Brock Biology of Microorganisms 2021 PDF Past Paper

Genetics of Bacteria and Archaea 9 I Mutation 299...

Genetics of Bacteria and Archaea 9 I Mutation 299 II Gene Transfer in Bacteria 306 III Gene Transfer in Archaea and Other Genetic Events 318 MICROBIOLOGYNOW Live Cell Imaging Captures Bacterial Promiscuity While often considered asexual, both Bacteria and Archaea plasmid encoding resistance to tetracycline appear red due evolve quickly through DNA exchange by horizontal transfer. to the labeled TetA resistance protein, whereas cells Increased fitness resulting from genetic promiscuity has tra- sensitive to tetracycline appear green due to their uptake of ditionally been monitored in the laboratory through changes the labeled antibiotic.2 Why are the sensitive cells still alive? in physiology. However, advances in microscopy and fluores- Remarkably, an efflux pump (Chapter 8) helps the sensitive cent labeling now make it possible to witness DNA cells buy just enough time to obtain the plasmid from their exchange using live cell imaging. resistant neighbors through a genetic event called conjuga- One fascinating example is the DNA uptake system tion. How long does this whole process take? Transfer of the (natural competence) in the bacterium Vibrio cholerae. Using plasmid only takes minutes, and sensitive cells can express an appendage called a pilus, V. cholerae is able to reach TetA in under 2 hours. outside of its cell, snatch pieces of free DNA, and transport These examples highlight the astounding power of hori- them into the cell. Thus, its pili effectively function as zontal gene transfer in prokaryotic cells as well as the variety “harpoons” fishing for free DNA. The top panel of the images of tools at their disposal to improve their fitness. Bacterial shown here is a time series of the “harpoon” (white arrow) genetics is indeed an exciting and dynamic discipline and has being cast from the cell into the environment, attaching to given birth to many of the genetic concepts we know today. free DNA (red), and then retracting back to the cell.1 Transfer of antibiotic resistance can also be monitored Sources: 1Ellison, C.K., et al. 2018. Retraction of DNA-bound type IV competence pili initiates DNA uptake during natural transformation using live cell imaging. The photo at the bottom shows a of Vibrio cholerae. Nat. Microbiol. 3: 773. mixed population of Escherichia coli cells surrounded by the 2 Nolivos, S., et al. 2019. Role of AcrAB-TolC multidrug efflux pump in drug- antibiotic tetracycline (labeled green). Cells possessing a resistance acquisition by plasmid transfer. Science 364: 778. 297 M09_MADI4790_16_GE_C09.indd 297 08/03/2021 16:41  298   UNIT 2 MOLECULAR BIOLOGY AND GENETICS The diversity and amazing ability of bacteria to survive ever-changing In this chapter we discuss the major themes of bacterial and environmental conditions and a host of stressors is controlled by archaeal genetics and the processes of gene transfer from one cell to their genetic makeup. While their asexual reproduction would sug- another (Figure 9.1). We first describe how changes arise in the gest that cells within Bacteria and Archaea do not exchange genes, the genome, which can be due to random errors in DNA replication or microbiologist Joshua Lederberg made a groundbreaking discovery from DNA damage, and then we consider how horizontal gene in 1946—like plants and animals, bacteria can also exchange genes! transfer (Figure 9.1; see also the photomicrograph of actual gene Lederberg’s groundbreaking work showcasing genetic recombination transfer on page 297) moves genes from one cell to another by in Bacteria not only earned him a Nobel Prize but also helped launch mechanisms uncoupled from reproduction. Microbiologists have the field of molecular biology and the use of Bacteria to study how leveraged aspects of these mutational and genetic exchange pro- 2 UNIT genes work in higher organisms, such as plants and animals. cesses to identify genes encoding exciting microbial processes such Understanding the varied mechanisms by which Bacteria and as chemotaxis, quorum sensing, and persistence that we discussed Archaea exchange genes has helped tackle the conundrum of how in Chapters 7 and 8. While changes to the genome underlie micro- these microbes can exhibit so much diversity and persist in habitats bial diversity and habitat adaptation, microorganisms also possess where other forms of life cannot. Gene exchange between microbes, mechanisms to maintain genomic stability, and we end this chapter along with genetic innovations that arise from random changes in by considering these. Taken together, both genomic change and a cell’s genetic blueprint, occur frequently and confer selectable genomic stability are important to the evolution of an organism and advantages that ultimately drive cell survival and genetic diversity. its competitive success in nature. Transduction Transducing particle UV-light-induced DNA damage Recipient cell Virus-infected cell Transformation Retracting pilus Conjugation Free DNA Pilus Fused vesicle Conjugation bridge Plasmid-donating Vesicles cell containing DNA Figure 9.1 Overview of bacterial and archaeal genetics. DNA mutations and mechanisms of DNA transfer contribute to the genetic diversity of Bacteria and Archaea. Transduction, transformation, and conjugation are the three known ways by which prokaryotic cells can exchange genes. M09_MADI4790_16_GE_C09.indd 298 08/03/2021 16:41  CHAPTER 9 Genetics of Bacteria and Archaea  299 I Mutation nucleotide sequence of its genome (Figure 9.2a). In addition, the observable properties of a mutant—its phenotype—may also be C hanges in the human world can be good, bad, or indifferent, and the same is true of the microbial world. Changes in the genetic blueprint of a microbe may fuel its evolutionary progress, altered relative to its parent (Figure 9.2b). This altered phenotype is called a mutant phenotype. The term “wild type” may be used to refer to an entire organism accelerate its death, or have no detectable effect on its biology. or just to the status of a particular gene that is under investigation. Mutant derivatives can be obtained either directly from a wild-type A ll organisms contain a specific sequence of nucleotides in their genome, their genetic blueprint. A mutation is a heritable strain or from another strain—referred to as a parental strain— previously derived from the wild type; for example, another mutant. 