNA is digested with a restriction
enzyme, yielding thousands of fragments of various sizes. The
fragments are called RFLPs (pronounced “rif-lip”), for restric-
tion fragment length polymorphisms. The different fragments are
then separated by gel electrophoresis. The fragments are put

Isolate DNA.

(a) Constructing a gene library

Fragment DNA
with restriction
enzymes.

Clone DNA
in a bacterial
artificial
chromosome
(BAC).

Sequence DNA fragments.

BAC

(b) Random sequencing

Assemble sequences.

Edit
sequences;
fill in gaps.C T T TGA C

4 5

6
2
3

Figure 9.15 Shotgun sequencing. In this technique, a genome is cut into pieces, and each piece is sequenced.
Then the pieces are fit together. There may be gaps if a specific DNA fragment was not sequenced.

Q Does this technique identify genes and their locations?

in a well at one end of a layer of agarose gel. Then an electrical
current is passed through the gel. While the charge is applied,
the different-sized RFLPs migrate through the gel at different
rates. The RFLPs are transferred onto a filter by blotting and
are exposed to a labeled probe made from the cloned gene of
interest, in this case the CF gene. The probe will hybridize to
this mutant gene but not to the normal gene. Fragments to
which the probe binds are identified by a colored dye. With
this method, any person’s DNA can be tested for the presence
of the mutated gene.

Genetic testing can now be used to screen for several hun-
dred genetic diseases. Such screening procedures can be per-
formed on prospective parents and also on fetal tissue. Two of
the more commonly screened genes are those associated with
inherited forms of breast cancer and the gene responsible for
Huntington’s disease. Genetic testing can help a physician pre-
scribe the correct medication for a patient. The drug herceptin,
for example, is effective only in breast cancer patients with a
specific nucleotide sequence in the HER2 gene.

Forensic Microbiology
For several years, microbiologists have used RFLPs in a method
of identification known as DNA fingerprinting to identify

bacterial or viral pathogens (Figure 9.17).
DNA chips (see Figure 10.18, page 288) or PCR microarrays

that can screen a sample for multiple pathogens at once are
now being used. In a DNA chip, up to 22 primers from dif-
ferent microorganisms can be used to initiate the PCR. A sus-
pect microorganism is identified if DNA is copied from one of
the primers. At the Centers for Disease Control and Prevention

CHAPTER 9 Biotechnology and DNA Technology 259

Gel

Human
DNA
fragments

Nitrocellulose
filter

Gel
DNA
transferred
to filter

Sealable
plastic bag

Labeled probes

Sponge

Nitrocellulose
filter

Salt solution

Gel

Paper towels

1 DNA containing the gene of interest is extracted from
human cells and cut into fragments by restriction
enzymes. Fragments are called restriction fragment
length polymorphisms, or RFLPs (pronounced “rif-lips”).

2 The fragments are separated according to size by gel
electrophoresis. Each band contains many copies of a
particular DNA fragment. The bands are invisible but can
be made visible by staining.

3 The DNA bands are transferred to a nitrocellulose filter by
blotting. The solution passes through the gel and filter to
the paper towels by capillary action.

4 This produces a nitrocellulose filter with DNA fragments
positioned exactly as on the gel.

5 The filter is exposed to a labeled probe for a specific gene.
The probe will base-pair (hybridize) with a short sequence
present on the gene.

6 The fragment containing the gene of interest is identified by a
band on the filter.

Restriction enzyme

Larger

Smaller

Gene of interest

Figure 9.16 Southern blotting.

Q What is the purpose of Southern blotting?

(CDC), PulseNet uses RFLPs to track outbreaks of foodborne
disease. In some cases, PCR using specific primers can be used
to track a bacterial strain to locate the source of an outbreak.

The genomics of pathogens has become a mainstay of mon-
itoring, preventing, and controlling infectious disease. The use
of genomics to trace a disease outbreak is described in the Clin-
ical Focus box on page 264. The new field of forensic microbi-
ology developed because hospitals, food manufacturers, and
individuals can be sued in courts of law and because microor-
ganisms can be used as weapons. In the 2001 anthrax attacks
in the United States, DNA fingerprints of Bacillus anthracis
were used to track the source and then the alleged attacker.

Northern Arizona University researchers determined that the
B. anthracis endospores used in a 1993 attack by a cult in Japan
were actually a nonpathogenic vaccine strain. No one was hurt
when those endospores were released. Currently, a DNA data-
base is being developed for microorganisms that could be used
in biological crimes.

Microbial forensics has been used in court. In the 1990s,
DNA fingerprints of HIV were used for the first time to obtain
a rape conviction. Since then, a physician was convicted of
injecting his former lover with HIV from one of his patients,
based on the DNA fingerprint of the HIV.

260 PART ONE Fundamentals of Microbiology

SEM 1 mm

E. coli isolates from
patients whose
infections were
not juice related

E. coli isolates from
patients who drank
contaminated juice

Apple juice
isolates

that make nanoparticles from a variety of elements, includ-
ing gold, silver, selenium, and cadmium. (Figure 9.18). Nano-
technology research is growing, with researchers developing
innovative ways of using bacteria to produce nanospheres for
potential drug targeting and delivery. Researchers with the
U.S. Department of Energy are using bacteria in nanoscale
electrical circuits to make hydrogen gas. Swedish researchers
are using Acetobacter xylinum to build cellulose nanofibers for
artificial blood vessels.

CHECK YOUR UNDERSTANDING

✓ 9-15, 9-16 How are shotgun sequencing, bioinformatics,
and proteomics related to genome projects?

✓ 9-17 What is Southern blotting?
✓ 9-18 Why do RFLPs result in a DNA fingerprint?

Agricultural Applications
The process of selecting for genetically desirable plants has
always been time-consuming. Conventional plant cross-breeding
is laborious and involves waiting for the planted seed to germi-
nate and the resulting plant to mature in order to learn whether
the plant has the desired traits. Plant breeding has been revo-
lutionized by the use of plant cells grown in culture. Clones of
plant cells, including cells that have been genetically altered by
rDNA techniques, can be grown in large numbers. These cells can
then be induced to regenerate whole plants, from which seeds
can be harvested.

Recombinant DNA can be introduced into plant cells in
several ways. Previously we mentioned protoplast fusion and
the use of DNA-coated “bullets.” The most elegant method,
however, makes use of a plasmid called the Ti plasmid
(Ti stands for tumor-inducing), which occurs naturally in
the bacterium Agrobacterium tumefaciens (TOO-mah-fas9ē-enz).
This bacterium infects certain plants, in which the Ti plasmid

Figure 9.17 DNA fingerprints used to track an infectious
disease. This figure shows the RFLP patterns of bacterial isolates
from an outbreak of Escherichia coli O157:H7. The isolates from apple
juice are identical to the patterns of isolates from patients who drank
the contaminated juice but different from those from patients whose
infections were not juice related.

Q What is forensic microbiology?

