13.

6 BACTERIAL PLASMIDS
Conjugation, the transfer of DNA between bacteria involving direct contact, depends on the presence of an extra piece of DNAknown as a plasmid. Plasmids play many important roles in the lives of procaryotes. They also have proved invaluable to microbiologists and molecular geneticists in constructing and transferring new genetic combinations and in cloning genes as described in chapter 14. Recall from chapter 3 that plasmids are small doublestranded DNA molecules that can exist independently of host chromosomes. They have their own replication origins and autonomously replicate and are stably inherited. Some plasmids are episomes, plasmids that can exist either with or without being integrated into host chromosomes. Although there are a variety of plasmid types, our concern here is with conjugative plasmids. These plasmids can transfer copies of themselves to other bacteria during the process of conjugation, which is discussed in section 13.7.
Bacterial Plasmids 335
35 53 Transposase recognizes the inverted repeats and cleaves at both ends of the transposable element, releasing it from its original site. Transposase carries the TE to a new site and cleaves the target DNA at staggered sites. The transposable element is inserted into the target site. Target DNA Transposase TE Transposable element DNA gap repair synthesis Transposable element Direct repeats IR IR IR IR IR IR IR IR TEA few hundred to several thousand base pairs in length TACGAATGCT ATGCT 35 53 TACGAATGCT TACGA 35 53 35 53 35 53 TACGA ATGCT

Figure 13.20 Simple Transposition. TE, transposable

element; IR, inverted repeat.
Resolvase catalyzes recombination between the two

elements. This resolves them into two separate structures, each with a copy of the transposable element. The gaps are filled in by DNA polymerase and sealed by DNA ligase. Transposase makes cuts at the arrows and the strands are exchanged and ligated together, forming a cointegrate. Target sequence Transposable element Target DNA + + 1 2 3

Figure 13.21 Replicative Transposition.

336 Chapter 13 Microbial Genetics:Mechanisms of Genetic Variation
IS1 Km Amp Sm, Su Cm IS1 Tn4 Tn3 RTF 38 base pair inverted repeat tnpA Transposase (MW 120 K) Resolvase (MW 21 K) -lactamase (MW 20 K) 38 base pair inverted repeat tnpR bla Target site flanking repeat Target site flanking repeat

Figure 13.22 The Tn3 Composite Transposon within an R Plasmid. Tn3 is

a replicative transposon that contains the gene for _-lactamase (bla), an enzyme that confers resistance to the antibiotic ampicillin (Amp).The arrows below the Tn3 genes indicate the direction of transcription.Tn3 can be found in the resistance plasmid R1, where it is inserted into another transposable element,Tn4. Tn4 carries genes that provide resistance to streptomycin (Sm) and sulfonamide (Su).The plasmid also carries resistance genes for kanamycin (Km) and chloramphenicol (Cm).The RTF region of R1 codes for proteins needed for plasmid replication and transfer.

Perhaps the best-studied conjugative plasmid is F factor. It plays a major role in conjugation in E. coli and was the first conjugative plasmid to be described (figure 13.23). The F factor is about 100 kilobases long and bears genes responsible for cell attachment and plasmid transfer between specific bacterial strains during conjugation. Most of the information required for plasmid transfer is located in the tra operon, which contains at least 28

genes. Many of these direct the formation of sex pili that attach the F_ cell (the donor cell containing an F plasmid) to an F_ cell (figure 13.24). Other gene products aid DNA transfer. In addition, the F factor has several segments called insertion sequences that assist plasmid integration into the host cell chromosome. Thus the F factor is an episome that can exist outside the bacterial chromosome or can be integrated into it (figure 13.25). 1. Define the following: episome, conjugative plasmid, transposition, transposase, and conjugative transposon. 2. Compare and contrast plasmids and transposable elements.Compare and contrast insertion sequences, composite transposons, and replicative transposons. 3. How might one demonstrate the presence of a plasmid in a host cell? 4. What is simple (cut-and-paste) transposition? What is replicative transposition? How do the two mechanisms of transposition differ? What happens to the target site during transposition? 5. What effect would you expect the existence of transposable elements and plasmids to have on the rate of microbial evolution? Give your reasoning. 6. How do multiple-drug-resistant plasmids often arise?
Bacterial Conjugation 337
F factor oriT Region of F factor with genes needed for conjugation Encodes pilin protein Encode proteins that are components of the type IV secretion system Encodes coupling protein Encodes relaxase oriT traM traJ traY traA traL traE traK traB traP traR traC traW traU traN traF traQ traH traG traS traT traD traI traX trbD trbG trbI trbC

trbE trbA trbB trbJ trbF trbH traV

Figure 13.23 The F plasmid. Genes that play a role in conjugation are shown, and some of their functions are
indicated.The plasmid also contains three insertion sequences and a transposon.The site for initiation of rolling-circle replication and gene transfer during conjugation is oriT. Figure 13.24 Bacterial Conjugation. An electron micrograph of two E. coli cells in an early stage of conjugation. The F+ cell to the right is covered with small pili or fimbriae, and a sex pilus connects the two cells.

