Bacterial Genetics

28 Chapter

Bacterial Genetics

1. Reproduction Versus Gene Transfer ................................... 896 2. Fate of the Incoming DNA After Uptake........................... 897

3. Transformation Is Gene Transfer by Naked DNA ......898 4. Gene Transfer by Virus— Transduction........................... 904 5. Transfer of Plasmids Between Bacteria................................... 908 6. Gene Transfer Among GramPositive Bacteria...................915 7. Archaeal Genetics ................917 8. Whole-Genome Sequencing............................. 918 Review Questions .......................923 Further Reading........................... 924

I

n many ways, bacterial genetics underlies molecular biology. The discovery of gene transfer in bacteria,

and, in particular, the involvement of plasmids in this, provided the foundations for molecular cloning. The genetics of bacteria is very different from that of higher organisms. Firstly, bacteria are generally haploid, with one copy of each gene on a single circular chromosome (unlike eukaryotes, which are diploid with multiple linear chromosomes). Secondly, gene transfer in bacteria is normally unidirectional; that is, a donor cell transfers genes to a recipient cell rather than two cells sharing genetic information to generate progeny as seen in the more familiar forms of reproduction in higher organisms. Gene transfer in bacteria occurs by three major mechanisms, which form the main topics of this chapter. 1. Reproduction Versus Gene Transfer Sex and reproduction are not at all the same thing in all organisms. In animals, reproduction normally involves sex, but in bacteria, and in many lower eukaryotes, these are two distinct processes. Bacteria divide by binary fission. First, they replicate their single chromosome and then the cell elongates and divides down the middle. No resort-

binary fission Simple form of cell division in which the cell replicates its DNA, elongates, and divides down the middle.

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Molecular Biology. DOI: https://doi.org/10.1016/B978-0-12-813288-3.00028-8 © 2019 Elsevier Inc. All rights reserved.

2. Fate of the Incoming DNA After Uptake

ing of the genes between two individuals (that is, no sex) is involved and so this is known as asexual or vegetative reproduction. From a biological perspective, sexual reproduction serves the purpose of reshuffling genetic information. This will sometimes produce offspring with combinations of genes superior to those of either parent (and, of course, sometimes worse!). Although bacteria normally grow and divide asexually, gene transfer may occur between bacterial cells. During sexual reproduction in higher organisms, germ-line cells from two parents fuse to form a zygote that contains equal amounts of genetic information from each parent. In contrast, in bacteria gene transfer is normally unidirectional and cell fusion does not occur. Genes from one bacterial cell are donated to another. We thus have a donor cell that donates DNA and a recipient cell that receives the DNA. The transfer of genes between bacteria fulfills a similar evolutionary purpose to the mingling of genes during sexual reproduction in higher organisms. However, mechanistically it is very different. Consequently, some scientists regard bacterial gene transfer as a primitive or aberrant form of sex, whereas others believe that it is quite distinct, and that use of the same terminology is misleading. Molecular biologists use bacteria together with their plasmids and viruses to carry most cloned genes, whether they are originally from cabbages or cockroaches. Consequently, a basic understanding of bacterial gene transfer is needed to understand the genetic engineering of plants and animals. Gene transfer in bacteria occurs by three basic mechanisms. Genes are only transferred by cell-to-cell contact in conjugation. In transduction, genes are transferred via virus particles, and in transformation, bacterial cells take up free molecules of DNA. Before considering these three mechanisms in detail, we will discuss what happens to the DNA after uptake, as similar considerations apply in all three cases.

897

In bacteria, cell division and the reshuffling of genetic information are completely separate processes.

Gene transfer between bacteria may involve uptake of naked DNA, transport of DNA via virus particles, or transfer of DNA via a specialized cell-tocell connection.

2. Fate of the Incoming DNA After Uptake Irrespective of its mode of entry, DNA that enters a bacterial cell has one of three possible fates. It may survive as an independent DNA molecule, it may be completely degraded, or part may survive by integration or recombination with the host chromosome before the rest is degraded. For incoming DNA to survive inside a bacterial cell as a self-replicating DNA molecule, it must be a replicon. In other words, it must have its own origin of replication and lack exposed ends. For survival in the vast majority of bacteria, this means that it must be circular. In those few bacteria, such as Borrelia and Streptomyces (see Chapter 4: Genes, Genomes, and DNA) with linear replicons, the ends must be properly protected. In eukaryotes, long-term survival of a linear DNA molecule requires a replication origin, a centromere sequence, and telomeres to protect the ends (see Chapter 4: Genes, Genomes, and DNA). A linear fragment of double-stranded DNA that enters a bacterial cell will normally be broken down by exonucleases that attack the exposed ends. For any of its genes to survive, they must be incorporated into the chromosome of the recipient cell by the process of recombination (see Chapter 27: Recombination). For recombination to occur, crossovers must form between regions of DNA of similar sequence—that is, homologous sequences. The two DNA molecules will swap DNA between two crossover points (Fig. 28.01). Consequently, if genes from incoming DNA are incorporated, the corresponding original genes of the recipient cell are lost. Such homologous recombination normally only occurs between closely related molecules of DNA—for example, DNA from two strains of the same bacterial species. Unrelated DNA may be incorporated by recombination provided it is

Incoming fragments of DNA will be destroyed by cells that receive them unless they form a replicon.

Incoming fragments of DNA may be preserved from destruction by recombination onto the host chromosome.

asexual or vegetative reproduction Form of reproduction in which there is no reshuffling of the genes between two individuals. conjugation Process in which genes are transferred from one bacterium to another by cell-to-cell contact. donor cell Cell that donates DNA to another cell. recipient cell Cell that receives DNA from another cell. sexual reproduction Form of reproduction that involves reshuffling of the genes between two individuals. transduction Process in which genes are transferred from one bacterium to another via virus particles. transformation (As used in bacterial genetics) Process in which genes are transferred into a cell as free molecules of DNA.

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FIGURE 28.01 Recombination Allows Survival of Transformed DNA In most cases, incoming linear DNA molecules are degraded by the host cell exonucleases. If there are homologous regions between incoming DNA and the host chromosome, crossing over may replace regions of the host chromosome with part of the incoming DNA.

g homologous DNA omin Inc a b

c

d

c

b

d

a

898

Crossover Host chromosome

HOMOLOGOUS RECOMBINATION (CROSSING OVER)

c

d

a

b

Some incoming genes are incorporated

Incoming circular DNA with its own origin of replication can survive without recombination.

Restriction enzymes degrade unmethylated foreign DNA, whether linear or circular.

surrounded by sequences that are related (Fig. 28.02). Another possibility is that the incoming DNA contains a transposon that can function in the recipient cell. If so, then the transposon may survive by abandoning the incoming DNA molecule and jumping into the chromosome of the new host cell. If the incoming DNA is a plasmid that can replicate on its own, recombination into the chromosome is not necessary for survival. For genetic engineering purposes, it is usually more convenient to avoid adding genes to the bacterial chromosome via recombination. Consequently, molecular biologists often put the genes they are working with onto plasmids (see Chapter 23: Plasmids). In addition to exonuclease attack, incoming DNA is often susceptible to restriction. This is a protective mechanism designed to destroy incoming foreign DNA. Most bacteria assume that foreign DNA is more likely to come from an enemy, such as a virus, than from a harmless relative, and they cut it into small fragments with restriction enzymes. This applies to both linear and circular DNA, since the degradative enzymes are endonucleases that cut DNA molecules in the middle (see Chapter 5: Manipulation of Nucleic Acids, for details). Only DNA that has been modified by methylating the appropriate recognition sequences is accepted as friendly. In genetic engineering, restriction negative host strains are used to surmount this obstacle.

3. Transformation Is Gene Transfer by Naked DNA The simplest way to transfer genetic information is for one cell to release DNA into the medium and for another cell to import it. The transfer of “pure” or “naked” DNA from the external medium into a bacterium is known as transformation (Fig. 28.03). By “naked,” we mean no other biological macromolecules, such as protein, are present to enclose or protect the DNA. No actual cell-to-cell contact happens during transformation, nor is the DNA packaged inside a virus particle. Bacterial cells can often take up naked DNA molecules and may incorporate the genetic information they carry.