The requirements to prove in a court of law the source of
a microbe are stricter than for the medical community. For
example, to prove intent to commit harm requires collecting
evidence properly and establishing a chain of custody of that
evidence. Microbial properties that are unimportant in pub-
lic health may be important clues in forensic investigations.
The genetic fingerprint of sexually transmitted pathogens, for
instance, has been used as evidence in sexual abuse and rape
cases. In the Clinic (page 242) offers another example of the use
of bacterial genomics in a criminal investigation. These devel-
opments suggest that the human microbiome may become an
important law enforcement tool. The American Academy of
Microbiology recently proposed professional certification in
forensic microbiology.

Nanotechnology
Nanotechnology deals with the design and manufacture of
extremely small electronic circuits and mechanical devices
built at the molecular level of matter. Molecule-sized robots
or computers can be used to detect contamination in food,
diseases in plants, or biological weapons. However, the small
machines require small (a nanometer is 10−9 meters; 1000 nm
fit in 1 mm) wires and components. Bacteria may provide the
needed small metals without producing the toxic waste associ-
ated with chemical manufacture. Bacteria have been isolated

Figure 9.18 Bacillus cells
growing on selenium form
chains of elemental selenium.

Q What might
bacteria provide for
nanotechnology?

Fingerprints, blood types, and DNA were once new to crime scene investigations (CSI). Each technique
uses unique profiles from the human
body to draw conclusions about a
person’s actions or whereabouts. Now the
microbiome might be the next CSI tool.

Even after we wash our hands,
certain bacteria persist. These microbes
can also be transferred to objects in
the home or office or to other people
we live with. But which microbes
commonly live on the body also varies
greatly throughout the population as a
whole—meaning that the microbiome
can become a unique identifier in certain
situations.

A research project called The Home
Microbiome Project followed seven
families and their pets over 6 weeks.
Researchers discovered distinct microbial

communities in each house. Couples
and their young children shared most
of their microbial community. When
three of the families moved, it took
less than a day for the new house to
have the same microbial population as
the old one.

In another study, it was shown that a
person’s “microbiome fingerprint” remains
fairly consistent over time. All this research
suggests that microbiome composition may
be the basis for a reliable forensic tool.
Microbiome profiles could be used to track
whether a person lived somewhere, used
a particular cell phone, or walked over a
surface. Humans also exchange microbes
during intercourse, so microbes on pubic
hair might also provide evidence of sexual
assault.

Microbiota, like this skin biofilm, may one day be another
crime scene “fingerprint.”

causes the formation of a tumorlike growth called a crown gall
(Figure 9.19). A part of the Ti plasmid, called T-DNA, integrates
into the genome of the infected plant. The T-DNA stimulates
local cellular growth (the crown gall) and simultaneously
causes the production of certain products used by the bacteria
as a source of nutritional carbon and nitrogen.

For plant scientists, the attraction of the Ti plasmid is that it
provides a vehicle for introducing rDNA into a plant (Figure 9.20).
A scientist can insert foreign genes into the T-DNA, put the
recombinant plasmid back into the Agrobacterium cell, and use
the bacterium to insert the recombinant Ti plasmid into a plant
cell. The plant cell with the foreign gene can then be used to
generate a new plant. With luck, the new plant will express the
foreign gene. Unfortunately, Agrobacterium does not naturally
infect grasses, so it cannot be used to improve grains such as
wheat, rice, or corn.

Noteworthy accomplishments of this approach are the
introduction into plants of resistance to the herbicide glypho-
sate. Normally, the herbicide kills both weeds and useful
plants by inhibiting an enzyme necessary for making certain
essential amino acids. Some Salmonella bacteria happen to
have this enzyme, but are resistant to the herbicide. When the
DNA for this enzyme is introduced into a crop plant, the crop

EXPLORING THE MICROBIOME Crime Scene Investigation and
Your Microbiome

Crown gall

Figure 9.19 Crown gall disease on a rose plant. The tumorlike
growth is stimulated by a gene on the Ti plasmid that Agrobacterium
tumefaciens inserted into a plant cell.

Q What are some of the agricultural applications of rDNA
technology? 261

262 PART ONE Fundamentals of Microbiology

becomes resistant to the herbicide, which then kills only the
weeds. The Bt gene from Bacillus thuringiensis has been inserted
into a variety of crop plants, including cotton and potatoes, so
insects that eat the plants will be killed. Resistance to drought,
viral infection, and several other environmental stresses has
also been engineered into crop plants.

Another example involves FlavrSavr™ tomatoes, which
stay firm after harvest because the gene for polygalacturonase
(PG), the enzyme that breaks down pectin, is suppressed. The
suppression was accomplished by antisense DNA technology.
First, a length of DNA complementary to the PG mRNA is syn-
thesized. This antisense DNA is taken up by the cell and binds
to the mRNA to inhibit translation. The DNA-RNA hybrid is
broken down by the cell’s enzymes, freeing the antisense DNA
to disable another mRNA.

An example of a genetically modified bacterium now in
agricultural use is Pseudomonas fluorescens that has been engi-
neered to produce Bt toxin, normally produced by Bacillus
thuringiensis. The genetically altered Pseudomonas, which pro-
duces much more toxin than B. thuringiensis, can be added to
plant seeds and in time will enter the vascular system of the
growing plant. Its toxin is ingested by the feeding insect larvae
and kills them (but is harmless to humans and other warm-
blooded animals).

1 The plasmid is removed
from the bacterium, and
the T-DNA is cut by a
restriction enzyme.

Ti
plasmid

T-DNA

Restriction
cleavage
site

2 Foreign DNA is cut
by the same enzyme.

3 The foreign DNA is
inserted into the T-DNA
of the plasmid.

Recombinant
Ti plasmid

4 The plasmid
is reinserted
into a bacterium.

Agrobacterium tumefaciens
bacterium

5 The bacterium is
used to insert the
T-DNA carrying the
foreign gene into the
chromosome of a
plant cell.

6 The plant cells
are grown in
culture.

7 A plant is generated from a cell
clone. All of its cells carry the
foreign gene and may express
it as a new trait.

Inserted T-DNA
carrying foreign
gene

Figure 9.20 Using the Ti plasmid as a vector for genetic modification in plants.

Q Why is the Ti plasmid important to biotechnology?

Animal husbandry has also benefited from rDNA technol-
ogy to develop disease-resistant food animals. Techniques for
making cattle resistant to bovine spongiform encephalopathy
and chickens and pigs resistant to avian influenza are currently
being researched.

Table 9.3 lists several rDNA products used in agriculture

and animal husbandry.

CHECK YOUR UNDERSTANDING

✓ 9-19 Of what value is the plant pathogen Agrobacterium?

Safety Issues and the Ethics of
Using DNA Technology
LEARNING OBJECTIVE

9-20 List the advantages of, and problems associated with, the
use of genetic modification techniques.

There will always be concern about the safety of any new tech-
nology, and genetic modification and biotechnology are cer-
tainly no exceptions. One reason for this concern is it’s nearly
impossible to prove that something is entirely safe under all
conceivable conditions. People worry that the same tech-
niques that can alter a microbe or plant to make them useful
to humans could also inadvertently make them pathogenic to

CHAPTER 9 Biotechnology and DNA Technology 263

TABLE 9.3 Some Agriculturally Important Products of rDNA Technology
Product Comments

AGRICULTURAL PRODUCTS

Button mushroom (Agaricus bisporus) Gene for polyphenyl oxidase, which causes browning, is deleted.