13.7 BACTERIAL CONJUGATION

The initial evidence for bacterial conjugation, the transfer of DNA by direct cell to cell contact, came from an elegant experiment performed by Joshua Lederberg and Edward Tatum in 1946. They mixed two auxotrophic strains, incubated the culture for several hours in nutrient medium, and then plated it on minimal medium. To reduce the chance that their results were due to simple reversion, they used double and triple auxotrophs on the assumption that two or three simultaneous reversions would be extremely rare. For example, one strain required biotin (Bio _), phenylalanine (Phe_), and cysteine (Cys_) for growth, and another needed threonine (Thr_), leucine (Leu_), and thiamine (Thi_). Recombinant prototrophic colonies appeared on the minimal medium after incubation (figure 13.26). Thus the chromosomes of the two auxotrophs were able to associate and undergo recombination. Lederberg and Tatum did not directly prove that physical contact of the cells was necessary for gene transfer. This evidence was provided by Bernard Davis (1950), who constructed a U tube consisting of two pieces of curved glass tubing fused at the base to form a U shape with a fritted glass filter between the halves. The filter allowed the passage of media but not bacteria. The U tube was filled with nutrient medium and each side inoculated with a different auxotrophic strain of E. coli (figure 13.27). During incubation, the medium was pumped back and forth through the filter to ensure medium exchange between the halves. After a 4 hour incubation, the bacteria were plated on minimal medium. Davis discovered that when the two auxotrophic strains were separated from each other by the fine filter, gene transfer could not take place. Therefore direct contact was required for the recombination that Lederberg and Tatum had observed. F factor-mediated conjugation is one of the best-studied conjugation systems. It is the focus of this section.
338 Chapter 13 Microbial Genetics:Mechanisms of Genetic Variation
IS

F factor 12 IS AB AB 12 O

Integrated F factor A IS 2 O 1 IS B O Bacterial chromosome

Figure 13.25 F Plasmid Integration. The reversible integration of an F plasmid or factor into a host bacterial
chromosome.The process begins with association between plasmid and bacterial insertion sequences.The O arrowhead (white) indicates the site at which oriented transfer of chromosome to the recipient cell begins. A, B, 1, and 2 represent genetic markers.

F__F_ Mating
In 1952 William Hayes demonstrated that the gene transfer observed by Lederberg and Tatum was polar. That is, there were definite donor (F_, or fertile) and recipient (F_, or nonfertile) strains, and gene transfer was nonreciprocal. He also found that in F _ _ F_ mating the progeny were only rarely changed with regard to auxotrophy (that is, chromosomal genes were not often transferred), but F_ strains frequently became F_. These results are readily explained in terms of the F factor previously described (figure 13.23). The F_ strain contains an extrachromosomal F factor carrying the genes for sex pilus formation and plasmid transfer. The sex pilus is used to establish contact between the F_ and F_ cells (figure 13.28a). Once contact is made, the pilus retracts, bringing the cells into close physical contact. The F_ cell then prepares for DNA transfer by assembling a type IV secretion apparatus, using many of the same genes used for sex pilus biogenesis; the sex pilus is embedded in the secretion structure (figure 13.29). The F factor then replicates by a rolling-circle mechanism (see figure 11.12). Replication is initiated by a complex of proteins called the relaxosome, which nicks one strand of the F factor at a site called oriT (for origin of transfer). Relaxase, an enzyme associated with the relaxosome, remains attached to the 5' end of the nicked strand. As F factor is replicated, the displaced strand and the attached relaxase enzyme move through the type IV secretion system to the recipient cell. Because the pilus is embedded in the secretion apparatus, it has been suggested that the DNA moves through a lumen in the pilus. However, studies of a related conjugation system, that of the plant pathogen Agrobacterium tumefaciens, provide strong evidence that the DNA does not move through the sex pilus. However, it should be noted that although the F factor system and the Agrobacterium system are related, there is one important difference between the two. The F factor system is used to transfer DNAfrom one bacterium to another, whereas the Agrobacterium system moves DNA from the bacterium into its plant host. Whatever the route of transfer, as the plasmid is transferred, the entering strand is copied to produce double-stranded DNA. The recombination frequency is low because chromosomal genes are rarely transferred with the independent F factor. Microorganism associations
with vascular plants: Agrobacterium (section 29.5)

Bacterial Conjugation 339

Bio- Phe- Cys- Thr+ Leu+ Thi+ Bio+ Phe+ Cys+ Thr- Leu- ThiBio+ Phe+ Cys+ Thr+ Leu+ Thi+ Prototrophic colonies Minimal medium without supplements Mixture

Figure 13.26 Evidence for Bacterial Conjugation.