3. Transformation Is Gene Transfer by Naked DNA

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FIGURE 28.02 Incorporation of Unrelated DNA

DNA unrelated to chromosome

Region of homology

Incoming DNA does not have to be entirely related to the host in order for recombination to occur. In some instances, the incoming DNA has regions that are related (purple) and regions that are totally unrelated (green). The regions of homology may be large enough to allow recombination, thus integrating an unrelated piece of DNA into the host chromosome. Receiving new genetic material may provide the host cell with a new trait that is desirable to changing environments. In organisms that make identical clones during reproduction, this strategy is critical to evolutionary survival.

c d

b

c

a

b a

d

Crossover Host chromosome

HOMOLOGOUS RECOMBINATION

Some incoming genes are incorporated

d

a

b

c DESTROY CELL AND PURIFY DNA

ADD DNA TO RECIPIENT CELL

RECOMBINATION

Chromosome

ORIGINAL BACTERIAL CELL

FRAGMENTS OF DNA

TRANSFORMED CELL

RECOMBINANT CELL

FIGURE 28.03 Gene Transfer by Transformation Under the right conditions, bacteria can take up pieces of naked DNA from the external environment. The fragment of DNA may pass through the outer cell layers without the aid of a protein or virus. Once inside the bacteria, the fragment of DNA must recombine with the chromosome to prevent degradation by exonucleases or restriction enzymes.

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Cells that have cell walls usually need some sort of treatment before they can take up DNA.

In practice, transformation is mostly a laboratory technique. The DNA is extracted from one organism by the experimenter and offered to other cells in culture. Cells able to take up DNA are said to be “competent.” Some species of bacteria readily take up external DNA without any pretreatment. Probably they use this ability to take up DNA under natural conditions. From time to time, bacteria in natural habitats die and disintegrate, which releases DNA that nearby cells may import. Other bacteria rarely undergo natural transformation and must first be treated to make them competent. Two different laboratory approaches are used to make bacteria competent for transformation. One method is to chill the bacterial cells in the presence of metal ions, especially high concentrations of Ca21, that damage their cell walls and then to heat shock them briefly. This loosens the structure of the cell walls and allows DNA to enter. Another method is electroshock treatment. Bacteria are placed in an “electroporator” and zapped with a high-voltage discharge that opens the cell wall and allows the DNA to get into the cell. Laboratory transformation techniques are an essential tool in genetic engineering. After genes or other useful segments of DNA have been cloned in the test tube, it is almost always necessary to put them into some bacterial cell for analysis or manipulation. Escherichia coli (E. coli) is normally treated by some variant of the Ca21/cold-shock treatment and does not require electroshock. Yeast cells may also be transformed. Since yeast has a very thick cell wall, electroshock is used. Conversely, animal cells, which lack cell walls, often take up DNA readily, and only require a mild chemical treatment.

3.1. Transformation As Proof That DNA Is the Genetic Material

The transfer of inherited characteristics due to the uptake of pure DNA was part of the original proof that DNA was the genetic information.

Transformation was first observed by Oswald Avery in 1944 and provided the earliest strong evidence that purified DNA carries genetic information and, therefore, that genes are made of DNA. Pneumococcus pneumoniae (now renamed Streptococcus pneumoniae) has two variants; one forms smooth colonies when grown on nutrient agar, the other has a rough appearance. The smooth variant has a capsule that surrounds the bacterial cell wall, whereas the bacteria in the rough colonies lack the capsule. The ability to make a capsule affects both colony shape and virulence as the capsule protects bacteria from the animal immune system. Thus, if smooth isolates of S. pneumoniae are injected into a live mouse, it dies of bacterial pneumonia. In contrast, rough strains are nonvirulent. Avery exploited this difference to prove that DNA from one strain could “transform” or change the other strain. Avery used DNA extracted from virulent strains of S. pneumonia. He purified the DNA and added it to harmless strains of the same bacterial species. Some of the harmless bacteria took up the DNA and were transformed into virulent strains. Hence, Avery named this process transformation (Fig. 28.04). (Strictly speaking, Avery’s transforming DNA could have interacted in some unknown way with the host chromosome to promote a genetic change. His experiment was therefore not absolute proof that DNA is the genetic material. Nonetheless, this is the most obvious interpretation and this observation convinced many scientists that genes were very likely made of DNA.) The use of viruses to transfer DNA into a bacterium provided more evidence that DNA was the genetic material that passed from one generation to the next. Special terminology is used when scientists use naked viral DNA during transformation. In a viral infection, the virus punctures a hole in the bacterial cell wall and injects DNA from the viral particle into the cytoplasm. The viral DNA induces the host to manufacture new viral particles. When viruses infect cells naturally, they often leave their protein coats behind and only the viral genome enters [see Chapter 24: Viruses, Viroids and Prions]. The term transfection (a hybrid of

competent cell Cell that is capable of taking up DNA from the surrounding medium. electroporator Device that uses a high-voltage discharge to make cells competent for taking up DNA. transfection Process in which purified viral DNA enters a cell by transformation. Often used to refer to entry of any DNA, even if not of viral origin, into an animal cell.

3. Transformation Is Gene Transfer by Naked DNA

SMOOTH COLONY VARIANT (has capsule)

Capsule layer

Gene for capsule

901

DEAD MOUSE

INJECT INTO MOUSE Cell wall KILL, EXTRACT DNA, AND PURIFY

Chromosome

ADD DNA TO ROUGH VARIANT

LIVE MOUSE ROUGH COLONY VARIANT (no capsule)

Cell wall INJECT INTO MOUSE Chromosome DEAD MOUSE Gene for capsule INJECT INTO MOUSE

FIGURE 28.04 Avery’s Experiment Avery isolated DNA from the virulent variant and added it to the rough variant of S. pneumonia. He noticed that the virulent DNA “transformed” or changed the rough variant into a smooth variant. To confirm that the bacteria were truly transformed, he exposed mice to the newly created smooth variants, and the mice died. Thus, the transformed bacteria had gained both the smooth appearance and virulence by taking up DNA from the original virulent strains.

transformation with infection) refers to the use of purified viral DNA in transformation. In this case, the experimenter purifies the viral genome from the virus particle and offers it to competent cells (Fig. 28.05). If taken up, purified viral DNA induces the cell to synthesize virus, illustrating that the virus coat is only necessary to protect the viral DNA outside the host cell and does not carry any of the virus genetic information. Transformation and transfection can also have two other meanings. Cancer specialists use the term “transformation” to refer to the changing of a normal cell into a cancer cell, even though in most cases no extra DNA enters the cell. (Note that alterations in the DNA are indeed involved in creating cancer cells, but as a result of mutation.) Supposedly to avoid ambiguity, researchers who use animal cells often use the term “transfection” to refer to the uptake of DNA (by transformation!) whether it is of viral origin or not.

3.2. Transformation in Nature More detailed investigation of S. pneumoniae and other gram-positive bacteria, including Bacillus, shows that they develop natural competence in dense cultures. competent cell Cell that is capable of taking up DNA from the surrounding medium. transformation (As used of cancer) Changing a normal cell into a cancer cell, even if no extra DNA enters the cell.

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FIGURE 28.05 Transfection During viral transfection, an experimenter first isolates pure viral DNA from virus particles. In this diagram, DNA is isolated from P1 virus. Next, the bacterial cell wall is made competent to take up naked DNA (usually by treating with calcium ions or by electroshock). The isolated DNA and the competent bacteria are mixed. If the bacteria take up the P1 DNA, the bacteria will start producing viral particles and burst to release the viral progeny. Thus, viral DNA alone can give the same end result as infection with whole virus particles.

DNA Protein coat

P1 virus particles PURIFY DNA

DNA

TRANSFECT ONLY DNA INTO CELL

Bacterial cell Chromosome

P1 MULTIPLIES

BURST

3. Transformation Is Gene Transfer by Naked DNA

903

FIGURE 28.06 Competence Pheromones

Cell wall Chromosome Precursor polypeptide

Shorter peptide or pheromone is cut out PHEROMONE IS SECRETED

Competence pheromone

Dense cultures of Streptococcus pneumoniae start producing competence pheromones that induce nearby cells to take up DNA. First, certain cells of the culture produce polypeptide precursors, which are digested into a small peptide, or competence pheromone. The small peptide is secreted from the producer cell and binds to a receptor on a nearby cell. The receptor then signals that cell to make proteins used in DNA uptake.

PHEROMONE BINDS TO RECEPTOR ON ANOTHER CELL

SIGNAL TO ACTIVATE GENES

Pheromone receptor ON

DNA UPTAKE GENES EXPRESSED

Endonuclease DNA receptor Exonuclease

Competence is induced by competence pheromones. (A pheromone is a hormone that travels between organisms, rather than circulating within the same organism.) Competence pheromones are short peptides that are secreted into the culture medium by dividing bacteria (Fig. 28.06). Only when the density of bacteria is high, pheromone Hormone or messenger molecule that travels between organisms, rather than circulating within the same organism.