Bt cotton and Bt corn Plants have toxin-producing gene from Bacillus thuringiensis; toxin kills insects that eat plants.

Genetically modified tomatoes, raspberries Antisense gene blocks pectin degradation, so fruits have longer shelf life.

Pseudomonas syringae, ice-minus bacterium Lacks normal protein product that initiates undesirable ice formation on plants.

RoundUp (glyphosate)-resistant crops Plants have bacterial gene; allows use of herbicide on weeds without damaging crops.

ANIMAL PRODUCTS

Aedes aegypti Male mosquito with a gene that causes larvae to die; used to control spread of Zika virus.

Atlantic salmon Salmon grow faster with a gene from Chinook salmon and promoter from another fish (pout).

GloFish® Brightly colored fluorescent aquarium fish with the color-protein genes from marine
invertebrates.

humans or otherwise dangerous to living organisms or could
create an ecological nightmare. Therefore, laboratories engaged
in rDNA research must meet rigorous standards of control to
avoid either accidental release of genetically modified organ-
isms into the environment or exposure of humans to any risk
of infection. To reduce risk further, microbiologists engaged in
genetic modification often delete from the microbes’ genomes
certain genes that are essential for growth in environments out-
side the laboratory. Genetically modified organisms intended
for use in the environment (in agriculture, for example) may be
engineered to contain “suicide genes”—genes that eventually
turn on to produce a toxin that kills the microbes, thus ensur-
ing that they will not survive in the environment for very long
after they have accomplished their task.

The safety issues in agricultural biotechnology are similar to
those concerning chemical pesticides: toxicity to humans and
to nonpest species. Although not shown to be harmful, geneti-
cally modified foods have not been popular with consumers.
In 1999, researchers in Ohio noticed that humans may develop
allergies to Bacillus thuringiensis (Bt) toxin after working in
fields sprayed with the insecticide. And an Iowa study showed
that the caterpillar stage of Monarch butterflies could be killed
by ingesting windblown Bt-carrying pollen that landed on
milkweed, the caterpillars’ normal food. Crop plants can be
genetically modified for herbicide resistance so that fields can
be sprayed to eliminate weeds without killing the desired crop.
However, if the modified plants pollinate related weed species,

weeds could become resistant to herbicides, making it more
difficult to control unwanted plants. An unanswered question
is whether releasing genetically modified organisms will alter
evolution as genes move to wild species.

These developing technologies also raise a variety of ethi-
cal issues. Genetic testing for diseases is becoming routine.
Who should have access to this information? Should employers
have the right to know the results of such tests? How can we be
assured that such information will not be used to discriminate
against certain groups? Should individuals be told they will get
an incurable disease? If so, when?

Genetic counseling, which provides advice and counseling
to prospective parents with family histories of genetic disease,
is becoming more important in considerations about whether
to have children.

There are probably just as many harmful applications of
a new technology as there are helpful ones. It is particularly
easy to imagine DNA technology being used to develop new
and powerful biological weapons. In addition, because such
research efforts are performed under top-secret conditions,
it is virtually impossible for the general public to learn of
them.

Perhaps more than most new technologies, molecular
genetics holds the promise of affecting human life in previ-
ously unimaginable ways. It is important that society and indi-
viduals be given every opportunity to understand the potential
impact of these new developments.

CLINICAL FOCUS Norovirus—Who Is Responsible for the
Outbreak?

As you read through this box, you will encounter a series of questions that microbiologists ask themselves as
they trace a disease outbreak. Whether the
microbiologist is called as an expert witness
in court will depend on whether a lawsuit
is filed. Try to answer each question before
going on to the next one.

1. On May 7, Nadia Koehler, a microbiologist
at a county health department, is noti-
fied of a gastroenteritis outbreak among
115 people. The case is defined as
vomiting and diarrhea and fever, cramps,
or nausea.
What information does Nadia need?

2. Nadia needs to find out where the ill
people have been in the past 48 hours.
After several interviews, Nadia finds
out that the ill people include 23 school
employees, 55 publishing company
employees, 9 employees of a social
service organization, and 28 other
people (see Figure A).
Now what does Nadia need to know?

3. Next, Nadia finds out what these
115 people have in common. In her
investigation, Nadia discovers that on
May 2, the school staff had been served

N
um

be
r

of
r

ep
or

te
d

ca
se

s
2

School
meal
served

Initial reports
of community
cases

3 4 5 6
Date

45
40
35
30
25
20
15

5
Reported community
cases

Social service group

School employees

Publishing company
employees

KEY

a party-sized sandwich catered by
a national franchise restaurant. On
May 3, the publishing company and
social service staff luncheons were
catered by the same restaurant. The
remaining 28 people ate sandwiches
at the same restaurant, at varying
times between these two days.
What does Nadia do next?

4. Nadia analyzes exposures to 16 food
items; the results show that eating
lettuce is significantly associated with
illness.
What is Nadia’s next step?

5. Nadia then requests a reverse-
transcription PCR (RT-PCR) using a
norovirus primer to be done on stool
samples (Figure B).
What did Nadia conclude?

6. RT-PCR confirmed norovirus infection.
Nadia’s next request is for a sequence
analysis to be performed on 21 stool
specimens. The results demonstrated
100% sequence homology for the 21
specimens.
What should Nadia do next?

7. Nadia learns that a food handler
employed by the restaurant had
experienced vomiting and diarrhea on
May 1. The food handler believes he had
acquired the illness from his child. The
child’s illness was traced to an ill cousin
who had been exposed to norovirus at
a child-care center. The food handler’s
vomiting ended by the early morning of
May 2, and he returned to work at the
restaurant later that morning.
What should Nadia look for now?

8. Now Nadia compares the virus strains
from the food handler to the ones
from the ill customers. She requests a
sequence analysis on viruses from the

1 2 3 4 5 6 7 8

213

food handler and eight ill customers.
They are identical to the strains
identified in step 6.
Where does Nadia look next?

9. Nadia looks for any areas in
the restaurant that still may be
contaminated by the norovirus. She
finds out that the lettuce was sliced
each morning by the food handler who
had been sick. Nadia’s inspection
reveals that the food preparation sink
is also used for handwashing. The sink
was not sanitized before and after
the lettuce was washed. The health
department closes the restaurant
until it can be cleaned with the proper
sanitizers.

Noroviruses are the most common
cause of outbreaks of acute gastroenteritis
worldwide. Annually, norovirus causes 20
million cases of gastroenteritis. During
2015, 316 norovirus outbreaks in the United
States were reported.

Source: Adapted from CDC, Foodborne Outbreak Online
Database (FOOD).

Figure B Results of PCR of patient
samples. Lane 1, 123-bp size ladders.
Lane 2, negative RT-PCR control; Lanes
3–8, patient samples. Norovirus is
identified by the 213-bp band of DNA.

Figure A Number of cases reported.