2 UNIT change in the base sequence of that genome, that is, a change that Figure 9.2b shows a plate of MacConkey agar (a culture medium that is passed from the mother cell to progeny cells. Mutations can lead contains a pH indicator that turns red if sugar is fermented) contain- to changes in the properties of an organism; some mutations are ing the sugar maltose. This medium shows the phenotypic difference beneficial, some are detrimental, but most are neutral and have between wild-type Escherichia coli and mutant derivatives in the no effect. Although the rate of spontaneous mutation is low maltose utilization pathway. (Section 9.3), the impressive characteristic of exponential growth in Depending on the mutation, a mutant strain may or may not differ Bacteria and Archaea ensures that mutations accumulate in a popula- in phenotype from its parent. By convention in bacterial genetics, the tion surprisingly fast. Moreover, whereas a single mutation typically genotype of an organism is designated by three lowercase letters fol- brings about only a small change in a cell, horizontal gene transfer lowed by a capital letter (all in italics) indicating a particular gene. For often generates much larger changes. Taken together, mutation and example, the hisC gene of E. coli encodes a protein called HisC that genetic exchange fuel the evolutionary process. functions in biosynthesis of the amino acid histidine (◀ Figure 6.27). We begin by considering the molecular mechanism of mutation Mutations in the hisC gene would be designated as hisC1, hisC2, and and the properties of mutant microorganisms. so on, the numbers referring to the order of isolation of the mutant strains. Each hisC mutation would be different, and each hisC muta- 9.1 Mutations and Mutants tion might affect the HisC protein in different ways. In order to appreciate the impact that genome changes can have on The phenotype of an organism is designated by a capital letter fol- a cell (or a virus), we start with a brief genetics primer. The genomes lowed by two lowercase letters, with either a plus or a minus super- of cells consist of double-stranded DNA. In viruses, by contrast, the script to indicate the presence or absence of that property. For genome may consist of double- or single-stranded DNA or RNA, example, a His + strain of E. coli is one that is capable of making its depending on the virus (Chapters 5 and 11). By convention, a strain own histidine, whereas a His - strain is not. The His- strain would of an organism or a virus isolated from nature is called the therefore require a histidine supplement for growth. A mutation in wild-type strain and therefore contains the wild-type genome. A the hisC gene leads to a His- phenotype if it eliminates the function cell or virus derived from the wild type whose genome carries of the HisC protein. Likewise, the lamB and malQ mutations in a change in nucleotide sequence is called a mutant. A mutant by Figure 9.2 lead to a Mal- phenotype, as the mutants can no longer definition differs from the wild-type strain in its genotype, the use maltose as an energy source. Pmal malK lamB malM WT Pmal malQ Pmal WT Wild type WT M1 malK malM Deletion M1 Mutant 1 Howard Shuman and Thomas Silhavy M1 M2 of lamB Pmal malQ M2 Mutant 2 Pmal M2 malK lamB malM Mutation in malQ Pmal malQ (a) (b) Figure 9.2 Wild-type versus mutant phenotype. (a) Genotype maps of Escherichia coli wild-type and maltose utilization mutants; P is the promoter region and mal and lam regions are structural genes. (b) Growth of wild-type E. coli and mutants on a plate of MacConkey agar, a differential medium. The medium contains maltose as the carbon source and a pH indicator that turns red if maltose is fermented. While the wild-type strain ferments maltose, mutant strains M1 and M2 are unable to ferment maltose due to a deletion of the lamB gene and a point mutation in the malQ gene, respectively. M09_MADI4790_16_GE_C09.indd 299 08/03/2021 16:41  300   UNIT 2 MOLECULAR BIOLOGY AND GENETICS Isolation of Mutants: Screening versus Selection pigmentation (for example, of phototrophic organisms) may have Virtually any characteristic of an organism can be changed by a selective advantage in nature. We can detect nonselectable muta- mutation. Some mutations are selectable, conferring some type of tions only by examining large numbers of colonies and looking advantage on organisms possessing them, whereas others are non- for the “different” ones, an often laborious process called selectable, even though they may lead to a very clear change in the screening. phenotype of an organism. A selectable mutation confers a clear As illustrated in Figure 9.2 (see also Figure 9.35), a thorough advantage on the mutant strain under certain environmental con- understanding of microbial physiology is the key to designing suc- ditions, so the progeny of the mutant cell are able to grow and cessful genetic screens for nonselectable mutations. In Figure 9.2b a genetic screen for Mal- mutants utilizes a culture medium contain- 2 replace the parent. A good example of a selectable mutation is UNIT drug resistance: An antibiotic-resistant mutant can grow in the ing a pH indicator that turns red if maltose is fermented (MacCon- presence of an antibiotic that inhibits or kills the parent key agar). Because the Mal- mutants are unable to utilize maltose, (Figure 9.3a) and is thus selected under these conditions. It is rela- they are unpigmented compared to the red wild-type strain. To iden- tively easy to detect and isolate selectable mutants by choosing tify mutants in biofilm formation, individual colonies can be grown the appropriate environmental conditions. Selection is therefore in liquid medium in a microtiter plate and a stain can be used to an extremely powerful genetic tool, allowing the isolation of a track whether cells can form a biofilm and adhere to the microtiter single mutant from a population containing millions