Lederberg and Tatums demonstration of genetic recombination using triple auxotrophs. See text for details.
Met Thr+ Leu+Thi+ Met + Thr LeuThi Pressure or suction Fritted glass filter

Figure 13.27 The U-tube Experiment. The U-tube experiment

used to show that genetic recombination by conjugation requires direct physical contact between bacteria. See text for details.

Hfr Conjugation
Not long after the discovery of F_ _ F_ mating, a second type of F factor-mediated conjugation was discovered. In this type of conjugation, the donor transfers chromosomal genes with great efficiency, but does not change the recipient bacteria into F_ cells. Because of the high frequency of recombinants produced by this mating, it is referred to as Hfr conjugation and the donor is called an Hfr strain. Although initially the mechanism of Hfr conjugation was not known, eventually it was determined that Hfr strains contain the F factor integrated into their chromosome, rather than free in the cytoplasm (figure 13.28b). When integrated, the F plasmids tra operon is still functional; the plasmid can direct the synthesis of pili, carry out rolling-circle replication, and transfer genetic material to an F_ recipient cell. However, rather than transferring itself, the F factor directs the transfer of host chromosome. DNAtransfer begins when the integrated F factor is nicked at its site of transfer origin. As it is replicated, the chromosome moves to the recipient (figure 13.28c). Because only part of the F factor is transferred, the F_ recipient does not become F_ unless the whole chromosome is transferred. Transfer of the entire chromosome with the integrated F factor requires about 100 minutes in E. coli, and the connection between the cells usually breaks before this process is finished. Thus a complete F factor usually is not transferred, and the recipient remains F_. As mentioned earlier, when an Hfr strain participates in conjugation, bacterial genes are frequently transferred to the recipient. Gene transfer can be in either a clockwise or counterclockwise direction around the circular chromosome, depending on the orientation of the integrated F factor. After the replicated donor chromosome enters the recipient cell, it may be degraded or incorporated into the F_ genome by recombination.

FConjugation
Because the F plasmid is an episome, it can leave the bacterial chromosome and resume status as an autonomous F factor. Sometimes during this process the plasmid makes an error in excision and picks up a portion of the chromosome. Because it is now genotypically distinct from the original F factor, it is called an Fplasmid (figure 13.30a). It is not unusual to observe the inclusion of one or more chromosomal genes in excised F plasmids.Acell containing an Fplasmid retains all of its genes, although some of them are on the plasmid. It mates only with an F_ recipient. F_ F_ conjugation is similar to F__F_ mating. Once again, the plasmid is transferred as it is copied by rolling-circle replication. However, bacterial genes on the chromosome usually are not transferred (figure 13.30b). Bacterial genes acquired during excision of the Fplasmid are transferred with it and need not be incorporated into the recipient chromosome to be expressed. The recipient becomes Fand is a partially diploid merozygote because

the same bacterial genes present on the Fplasmid are also found on the recipients chromosome. In this way specific bacterial genes may spread rapidly throughout a bacterial population. Fconjugation is very important to the microbial geneticist. A partial diploids behavior shows whether the allele carried by an Fplasmid is dominant or recessive to the chromosomal gene. The formation of Fplasmids also is useful in mapping the chromosome because if two genes are picked up by an F factor they must be neighbors.

Other Examples of Bacterial Conjugation

Although most research on plasmids and conjugation has been done using E. coli and other gram-negative bacteria, self-transmissible
340 Chapter 13 Microbial Genetics:Mechanisms of Genetic Variation
F factor Bacterial chromosome Coupling factor Type IV secretion system Secretion system F+ cell F+ cell Relaxosome Relaxase Origin of transfer F+ cell Recipient cell (F) Relaxosome makes a cut at the origin of transfer and begins to separate one DNA strand. The intact strand is replicated by the rolling circle mechanism. Sex pilus makes contact with F- recipient cell. Sex pilus contracts bringing cells together. Type IV secretion system is constructed and joins two cells. Accessory proteins of the relaxosome are released. The DNA/relaxase complex is recognized by the coupling factor and transferred to the secretion system. The secretion system pumps the DNA/relaxase complex into the recipient cell. As the DNA enters, the F-factor DNA is replicated to become double stranded. Coupling factor Frecipient (a) F+ x F- conjugation Bacterial chromosome Origin of transfer Origin of

transfer F factor Integration of F factor into chromosome F+ cell Hfr cell lac+ pro+ lac+ pro+ (a) F_ _ F_ conjugation (b) Insertion of F factor into chromosome

Bacterial Conjugation 341

Origin of transfer (toward lac+) Sex pilus makes contact with F cell and contracts to pull Hfr and F cell together. Type IV secretion system connects cells. New strand synthesized by rolling circle replication Transfer of Hfr chromosome Hfr cell Hfr cell F cell F recipient lac+ pro+ lac+ pro+ lac+ pro+ lac+ pro+ lac+ pro+ lac+ pro+ lac+ pro lac+ pro+ lac pro lac pro Conjugation for longer times Conjugation for short time Recombination between exogenote and endogenote Recombination between exogenote and endogenote Hfr cell F recipient lac lac+ pro+ lac+ pro lac pro lac+ pro+