264

CHAPTER 9 Biotechnology and DNA Technology 265

Like the invention of the microscope, the development of
DNA techniques is causing profound changes in science, agri-
culture, and human health care. With this technology not
quite 50 years old, it is difficult to predict exactly what changes
will occur. However, it is likely that within another 30 years,
many of the treatments and diagnostic methods discussed
in this book will have been replaced by far more powerful

Study Outline

techniques based on the unprecedented ability to manipulate
DNA precisely.

CHECK YOUR UNDERSTANDING

✓ 9-20 Identify two advantages and two problems associated
with genetically modified organisms.

Go to @MasteringMicrobiology for Interactive Microbiology, In the Clinic
videos, MicroFlix, MicroBoosters, 3D animations, practice quizzes, and more.

Introduction to Biotechnology (pp. 243–245)
1. Biotechnology is the use of microorganisms, cells, or cell

components to make a product.

Recombinant DNA Technology (p. 243)
2. Closely related organisms can exchange genes in natural

recombination .

3. Genes can be transferred among unrelated species via laboratory
manipulation, called rDNA technology.

4. Recombinant DNA is DNA that has been artificially manipulated
to combine genes from two different sources.

An Overview of Recombinant DNA Procedures (pp. 243–245)
5. A desired gene is inserted into a DNA vector, such as a plasmid or a

viral genome.

6. The vector inserts the DNA into a new cell, which is grown to form
a clone.

7. Large quantities of the gene product can be harvested from the
clone.

Tools of Biotechnology (pp. 245–248)

Selection (p. 245)
1. Microbes with desirable traits are selected for culturing by artificial

selection .

Mutation (p. 245)
2. Mutagens are used to cause mutations that might result in a

microbe with desirable traits.

3. Site-directed mutagenesis is used to change a specific codon in a gene.

Restriction Enzymes (pp. 245–246)
4. Prepackaged kits are available for rDNA techniques.

5. A restriction enzyme recognizes and cuts only one particular
nucleotide sequence in DNA .

6. Some restriction enzymes produce sticky ends, short stretches of
single-stranded DNA at the ends of the DNA fragments.

7. Fragments of DNA produced by the same restriction enzyme will
spontaneously join by base pairing. DNA ligase can covalently link
the DNA backbones.

Vectors (pp. 246–247)
8. Vectors are DNA used to transfer other DNA between cells.

9. A plasmid containing a new gene can be inserted into a cell by
transformation .

10. A virus containing a new gene can insert the gene into a cell.

Polymerase Chain Reaction (pp. 247–248)
11. The polymerase chain reaction (PCR) is used to make multiple

copies of a desired piece of DNA enzymatically.

12. PCR can be used to increase the amounts of DNA in samples to
detectable levels. This may allow sequencing of genes, the diagnosis
of genetic diseases, or the detection of viruses.

Techniques of Genetic Modification (pp. 248–254)

Inserting Foreign DNA into Cells (pp. 249–250)
1. Cells can take up naked DNA by transformation . Chemical

treatments are used to make cells that are not naturally competent
take up DNA .

2. Pores made in protoplasts and animal cells by electric current in the
process of electroporation can provide entrance for new pieces of DNA .

3. Protoplast fusion is the joining of cells whose cell walls have been
removed.

4. Foreign DNA can be introduced into plant cells by shooting DNA-
coated particles into the cells or by using a thin micropipette.

Obtaining DNA (pp. 250–252)
5. Genomic libraries can be made by cutting up an entire genome

with restriction enzymes and inserting the fragments into bacterial
plasmids or phages.

6. Complementary DNA (cDNA) made from mRNA by reverse
transcription can be cloned in genomic libraries.

7. Synthetic DNA can be made in vitro by a DNA synthesis machine.

Selecting a Clone (pp. 252–253)
8. Antibiotic-resistance markers on plasmid vectors are used to

identify cells containing the engineered vector by direct selection .

9. In blue-white screening, the vector contains the genes for amp and
β-galactosidase.

10. The desired gene is inserted into the β-galactosidase gene site,
destroying the gene.

11. Clones containing the recombinant vector will be resistant to
ampicillin and unable to hydrolyze X-gal (white colonies).

12. Clones containing foreign DNA can be tested for the desired gene
product.

13. A short piece of labeled DNA called a DNA probe can be used to
identify clones carrying the desired gene.

266 PART ONE Fundamentals of Microbiology

Study Questions

11. Bioinformatics is the use of computer applications to study genetic
data; proteomics is the study of a cell’s proteins.

12. Southern blotting can be used to locate a gene in a cell.

13. DNA probes can be used to quickly identify a pathogen in body
tissue or food.

14. Forensic microbiologists use DNA fingerprinting to identify the
source of bacterial or viral pathogens.

15. Bacteria may be used to make nano-sized materials for
nanotechnology machines.

Agricultural Applications (pp. 260–262)
16. Cells from plants with desirable characteristics can be cloned

to produce many identical cells. These cells can then be used to
produce whole plants from which seeds can be harvested.

17. Plant cells can be modified by using the Ti plasmid vector. The
tumor-producing T genes are replaced with desired genes, and
the rDNA is inserted into Agrobacterium. The bacterium naturally
transforms its plant hosts.

18. Antisense DNA can prevent expression of unwanted proteins.

Safety Issues and the Ethics of Using DNA
Technology (pp. 262–265)
1. Strict safety standards are used to avoid the accidental release of

genetically modified microorganisms.

2. Some microbes used in rDNA cloning have been altered so that
they cannot survive outside the laboratory.

3. Microorganisms intended for use in the environment may be
modified to contain suicide genes so that the organisms do not
persist in the environment.

4. Genetic testing raises a number of ethical questions: Should
employers have access to a person’s genetic records? Will genetic
information be used to discriminate against people? Will genetic
counseling be available to everyone?

5. Genetically modified crops must be safe for consumption and for
release in the environment.

Making a Gene Product (pp. 253–254)
14. E. coli is used to produce proteins using rDNA because E. coli is

easily grown and its genomics are well understood.

15. Efforts must be made to ensure that E. coli’s endotoxin does not
contaminate a product intended for human use.

16. To recover the product, E. coli must be lysed, or the gene must be
linked to a gene that produces a naturally secreted protein.

17. Yeasts can be genetically modified and are likely to secrete a gene
product continuously.

18. Genetically modified mammalian cells can be grown to produce
proteins such as hormones for medical use.

19. Genetically modified plant cells can be grown and used to produce
plants with new properties.

Applications of DNA Technology (pp. 254–262)
1. Cloned DNA is used to produce products, study the cloned DNA,

and alter the phenotype of an organism.

Therapeutic Applications (pp. 255–256)
2. Synthetic genes linked to the β-galactosidase gene (lacZ) in a

plasmid vector were inserted into E. coli, allowing E. coli to
produce and secrete the two polypeptides used to make human
insulin.

3. Cells and viruses can be modified to produce a pathogen’s surface
protein, which can be used as a vaccine.

4. DNA vaccines consist of rDNA cloned in bacteria.

5. Gene therapy can be used to cure genetic diseases by replacing the
defective or missing gene.

6. RNAi may be useful to prevent expression of abnormal proteins.

Genome Projects (pp. 256–257)
7. Nucleotide sequences of genomes from more than 1000 organisms,

including humans, have been completed.