or even bil- plate (see Figure 9.35). If possible, selection is almost always the lions of parental cells. preferred strategy over screening for obtaining mutants in a genetic Mastering Some examples of important nonselectable mutations are sec- experiment because selective conditions typically place such severe Microbiology ondary metabolite production loss in an antibiotic-producing restraints on the population that mutants are easily detectable. Art Activity: Figure 9.4 organism, loss of attachment ability in a biofilm-forming organ- Examples of common classes of mutants and the means by which Screening for nutritional ism, and loss of color in a pigmented organism (Figure 9.3b, c). they are detected are listed in Table 9.1. auxotrophs All three types of mutants usually have neither an advantage nor a disadvantage over their parent cells when grown in the labora- Isolation of Nutritional Auxotrophs tory, although antibiotic production ( ▶ Section 16.12), biofilm Although screening is more tedious than selection, useful meth- formation (◀ Sections 4.9 and 8.10, and ▶ Section 20.4), and ods have been developed for screening large numbers of colonies for certain types of mutations. For instance, nutritionally defective mutants can be detected by the technique of replica plating (Figure 9.4). A colony from a master plate can be transferred onto an agar plate lacking the nutrient by using a sterile loop, tooth- pick, or even a robotic arm. Parental colonies will grow normally, whereas those of the mutant will not. Thus, the inability of a colony to grow on a medium lacking the nutrient signals that it is a mutant. The colony on the master plate corresponding to the vacant spot on the replica plate can then be picked, purified, and characterized. A mutant strain with an additional nutritional requirement above that of the wild type or parental strain from which it was T.D. Brock derived is called an auxotroph (Table 9.1), and the strain from which an auxotroph originates is called a prototroph. For instance, (a) mutants of E. coli with His- and Mal- (Figure 9.2) phenotypes are histidine and maltose auxotrophs, respectively, while the parental His + and Mal + strains from which the auxotrophs were derived are the prototrophs of such strains. As described earlier, many dif- Shiladitya DasSarma ferent mutations can lead to a strain showing a His - or Mal- phe- Priya DasSarma and Steven R. Spilatro notype, and thus an initial step in characterizing the genetics of a metabolic pathway (such as histidine biosynthesis and maltose catabolism) would be the isolation of several His - or Mal- strains (b) (c) followed by their comparative genetic analyses (Figure 9.2). This comparative analysis process, called complementation, is discussed Figure 9.3 Selectable and nonselectable mutations. (a) Development of antibiotic- in Section 9.5. resistant mutants, a type of easily selectable mutation, within the inhibition zone of an antibiotic assay disk. (b) Nonselectable mutations. UV-radiation-induced nonpig- mented mutants of Serratia marcescens. The wild type has a dark red pigment. The Check Your Understanding white or colorless mutants make no pigment. (c) Colonies of mutants of a species of Distinguish between a mutation and a mutant. Halobacterium, a member of the Archaea. The lower pink wild-type colony contains Distinguish between screening and selection. intracellular gas vesicle nanoparticles that regulate buoyancy and also scatter light, resulting in a milky appearance. The upper red-orange colony is a mutant lacking How does an auxotroph differ from a prototroph? gas vesicles and is translucent as a result. M09_MADI4790_16_GE_C09.indd 300 08/03/2021 16:41  CHAPTER 9 Genetics of Bacteria and Archaea  301 TABLE 9.1 Some examples of mutants Phenotype Nature of change Detection of mutant Auxotroph Loss of enzyme in biosynthetic pathway Inability to grow on medium lacking the nutrient Temperature-sensitive Alteration of an essential protein so it is more heat-sensitive Inability to grow at a high temperature that normally ­supports growth Cold-sensitive Alteration of an essential protein so it is inactivated at low Inability to grow at a low temperature that normally temperature ­supports growth 2 UNIT Drug-resistant Detoxification of drug or alteration of drug target or Growth on medium containing a normally inhibitory permeability to drug concentration of the drug Rough colony Loss or change in lipopolysaccharide layer Granular, irregular colonies instead of smooth, glistening colonies Nonencapsulated Loss or modification of surface capsule Small, rough colonies instead of larger, smooth colonies Nonmotile Loss of flagella or nonfunctional flagella Compact instead of flat, spreading colonies; lack of motility by microscopy Pigmentless Loss of enzyme in biosynthetic pathway leading to loss of Presence of different color or lack of color one or more pigments Sugar fermentation Loss of enzyme in degradative pathway Lack of color change on agar containing sugar and a pH indicator Virus-resistant Loss of virus receptor Growth in presence of large amounts of virus 9.2 Molecular Basis of Mutation agents and include mutations made deliberately by humans. Induced mutations can result from exposure to natural radiation (cosmic rays Mutations can be either spontaneous or induced events. and so on) that alters the structure of bases in the DNA, or from a Spontaneous mutations are those that occur without external inter- variety of chemicals that chemically modify DNA (Section 9.4). vention, and most result from occasional errors in the pairing of Mutations that change only one base pair are called point bases by DNA polymerase during DNA replication (Chapter 6). mutations and occur when a single base-pair substitution occurs in Induced mutations, by contrast, are those caused by environmental Auxotrophs Sterile toothpick 1. Pick and transfer colonies Complete medium 2. Incubate and All colonies grow to fresh medium. examine plates. Auxotrophs Derek J. Fisher Master plate; growth on complete medium Selective medium Mutants do not grow Figure 9.4 Screening for nutritional auxotrophs. The replica-plating method can be used for the detection of