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Transformation occurs among certain bacteria in the natural environment.

will the pheromones reach sufficient levels to trigger competence. This mechanism is presumably meant to ensure that any DNA taken up will come from related bacteria as competence is only induced when there are many nearby cells of the same species. Natural competence is not merely due to random entry of DNA, but involves the induction of a variety of genes whose products take part in DNA uptake. First, DNA is bound by cell-surface receptors (Fig. 28.07). Then the bound DNA is cut into shorter segments by endonucleases, and one of the strands is completely degraded by an exonuclease. Only the resulting short single-stranded segments of DNA enter the cell. Part of the incoming DNA may then displace the corresponding region of the host chromosome by recombination. Note that in the case of artificially induced competence, the mechanism is quite different. Double-stranded DNA enters the cell through a cell wall that is seriously damaged. Indeed, many, perhaps the majority, of the cells that are made artificially competent are killed by the treatment. It is the few survivors who take up the DNA. Before succumbing to the myth that natural gene transfer is peaceful and tranquil compared to brutal laboratory methods, some recent findings deserve attention. Firstly, in S. pneumoniae competent cells practice “fratricide.” They actively kill and lyse both noncompetent cells of their own species and those of closely related species. This gives them access to their victims DNA, which they then take up. Even more aggressive is the gram-negative Vibrio cholerae, the causative agent of cholera (see Box 28.01).

4. Gene Transfer by Virus—Transduction

Transduction is when viruses pick up fragments of current host DNA and carry them to another host cell.

When a virus succeeds in infecting a bacterial cell, it manufactures more virus particles, each of which should contain a new copy of the virus genome. Occasionally, viruses make mistakes in packaging DNA, and fragments of bacterial DNA get packaged into the virus particle. From the viewpoint of the virus, this results in a defective particle. Nonetheless, such a virus particle, carrying bacterial DNA, may infect another bacterial cell. If so, instead of injecting viral genes, it injects DNA from the previous bacterial victim. This mode of gene transfer is known as transduction. Bacterial geneticists routinely carry out gene transfer between different but related strains of bacteria by transduction using bacterial viruses, or bacteriophages (phages for short). If the bacterial strains are closely related the incoming DNA is accepted as “friendly” and is not destroyed by restriction. In practice, transduction is the simplest way to replace a few genes of one bacterial strain with those of a close relative. To perform transduction, a bacteriophage is grown on a culture of the donor bacterial strain. These bacteria are destroyed by the phage, leaving behind only DNA fragments that carry some of their genes and that are packaged inside phage particles. If required, this phage sample can be stored in the fridge for weeks or months before use. Later, the phage is mixed with a recipient bacterial strain and the viruses infect the bacteria, injecting their DNA. Most recipients get genuine phage DNA and are killed. However, a few get donor bacterial DNA and are successfully transduced (Fig. 28.09).

4.1. Generalized Transduction There are two distinct types of transduction. In generalized transduction, fragments of bacterial DNA are packaged more or less at random in the phage particles. This is the case for bacteriophage P1 as described earlier (Fig. 28.09). Consequently, all genes have roughly the same chance of being transferred. In specialized transduction, certain regions of the bacterial DNA are carried preferentially—discussed later. generalized transduction Type of transduction where fragments of bacterial DNA are packaged at random and all genes have roughly the same chance of being transferred. specialized transduction Type of transduction where certain regions of the bacterial DNA are carried preferentially.

4. Gene Transfer by Virus—Transduction

Long piece of double-stranded DNA

Endonuclease Receptor Exonuclease

DNA BOUND

DNA is cut by endonuclease

ADDITIONAL DNA CUTS MAKE SMALL FRAGMENTS

EXONUCLEASE DEGRADES ONE STRAND OF HELIX AND OTHER STRAND ENTERS

SHORT SINGLE-STRANDED DNA IS TAKEN INTO CELL

905

FIGURE 28.07 Mechanism of Natural Competence A cell that is naturally competent takes DNA into its cytoplasm by a protein-mediated process. First, the long molecule of double-stranded DNA is recognized by a receptor on the surface of the competent cell. A cell-surface endonuclease digests the DNA into small fragments. An exonuclease then degrades one strand of the DNA. The remaining single-stranded fragment is taken into the cytoplasm of the bacterium.

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Box 28.01 Vibrio Stabs Other Cells to Death to Steal Their DNA V. cholerae is notorious as the causative agent of cholera. Every so often new strains of cholera with altered characteristics emerge and spread causing new epidemics. These are the result of horizontal gene transfer (HGT) between different Vibrio strains in their natural habitat—coastal waters and estuaries. Some HGT is due to the integration of bacteriophages into the host cell genome. Indeed, the genes for choleratoxin are carried on the CTX phage that integrates into the bacterial chromosome. Other HGT events are due to transformation. These include surface alterations that protect from the human immune system, such as converting V. cholerae El Tor to V. cholerae Bengal. This new strain began a worldwide epidemic starting in 1992 in India. The mechanism of transformation in Vibrio is extremely aggressive and involves assassination of the cell that is the source of the DNA. The victim is stabbed by a structure known as a type VI secretion system (T6SS). This kills the target cell and results in the release of its DNA. The DNA is then imported into the attacking cell by a type IV pilus together with other components (Fig. 28.08). DNA-uptake machinery chitin PilA pilABCD

pilMNOPQ

TfoX CRP cAMP

qstR

comEA

comEC

ComEA

External DNA

ComEC

HapR

QstR protein VipA/B

High cell density

T6SS genes

IM

OM

Type VI secretion system

FIGURE 28.08 Vibrio Natural DNA Uptake Natural uptake of DNA in Vibrio depends on the possession of a type VI secretion system (T6SS, purple) as well as dedicated DNA uptake machinery consisting of a type IV pilus (blue) plus two competence proteins (green). Genes encoding these components are shown as wide, color-coded arrows. High cell density plus the presence of chitin induce these structures via the TfoX and QstR regulators. Activation of the Tfox protein also needs cyclic AMP to bind via CRP protein (see Chapter16: Regulation of Transcription in Prokaryotes, Section 16.6 for details of the cyclic AMP system). Solid black arrows indicate positive regulation. Dashed arrow indicates expression of QstR protein. Credit: Fig. 1 in Metzger, L.C., Blokesch, M., 2016. Regulation of competence-mediated horizontal gene transfer in the natural habitat of Vibrio cholerae. Curr. Opin. Microbiol. 30, 17.

Bacteria possess a variety of secretion systems that can both export and/or import proteins and/or nucleic acids. The T6SS is structurally related to the contractile tails of certain bacteriophage and operates in a similar manner. The T6SS has a contractile sheath that pushes the spike into the victim upon contraction. Toxic proteins then enter and kill the victim. The structures that take part in this process are all induced in response to chitin. Remember that chitin is the polymer found in the hard coats of insects and crustaceans. Because of this, chitin is the most common polymer in aquatic habitats. Vibrio often associates with the surfaces of marine crustaceans and can digest chitin using secreted enzymes. As might be expected, it is actually the soluble fragments of chitin breakdown that are responsible for inducing transformation, not the original insoluble polymer. As Fig. 28.08 indicates, the induction of the DNA uptake machinery also requires high cell density, as monitored by the HapR regulator. This ensures that plentiful DNA donor cells are nearby before major resources are invested.

For a bacterial virus to transduce, several conditions must be met. In particular, the phage must not degrade the bacterial DNA. For example, phage T4 normally destroys the DNA of E. coli after infection. However, mutants of T4 that have lost the ability to degrade host DNA work well as transducing phages. The packaging

4. Gene Transfer by Virus—Transduction

PHAGE INFECTS DONOR CELL

AN OCCASIONAL PHAGE PACKAGES BACTERIAL DNA

PHAGE WITH BACTERIAL DNA INFECTS RECIPIENT CELL

907

DONOR DNA ENTERS RECIPIENT CELL

FIGURE 28.09 Principle of Transduction Occasionally, when a phage infects a bacterium, one of the virus coats will be packaged with host bacterial DNA (pink). The defective phage particle infects a nearby cell where it injects the bacterial DNA. This cell will survive since it is not injected with viral DNA. The incoming DNA may recombine with the host chromosome, thus this cell may gain new genetic information.

mechanism is also critical. Some phages, such as lambda, use specific recognition sequences when packaging their DNA into the virus particle and so will not package random fragments of DNA (see Section 28.4.2). In other cases, packaging depends on the amount of DNA the head of the virus particle can hold. Such “headful packaging” is essential for generalized transduction. Two examples of generalized transducing phages are P1, which works on E. coli, and P22, which infects Salmonella. The ratio of transducing particles to live virus is about 1:100 in both cases; that is, for every 100 virus particles made, one will contain bacterial host DNA. The likelihood of the transduced DNA recombining into the recipient chromosome is roughly 12 in 100. P1 can package approximately 2% of the E. coli chromosome (about 90 kb of DNA), whereas P22 is smaller and can carry only 1% of the Salmonella chromosome. Taken all together, about 1 in 500,000 P1 particles will successfully transduce any particular gene on the E. coli chromosome. This may seem a low probability, but as both typical bacterial cultures and preparations of P1 contain about 109 per mL, transduction happens at useful frequencies in practice. P1 can also transduce DNA from E. coli into certain other gram-negative bacteria, such as Klebsiella.