8. This leads to determining the proteins produced in a cell.

Scientific Applications (pp. 257–260)
9. DNA can be used to increase understanding of DNA, for genetic

fingerprinting, and for gene therapy.

10. DNA sequencing machines are used to determine the
nucleotide base sequence of restriction fragments in shotgun
sequencing.

For answers to Knowledge and Comprehension questions, turn to the
Answers tab at the back of the textbook.

Knowledge and Comprehension

Review
1. Compare and contrast the following terms:

a. cDNA and gene

b. RFLP and gene

c. DNA probe and gene

d. DNA polymerase and DNA ligase

e. rDNA and cDNA

f. genome and proteome

2. Differentiate the following terms. Which one is “hit and miss”—
that is, does not add a specific gene to a cell?
a. protoplast fusion
b. gene gun
c. microinjection
d. electroporation

CHAPTER 9 Biotechnology and DNA Technology 267

3. Some commonly used restriction enzymes are listed in Table 9.1
on page 246.
a. Indicate which enzymes produce sticky ends.
b. Of what value are sticky ends in making rDNA?

4. Suppose you want multiple copies of a gene you have synthesized.
How would you obtain the necessary copies by cloning? By PCR?

5. DRAW IT Using the following map of plasmid pMICRO,
diagram the locations of the restriction fragments that result from
digesting pMICRO with EcoRI, HindIII, and both enzymes together
following electrophoresis. Which enzyme makes the smallest
fragment containing the tetracycline resistance gene?

S
iz

e
la

dd
er

E
co

R
I d

ig
es

H
in

dI
II

di
ge

D
ou

bl
e

di
ge
st

Base
pairs

2517

1517

200

1000
900
800
700
600
500

400

300

200

100

tet

HindIII

100 bp
200 bp

650 bp

550 bp

1300 bp

HindIII
EcoRI
EcoRI
EcoRI

6. Describe an rDNA experiment in two or three sentences. Use
the following terms: intron, exon, DNA, mRNA, cDNA, RNA
polymerase, reverse transcriptase.

7. List at least two examples of the use of rDNA in medicine and in
agriculture.

8. You are attempting to insert a gene for saltwater tolerance into a
plant by using the Ti plasmid. In addition to the desired gene, you
add a gene for tetracycline resistance (tet) to the plasmid. What is
the purpose of the tet gene?

9. How does RNAi “silence” a gene?

10. NAME IT This virus family, normally associated with AIDS, may
be useful for gene therapy.

Multiple Choice
1. Restriction enzymes were first discovered with the observation that

a. DNA is restricted to the nucleus.
b. phage DNA is destroyed in a host cell.
c. foreign DNA is kept out of a cell.
d. foreign DNA is restricted to the cytoplasm.
e. all of the above

2. The DNA probe, 3’-GGCTTA, will hybridize with which of the
following?
a. 5’-CCGUUA
b. 5’-CCGAAT
c. 5’-GGCTTA

d. 3’-CCGAAT
e. 3’-GGCAAU

3. Which of the following is the fourth basic step to genetically
modify a cell?
a. transformation
b. ligation
c. plasmid cleavage
d. restriction-enzyme digestion of gene
e. isolation of gene

4. The following enzymes are used to make cDNA. What is the second
enzyme used to make cDNA?
a. reverse transcriptase
b. ribozyme
c. RNA polymerase
d. DNA polymerase

5. If you put a gene in a virus, the next step in genetic modification
would be
a. insertion of a plasmid.
b. transformation.
c. transduction.

d. PCR.
e. Southern blotting.

6. You have a small gene that you want replicated by PCR. You add
radioactively labeled nucleotides to the PCR thermal cycler. After
three replication cycles, what percentage of the DNA single strands
are radioactively labeled?
a. 0%
b. 12.5%
c. 50%

d. 87.5%
e. 100%

Match the following choices to the statements in questions 7 through 10.
a. antisense
b. clone
c. library

d. Southern blot
e. vector

7. Pieces of human DNA stored in yeast cells.

8. A population of cells carrying a desired plasmid.

9. Self-replicating DNA for transmitting a gene from one organism to
another.

10. DNA that hybridizes with mRNA.

268 PART ONE Fundamentals of Microbiology

Clinical Applications and Evaluation
1. PCR has been used to examine oysters for

the presence of Vibrio cholerae. Oysters from
different areas were homogenized, and DNA
was extracted from the homogenates. The DNA
was digested by the restriction enzyme HincII.
A primer for the hemolysin gene of V. cholerae
was used for the PCR reaction. After PCR, each
sample was electrophoresed and stained with
a probe for the hemolysin gene. Which of
the oyster samples were (was) positive for
V. cholerae? How can you tell? Why look for
V. cholerae in oysters? What is the advantage
of PCR over conventional biochemical tests to
identify the bacteria?

A B C

2. Using the restriction enzyme EcoRI, the following gel electrophoresis
patterns were obtained from digests of various DNA molecules from
a transformation experiment. Can you conclude from these data that
transformation occurred? Explain why or why not.

Origin

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ec

to
r

V
ec

to
r

+
n

ew
g

en
e

N
ew

g
en

O
rig

in
al

c
el

l’s
D

N
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T
ra

ns
fo

rm
ed

ce
ll’

s
D

N
A

Analysis
1. Design an experiment using vaccinia virus to make a vaccine

against the AIDS virus (HIV).

2. Why did the use of DNA polymerase from the bacterium Thermus
aquaticus allow researchers to add the necessary reagents to tubes in
a preprogrammed heating block?

3. The following picture shows bacterial colonies growing on X-gal
plus ampicillin in a blue-white screening test. Which colonies have
the recombinant plasmid? The small satellite colonies do not have
the plasmid. Why did they start growing on the medium 48 hours
after the larger colonies?

Cover
Brief
Contents
Title Page
Copyright Page
About the Authors
Digital Authors
Preface
Acknowledgments

Contents

Features
ASM Recommended Curriculum Guidelinesfor Undergraduate Microbiology
PART ONE Fundamentals of Microbiology