nutritional mutants. Colonies from the master plate are transferred using a sterile toothpick to a gridded plate containing different media for selection. The colonies not appearing on the selective medium are labeled as auxotrophs. The selective medium lacked one nutrient (the amino acid leucine) present in the master plate. Therefore, the colonies on the complete medium plate that are not represented on the selective medium plate are leu- cine auxotrophs (Leu-). M09_MADI4790_16_GE_C09.indd 301 08/03/2021 16:41  302   UNIT 2 MOLECULAR BIOLOGY AND GENETICS the DNA. Many point mutations do not actually cause any pheno- results in an amino acid change within the polypeptide from tyro- typic change (although the organism’s genotype has changed). How- sine to asparagine at a specific site. This is called a missense ever, as for all mutations, any phenotypic change that results from ­mutation because the informational “sense” (the precise sequence a point mutation depends on exactly where in the genome the muta- of amino acids) in the polypeptide has changed. If the change is at tion occurs and the nature of the nucleotide change, as we will see. a critical location in the polypeptide chain, the protein could be inactive, have reduced activity, or have modified activity. However, Base-Pair Substitutions: Missense, Nonsense, not all missense mutations lead to nonfunctional proteins. The out- and Silent Mutations come depends on where the substitution lies in the polypeptide chain and on how it affects protein folding and activity. While muta- 2 If a point mutation is within the region of a gene that encodes a UNIT polypeptide, any change in the phenotype of the cell is most likely the tions in the active site of an enzyme are more likely to destroy cata- result of a change in the amino acid sequence of that polypeptide. The lytic activity than mutations in other regions of the protein, some error in the DNA is transcribed into mRNA, and the erroneous mRNA active site mutations can alter an enzyme’s substrate specificity. This in turn is translated to yield a polypeptide. In interpreting the results type of change can result in a mutant enzyme with differing activity of a mutation, we must first recall that the genetic code is degenerate from the wild-type enzyme, and such events have contributed to the (◀ Section 6.9 and Table 6.4). Consequently, not all mutations in the spectacular metabolic diversity we see in the microbial world base sequence encoding a polypeptide will change the polypeptide (Chapters 3 and 14). sequence. This is illustrated in Figure 9.5, which shows several possible Another possible outcome of a base-pair substitution is the forma- results when the DNA that encodes a single tyrosine codon in a poly- tion of a stop (nonsense) codon in the DNA. This results in prema- peptide is mutated. First, a change in the RNA from UAC to UAU ture termination of translation, leading to an incomplete would have no apparent effect because UAU is also a tyrosine codon. polypeptide (Figure 9.5). Mutations of this type are called ­nonsense Although they do not affect the sequence of the encoded polypeptide, mutations because the change is from a sense (coding) codon to a such changes in the DNA are considered one type of silent mutation, nonsense (stop) codon (◀ Table 6.4). Unless the nonsense mutation that is, a mutation that does not affect the phenotype of the cell. Note is very near the end of the gene, the product is incompletely made; that silent mutations in coding regions are almost always in the third such truncated proteins are either inactive or, at the very least, lack base of the codon (arginine and leucine can also have silent mutations normal activity. in the first position) because of genetic code degeneracy. Other terms are occasionally used in microbial genetics to describe Changes in the first or second base of the codon more often lead the precise type of base substitution in a point mutation. ­Transitions to significant changes in the amino acid sequence of the polypep- are mutations in which one purine base (A or G) is substituted for tide. For instance, a single base change from UAC to AAC (Figure 9.5) another purine, or one pyrimidine base (C or T) is substituted for another pyrimidine. Transversions are point mutations in which a purine base is substituted for a pyrimidine base, or vice versa. 5¿...T A C... DNA...A T G... 5¿ Frameshifts and Other Insertions or Deletions Because the genetic code is read from one end of the nucleic acid M U TAT I O N S Normal DNA replication in consecutive blocks of three bases (codons), any deletion or insertion of a single base pair results in a shift in the reading frame. These frameshift mutations often have serious conse- quences. Single base insertions or deletions change the primary A AC TAG TAT 5¿ T A C sequence of the encoded polypeptide, typically in a major way DNA T TG ATC ATA ATG (Figure 9.6). Such microinsertions or microdeletions can result Transcription of from DNA polymerase replication errors. Insertion or deletion of light green strand two base pairs also causes a frameshift. However, insertion or dele- tion of three base pairs does not cause a frameshift but does add AAC UAG UAU 5¿ U A C mRNA or remove a codon; this results in the addition or deletion of a Asparagine Stop Tyrosine Tyrosine single amino acid in the polypeptide sequence. Although an codon codon codon codon amino acid addition or deletion may well be deleterious to pro- Translation tein function, it is usually not as serious a problem as a frameshift, which scrambles the entire polypeptide sequence downstream of the mutation. Faulty Incomplete Normal Normal protein protein protein protein Protein Insertions or deletions can also result in the gain or loss of hun- Missense Nonsense Silent Wild type dreds or even thousands of base pairs. Such changes inevitably result mutation mutation mutation Change in complete loss of gene function. Some deletions are so large that they may include several genes. If any of the deleted genes are essen- Figure 9.5 Possible effects of base-pair substitution in a gene encoding a protein. tial, the mutation will be lethal. Such deletions cannot be restored