Some viruses can carry fragments of host DNA. In generalized transduction, random pieces of the host DNA are packaged and injected into a different host.

4.2. Specialized Transduction During specialized transduction, certain specific regions of the bacterial chromosome are favored. This is due to integration of the bacteriophage into the host chromosome (see Chapter 24: Viruses, Viroids, and Prions). If the virus enters its lytic cycle and manufactures virus particles, those bacterial genes nearest the virus integration site are most likely to be incorrectly packaged into the viral particles. As discussed in Chapter 24, Viruses, Viroids, and Prions, when bacteriophage lambda (or λ) infects E. coli, it sometimes inserts its DNA into the bacterial chromosome (Fig. 28.10). This occurs at a single specific location, known as the lambda attachment site (attλ), which headful packaging Type of virus packaging mechanism that depends on the amount of DNA the head of the virus particle can hold (as opposed to using specific recognition sequences). lambda (or λ) Specialized transducing phage of E. coli that may insert its DNA into the bacterial chromosome. lambda attachment site (attλ) Site where lambda inserts its DNA into the bacterial chromosome. P1 A generalized transducing phage of E. coli. P22 A generalized transducing phage of Salmonella.

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908

bio

e

BACTERIAL CHROMOSOME

ge ne

a tt λ

lg ga

en

+ Lambda DNA

ga l

e en

ge ne

g bio

at t

att

R

L

Lambda DNA

FIGURE 28.10 Integration of Lambda into the E. coli Chromosome When bacteriophage lambda infects a host E. coli cell, it can integrate its phage DNA into the chromosome. The phage DNA will only integrate at a site called attλ, which is found between the bio gene and gal gene of the chromosome. Once integrated, the phage is referred to as a prophage.

Specialized transduction occurs in viruses that integrate into host DNA at specific sites in the host chromosome. Only adjacent genes to the integration site are transduced.

lies between the gal and bio genes. The integrated virus DNA is referred to as a prophage. When lambda is induced, it excises its DNA from the chromosome and goes into lytic mode. The original donor cell is destroyed and several hundred virus particles containing lambda DNA are produced. Just like generalized transducing phages, a small fraction of lambda virus particles contain bacterial DNA. There are, however, two major differences. First, only chromosomal genes next to the lambda attachment site are transduced. Second, the specialized transducing particles contain a hybrid-DNA molecule comprising both lambda and chromosomal DNA (Fig. 28.11). This hybrid molecule results from mistakes during excision of the lambda prophage. Chromosomal DNA to the right or to the left of the prophage, but not both, may be included in the transducing phage. In practice, this means that either the gal or bio genes are picked up. Mistakes in excision of lambda only occur at a rate of 1 in a million relative to correct excision. Furthermore, the defective excision must generate a segment of DNA approximately the same length as the lambda genome in order for it to fit into the phage head. Consequently, specialized transducing particles arise only at extremely low frequency. However, once a lambda-transducing phage has been created, it may reintegrate its DNA into the chromosome of another host cell. This may occur either in the attλ site or into the chromosomal copy of the gene (usually gal or bio) carried by the lambda-transducing phage. Inducing this defective prophage DNA will give a second generation of transducing phage particles at a much higher frequency. The properties of lambda-transducing phages depend on which lambda genes were lost in exchange for chromosomal DNA. The λdgal-transducing phages lack lambda genes needed for making head and tail components and instead, the virus contains the E. coli gal gene. These are therefore “defective” (hence, the “d” in λdgal). Defective phage may be grown together with a wild-type lambda as a helper phage, which provides the missing functions. In the case of λdgal, helper phage would make the head and tail components. Conversely, the λpbio-transducing phages lack the lambda int gene, which integrates the phage DNA into the attλ site, and instead contains the bio gene from E. coli. Since the phage cannot integrate, λpbio must enter the lytic phase and are thus obligate plaque formers (hence, the “p” in λpbio). If wild-type helper phage is added, the int function is restored, and the phage forms lysogens. Cloning vectors derived from lambda are widely used in genetic engineering (see Chapter 7: Cloning Genes for Synthetic Biology). Since the cloned DNA replaces many of the lambda genes, such vectors need to be grown in the presence of helper phages.

5. Transfer of Plasmids Between Bacteria The transfer of genetic information between two bacterial cells may occur via cellto-cell contact. This process is known as bacterial conjugation and typically depends on the presence of plasmids. Transferability is the ability of certain plasmids to move from one bacterial cell to another. Many medium-sized plasmids, such as the F-type and P-type plasmids, are able to move and are referred to as Tra1 (transferpositive) or self-transferable. For transfer to occur, the bacterial cell containing the plasmid must make physical contact with a suitable recipient cell. During bacterial conjugation DNA moves in one direction only, from the plasmid-carrying donor to

defective phage Mutant phage that lacks genes for making virus particles. helper phage Phage that provides the necessary genes so allowing a defective phage to make virus particles. prophage Virus DNA that is integrated into the host chromosome. Tra1 Transfer-positive (refers to a plasmid capable of self-transfer). transferability Ability of certain plasmids to move themselves from one bacterial cell to another.

5. Transfer of Plasmids Between Bacteria

gal gene

att L

Lambda DNA

ga l

ge ne

Lambda DNA

L att

INFREQUENT PACKAGING

909

FREQUENT PACKAGING

Lambda DNA

at tR

BACTERIAL CHROMOSOME

g bio

e en

INFREQUENT PACKAGING

Lambda DNA

att R

bio gene

FIGURE 28.11 Packaging of Host DNA During Transduction by Lambda When lambda phage enters its lytic cycle and makes phage particles, it usually packages the lambda DNA between the attL and attR sites. Occasionally, a mistake will occur, and part of the bacterial chromosome DNA will be packaged. Since lambda DNA normally integrates between the gal and bio genes of the E. coli chromosome, the defective lambda particles will most likely contain one or other of these genes.

the recipient (Fig. 28.12). The donor cell manufactures a sex pilus that binds to the recipient and draws the two cells together. Next, a conjugation bridge forms between the two cells and provides a channel for DNA to move from donor to recipient. In real life, mating bacteria tend to cluster together in groups of 510 (Fig. 28.13). The genes for formation of the sex pilus and conjugation bridge and for overseeing the DNA transfer process are known as tra genes and are all found on the plasmid itself. Since plasmid transfer requires over 30 genes, only medium or large plasmids possess this ability. Very small plasmids, such as the ColE plasmids, do not have enough DNA to accommodate the genes needed. The most famous self-transferable plasmid is the F-plasmid of E. coli, which is approximately 100 kbp long. Donor cells are sometimes known as F1 or “male” and recipient cells as F2 or “female” and conjugation is sometimes referred to as bacterial mating. Note, however, that the “sex” of a bacterial cell is determined by the presence or absence of a plasmid and that DNA transfer is unidirectional, from donor to recipient. When a recipient cell has received the F-plasmid, it becomes F1. From a human perspective it has been transmuted from “female” into a “male”! Thus, bacterial mating is not at all equivalent to sexual reproduction among higher organisms. Although we talk about “plasmid transfer,” in reality both the donor cell and the recipient cell end up with a copy of the plasmid. Thus the transfer mechanism includes the synthesis of a second copy of the plasmid. Plasmid DNA transfer involves replication by the rolling circle mechanism (Fig. 28.14). First, one of the two strands of the double-stranded DNA of the plasmid opens up at the origin of transfer. This linearized single strand of DNA moves through the conjugation bridge from the donor into the recipient cell. An unbroken single-stranded circle of plasmid DNA remains inside the donor cell. This is used as a template for the synthesis of a new second strand to replace the one that just left. As the linear single strand of plasmid DNA enters the female cell, a new complementary strand of DNA is made

Transferable plasmids move from one cell to another via the conjugation bridge.