1 The Microbial World and You
Microbes in Our Lives
The Microbiome
Naming and Classifying Microorganisms
Nomenclature
Types of Microorganisms
Classification of Microorganisms
A Brief History of Microbiology
The First Observations
The Debate over Spontaneous Generation
The First Golden Age of Microbiology
The Second Golden Age of Microbiology
The Third Golden Age of Microbiology
Microbes and Human Welfare
Recycling Vital Elements
Sewage Treatment: Using Microbes to Recycle Water
Bioremediation: Using Microbes to Clean Up Pollutants
Insect Pest Control by Microorganisms
Biotechnology and Recombinant DNA Technology
Microbes and Human Disease
Biofilms
Infectious Diseases
Emerging Infectious Diseases
Study Outline
Study Questions
2 Chemical Principles
The Structure of Atoms
Chemical Elements
Electronic Configurations
How Atoms Form Molecules: Chemical Bonds
Ionic Bonds
Covalent Bonds
Hydrogen Bonds
Molecular Mass and Moles
Chemical Reactions
Energy in Chemical Reactions
Synthesis Reactions
Decomposition Reactions
Exchange Reactions
The Reversibility of Chemical Reactions
IMPORTANT BIOLOGICAL MOLECULES
Inorganic Compounds
Water
Acids, Bases, and Salts
Acid–Base Balance: The Concept of pH
Organic Compounds
Structure and Chemistry
Carbohydrates
Lipids
Proteins
Nucleic Acids
Adenosine Triphosphate (ATP)
Study Outline
Study Questions
3 Observing Microorganisms Through a Microscope
Units of Measurement
Microscopy: The Instruments
Light Microscopy
Two-Photon Microscopy
Super-Resolution Light Microscopy
Scanning Acoustic Microscopy
Electron Microscopy
Scanned-Probe Microscopy
Preparation of Specimens for Light Microscopy
Preparing Smears for Staining
Simple Stains
Differential Stains
Special Stains
Study Outline
Study Questions
4 Functional Anatomy of Prokaryotic and Eukaryotic Cells
Comparing Prokaryotic and Eukaryotic Cells: An Overview
THE PROKARYOTIC CELL
The Size, Shape, and Arrangement of Bacterial Cells
Structures External to the Cell Wall
Glycocalyx
Flagella and Archaella
Axial Filaments
Fimbriae and Pili
The Cell Wall
Composition and Characteristics
Cell Walls and the Gram Stain Mechanism
Atypical Cell Walls
Damage to the Cell Wall
Structures Internal to the Cell Wall
The Plasma (Cytoplasmic) Membrane
The Movement of Materials across Membranes
Cytoplasm
The Nucleoid
Ribosomes
Inclusions
Endospores
THE EUKARYOTIC CELL
Flagella and Cilia
The Cell Wall and Glycocalyx
The Plasma (Cytoplasmic) Membrane
Cytoplasm
Ribosomes
Organelles
The Nucleus
Endoplasmic Reticulum
Golgi Complex
Lysosomes
Vacuoles
Mitochondria
Chloroplasts
Peroxisomes
Centrosome
The Evolution of Eukaryotes
Study Outline
Study Questions
5 Microbial Metabolism
Catabolic and Anabolic Reactions
Enzymes
Collision Theory
Enzymes and Chemical Reactions
Enzyme Specificity and Efficiency
Naming Enzymes
Enzyme Components
Factors Influencing Enzymatic Activity
Feedback Inhibition
Ribozymes
Energy Production
Oxidation-Reduction Reactions
The Generation of ATP
Metabolic Pathways of Energy Production
Carbohydrate Catabolism
Glycolysis
Additional Pathways to Glycolysis
Cellular Respiration
Fermentation
Lipid and Protein Catabolism
Biochemical Tests and Bacterial Identification
Photosynthesis
The Light-Dependent Reactions: Photophosphorylation
The Light-Independent Reactions: The Calvin-Benson Cycle
A Summary of Energy Production Mechanisms
Metabolic Diversity among Organisms
Photoautotrophs
Photoheterotrophs
Chemoautotrophs
Chemoheterotrophs
Metabolic Pathways of Energy Use
Polysaccharide Biosynthesis
Lipid Biosynthesis
Amino Acid and Protein Biosynthesis
Purine and Pyrimidine Biosynthesis
The Integration of Metabolism
Study Outline
Study Questions
6 Microbial Growth
The Requirements for Growth
Physical Requirements
Chemical Requirements
Biofilms
Culture Media
Chemically Defined Media
Complex Media
Anaerobic Growth Media and Methods
Special Culture Techniques
Selective and Differential Media
Enrichment Culture
Obtaining Pure Cultures
Preserving Bacterial Cultures
The Growth of Bacterial Cultures
Bacterial Division
Generation Time
Logarithmic Representation of Bacterial Populations
Phases of Growth
Direct Measurement of Microbial Growth
Estimating Bacterial Numbers by Indirect Methods
Study Outline
Study Questions
7 The Control of Microbial Growth
The Terminology of Microbial Control
The Rate of Microbial Death
Actions of Microbial Control Agents
Alteration of Membrane Permeability
Damage to Proteins and Nucleic Acids
Physical Methods of Microbial Control
Heat
Filtration
Low Temperatures
High Pressure
Desiccation
Osmotic Pressure
Radiation
Chemical Methods of Microbial Control
Principles of Effective Disinfection
Evaluating a Disinfectant
Types of Disinfectants
Microbial Characteristics and Microbial Control
Study Outline
Study Questions
8 Microbial Genetics
Structure and Function of the Genetic Material
Genotype and Phenotype
DNA and Chromosomes
The Flow of Genetic Information
DNA Replication
RNA and Protein Synthesis
The Regulation of Bacterial Gene Expression
Pre-transcriptional Control
Post-transcriptional Control
Changes in Genetic Material
Mutation
Types of Mutations
Mutagens
The Frequency of Mutation
Identifying Mutants
Identifying Chemical Carcinogens
Genetic Transfer and Recombination
Plasmids and Transposons
Transformation in Bacteria
Conjugation in Bacteria
Transduction in Bacteria
Genes and Evolution
Study Outline
Study Questions
9 Biotechnology and DNA Technology
Introduction to Biotechnology
Recombinant DNA Technology
An Overview of Recombinant DNA Procedures
Tools of Biotechnology
Selection
Mutation
Restriction Enzymes
Vectors
Polymerase Chain Reaction
Techniques of Genetic Modification
Inserting Foreign DNA into Cells
Obtaining DNA
Selecting a Clone
Making a Gene Product
Applications of DNA Technology
Therapeutic Applications
Genome Projects
Scientific Applications
Agricultural Applications
Safety Issues and the Ethics of Using DNA Technology
Study Outline
Study Questions

PART TWO A Survey of the Microbial World

10 Classification of Microorganisms
The Study of Phylogenetic Relationships
The Three Domains
A Phylogenetic Tree
Classification of Organisms
Scientific Nomenclature
The Taxonomic Hierarchy
Classification of Prokaryotes
Classification of Eukaryotes
Classification of Viruses
Methods of Classifying and Identifying Microorganisms
Morphological Characteristics
Differential Staining
Biochemical Tests
Serology
Phage Typing
Fatty Acid Profiles
Flow Cytometry
DNA Sequencing
DNA Fingerprinting
Nucleic Acid Hybridization
Putting Classification Methods Together
Study Outline
Study Questions
11 The Prokaryotes: Domains Bacteria and Archaea
The Prokaryotic Groups
DOMAIN BACTERIA
Gram-Negative Bacteria
Proteobacteria
The Nonproteobacteria Gram-Negative Bacteria
The Gram-Positive Bacteria
Firmicutes (Low G + C Gram-Positive Bacteria)
Tenericutes
Actinobacteria (High G + C Gram-Positive Bacteria)
DOMAIN ARCHAEA
Diversity within the Archaea
MICROBIAL DIVERSITY
Discoveries Illustrating the Range of Diversity
Study Outline
Study Questions
12 The Eukaryotes: Fungi, Algae, Protozoa, and Helminths
Fungi
Characteristics of Fungi
Medically Important Fungi
Fungal Diseases
Economic Effects of Fungi
Lichens
Algae
Characteristics of Algae
Selected Phyla of Algae
Roles of Algae in Nature
Protozoa
Characteristics of Protozoa
Medically Important Protozoa
Slime Molds
Helminths
Characteristics of Helminths
Platyhelminths
Nematodes
Arthropods as Vectors
Study Outline
Study Questions
13 Viruses, Viroids, and Prions
General Characteristics of Viruses
Host Range
Viral Size
Viral Structure
Nucleic Acid
Capsid and Envelope
General Morphology
Taxonomy of Viruses
Isolation, Cultivation, and Identification of Viruses
Growing Bacteriophages in the Laboratory
Growing Animal Viruses in the Laboratory
Viral Identification
Viral Multiplication
Multiplication of Bacteriophages
Multiplication of Animal Viruses
Viruses and Cancer
The Transformation of Normal Cells into Tumor Cells
DNA Oncogenic Viruses
RNA Oncogenic Viruses
Viruses to Treat Cancer
Latent Viral Infections
Persistent Viral Infections
Plant Viruses and Viroids
Prions
Study Outline
Study Questions