Three different protein products are possible from changes in the DNA for a single codon. The possible phenotypes are wild type and strains harboring missense, non- through further mutations, but only through genetic recombination sense, or silent mutations. (Sections 9.5–9.11). Large insertions and deletions may arise as a M09_MADI4790_16_GE_C09.indd 302 08/03/2021 16:41  CHAPTER 9 Genetics of Bacteria and Archaea  303 DNA mRNA Reading they function as suppressor mutations—mutations that compensate frame for the effect of the original mutation. Several classes of suppressor...GTGCCCTGTT......GUG CCC UGU U... +1 mutations are known. These include (1) a mutation somewhere else...CACGGGACAA... in the same gene that restores enzyme function, such as a second Transcription Insertion off of light frameshift mutation near the first that restores the original reading of C:G pair green strands Codons frame (Figure 9.6); (2) a mutation in another gene that restores the function of the original mutated gene; and (3) a mutation in another...GTGCCTGTT......CACGGACAA......GUG CCU GUU... 0 gene that results in the production of an enzyme that can replace the nonfunctional one. 2 UNIT Deletion Normal protein Suppressors can be best illustrated by mutations in tRNAs. Non- of C:G pair sense mutations can be suppressed by changing the anticodon...GTGCTGTT... sequence of a tRNA molecule so that it now recognizes a stop codon...CACGACAA......GUG CUG UU... –1 (Figure 9.7). Such an altered tRNA is called a suppressor tRNA and will insert the amino acid it carries at the stop codon that it now reads. Figure 9.6 Shifts in the reading frame of mRNA caused by insertions or deletions. Suppressor tRNA mutations exist in all three domains of life, and The reading frame in mRNA is established by properly positioning the message on their activity has been observed in both Bacteria and Archaea by the ribosome. The mRNA is read beginning at the 5′ end (toward the left in the introducing reporter genes (◀ Section 8.1) containing nonsense figure) and proceeds by units of three bases (codons). The normal reading frame is referred to as the 0 frame, that missing a base the - 1 frame, and that with an extra mutations. During these experiments, complete translation and base the + 1 frame. activity of the corresponding reporter protein was observed despite the presence of a stop codon within the coding region of the reporter. Suppressor tRNA mutations would be lethal unless a cell has more result of errors during genetic recombination. In addition, many than one tRNA gene for a particular codon. One tRNA gene may then large insertion mutations are due to the insertion of specific DNA be mutated to produce a suppressor, while the other gene’s product sequences called transposable elements (Section 9.11). performs the original function. Most cells have multiple genes for We will discuss the effects of mutations on the evolution of bacte- rial genomes in Chapter 13....GTC... Check Your Understanding DNA Do missense mutations occur in genes encoding tRNA? Why or why not? Nonsense mutation Why do frameshift mutations generally have more serious from G:A transition ­consequences than missense mutations?...ATC... * 9.3 Reversions and Mutation Rates Transcription of The rates at which the different kinds of mutations occur vary widely. mutated DNA Some mutations occur so rarely that they are almost impossible to...UAG......UAG... detect, whereas others occur so frequently that they present difficul- * * ties for an experimenter trying to maintain a genetically stable stock mRNA 5¿ 3¿ 5¿ 3¿ culture. Sometimes a second mutation can reverse the effect of an Nonsense mutation Nonsense + tRNA suppressor mutations initial mutation. Furthermore, all organisms possess a variety of sys- Translation tems for DNA repair. Consequently, the observed mutation rate depends not only on the frequency of DNA changes but also on the Gln efficiency of DNA repair. Gln Gln These tRNAs This suppressor cannot pair tRNA can pair Reversions (Back Mutations) and Suppressors with UAG codon with UAG codon GUC Point mutations are typically reversible, a process known as GUC AUC ­reversion. A revertant is therefore a strain in which the original phe- notype that was changed by mutation is restored by a second muta- Protein H 2N COOH H2N COOH tion. Revertants can be of two types, same site or second site. In Truncated protein Wild-type protein same-site revertants, the mutation that restores activity is at the same Figure 9.7 Suppression of nonsense mutations. Introduction of a nonsense mutation site as the original mutation. If the back mutation is not only at the in a gene encoding a protein results in the incorporation of a stop codon (indicated by same site but also restores the original sequence, it is called a true the *) in the corresponding mRNA. This single mutation leads to the production of a revertant. truncated polypeptide. The mutation is suppressed if a second mutation occurs in the In second-site revertants, the mutation is at a different site in the anticodon of a tRNA, a tRNA charged with glutamine in this example, which allows DNA. Second-site mutations can restore a wild-type phenotype if the mutated tRNA or suppressor tRNA to bind to the nonsense codon. M09_MADI4790_16_GE_C09.indd 303 08/03/2021 16:41  304   UNIT 2 MOLECULAR BIOLOGY AND GENETICS tRNAs, and so suppressor mutations are reasonably common, at least be done most effectively by increasing the pool of mutations. As in microorganisms. Sometimes the amino acid inserted by the sup- we see in the next section, it is possible to greatly increase the pressor tRNA is identical to the original amino acid and the protein mutation rate by treatment with mutagenic agents. In addition, is fully active. In other cases, however, a different amino acid is the mutation rate may change under certain circumstances, such inserted and a protein that is only partially active may be produced. as when cells are placed under high-stress conditions. Mutation Rates Check Your Understanding For most microorganisms, errors in DNA replication occur at a fre- Why are suppressor tRNA mutations not lethal? quency of 10 - 6 to 10 - 7 per thousand bases