Figure 13.28 F Factor-Mediated Conjugation. The F factor encodes proteins for building the sex pilus and
proteins needed to construct the type IV secretion system that will transfer DNA from the donor to the F _ recipient. One protein, the coupling factor, is thought to guide the DNA to the secretion system. (a) During F_ _ F_ conjugation, only the F factor is transferred because the plasmid is extrachromosomal.The recipient cell becomes F_. (b) Integration of the F factor into the chromosome creates an Hfr cell. (c) During Hfr _ F_ conjugation, some plasmid genes and some chromosomal genes are transferred to the recipient. Note that only a portion of the F factor moves into the recipient. Because the entire plasmid is not transferred, the recipient remains F _. In addition, the incoming DNA must recombine into the recipients chromosome if it is to be stably maintained.
(c) Hfr _ F_ conjugation

342 Chapter 13 Microbial Genetics:Mechanisms of Genetic Variation

plasmids are present in gram-positive bacterial genera such as Bacillus, Streptococcus, Enterococcus, Staphylococcus, and Streptomyces. Much less is known about these systems. It appears that fewer transfer genes are involved, possibly because a sex pilus may not be required for plasmid transfer. For example, Enterococcus faecalis recipient cells release short peptide chemical signals that activate transfer genes in donor cells containing the proper plasmid. Donor and recipient cells directly adhere to one another through special plasmid-encoded proteins released by the activated donor cell. Plasmid transfer then occurs. 1. What is bacterial conjugation and how was it discovered? 2. Distinguish between F_, Hfr, and F_ strains of E. coli with respect to their physical nature and role in conjugation. 3. Describe in some detail how F__F_ and Hfr conjugation processes proceed, and distinguish between the two in terms of mechanism and the final results. 4. What is Fconjugation and why is it so useful to the microbial geneticist? How does the Fplasmid differ from a regular F plasmid?

13.8 DNA TRANSFORMATION

The second way DNA can move between bacteria is through transformation, discovered by Fred Griffith in 1928. Transformation is the uptake by a cell of a naked DNA molecule or fragment from the medium and the incorporation of this molecule into the recipient chromosome in a heritable form. In natural transformation the DNA comes from a donor bacterium. The process is random, and any portion of a genome may be transferred between bacteria. DNA as genetic material (section 11.1)
TraA (pilin) Tip C D
coupling

ATP ADP+P ATP ADP+P TraQ, X L Sex pilus OM P LPS PG PM

Figure 13.29 The Type IV Secretion System Encoded by F Factor. The F factor-encoded type IV secretion system is
composed of numerous Tra proteins, including TraA proteins,

which form the sex pilus, and TraD, which is the coupling factor. Some Tra proteins are located in the plasma membrane (PM), others extend into the periplasm (P) and pass through the peptidoglycan layer (PG) into the outer membrane (OM) and its lipopolysaccharide (LPS) layer.
De-integration including part of bacterial chromosome A A A AA A a a a (a) (b) F_ plasmid begins replication and transfer Cells connected by type IV secretion system F_ plasmid replicated and transferred Hfr F_ F_ F_ F_ F_

Figure 13.30 F' Conjugation. (a) Due to an error in excision,

the A gene of an Hfr cell is picked up by the F factor. (b) The A gene is then transferred to a recipient during conjugation. See text for explanation. DNA Transformation 343

When bacteria lyse, they release considerable amounts of DNA into the surrounding environment. These fragments may be relatively large and contain several genes. If a fragment contacts a competent cell, a cell that is able to take up DNA and be transformed, the DNAcan be bound to the cell and taken inside (figure 13.31a). The transformation frequency of very competent cells is around 10_3 for most genera when an excess of DNAis used. That is, about one cell in every thousand will take up and integrate the gene. Competency is a complex phenomenon and is dependent on several conditions. Bacteria need to be in a certain stage of growth; for example, Streptococcus pneumoniae becomes competent during the exponential phase when the population reaches about 10 7 to 108 cells per ml. When a population becomes competent, bacteria such as S. pneumoniae secrete a small protein called the competence factor that stimulates the production of 8 to 10 new proteins required for transformation. Natural transformation has been discovered so far only in certain genera including Streptococcus, Bacillus, Thermoactinomyces, Haemophilus, Neisseria, Moraxella, Acinetobacter, Azotobacter, Helicobacter, and Pseudomonas. Gene transfer by this process occurs in soil and aquatic ecosystems and may be an important route of genetic exchange in biofilm and other microbial communities. The mechanism of transformation has been intensively studied in S. pneumoniae (figure 13.32). A competent cell binds a double-stranded DNA fragment if the fragment is moderately large; the process is random, and donor fragments compete with each other. The DNA then is cleaved by endonucleases to doublestranded fragments about 5 to 15 kilobases in size. DNA uptake requires energy expenditure. One strand is hydrolyzed by an