PART THREE Interaction between Microbe and Host

14 Principles of Disease and Epidemiology
Pathology, Infection, and Disease
Human Microbiome
Relationships between the Normal Microbiota and the Host
Opportunistic Microorganisms
Cooperation among Microorganisms
The Etiology of Infectious Diseases
Koch’s Postulates
Exceptions to Koch’s Postulates
Classifying Infectious Diseases
Occurrence of a Disease
Severity or Duration of a Disease
Extent of Host Involvement
Patterns of Disease
Predisposing Factors
Development of Disease
The Spread of Infection
Reservoirs of Infection
Transmission of Disease
Healthcare-Associated Infections (HAIs)
Microorganisms in the Hospital
Compromised Host
Chain of Transmission
Control of Healthcare-Associated Infections
Emerging Infectious Diseases
Epidemiology
Descriptive Epidemiology
Analytical Epidemiology
Experimental Epidemiology
Case Reporting
The Centers for Disease Control and Prevention (CDC)
Study Outline
Study Questions
15 Microbial Mechanisms of Pathogenicity
How Microorganisms Enter a Host
Portals of Entry
The Preferred Portal of Entry
Numbers of Invading Microbes
Adherence
How Bacterial Pathogens Penetrate Host Defenses
Capsules
Cell Wall Components
Enzymes
Antigenic Variation
Penetration into the Host
Biofilms
How Bacterial Pathogens Damage Host Cells
Using the Host’s Nutrients: Siderophores
Direct Damage
Production of Toxins
Plasmids, Lysogeny, and Pathogenicity
Pathogenic Properties of Viruses
Viral Mechanisms for Evading Host Defenses
Cytopathic Effects of Viruses
Pathogenic Properties of Fungi, Protozoa, Helminths, and Algae
Fungi
Protozoa
Helminths
Algae
Portals of Exit
Study Outline
Study Questions
16 Innate Immunity: Nonspecific Defenses of the Host
The Concept of Immunity
FIRST LINE OF DEFENSE: SKIN AND MUCOUS MEMBRANES
Physical Factors
Chemical Factors
Normal Microbiota and Innate Immunity
SECOND LINE OF DEFENSE
Formed Elements in Blood
The Lymphatic System
Phagocytes
Actions of Phagocytic Cells
The Mechanism of Phagocytosis
Inflammation
Vasodilation and Increased Permeability of Blood Vessels
Phagocyte Migration and Phagocytosis
Tissue Repair
Fever
Antimicrobial Substances
The Complement System
Interferons
Iron-Binding Proteins
Antimicrobial Peptides
Other Factors
Study Outline
Study Questions
17 Adaptive Immunity: Specific Defenses of the Host
The Adaptive Immune System
Dual Nature of the Adaptive Immune System
Overview of Humoral Immunity
Overview of Cellular Immunity
Cytokines: Chemical Messengers of Immune Cells
Antigens and Antibodies
Antigens
Humoral Immunity: Antibodies
Humoral Immunity Response Process
Activation and Clonal Expansion of Antibody-Producing Cells
The Diversity of Antibodies
Results of the Antigen–Antibody Interaction
Cellular Immunity Response Process
Antigen-Presenting Cells (APCs)
Classes of T Cells
Nonspecific Cells and Extracellular Killing by the Adaptive Immune System
Immunological Memory
Types of Adaptive Immunity
Study Outline
Study Questions
18 Practical Applications of Immunology
Vaccines
Principles and Effects of Vaccination
Types of Vaccines and Their Characteristics
Vaccine Production, Delivery Methods, and Formulations
Diagnostic Immunology
Use of Monoclonal Antibodies
Precipitation Reactions
Agglutination Reactions
Neutralization Reactions
Complement-Fixation Reactions
Fluorescent-Antibody Techniques
Enzyme-Linked Immunosorbent Assay (ELISA)
Western Blotting (Immunoblotting)
The Future of Diagnostic and Therapeutic Immunology
Study Outline
Study Questions
19 Disorders Associated with the Immune System
Hypersensitivity
Allergies and the Microbiome
Type I (Anaphylactic) Reactions
Type II (Cytotoxic) Reactions
Type III (Immune Complex) Reactions
Type IV (Delayed Cell-Mediated) Reactions
Autoimmune Diseases
Cytotoxic Autoimmune Reactions
Immune Complex Autoimmune Reactions
Cell-Mediated Autoimmune Reactions
Reactions to Transplantation
Immunosuppression to Prevent Transplant Rejection
The Immune System and Cancer
Immunotherapy for Cancer
Immunodeficiencies
Congenital Immunodeficiencies
Acquired Immunodeficiencies
Acquired Immunodeficiency Syndrome (AIDS)
The Origin of AIDS
HIV Infection
Diagnostic Methods
HIV Transmission
AIDS Worldwide
Preventing and Treating AIDS
Study Outline
Study Questions
20 Antimicrobial Drugs
The History of Chemotherapy
Antibiotic Use and Discovery Today
Spectrum of Antimicrobial Activity
The Action of Antimicrobial Drugs
Inhibiting Cell Wall Synthesis
Inhibiting Protein Synthesis
Injuring the Plasma Membrane
Inhibiting Nucleic Acid Synthesis
Inhibiting the Synthesis of Essential Metabolites
Common Antimicrobial Drugs
Antibacterial Antibiotics: Inhibitors of Cell Wall Synthesis
Inhibitors of Protein Synthesis
Injury to Membranes
Nucleic Acid Synthesis Inhibitors
Competitive Inhibition of Essential Metabolites
Antifungal Drugs
Antiviral Drugs
Antiprotozoan and Antihelminthic Drugs
Tests to Guide Chemotherapy
The Diffusion Methods
Broth Dilution Tests
Resistance to Antimicrobial Drugs
Mechanisms of Resistance
Antibiotic Misuse
Cost and Prevention of Resistance
Antibiotic Safety
Effects of Combinations of Drugs
Future of Chemotherapeutic Agents
Study Outline
Study Questions