A single strand of newly made DNA is transferred from the donor to the recipient cell during conjugation.

conjugation bridge Junction that forms between two cells and provides a channel for DNA to move from donor to recipient during conjugation. F-plasmid Fertility plasmid that allows E. coli to donate DNA by conjugation. sex pilus Protein filament made by donor bacteria that binds to a suitable recipient and draws the two cells together.

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FIGURE 28.12 Bacterial Conjugation

FORMATION OF MATING PAIRS Basal structure Chromosome

1

Certain plasmids, called Tra or transfer-positive, are able to move a copy of their DNA into a different cell through a mechanism called bacterial conjugation. First, the cell containing a Tra1 plasmid manufactures a rod-like extension on the surface of the outer membrane called a sex pilus. The sex pilus binds to a nearby cell and pulls the two cells together by retracting. Once the cells are in contact, the basal structure of the pilus makes a connection between the two cells known as the conjugation bridge. This connects the cytoplasm of the two cells, so the plasmid can transfer a copy of itself to the recipient cell.

Sex pilus Transferable plasmid Chromosome

DONOR CELL

RECIPIENT CELL

A

FORMATION OF A CONJUGATION BRIDGE DONOR CELL

Conjugation bridge Plasmid transfers as a single strand of DNA RECIPIENT CELL

B

using the incoming strand as template. Thus, only one strand of plasmid DNA is transferred from the donor to the recipient. The detailed physical mechanism of DNA transfer via the conjugation bridge was only solved relatively recently. The earliest proposals were that DNA traveled

FIGURE 28.13 Conjugating Cells of E. coli False-color transmission electron micrograph (TEM) of a male E. coli bacterium (bottom-right) conjugating with two females. This male has attached two F-pili to each of the females. The tiny bodies covering the F-pili are bacteriophage MS2, a virus that attacks only male bacteria and binds specifically to F-pili. Magnification: 3 11,250. Credit: Dr. L. Caro, Photo Researchers, Inc.

FIGURE 28.14 Plasmid Transfer Involving Rolling Circle Replication

REPLICATION Origin of transfer

Single-strand nick is made

Single-strand enters recipient cell 5'-end

F-plasmid

A

F-plasmid

Synthesis of new DNA complementary to unbroken strand

Double-stranded DNA

TRANSFER F- plasmid

DONOR CELL

Complementary strand synthesized in donor

Chromosome

5' 3' 5'

Complementary strand synthesized in recipient

B

RECIPIENT CELL

(A) During bacterial conjugation, the F-plasmid of E. coli is transferred to a new cell by rolling circle replication. First, one strand of the F-plasmid is nicked at the origin of transfer. The two strands start to separate and synthesis of a new strand starts at the origin (green strand). (B) The singlestrand of F-plasmid DNA that is displaced (pink strand) crosses the conjugation bridge and enters the recipient cell. The second strand of the F-plasmid is synthesized inside the recipient cell. Once the complete plasmid has been transferred, it is re-ligated to form a circle once again.

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FIGURE 28.15 Basal Structure of the Sex Pilus The basal structure of the sex pilus resembles a type IV secretion system. It crosses both the inner and outer membranes and its central channel is large enough for the transit of proteins or DNA. The details of individual components vary somewhat between organisms, depending on the specific role of the system. This diagram is a simplified version showing the common core structures.

Shaft of pilus

Outer layer

Outer membrane

Peptidoglycan Inner membrane

Coupling protein uses ATP

Plasmids unable to transfer themselves may be able to hitchhike using the transfer systems of other plasmids.

Inner layer

through the central channel of the sex pilus itself. Although this is incorrect, the DNA does in fact travel through the central channel of the basal structure on which the pilus is built. When the sex pilus is assembled, its protein subunits travel through the channel in the basal structure (also known as the transfer apparatus). After donor and recipient have made contact, the pilus is retracted and the pilus subunits return through the same channel. This brings the two cells into close contact and leaves the basal structure bridging the inner and outer membranes of the donor and in contact with the recipient. DNA then moves through the channel of the basal structure into the recipient. The basal structure belongs to the family of type IV secretion systems. These are used by a variety of bacteria for protein secretion as well as DNA uptake and DNA transfer (Fig. 28.15). Although small plasmids such as ColE are not self-transferable, they are often mobilizable (Mob1). A transferable plasmid, such as the F-plasmid, can mobilize the ColE plasmid if they both inhabit the same cell. The F-plasmid oversees conjugation and forms the conjugation bridge and the ColE plasmid is transferred through this. The mob (mobilization) genes of the ColE plasmid are responsible for making a single-stranded nick at the origin of transfer of ColE and for unwinding the strand to be transferred. Other small plasmids are neither self-transferable nor mobilizable.

5.1. Transfer of Chromosomal Genes Requires Plasmid Integration

Transferable plasmids sometimes move chromosomal DNA from one cell to another.

Although many plasmids allow the cells carrying them to conjugate, usually only the plasmid itself is transferred through the conjugation bridge. But occasionally, plasmids mediate transfer of the host chromosome when they move from one bacterial cell to another. Plasmids, such as the F-plasmid of E. coli, that enable a cell to donate host chromosomal DNA are called fertility plasmids. In order to transfer chromosomal genes, a plasmid must first physically integrate itself into the chromosome of the bacterium. This event requires pairs of identical (or nearly identical) DNA sequences, one on the plasmid and the other on the chromosome. For example, the F-plasmid uses insertion sequences (see Chapter 25: Mobile DNA) for integration into the chromosome of E. coli (Fig. 28.16). A variety of different insertion sequences are found on the chromosome of E. coli and in its plasmids and viruses. The F-plasmid has three insertion sequences (Fig. 28.17): Two copies of IS3 and a single copy of IS2. The chromosome of a typical laboratory strain of E. coli has 13 copies of IS2 and 6 copies of IS3 scattered

fertility plasmid Plasmid that enables a cell to donate DNA by conjugation. insertion sequence A simple transposon consisting only of inverted repeats surrounding a gene that encodes transposase.

5. Transfer of Plasmids Between Bacteria

913

IS IS IS

d s D NA

IS

Bacterial chromosome

F-plasmid ds DNA

F-plasmid DNA

FIGURE 28.16 Integration of F-Plasmid Into Chromosome If recombination occurs between two insertion sequences, one on the F-plasmid and one on the host bacterial chromosome, the entire F-plasmid becomes integrated into the chromosome.

IS 3 es

IS3

Tra ns fe r

n ge

FIGURE 28.17 Insertion Sequences on F-Plasmid and Chromosome

Tn1 00 0

IS2

F-plasmid 100 kb

Re

ori T Origin of transfer pli

cat io

n gen

es

ori V

Origin of vegetative replication

around more or less at random. Integration of the F-plasmid may occur in either orientation at any of these 19 sites. When an F-plasmid that is integrated into the chromosome transfers itself by conjugation, it drags along the chromosomal genes to which it is attached (Fig. 28.18). Just as for the unintegrated F-plasmid, only a single strand of the DNA moves and the recipient cell has to make the complementary strand itself. Bacteria with an F-plasmid integrated into the chromosome are known as Hfr strains because they transfer chromosomal genes at high frequency. A prolonged mating of 90 minutes or so is needed to transfer the whole chromosome of E. coli. More often, bacteria break off after a shorter period of, say, 1530 minutes, and only part of the chromosome is transferred. Since different Hfr strains have their F-plasmids inserted at different sites on the bacterial chromosome, transfer of chromosomal genes begins at different points. In addition, the F-plasmid may be inserted in either orientation. Consequently, chromosomal gene transfer may be either clockwise or counterclockwise for any particular Hfr strain.

Insertion sequences are scattered throughout the F-plasmid and chromosome of E. coli. The Fplasmid has two IS3 elements and one IS2 element. Even more copies of IS2 and IS3 are found on the chromosome (not shown). A recombination event between any of the chromosomal IS2 or IS3 elements and the corresponding element on the F-plasmid will integrate the entire F-plasmid into the chromosome. Tn1000 (also known as γδ) is another insertion sequence, although not generally involved in F-plasmid integration in E. coli.

In order to mobilize chromosomal DNA, the plasmid must first integrate into the chromosome.

Hfr-strain Bacterial strain that transfers chromosomal genes at high frequency due to an integrated fertility plasmid (F-plasmid).