envelope-associated exonuclease during uptake; the other strand associates with small proteins and moves through the plasma membrane. The single-stranded fragment can then align with a homologous region of the genome and be integrated, probably by a mechanism similar to that depicted in figure 13.18. Transformation in Haemophilus influenzae, a gram-negative bacterium, differs from that in S. pneumoniae in several respects. H. influenzae does not produce a competence factor to stimulate the development of competence, and it takes up DNA from only closely related species (S. pneumoniae is less particular about the source of its DNA). Double-stranded DNA, complexed with proteins, is taken in by membrane vesicles. The specificity of H. influenzae transformation is due to a special 11 base pair sequence (5AAGTGCGGTCA3) that is repeated over 1,400 times in H. influenzae DNA. DNAmust have this sequence to be bound by a competent cell. The protein complexes that take up free DNA must be able to move it through gram-negative and gram-positive walls, which may be both thick and complex. As expected, the machinery is quite large and complicated and appears related to protein secretion systems. Figure 13.33a shows a schematic diagram of the complex used by the gram-negative bacterium Neisseria gonorrhoeae. PilQ aids in the movement across the outer membrane, and the pilin complex PilE moves the DNA through the periplasm and peptidoglycan. ComE is a DNAbinding protein; N is the nuclease that degrades one strand before the DNA enters the cytoplasm through the transmembrane channel formed by
Uptake of DNA Uptake of plasmid OR Bacterial chromosome Degradation DNA fragments DNA plasmid Stable transformation Stable transformation Unsuccessful transformation Integration by nonreciprocal recombination Bacterial chromosome (b) Transformation with a plasmid (a) Transformation with DNA fragments

Figure 13.31 Bacterial Transformation. Transformation

with (a) DNA fragments and (b) plasmids. Transformation with a plasmid often is induced artificially in the laboratory. The transforming DNA is in purple and integration is at a homologous region of the genome. 344 Chapter 13 Microbial Genetics:Mechanisms of Genetic Variation
DNA fragment binds to a cell surface receptor. An extracellular endonuclease cuts the DNA into smaller fragments. One strand is degraded and a single strand is transported into

Uptake the cell. system The DNA strand aligns itself with a homologous region on the bacterial chromosome. The DNA strand is incorporated into the bacterial chromosome via homologous recombination. The heteroduplex DNA is repaired in a way that changes lac_ strand to create a lac+ gene. Transformed cell lac_ lac+ lac+ lac_ lac_ lac+ lac+ lac_ lac+ Receptor Heteroduplex

Figure 13.32 Bacterial Transformation as Seen in S.pneumoniae. ComA. The machinery in the gram-positive bacterium Bacillus subtilis is depicted in figure 13.33b. It is localized to the poles of the cell, and as can be seen, many of the components are similar to those of N. gonorrhoeae: the pilin complex (ComGC), DNAbinding protein (ComEA), nuclease (N), and channel protein (ComEC). ComFA is a DNA translocase that moves the DNA into the cytoplasm. A gram-negative equivalent of ComFA has not been identified yet in N. gonorrhoeae. Microbial geneticists exploit transformation to move DNA (usually recombinant DNA) into cells. However, as already noted, many species, including E. coli, are not naturally transformation competent. Fortunately, these bacteria can be made artificially competent by certain treatments. Two common techniques are electrical shock and exposure to calcium chloride. Both approaches render the cell membrane more permeable to DNA and both have been used to make artificially competent E. coli cells. To increase the transformation frequency with E. coli, strains that lack one or more nucleases are used. These strains are especially important when transforming the cells with linear DNA, which is vulnerable to attack by nucleases. It is easier to transform bacteria with plasmid DNA since plasmids are not as easily degraded as linear fragments and can replicate within the host (figure 13.31 b). 1. Define transformation and competence. 2. Describe how transformation occurs in S. pneumoniae. How does the process differ in H. influenzae? 3. Discuss two ways in which artificial transformation can be used to place functional genes within bacterial cells.
Outer membrane

ComGC Plasma membrane Plasma membrane ComFA ComEC ComA ComA ComEA ComE PilQ PilQ ComEC N N Peptidoglycan PilE Peptidoglycan (a) (b)

Figure 13.33 DNA Uptake Systems. (a) DNA uptake

machinery in N.gonorrhoeae. (b) Uptake machinery in B.subtilis. See text for details. Transduction 345