during a single round 2 Which class of mutation, missense or nonsense, UNIT of replication. A typical gene has about 1000 base pairs. Therefore, is more ­common, and why? the frequency of a mutation in a given gene is also in the range of 10 - 6 to 10 - 7 per round of replication. For instance, in a bacterial culture having 108 cells>ml, there are likely to be a number of dif- 9.4 Mutagenesis ferent mutants for any given gene in each milliliter of culture. The spontaneous rate of mutation is very low, but a variety of chemi- Eukaryotes with very large genomes tend to have replication error cal, physical, and biological agents can increase the mutation rate rates about 10-fold lower than typical bacteria, whereas DNA and are therefore said to induce mutations. These agents are called viruses, especially those with very small genomes, may have error mutagens. Because Bacteria and Archaea are often exposed to muta- rates 100-fold to 1000-fold higher than those of cellular organisms. gens, not only intentionally in the laboratory but also accidentally RNA viruses have even higher error rates due to less effective poly- in their environment, we discuss some of the major categories of merase proofreading (◀ Section 6.4) and the lack of RNA repair mutagens and their activities here. mechanisms. Single base errors during DNA replication are more likely to lead Chemical Mutagens and Radiation to missense mutations than to nonsense mutations because most single base substitutions yield codons that encode other amino acids An overview of some of the major chemical mutagens and their (◀ Table 6.4). The next most frequent type of codon change caused modes of action is given in Table 9.2. Several classes of chemical muta- by a single base change leads to a silent mutation. This is because for gens exist. The nucleotide base analogs are molecules that resemble the the most part alternate codons for a given amino acid differ from each purine and pyrimidine bases of DNA in structure but display faulty other by a single base change in the “silent” third position. A given base-pairing properties (Figure 9.8). If a base analog is incorporated codon can be changed to any of 27 other codons by a single base into DNA in place of the natural base, the DNA may replicate nor- substitution, and on average, about two of these will be silent muta- mally most of the time. However, DNA replication errors occur at tions, one a nonsense mutation, and the rest missense mutations. higher frequencies at these sites due to incorrect base pairing. The Unless a mutation can be selected for, its experimental detection result is the incorporation of a mismatched base into the new strand is difficult, and much of the skill of the microbial geneticist of DNA and thus introduction of a mutation. During subsequent requires increasing the efficiency of mutation detection. This can segregation of this strand in cell division, the mutation is revealed. TABLE 9.2 Chemical and physical mutagens and their modes of action Agent Action Result Base analogs 5-Bromouracil Incorporated like T; occasional faulty pairing with G AT S GC and occasionally GC S AT 2-Aminopurine Incorporated like A; faulty pairing with C AT S GC and occasionally GC S AT Chemicals that react with DNA Nitrous acid (HNO2) Deaminates A and C AT S GC and GC S AT Hydroxylamine (NH2OH) Reacts with C GC S AT Alkylating agents Monofunctional (for example, ethyl methanesulfonate) Puts methyl on G; faulty pairing with T GC S AT Bifunctional (for example, mitomycin, nitrogen mustards, Cross-links DNA strands; faulty region excised by DNase Both point mutations and deletions nitrosoguanidine) Intercalating agents Acridines, ethidium bromide Inserts between two base pairs Microinsertions and microdeletions Radiation Ultraviolet (UV) Pyrimidine dimer formation Repair may lead to error or deletion Ionizing radiation (for example, X-rays) Free-radical attack on DNA, breaking chain Repair may lead to error or deletion M09_MADI4790_16_GE_C09.indd 304 08/03/2021 16:41  CHAPTER 9 Genetics of Bacteria and Archaea  305 Electromagnetic spectrum Analog Substitutes for Ionizing O O X-rays H Br H CH3 Microwave N N Radar Cosmic Television O N O N Gamma Radio H H Wavelength –6 10 10–4 10–2 100 102 104 106 108 1010 (nm) 2 UNIT 5-Bromouracil Thymine (a) H2N N N N N H2N N N N H N H 200 nm 400 nm 600 nm 800 nm Ultraviolet (UV) Visible Infrared (IR) 2-Aminopurine Adenine (b) Figure 9.9 Wavelengths of radiation. Ultraviolet radiation consists of wavelengths just shorter than visible light. For any electromagnetic radiation, the shorter the Figure 9.8 Nucleotide base analogs. Structure of two common nucleotide base wavelength, the higher the energy. DNA absorbs strongly at 260 nm. analogs used to induce mutations compared with the normal nucleic acid bases for which they substitute. (a) 5-Bromouracil can base-pair with guanine, causing AT to GC substitutions. (b) 2-Aminopurine can base-pair with cytosine, causing AT to GC is due primarily to its effect on DNA. Conversely, ionizing radiation substitutions. is more powerful than UV radiation and includes short-wavelength radiation such as X-rays, cosmic rays, and gamma rays (Figure 9.9). These rays cause water and other substances to ionize, resulting in the formation of free radicals such as the hydroxyl radical (OH # , Other chemical mutagens induce chemical modifications in one base or another, resulting in faulty base pairing or related changes ◀ Section 4.16) that can damage macromolecules in the cell, (Table 9.2). For example, alkylating agents (chemicals that react including DNA. This causes double-stranded and single-stranded with amino, carboxyl, and hydroxyl groups by substituting them breaks that may lead to rearrangements or large deletions. with alkyl groups) such as nitrosoguanidine are powerful mutagens and generally induce mutations at higher frequency than base ana- DNA Repair and the SOS System logs. Unlike base analogs, which have an effect only when incorpo- By definition, a mutation is a heritable change in the genetic ­material. rated during DNA replication, alkylating agents can introduce Therefore, if damaged DNA can be corrected before the cell divides, changes even