CHAPTER TWENTY EIGHT
Bacterial Genetics

Chromos

om al D

N

a

A

ori T origin of transfer

INTEGRATED F-PLASMID Single-stranded nick is made at ori T

SINGLE STRAND IS UNROLLED AND ENTERS RECIPIENT

N

f e d c b a

Front of F-plasmid

A

om al D

An integrated F-plasmid can still induce bacterial conjugation and rolling circle transfer of DNA into another bacterial cell. Since rolling circle replication does not stop until the entire circle is replicated, the attached chromosome is also transferred into the recipient cell. First, a single-stranded nick is made at the oriT, or transfer origin of the integrated plasmid. The free 50 end (black triangle) enters the recipient cell through the conjugation bridge. Notice that the transfer of the single-stranded DNA does not end with the Fplasmid DNA and continues into the chromosomal DNA. Genes closest to the site of plasmid integration are transferred first (in the order a, b, c, d, e, f, in this example). The amount of chromosomal DNA that is transferred depends on how long the two bacteria remain attached by the conjugation bridge.

f e d c b

id sm pla F-

FIGURE 28.18 Transfer of Chromosomal Genes by F-Plasmid

Chromos

914

ori T enters recipient first

Rear of F-plasmid

Hfr strains were used in earlier times to identify the order of genes on the E. coli chromosome. To monitor whether the recipient has received a particular gene, the donor and recipient strains must have different alleles of this gene that can be distinguished phenotypically, usually by their growth properties. For example, the recipient might have a mutation in the lac operon that prevents growth on lactose as carbon source. The donor Hfr strain would have an allele that restores the ability to use lactose. Using this method, genetic maps were constructed by two major approaches. First, the cotransfer frequency of two genes was measured. If two genes were close to each other, a donor Hfr strain would transfer them together at high frequency. Conversely, if two genes were far apart on the chromosome, an Hfr strain would usually only transfer one of them, and the cotransfer frequency would be low. Secondly, time-of-entry measurements were made to determine gene order. Hfr strains transfer chromosomal genes starting where the F-plasmid is integrated and proceeding sequentially around the circular chromosome (Fig. 28.19). The length of time it takes for a gene to enter the recipient gives an estimate of its relative distance from the origin of transfer of the Hfr strain. For time of entry mapping the site and orientation of the F-plasmid must be known. In addition, mutations in the genes being studied (a, b, c, and d) must give recognizable phenotypes. Finally, the recipient must be resistant to some antibiotic (e.g., streptomycin) so that it can be selected on medium that prevents growth of the Hfr strain. Different Hfr strains will transfer the same genes in different orders and at different times, depending on their location relative to the integration site of the F-plasmid. cotransfer frequency

Frequency with which two genes remain associated during transfer of DNA between bacterial cells.

6. Gene Transfer Among Gram-Positive Bacteria

Hfr 1

Hfr 2

0/100

0/100

a

FIGURE 28.19 Time of Entry by Conjugation

F a

b

915

b

c

c

75

25

d

d

Bacterial chromosome

Bacterial chromosome

75

25

F

ge n ed

c

b gen e

ea

0

ge ne

ge n ea

5 10 15 20 Time (minutes)

ge n

c

gen eb

ge ne

ge n

0

Number of recipients receiving gene

50

ed

Number of recipients receiving gene

50

5 10 15 20 Time (minutes)

F-plasmids can excise themselves from the chromosome by reversing the integration process. Sometimes they excise carrying pieces of chromosomal DNA, which creates F0 - or F-prime plasmids. This typically occurs by recombination between a different pair of IS sequences than used during integration. Such F0 -plasmids may be transferred to F-minus recipients, carrying with them the chromosomal segment from their previous host. If the chromosomal segment is homologous, the F0 can reintegrate via homologous recombination. Historically, F-primes were used to carry part of the lacZ gene in the alpha-complementation method for screening recombinant plasmids (see Chapter 7: Cloning Genes for Synthetic Biology).

To determine the time of entry by conjugation, the Hfr strain is mixed with a recipient strain carrying a defective copy of a particular gene, “a.” After conjugation has proceeded for a specific time, a sample of the mixture is removed. This is plated on agar, which prevents growth of the Hfr and only allows growth of strains carrying the wild-type version of gene “a.” Survivors are derivatives of the recipient that have gained the wild-type version of gene “a” from the Hfr. This is repeated for several time points. The whole procedure is then repeated for the other genes. In strain Hfr 1 (left panel), the integrated F-plasmid is closest to gene “d” and only begins transferring gene “a” after about 20 min. In strain Hfr 2 (right panel), the F-plasmid is integrated closer to gene “a,” which therefore begins to appear in the recipient as early as 5 min after transfer begins.

6. Gene Transfer Among Gram-Positive Bacteria Traditionally, the bacteria are divided into two major groups: The gram-negative and the gram-positive bacteria. This division was originally based on their response to the gram stain. The differences in staining reflect differences in the chemical composition and structure of the cell envelope. The envelope of gram-negative bacteria consists of the following layers (from inside to outside): Cytoplasmic membrane, cell wall (peptidoglycan), and outer membrane (Fig. 28.20). The envelope of grampositive bacteria is simpler and lacks the outer membrane. Both kinds of bacteria sometimes have an extra protective layer, the capsule, on the very outside. The gram-negative bacterium E. coli is widely used as a host for cloning and expressing genes from a variety of other organisms. The synthesis of large amounts of a purified recombinant protein from a cloned gene is often desirable. Secretion of recombinant protein into the culture medium would be very convenient as this avoids the need to purify it away from all the other proteins inside the bacterial cell. However, the complex envelope of gram-negative bacteria is a major hindrance in

Gram-negative bacteria, including E. coli, have an extra outer membrane.

alpha-complementation Assembly of functional beta-galactosidase from N-terminal alpha fragment plus the remaining part of the protein. F0 or F-prime plasmid F-plasmid of E. coli that has excised itself from the host chromosome and contains segments of the host DNA in addition to the regular plasmid DNA. gram-negative bacteria Major division of bacteria that possess an extra outer membrane lying outside the cell wall. gram-positive bacteria Major division of bacteria that lack an extra outer membrane lying outside the cell wall. outer membrane Extra membrane lying outside the cell wall in gram-negative but not gram-positive bacteria.

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FIGURE 28.20 Differences in Envelopes of Gram-Negative and Gram-Positive Bacteria The outer surfaces of gram-positive and gram-negative bacteria have different structures. (A) In gramnegative bacteria, such as E. coli, there are three surface layers. The outermost layer, called the outer membrane, is a lipid bilayer that contains various proteins embedded within the lipids, and an outer coating of lipopolysaccharide. Next, within the periplasmic space, the cell wall contains a single layer of peptidoglycan. Lipoproteins connect this cell wall to the outer membrane. The layer closest to the cytoplasm, called the inner membrane, is a lipid bilayer embedded with various proteins. (B) The outer surface of grampositive bacteria only has two layers, a thick wall of peptidoglycan plus teichoic acid surrounding the cell membrane.

GRAM-NEGATIVE ENVELOPE

Lipopolysaccharide

Outer membrane

Lipoprotein

Peptidoglycan (single layer wall)

Inner membrane

Protein

A

Phospholipids

GRAM-POSITIVE ENVELOPE

Several layers of peptidoglycan plus teichoic acid (red)

Inner membrane

Protein

B

Transfer of plasmids between gram-positive bacteria is often promoted by pheromones.

Periplasmic space

Phospholipids

the export of proteins into the culture medium. In contrast, secretion across the simpler gram-positive envelope is easier. Indeed, many gram-positive bacteria, such as Bacillus, excrete proteins into the culture medium naturally. As a result, there is considerable interest in using gram-positive bacteria as hosts in genetic engineering. Unfortunately, the genetics of gram-positive bacteria is far behind that of the intensively studied E. coli and its relatives. Nonetheless, mechanisms of gene transfer are available in gram-positive bacteria. Self-transmissible plasmids are widespread among gram-positive bacteria and many of these plasmids are rather promiscuous. Since the cell envelope is simpler in gram-positive bacteria, plasmid transfer is also simpler and a sex pilus is not needed. Apparently, barely a dozen genes are required to encode the transfer functions. Some gram-positive bacteria, such as Enterococcus, secrete mating pheromones into the culture medium. These are short peptides (7 or 8 amino acids long) that induce the tra genes of transferable plasmids by binding to a pheromone receptor protein. Only bacteria that lack a particular plasmid, secrete the corresponding pheromone, consequently the plasmid only expresses its transfer genes when a suitable recipient is nearby. Different pheromones are specific for different plasmids. In Enterococcus, two different peptide signal molecules, the pheromone and an inhibitor peptide, compete for binding to the same site on the PrgX receptor protein. Cells lacking the plasmid secrete the pheromone and plasmid-bearing cells

7. Archaeal Genetics

Mating pheromone Pheromone binds to receptor Tra+ plasmid Chromosome

Gram + cell without plasmid

Gram + cell with transferable plasmid

MATING PAIR FORMS; PLASMID IS TRANSFERRED

917

FIGURE 28.21 Pheromones Induce Mating in Gram-Positive Bacteria In gram-positive bacteria such as Enterococcus, cells without plasmids secrete pheromones to attract bacteria with transferable (Tra1) plasmids. Mating pheromones bind to receptors on the surface of cells containing Tra1 plasmids. Binding the receptor activates the transfer genes to form a conjugation bridge and transfer the plasmid by rolling circle replication. Each pheromone is specific and only attracts bacteria with certain plasmids.