13.9 TRANSDUCTION
The third mode of bacterial gene transfer is transduction. It is a frequent mode of horizontal gene transfer in nature and is mediated by viruses. The morphology and life cycle of bacterial viruses or bacteriophages is not discussed in detail until chapter 17. Nevertheless, it is necessary to briefly describe the life cycle here as background for a consideration of their role in gene transfer. Viruses are structurally simple, often composed of just a nucleic acid genome protected by a protein coat called the capsid. They are unable to replicate autonomously. Instead, they infect and take control of a host cell, forcing the host to make many copies of the virus. Viruses that infect bacteria are called bacteriophages, or phages for short. Some phages are replicated by their bacterial host immediately after entry. After the number of replicated phages reaches a certain number, they cause the host to lyse, so they can be released and infect new host cells ( figure 13.34). These phages are called virulent bacteriophages and the process is called the lytic cycle. Other bacteriophages do not immediately kill their host. Many of these viruses enter the host bacterium and, instead of replicating, insert their genomes into the bacterial chromosome. Once inserted, the viral genome is called a prophage. The host bacterium is unharmed by this, and the phage genome is passively replicated as the host cells genome is replicated. These bacteriophages are called temperate bacteriophages and the relationship between these viruses and their host is called lysogeny (figure 13.34). Bacteria that have been lysogenized are called lysogens. Temperate phages can remain inactive in their hosts for many generations. However, they can be induced to switch to a lytic cycle of growth under certain conditions, including UV irradiation. When this occurs, the prophage is excised from the bacterial genome and the lytic cycle proceeds. Transduction is the transfer of bacterial genes by viruses. Bacterial genes are incorporated into a phage capsid because of errors made during the virus life cycle. The virus containing these genes then injects them into another bacterium, completing the transfer. There are two different kinds of transduction: generalized and specialized.

Generalized Transduction
Generalized transduction occurs during the lytic cycle of virulent and some temperate phages and can transfer any part of the bacterial genome (figure 13.35). During the assembly stage, when the viral chromosomes are packaged into protein capsids, random fragments of the partially degraded bacterial chromosome also may be packaged by mistake. Because the capsid can contain only a limited quantity of DNA, the viral DNA is left behind. The quantity of bacterial DNAcarried depends primarily on the size of the capsid. The P22 phage of Salmonella enterica serovar Typhimurium usually carries about 1% of the bacterial genome; the P1 phage of E. coli and a variety of gram-negative bacteria carries about 2.0 to 2.5% of the genome. The resulting virus particle often injects the DNAinto another bacterial cell but cannot initiate a lytic cycle. This phage is known as a generalized transducing particle or phage and is simply a carrier of genetic information from the original bacterium to another cell. As in transformation, once the DNA has been injected, it must be incorporated into the recipient cells chromosome to preserve the transferred genes. The DNA remains double stranded during
Phage DNA Bacterial chromosome Phage injects its DNA into cytoplasm. Exposure to stress such as UV light triggers excision from host chromosome. New phages can bind to bacterial cells. Cell lyses and releases the new phages. Phage DNA directs the synthesis of many new phages. Phage DNA integrates into host chromosome. Prophage DNA is copied when cell divides. Prophage Lytic cycle Lysogenic cycle

Figure 13.34 Lytic and Lysogenic Cycles of Temperate Phages. Virulent phages undergo only the lytic cycle. Temperate phages
have two phases to their life cycles.The lysogenic cycle allows the genome of the virus to be replicated passively as the host cells genome is replicated. Certain environmental factors such as UV light can cause a switch from the lysogenic cycle to the lytic cycle. In the lytic cycle, new virus particles are made and released when the host cell lyses.Virulent phages are limited to just the lytic cycle. 346 Chapter 13 Microbial Genetics:Mechanisms of Genetic Variation
Crossing over Phage infects bacterial cell. Host DNA is hydrolyzed into pieces, and phage

DNA and proteins are made. Phages assemble; occasionally a phage carries a piece of the host cell chromosome. Transducing phage injects its DNA into a new recipient cell. The transduced DNA is recombined into the chromosome of the recipient cell. The recombinant bacterium has a genotype (his+lys_ ) that is different from recipient bacterial cell (his_ lys_ ). Recombinant bacterium Transducing phage with host DNA Phage DNA Recipient cell (his_ lys_ ) his+ his+ his+ his+ his+ his_ lys+ lys+ lys+ lys_ lys_

Figure 13.35 Generalized Transduction in Bacteria. transfer, and both strands are integrated into the endogenotes genome.About 70 to 90% of the transferredDNAis not integrated but often is able to survive temporarily and be expressed. Abortive transductants are bacteria that contain this nonintegrated, transduced DNA and are partial diploids. Generalized transduction was discovered in 1951 by Joshua Lederberg and Norton Zinder during an attempt to show that conjugation, discovered several years earlier in E. coli, could occur in other bacterial species. Lederberg and Zinder were repeating the earlier experiments with S. enterica serovar Typhimurium. They found that incubation of a mixture of two multiply auxotrophic strains yielded prototrophs at the level of about one in 105. This seemed like good evidence for bacterial recombination, and indeed it was, but their initial conclusion that the transfer resulted from conjugation was not borne out. When these investigators performed the U-tube experiment (figure 13.27) with Salmonella, they still recovered prototrophs. The filter in the U tube had pores that were small enough to block the movement of bacteria between the two sides but allowed phage P22 to pass. Lederberg and Zinder had intended to confirm that conjugation was present in another bacterial species but instead discovered a completely new mechanism of bacterial gene transfer. This seemingly routine piece of research led to surprising and important results. A scientist must always keep an open mind about results and be prepared for the unexpected.

