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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 Genetics

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▶ Plasmids exist in cells separate from chromosomes.

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CHAPTER 8 Microbial Genetics 205

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 ProteinDNA

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 Phenotype 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.

205 224 227 234

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

mRNA

Protein

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

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 the 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 treatment 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.

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KEY CONCEPTS

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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? Q

CHECK YOUR UNDERSTANDING

✓ 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

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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

Parental strand

3¿ end Daughter

strand forming

5¿ end Daughter

strand Parental strand

Parental strand

G

Replication fork

C

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

CG

G

KEY

T

T

C

1

2

2

3

3

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 O

P

O

OH

OH

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

P

C G

TA

CG 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? Q

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

OH

Sugar

Phosphate

OH

OHOH

T

C G C G

P

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?

Q

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.

1

Proteins stabilize the unwound parental DNA.

2

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.

5

DNA polymerase

Primase RNA primer

DNA ligase joins the discontinuous fragments of the lagging strand.

6

DNA polymerase

DNA ligaseOkazaki fragment

Parental strand

5¿

3¿

5¿

3¿

REPLICATION

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.

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CHECK YOUR UNDERSTANDING

✓ 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.