5'

secrete the inhibitor. When the inhibitor peptide binds to PrgX, it acts as a repressor and prevents transcription from the promoter of prgQ. Conversely, when the pheromone binds to PrgX, it dissociates from the promoter and the prgQ gene is derepressed and activates the plasmid-transfer system. This occurs when a plasmid free recipient cell comes close to a cell carrying a plasmid. The final result is cell aggregation and plasmid transfer (Fig. 28.21). Gram-positive bacteria also harbor conjugative transposons (e.g., Tn916 of Enterococcus). These can transfer themselves from one bacterial cell to another (see Chapter 25: Mobile DNA). These elements excise themselves temporarily from the chromosome of the donor cell before conjugation. Once inside the recipient, they reinsert themselves into the bacterial chromosome.

7. Archaeal Genetics There are two genetically distinct lineages of prokaryotes, the “normal” bacteria (previously Eubacteria) and the Archaea (previously Archaebacteria). Although both are prokaryotic cells without a nucleus, the bacteria and Archaea are not related to each other genetically. Together with the eukaryotes (strictly, the nuclear genome of eukaryotes) they comprise the three domains of life. (See Chapter 29: Molecular Evolution, for further discussion of these relationships.) The bacteria include most typical bacteria found in normal environments, including both the gram-negative and gram-positive bacteria discussed earlier. The Archaea include the methane producing “bacteria,” or methanogens, and a variety of less well-known prokaryotes found in extreme environments. Many have strange biochemical pathways and are adapted to extremes of temperature, pH, or salinity. This makes the Archaea an attractive source of novel enzymes or proteins with unusual properties

Archaea are genetically distinct from Bacteria and often live under unusual or extreme conditions.

Archaea (or Archaebacteria) Type of prokaryote forming a genetically distinct domain of life. Includes many cells that grow under extreme conditions. conjugative transposon Transposon that can transfer itself from one bacterial cell to another by conjugation. Eubacteria Officially Bacteria—the typical bacteria that have peptidoglycan in their cell walls and form a separate domain from the Archaea.

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Gene transfer in Archaea is widespread but still poorly understood.

and/or resistance to extreme conditions. There are many possible industrial uses for enzymes capable of withstanding extreme temperatures, for example. Although many complete genome sequences are available for members of the Archaea, development of systems for gene transfer has lagged way behind the bacteria. There are many practical problems, including the need to grow many Archaea under extreme conditions. For example, most extreme thermophiles grow at temperatures high enough to melt agar. Obtaining colonies on solid media has required the development of alternative materials. Another major problem is choice of a selectable marker. Most standard antibiotics do not affect Archaea due to their unusual biochemistry. For example, Archaea do not have cell walls made of peptidoglycan and are therefore not susceptible to penicillin derivatives. In addition, many resistance proteins from normal organisms are denatured at the extremes of temperature, salinity, or pH under which many Archaea grow. Novobiocin (a DNA gyrase inhibitor—see Chapter 10: Cell Division and DNA Replication) and mevinolin (an inhibitor of the isoprenoid pathway) have been used to inhibit halophiles, and puromycin and neomycin (both protein synthesis inhibitors—see Chapter 13: Protein Synthesis) will inhibit methanogens. It has been possible to express the lacZ reporter gene in methanogens under control of an archaeal promoter. However, staining of β-galactosidase with Xgal requires exposure to air, which kills methanogens! Consequently, colonies must first be replicated and one set sacrificed for analysis. Transformation procedures now exist for getting DNA into several Archaea. Some rely on removal of divalent cations, especially Mg21, which results in the disassembly of the glycoprotein layer surrounding many archaeal cells. (Note the contrast with the corresponding procedures for typical bacteria, which involve coldshock in the presence of excess divalent cations!) However, those Archaea that can survive the removal of high salt or being exposed to oxygen can often be transformed by electroporation. Viruses have been discovered that infect many Archaea. So far, only one, the ΨM1 phage of Methanobacterium thermoautotrophicum, has been shown to transduce the genes of its host bacterium. Unfortunately, this is of no practical use because of the low burst size—about six phage particles are liberated per cell after infection. The SSV1 phage of Sulfolobus solfataricus integrates into the bacterial chromosome and may be of future use. Plasmids have been found in several Archaea and some have been developed into cloning vectors (Fig. 28.22). However, only the plasmids of Sulfolobus (and related Archaea) are self-transferable, as far as is known. Conjugation in Sulfolobus relies on type IV pili to bring the cells together. The process of DNA transfer is still mysterious although it occurs between genetically identical cells and may be bidirectional. This differs from typical bacterial conjugation, which involves one-way transfer of DNA from a cell containing a plasmid to a recipient cell that lacks a plasmid. The recently discovered CedAB protein complex is located in the in Sulfolobus cell membrane and takes part in DNA uptake. Whether single or double-stranded DNA is transferred in Sulfolobus is unknown; however, DNA that is transferred can take part in homologous recombination. The CedB protein uses hydrolysis of adenosine triphosphate (ATP) to energize DNA uptake. Conjugation has also been reported in some halobacteria that form conjugation bridges without the participation of fertility plasmids. This system is still obscure. Neither type of archaeal conjugation has so far been developed into the sort of useful gene-transfer system found in well-studied bacteria such as E. coli or Bacillus.

8. Whole-Genome Sequencing The techniques for gene transfer described in this chapter have allowed the construction of detailed genetic maps and convenient gene transfer systems for E. coli and a few other well-investigated bacteria. However, for the vast majority of

8. Whole-Genome Sequencing

Crenarchaeota

919

Euryarchaeota

Halobacterium

Haloferax

Haloarcula

Halorubrum

Archaeoglobus Methanobacterium

Sulfolobus Thermoproteus

Methanospirillum

Pyrococcus

Methanosarcina

Desulfurococcus

Methanolobus Methanococcus

Pyrodictium

Methanopyrus Transformation Archaea

Eubacteria

Eukaryotes

Transduction Conjugation

FIGURE 28.22 Groups of Archaea and Their Gene-Transfer Mechanisms Phylogenetic tree of the Archaea lineage illustrating that different types of gene transfer can occur. The green zone contains salt tolerant organisms, the blue zone indicates methane producers and the red zone contains Archaea that grow at extremely high temperatures. Some Archaea use transformation whereas others use conjugation. Rare cases of viral transduction also occur. The modes of gene transfer seen within each family do not correlate well with either lifestyle or evolutionary relationships. The Crenarchaeota and the Euryarchaeota are the two major branches of the Archaea.

microorganisms, no “classical” genetics exists. Nowadays these are largely being investigated by more modern techniques, such as gene cloning and DNA sequencing. Since the development of rapid automated techniques for sequencing DNA (see Chapter 8: DNA Sequencing) many whole genomes have been totally sequenced. The first genome sequence to be finished was from the bacterium Hemophilus influenzae in 1995. Since then, thousands of bacterial genomes have been sequenced. Sequence comparison with genes of well-investigated organisms allows provisional identification of many genes. However, even in E. coli, the function of about a third of the genes remains uncertain. Whole-genome sequencing of pathogenic bacteria and comparison with their harmless relatives often reveals extra blocks of genes responsible for causing disease. Many virulence genes are carried on plasmids as discussed in Chapter 23, Plasmids. Others are found clustered together in regions of the chromosome known as “pathogenicity islands.” Most genes of Salmonella, as well as their order around the chromosome, correspond to those of its close relative E. coli, as would be expected. However, extra segments of DNA are found in Salmonella that are lacking in E. coli. Some of these are pathogenicity islands (Fig. 28.23). Such extra regions are often flanked by inverted repeats, implying that the whole region was inserted into the chromosome by transposition at some period in the evolutionary past. In agreement with this idea, such islands are often found in some strains of a particular species but not others. In addition, these islands tend to have different G/C to A/T ratios and/or codon usage frequencies from the rest of the chromosome, suggesting their origin in some other organism. Conversely, E. coli possesses a few DNA pathogenicity island Region of bacterial chromosome containing clustered genes for virulence.