Specialized Transduction
In specialized transduction, the transducing particle carries only specific portions of the bacterial genome. Specialized transduction

is made possible by an error in the lysogenic life cycle of phages that insert their genomes into a specific site in the host chromosome. When a prophage is induced to leave the host chromosome, excision is sometimes carried out improperly. The resulting phage genome contains portions of the bacterial chromosome (about 5 to 10% of the bacterial DNA) next to the integration site, much like the situation with Fplasmids (figure 13.36).Atransducing phage genome usually is defective and lacks some part of its attachment site. The transducing particle will inject bacterial genes into another bacterium, even though the defective phage cannot reproduce without assistance. The bacterial genes may become stably incorporated under the proper circumstances. The best-studied example of specialized transduction is carried out by the E. coli phage lambda. The lambda genome inserts into the host chromosome at specific locations known as attachment or att sites (figure 13.37, see also figures 17.19 and 17.22). The phage att sites and bacterial att sites are similar and can complex with each other. The att site for lambda is next to the gal and bio genes on the E. coli chromosome; consequently, specialized transducing lambda phages most often carry these bacterial genes. The lysate, or product of cell lysis, resulting from the induction of lysogenized E. coli contains normal phage and a few defective transducing particles. These particles are called either lambda dgal because they carry the galactose utilization genes or lambda dbio because they carry the bio from the other side of the att site (figure 13.37). Because these lysates contain only a few transducing particles, they often are called low-frequency transduction lysates (LFT lysates). Whereas the normal phage has a complete att site, defective transducing particles have a nonfunctional hybrid integration site that is part bacterial and part phage in origin. Integration of the defective phage chromosome does not readily take place. Transducing phages also may have lost some genes essential for reproduction. Stable transductants can arise only if there is a double cross-over event on each side of the gal site (figure 13.37). Temperate bacteriophages and lysogeny (section 17.5) Defective lambda phages carrying the gal or bio genes can integrate if there is a normal lambda phage in the same cell.We will continue our discussion of this with a phage carrying the gal gene.
Transduction 347
Lysogenized cell with prophage Induction Prophage Rare-deintegration that includes some bacterial genes Replication of defective virus DNA with incorporated host genes Assembly and release of transducing phage particles Infection of recipient cell Integration as prophage Crossover to integrate bacterial genes Bacterial chromosome containing both virus and donor DNA Bacterial chromosome

containing only donor DNA

Figure 13.36 Specialized Transduction by a Temperate Bacteriophage. Recombination can produce two
types of transductants. 348 Chapter 13 Microbial Genetics:Mechanisms of Genetic Variation
Integration to form prophage att sites gal+ 3 2 1 1 2 gal+ 3 bio+ Normal excision Error in excision 1 21 2 3 gal+ 3 gal+ 1 2 3 gal+ gal+ 3 2 1 dgal 1 2 123 gal+ gal gal+ gal dgal 3 1212 Unstable transductant gal+ gal gal+ 1 2 Stable transductant

Figure 13.37 The Mechanism of Transduction for Phage Lambda and E. coli. Integrated lambda phage
lies between the gal and bio genes.When it excises normally (top left), the new phage is complete and contains no bacterial genes. Rarely excision occurs asymmetrically (top right), and either the gal or bio genes are picked up and some phage genes are lost (only aberrant excision involving the gal genes is shown).The result is a defective lambda phage that carries bacterial genes and can transfer them to a new recipient.

The normal phage integrates, yielding two bacterial/phage hybrid att sites where the defective lambda dgal phage can insert (figure 13.37). It also supplies the genes missing in the defective phage. The normal phage in this instance is termed the helper phage because it aids integration and reproduction of the defective phage. These transductants are unstable because the prophages can be induced to excise by agents such asUVradiation. Excision, however, produces a lysate containing a fairly equal mixture of defective lambda dgal phage and normal helper phage. Because it is very effective in transduction, the lysate is called a high-frequency transwil92913_
Mapping the Genome 349

duction lysate (HFT lysate). Reinfection of bacteria with this mixture will result in the generation of considerably more transductants.