PART FOUR Microorganisms and Human Disease

21 Microbial Diseases of the Skin and Eyes
Structure and Function of the Skin
Mucous Membranes
Normal Microbiota of the Skin
Microbial Diseases of the Skin
Bacterial Diseases of the Skin
Viral Diseases of the Skin
Fungal Diseases of the Skin and Nails
Parasitic Infestation of the Skin
Microbial Diseases of the Eye
Inflammation of the Eye Membranes: Conjunctivitis
Bacterial Diseases of the Eye
Other Infectious Diseases of the Eye
Study Outline
Study Questions
22 Microbial Diseases of the Nervous System
Structure and Function of the Nervous System
Bacterial Diseases of the Nervous System
Bacterial Meningitis
Tetanus
Botulism
Leprosy
Viral Diseases of the Nervous System
Poliomyelitis
Rabies
Arboviral Encephalitis
Fungal Disease of the Nervous System
Cryptococcus neoformans Meningitis (Cryptococcosis)
Protozoan Diseases of the Nervous System
African Trypanosomiasis
Amebic Meningoencephalitis
Nervous System Diseases Caused by Prions
Bovine Spongiform Encephalopathy and Variant Creutzfeldt-Jakob Disease
Diseases Caused by Unidentified Agents
Study Outline
Study Questions
23 Microbial Diseases of the Cardiovascular and Lymphatic Systems
Structure and Function of the Cardiovascular and Lymphatic Systems
Bacterial Diseases of the Cardiovascular and Lymphatic Systems
Sepsis and Septic Shock
Bacterial Infections of the Heart
Rheumatic Fever
Tularemia
Brucellosis (Undulant Fever)
Anthrax
Gangrene
Systemic Diseases Caused by Bites and Scratches
Vector-Transmitted Diseases
Viral Diseases of the Cardiovascular and Lymphatic Systems
Burkitt’s Lymphoma
Infectious Mononucleosis
Other Diseases and Epstein-Barr Virus
Cytomegalovirus Infections
Chikungunya
Classic Viral Hemorrhagic Fevers
Emerging Viral Hemorrhagic Fevers
Protozoan Diseases of the Cardiovascular and Lymphatic Systems
Chagas Disease (American Trypanosomiasis)
Toxoplasmosis
Malaria
Leishmaniasis
Babesiosis
Helminthic Disease of the Cardiovascular and Lymphatic Systems
Schistosomiasis
Disease of Unknown Etiology
Kawasaki Syndrome
Study Outline
Study Questions
24 Microbial Diseases of the Respiratory System
Structure and Function of the Respiratory System
Normal Microbiota of the Respiratory System
MICROBIAL DISEASES OF THE UPPER RESPIRATORY SYSTEM
Bacterial Diseases of the Upper Respiratory System
Streptococcal Pharyngitis (Strep Throat)
Scarlet Fever
Diphtheria
Otitis Media
Viral Disease of the Upper Respiratory System
The Common Cold
MICROBIAL DISEASES OF THE LOWER RESPIRATORY SYSTEM
Bacterial Diseases of the Lower Respiratory System
Pertussis (Whooping Cough)
Tuberculosis
Bacterial Pneumonias
Melioidosis
Viral Diseases of the Lower Respiratory System
Viral Pneumonia
Respiratory Syncytial Virus (RSV)
Influenza (Flu)
Fungal Diseases of the Lower Respiratory System
Histoplasmosis
Coccidioidomycosis
Pneumocystis Pneumonia
Blastomycosis (North American Blastomycosis)
Other Fungi Involved in Respiratory Disease
Study Outline
Study Questions
25 Microbial Diseases of the Digestive System
Structure and Function of the Digestive System
Normal Microbiota of the Digestive System
Bacterial Diseases of the Mouth
Dental Caries (Tooth Decay)
Periodontal Disease
Bacterial Diseases of the Lower Digestive System
Staphylococcal Food Poisoning (Staphylococcal Enterotoxicosis)
Shigellosis (Bacillary Dysentery)
Salmonellosis (Salmonella Gastroenteritis)
Typhoid Fever
Cholera
Noncholera Vibrios
Escherichia coli Gastroenteritis
Campylobacteriosis (Campylobacter Gastroenteritis)
Helicobacter Peptic Ulcer Disease
Yersinia Gastroenteritis
Clostridium perfringens Gastroenteritis
Clostridium difficile–Associated Diarrhea
Bacillus cereus Gastroenteritis
Viral Diseases of the Digestive System
Mumps
Hepatitis
Viral Gastroenteritis
Fungal Diseases of the Digestive System
Protozoan Diseases of the Digestive System
Giardiasis
Cryptosporidiosis
Cyclosporiasis
Amebic Dysentery (Amebiasis)
Helminthic Diseases of the Digestive System
Tapeworms
Hydatid Disease
Nematodes
Study Outline
Study Questions
26 Microbial Diseases of the Urinary and Reproductive Systems
Structure and Function of the Urinary System
Structure and Function of the Reproductive Systems
Normal Microbiota of the Urinary and Reproductive Systems
DISEASES OF THE URINARY SYSTEM
Bacterial Diseases of the Urinary System
Cystitis
Pyelonephritis
Leptospirosis
DISEASES OF THE REPRODUCTIVE SYSTEMS
Bacterial Diseases of the Reproductive Systems
Gonorrhea
Nongonococcal Urethritis (NGU)
Pelvic Inflammatory Disease (PID)
Syphilis
Lymphogranuloma Venereum (LGV)
Chancroid (Soft Chancre)
Bacterial Vaginosis
Viral Diseases of the Reproductive Systems
Genital Herpes
Genital Warts
AIDS
Fungal Disease of the Reproductive Systems
Candidiasis
Protozoan Disease of the Reproductive Systems
Trichomoniasis
Study Outline
Study Questions

PART FIVE Environmental and Applied Microbiology

27 Environmental Microbiology
Microbial Diversity and Habitats
Symbiosis
Soil Microbiology and Biogeochemical Cycles
The Carbon Cycle
The Nitrogen Cycle
The Sulfur Cycle
Life without Sunshine
The Phosphorus Cycle
The Degradation of Synthetic Chemicals in Soil and Water
Aquatic Microbiology and Sewage Treatment
Aquatic Microorganisms
The Role of Microorganisms in Water Quality
Water Treatment
Sewage (Wastewater) Treatment
Study Outline
Study Questions
28 Applied and Industrial Microbiology
Food Microbiology
Foods and Disease
Industrial Food Canning
Aseptic Packaging
Radiation and Industrial Food Preservation
High-Pressure Food Preservation
The Role of Microorganisms in Food Production
Industrial Microbiology and Biotechnology
Fermentation Technology
Industrial Products
Alternative Energy Sources Using Microorganisms
Biofuels
Industrial Microbiology and the Future
Study Outline
Study Questions

Answers to Knowledge and Comprehension Study Questions
Appendix A Metabolic Pathways
Appendix B Exponents, Exponential Notation, Logarithms, and Generation Time
Appendix C Methods for Taking Clinical Samples
Appendix D Pronunciation Rules and Word Roots
Appendix E Classification of Prokaryotes According to Bergey’s Manual
Glossary

A
B
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E
F
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Credits
Trademark Attributions
Index

A
B
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