in nonreplicating DNA. Both base analogs and alkylat- no mutation will occur. While cells have a variety of different DNA ing agents tend to induce base-pair substitutions (Section 9.2). repair processes to correct mistakes (◀ Section 6.4) or repair ­damage, Another group of chemical mutagens, the acridines, are planar some are error-prone and the repair process itself introduces molecules that function as intercalating agents. These mutagens the mutation. Some types of DNA damage, especially large-scale become inserted between two DNA base pairs and push them apart. ­damage from highly mutagenic chemicals or large doses of radia- Then, during replication, this abnormal conformation can trigger tion, may cause lesions that interfere with replication. If such lesions single base insertions or deletions. Thus, acridines typically induce are not removed before replication occurs, DNA replication will stall frameshift rather than point mutations (Section 9.2). Ethidium bro- and lethal breaks in the chromosome will result. mide, which is commonly used to detect DNA in gel electrophoresis, In Bacteria, stalled replication or major DNA damage activates the is also an intercalating agent and therefore a mutagen. SOS repair system. The SOS system initiates a number of DNA Nonionizing and ionizing radiation are two forms of electromag- repair processes, some of which are error-free. However, the SOS netic radiation that are highly mutagenic (Figure 9.9). Ultraviolet system also allows DNA repair to occur without a template, that is, (UV) radiation is widely used to generate mutations because the with random incorporation of nucleotide precursors (deoxyribo- purine and pyrimidine bases of nucleic acids absorb UV radiation nucleotide triphosphates [dNTPs]). As might be expected, this strongly (the absorption maximum for DNA and RNA is at 260 nm). results in many errors and hence many mutations. However, muta- The primary mutagenic effect is the production of pyrimidine dimers, tions induced by the SOS repair system are better than the alterna- in which two adjacent pyrimidine bases (cytosine or thymine) on tive (death of the cell), as mutations can often be corrected while the same strand of DNA become covalently bonded to one another. chromosome breaks usually cannot. This either greatly impedes DNA polymerase activity or greatly In Escherichia coli the SOS repair system controls the transcription increases the probability of DNA polymerase misreading the of approximately 40 genes located throughout the chromosome that sequence at this point. Thus the killing of cells by UV radiation M09_MADI4790_16_GE_C09.indd 305 08/03/2021 16:41  306   UNIT 2 MOLECULAR BIOLOGY AND GENETICS Degraded LexA RecA activates LexA Patricia Foster and RecA DNA damage protease activity; protein Sarita Mallik activates RecA. LexA degraded. Error-prone DNA Active polymerase IV RecA 2 Olex dinB UNIT UvrA protein: Olex uvrA Error-free DNA repair LexA LexA Olex recA protein represses. Olex umuCD UmuCD proteins: Error-prone DNA repair Olex lexA LexA causes partial repression of recA. lex lexA operator structural gene Figure 9.10 SOS response to DNA damage. DNA RecA protein is produced even in the presence of LexA micrograph showing cells stained with DAPI (blue) and damage activates RecA protein, which in turn acti- protein. When LexA is inactivated, DNA repair genes DNA polymerase IV (yellow, in the nucleoid region). vates the protease activity of LexA, resulting in self- are highly transcribed. Inset photos: Both photos show Expression of dinB requires not only the loss of LexA cleavage. LexA normally represses the activities of DNA polymerase IV localization to the nucleoid during repression but also the protein RpoS, an RNA poly- RecA, the genes uvrA and umuCD that encode DNA the SOS response in Escherichia coli. Cells containing merase sigma factor whose synthesis is triggered by repair functions (the UmuCD proteins are part of DNA a fluorescently tagged DNA polymerase IV (DinB) were various stress responses. polymerase V), and dinB, which encodes DNA poly- treated with an antibiotic to induce DNA damage. Left: merase IV. However, repression is not complete. Some phase-contrast micrograph. Right: fluorescence participate in DNA damage tolerance and DNA repair. Not only to horizontal gene transfer, which we discuss next. Thus, uptake and does the SOS system form a regulon, but the general stress response or recombination of foreign DNA can also be used by Bacteria and RpoS regulon also plays a role in the repair system (◀ Sections 7.3 Archaea to fix chromosomal breaks. As we shall see in Part II, not and 7.9). In DNA damage tolerance, DNA lesions remain in the only does this form of DNA repair help with cell survival, but it also DNA, but are bypassed by specialized DNA polymerases that can can increase cell diversity. move past DNA damage—a process called translesion synthesis. Even if no template is available to allow insertion of the correct bases, it Check Your Understanding is less dangerous to cell survival in the long run to fill the gap than How do mutagens cause mutations? to let it remain. Consequently, translesion synthesis generates many What is meant by “error-prone” DNA repair? errors. In E. coli, in which the process of mutagenesis has been stud- ied in great detail, the two error-prone repair polymerases are DNA polymerase V, an enzyme encoded by the umuCD genes, and DNA polymerase IV, encoded by dinB (Figure 9.10). Both are induced as part of the SOS repair system. II Gene Transfer in Bacteria The master regulators of the SOS system are the proteins LexA and RecA. LexA is a repressor that normally prevents expression of the SOS L ateral gene flow within and between species of Bacteria is highly dynamic and facilitated by at least three different mechanisms. Such genetic exchange is distinct from system. The RecA protein, which normally functions in genetic recom- bination (Section 9.5), is activated by the presence of DNA damage, traditional mother-to-daughter inheritance and allows cells in particular by the single-stranded DNA that results when replication to rapidly acquire new characteristics and increase their stalls. The activated form