LFT lysates and those produced by generalized transduction have one transducing particle in 105 or 106 phages; HFTlysates contain transducing particles with a frequency of about 0.1 to 0.5. 1. Briefly describe the lytic and lysogenic viral reproductive cycles.Define lysogeny, lysogen, temperate phage, prophage, and transduction. 2. Describe generalized transduction,how it occurs, and the way in which it was discovered.What is an abortive transductant? 3. What is specialized transduction and how does it come about? Distinguish between LFT and HFT lysates and describe how they are formed. 4. How might one tell whether horizontal gene transfer was mediated by generalized or specialized transduction? 5. Why doesnt a cell lyse after successful transduction with a temperate phage? 6. Describe how conjugation, transformation, and transduction are similar. How are they different?

13.10 MAPPING THE GENOME

Before the advent of genome sequencing, microbial geneticists only had one general approach for elucidating the organization of genes in a bacterial chromosometo carry out linkage analysis. Such analyses yield a genetic map showing the position of genes relative to each other. Genetic mapping using linkage analysis is a very complex task. This section surveys approaches to mapping the bacterial genome, using E. coli as an example. All three modes of gene transfer and recombination have been used in mapping. Hfr conjugation is frequently used to map the relative location of bacterial genes. This technique rests on the observation that during conjugation, the chromosome moves from donor to recipient at a constant rate. In an interrupted mating experiment the conjugation bridge is broken and Hfr _ F_ mating is stopped at various intervals after the start of conjugation by mixing the culture vigorously in a blender (figure 13.38a). The order and timing of gene transfer can be determined because they are a direct reflection of the order of genes on the bacterial chromosome (figure 13.38b). For example, extrapolation of the curves in figure 13.38 b back to the x-axis gives the time at which each gene just began to enter the recipient. The result is a circular chromosome map with distances expressed in terms of the minutes elapsed until a gene is transferred. This technique can fairly precisely locate genes 3 minutes or more apart. The heights of the plateaus in figure 13.38 b are lower for genes that are more distant from the F factor (the origin of transfer) because there is an ever-greater chance that the conjugation bridge will spontaneously break before these genes are transfered. Because of the relatively large size of the E. coli genome, it is not possible to generate a map from one Hfr strain. Therefore several Hfr strains with the F plasmid integrated at different locations must be used and their maps superimposed on one another. The overall map is adjusted to 100 minutes, although complete transfer may require somewhat more than 100 minutes. In a sense, minutes are an indication of map distance and not strictly a measure of time. Zero time is set at the threonine (thr) locus. Gene linkage, or the proximity of two genes on a chromosome, can be determined from transformation by measuring the frequency with which two or more genes simultaneously transform a recipient cell. Consider the case for cotransformation by two genes. In theory, a bacterium could simultaneously receive two genes, each carried on a separate DNA fragment. However,

it is much more likely that genes residing on the same fragment will be simultaneously transferred. If two genes are closely linked on the chromosome, then they should be able to cotransform. The closer the genes are together, the more often they will be carried on the same fragment and the higher will be the frequency of cotransformation. If genes are spaced a great distance apart, they will be carried on separate DNA fragments and the frequency of double transformants will equal the product of the individual transformation frequencies. Generalized transduction can be used to obtain linkage information in much the same way as transformation. Linkages usually are expressed as cotransduction frequencies, using the argument that the closer two genes are to each other, the more likely they both will reside on the DNA fragment incorporated into a single phage capsid. The E. coli phage P1 is often used in such mapping because it can randomly transduce up to 1 to 2% of the genome (figure 13.39). Specialized transduction is used to find which phage attachment site is close to a specific gene. The relative locations of specific phage att sites are known from conjugational mapping, and the genes linked to each att site can be determined by means of specialized transduction. These data allow precise placement of genes on the chromosome. Asimplified genetic map of E. coli K12 is given in figure 13.40. Because conjugation data are not high resolution and cannot be used to position genes that are very close together, the map was developed using several mapping techniques. Interrupted mating data were combined with those from cotransduction and cotransformation studies. Data from recombination studies also were used. New genetic markers in the E. coli genome were located within a relatively small region of the genome (10 to 15 minutes long) using a series of Hfr strains with F factor integration sites scattered throughout the genome. Once the genetic marker was located with respect to several genes in the same region, its position relative to nearby neighbors was more accurately determined using transformation and transduction studies. Such analyses are no longer performed in E. coli and other microbes for which a genome sequence has been published. Using these techniques, researchers mapped about 2,200 genes of E. coli K12 and compared this with the actual nucleotide sequence of the genome (i.e., a physical map of the genome). Genome sequencing has revealed about 4,300 possible genes. Thus genetic analysis defined over half of the potential genes. The genetic map approximates the physical map, but they do not correspond perfectly. This is because the genetic map is derived from genetic linkage frequencies that do not correlate exactly with the number of nucleotides that separate two genes. Roughly speaking, one minute of the E. coli genetic map corresponds to 40 kilobases of DNA sequence.