of RecA then stimulates LexA to inactivate competitive fitness. itself by self-cleavage. This leads to derepression of the SOS system and the coordinate expression of proteins that participate in DNA repair. Because some of the DNA repair mechanisms of the SOS system— C omparative genomic analyses of closely related microbes that exhibit different phenotypes have revealed distinct genome dif- ferences. Often these idiosyncratic differences result from horizontal such as DNA polymerases IV and V—are inherently error-prone, many gene transfer, the movement of genes between cells that are not direct mutations arise. However, once the original DNA damage has been descendants of one another ( ▶ Section 13.9). Horizontal gene repaired, the SOS regulon is repressed and further mutagenesis ceases. transfer allows cells to quickly acquire new characteristics and fuels Besides controlling some DNA repair systems and error-prone metabolic diversity. polymerases, the SOS system also regulates processes that contribute M09_MADI4790_16_GE_C09.indd 306 08/03/2021 16:41  CHAPTER 9 Genetics of Bacteria and Archaea  307 Three mechanisms of genetic exchange are known in bacteria: by itself (but only if it possesses its own origin of replication, such (1) transformation, in which free DNA released from one cell is taken as a plasmid or phage genome); or (3) it may recombine with the up by another (Section 9.6); (2) transduction, in which DNA transfer recipient cell’s chromosome. is mediated by a virus (Section 9.7); and (3) conjugation, in which DNA transfer requires cell-to-cell contact and a conjugative plasmid 9.5 Genetic Recombination in the donor cell (Sections 9.8 and 9.9). While these processes were Recombination is the physical exchange of DNA between genetic presented in Figure 9.1, they are compared and contrasted in elements (structures that carry genetic information). Here we focus Figure 9.11. It should be noted that DNA transfer typically occurs in on homologous recombination, a process that results in genetic only one direction, from donor to recipient. 2 UNIT exchange between homologous DNA sequences from two different Before discussing the mechanisms of transfer, we consider the fate sources. Homologous DNA sequences are those that have nearly of transferred DNA. Regardless of how it was transferred, DNA that the same sequence; therefore, bases can pair over an extended enters the cell by horizontal gene transfer faces three possible fates: length of the two DNA molecules to facilitate exchange. This type (1) It may be degraded by the recipient cell’s restriction enzymes or of recombination is often essential to a cell’s ability to retain DNA other DNA destruction systems (Section 9.12); (2) it may replicate following genetic exchange and is behind the well-known phe- nomenon of “crossing over” in the genetics of eukaryotes. Transformation Transduction Donor Molecular Events in Homologous Recombination cells The RecA protein, previously mentioned in regard to the SOS repair system (Section 9.4 and Figure 9.10), is the key to homologous recombination. RecA is essential in nearly every homologous recom- bination pathway. RecA-like proteins have been identified in all Virus injection; Bacteria examined, as well as in the Archaea and most Eukarya. chromosome Lysis of disruption A molecular mechanism for homologous recombination between donor cell; two DNA molecules is shown in Figure 9.12. An enzyme that cuts DNA released DNA in the middle of a strand, called an endonuclease, begins the process by nicking one strand of the donor DNA molecule. This nicked strand is separated from the other strand by proteins with helicase activity; the resulting single-stranded segment binds single- strand binding protein (◀ Section 6.3) and then RecA. This results in a complex that promotes base pairing with the complementary sequence in the recipient DNA molecule. Base pairing, in turn, dis- places the other strand of the recipient DNA molecule (Figure 9.12) Viruses and is appropriately called strand invasion. containing The base pairing of one strand from each of the two DNA molecules Donor DNA donor DNA over long stretches generates recombination intermediates containing Recipient long heteroduplex regions, where each strand has originated from a cells different chromosome. After DNA strands at the branch or crossover regions are joined, the linked molecules are then resolved (separated) by enzymes that cut and rejoin the previously unbroken strands of both original DNA molecules. Depending on the orientation of the junction during resolution, two types of products—referred to as Conjugation “patches” or “splices”—are formed that differ in the conformation of Plasmid transfer Chromosome transfer the heteroduplex regions remaining after resolution (Figure 9.12). Plasmid-containing Donor cell with donor cell integrated plasmid Effect of Homologous Recombination on Genotype For homologous recombination to generate new genotypes, the two homologous sequences must be related but genetically distinct. This is obviously the case in a diploid eukaryotic cell, which has two sets of Recipient chromosomes, one from each parent. However, in bacteria, genetically cells distinct but homologous DNA molecules are brought together in dif- ferent ways. Genetic recombination in bacteria occurs after fragments of homologous DNA from a donor chromosome are transferred to a recipient cell by transformation, transduction, or conjugation. It is Figure 9.11 Processes by which DNA is transferred from donor to recipient bacte- only after the transfer event, when the DNA fragment from the donor rial cell. Just the initial steps in transfer are shown. Note that conjugation requires is in the recipient cell, that homologous recombination occurs. cell–cell contact, whereas transduction and transformation do not. M09_MADI4790_16_GE_C09.indd 307 08/03/2021 16:41  308   UNIT 2 MOLECULAR BIOLOGY AND GENETICS 5¿ D E F 3¿ Donor 3¿ D¿ E' F¿ 5¿ DNA 1. Endonuclease nicks DNA. DNA from Trp+ cells 5¿ D E F 3¿ 3¿ D¿ E¿ F¿ 5¿ Nick 2. Binding of SSB protein SSB protein 2 UNIT 5¿ D E F 3¿ 3¿ D¿ E¿ F¿ 5¿ Agar medium Agar medium lacking tryptophan lacking tryptophan 3¿ d e f 5¿ Recipient