The bacterium Hemophilus influenzae was the first organism to have its DNA completely sequenced.

Virulence genes are often clustered together forming “islands.”

CHAPTER TWENTY EIGHT
Bacterial Genetics

FIGURE 28.23 Pathogenicity Islands of Salmonella

h

e

i j

c

d

Harmless bacterium e.g., E. coli

k l

b a

Comparison of the E. coli genome with its close relative, Salmonella, reveals large regions of DNA that have no homology (orange). The remaining regions have similar genes that are in identical order. For example, Salmonella genes d through j are clustered together in the exact same order as E. coli genes d through j. Since Salmonella is pathogenic and E. coli is not, the regions of no homology probably encode the genes required for pathogenicity; therefore, they are termed pathogenicity islands. The islands are flanked by inverted repeats, suggesting the DNA may have been acquired through transposition. (Note: This figure is not drawn to scale; the pathogenicity islands are greatly exaggerated relative to the rest of the chromosome for purposes of illustration.)

g

f

m

920

Bacterial chromosomes f

g

i j

d

Related pathogenic relative e.g., Salmonella typhi

Pathogenicity islands

c

k m

a

b

Horizontal transfer of genes is especially significant in bacteria.

l

Differences in G/C and A/T ratios reveal segments of chromosomes with foreign origins.

Inverted repeats

h

e

segments missing in Salmonella. Interestingly, one of these is the area including the lac operon and a few surrounding genes. Thus, the classic lac operon, the moststudied “typical” gene of the “standard organism” is probably a relatively recent intruder into the E. coli genome! Pathogenicity islands are simply the best-known case of “specialization islands.” These are blocks of contiguous genes, presumed to have a “foreign” origin, which contribute to some specialized function that is not needed for simple survival. Not surprisingly, medical relevance has drawn most human interest. Other examples include genes encoding pathways for the biodegradation of aromatic hydrocarbons, herbicides, and other products of human industry and pollution. Movement of genes “sideways” is designated lateral or horizontal gene transfer in distinction to the “vertical” transfer of genes from ancestors to their direct descendants. HGT can occur by conjugation, natural transformation, viral transduction, or transposon jumping. HGT may occur between closely related organisms or those far apart taxonomically. Estimates suggest that in typical bacteria around 5% of the genes have been obtained by lateral gene transfer, and in rare cases up to 25%. Thermotoga is a bacterium adapted to life at very high temperatures and which consequently shares its habitat with several Archaea. Thermotoga has apparently gained around 25% of its genes by transfer from thermophilic Archaea such as Archaeoglobus and Pyrococcus. When we remember that the F-plasmid of E. coli

horizontal gene transfer Movement of genes sideways between unrelated organisms. Same as lateral gene transfer.

8. Whole-Genome Sequencing

921

can mediate DNA transfer into yeast (see Chapter 23: Plasmids), these results are perhaps not so surprising.

8.1. Bacterial Genome Assembly and Transplantation The Venter Institute has performed an intriguing set of genetic manipulations intended to pave the way for the synthesis of artificial life. They have shown that it is possible to transform a whole bacterial genome into a suitable recipient cell. For

** * **

Elements for yeast propagation and genome transplantation BssH II

*

Asc I

* 811–

9

900

–79

701

WM4

94D

****

BssH II 1

1–

90

1,077,947 1–

00

70

0

10

60

*

*

A G C T CG

Oligonucleotides

0

,00

200

1001–1

TG CT A ATCAA CGCA GA TG CAACT AG TC GCAT AT CATG

TA

CG

*

Oligonucleotide synthesizer

501–600

Yeast

800,000

104

1,080 bp cassettes (1,078) (Assemble 109X)

10,080 bp assemblies (109) (Assemble 11X)

100,000 bp assemblies (11) (Assemble 1X) 00

00

2–1

Asc I

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FIGURE 28.24 Assembly of Synthia Venter The whole genome for a synthetic organism called Mycoplasma mycoides was assembled in yeast. First, segments of DNA about 1000 bp in length were chemically synthesized using an oligonucleotide synthesizer. These were combined into groups of 10 to create the 10 kb fragments (blue arrows). The 10 kb pieces were combined into groups of 10 to create 100 kb fragments (green arrows). Finally, the 11 fragments were combined into one genome (red circle). The pieces were assembled using homologous recombination in yeast. Credit: Gibson, D.G., et al., 2010. Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329, 5256.

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this they used the bacteria of the genus Mycoplasma, which has one of the smallest bacterial genomes (just under 600,000 nucleotides in length). The genome from one species of Mycoplasma was purified and then transformed into a cell of another Mycoplasma species. The incoming chromosome was selected by antibiotic resistance and displaced the resident chromosome. Technically, this “genome transplantation” converted one species of the genus Mycoplasma to another. The next step was to synthesize the whole genome of Mycoplasma chemically and then insert it into a cell. This was done in several hierarchical stages. First, about 100 segments of DNA of 50007000 bases long were chemically synthesized. These had overlapping sequences at their ends that allowed them to be joined together by recombination in E. coli. Assembly proceeded via units of 24, 72, and 144 kb (quarter genomes) all carried on bacterial artificial chromosomes. Final assembly of the four quarters into a complete genome was performed in yeast. The genome was then transplanted into a Mycoplasma host cell and selected as before. Artificial “watermark” sequences were included in the artificially assembled genome to verify its presence. Several variants of this procedure have been carried out. One of the most recent versions (Fig. 28.24) contains the rather larger genome (1.08 megabases) of Mycoplasma mycoides chemically synthesized and modified to contain “watermark” sequences including the authors’ names and famous quotes, one being “What I cannot build, I cannot understand,” by the physicist Richard Feynman. This cell has been nicknamed Synthia Venter! The next issue to be tackled by whole genome assembly was the question of the minimal genome. That is: What is the fewest number of genes needed for a selfreplicating cell? Starting from Mycoplasma mycoides JCVI-syn1.0 (as depicted in Fig. 28.24), as many genes as possible were deleted. Genes were retained based on two major factors. Firstly, knowledge of molecular biology provided a list of genes needed to synthesize and process macromolecules. Secondly, exhaustive mutagenesis of the Mycoplasma genome by transposon insertions revealed which genes are essential. A third generation construct, JCVI-syn3.0 had a genome smaller than any naturally existing cell. JCVI-syn3.0 has 531 kbp of DNA with 473 genes (438 encoding proteins and 35 encoding RNA). Note that Mycoplasma mycoides JCVI-syn3.0 is grown in rich medium that supplies most small molecules needed for growth. Consequently, many genes that would be essential for growth in minimal medium or that would be needed to use growth substrates other than glucose have been deleted. Of the 438 protein-encoding genes, 149 (31% of the genome!) are of unknown function but were found to be essential by transposon mutagenesis. Many of these genes have homologs in other organisms, implying that they encode proteins essential for a wide range of organisms, despite their presently unknown function.

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In bacteria reproduction and sex are two distinct processes. Gene transfer between bacteria may occur by uptake of unprotected DNA, movement of DNA inside virus particles, or specialized cell-to-cell DNA transfer. DNA that enters a bacterial cell may survive on its own if it is a complete replicon. Otherwise, it will be degraded unless it is recombined into the host chromosome. Gene transfer by the uptake of unprotected or “naked” DNA is known as transformation. The transfer of inherited characters by transformation was part of the original proof that DNA (not protein) is the genetic material. Transformation occurs in certain bacteria under natural conditions.

Review Questions

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Gene transfer between bacteria by DNA in virus particles is known as transduction. In generalized transduction, random fragments of bacterial DNA are carried by virus particles. In specialized transduction, specific regions of the bacterial chromosome are preferentially packaged in virus particles. Many plasmids can transfer themselves between bacterial cells by a process known as conjugation. Transfer of chromosomal genes by plasmids requires integration of the plasmid into the bacterial chromosome. Mating pheromones secreted into the culture medium often regulate plasmid transfer between gram-positive bacteria. Conjugative transposons can both transpose between DNA molecules and transfer themselves between gram-positive bacteria by conjugation. Gene transfer in Archaea is common but still poorly investigated. Both plasmids and viruses exist that can transfer genes in these organisms. For most bacteria, genetic information has been gathered by sequencing the whole genome. Genome islands are blocks of contiguous genes usually with a “foreign” origin that perform some specialized function, such as virulence or biodegradation. Whole bacterial genomes have been chemically synthesized and successfully inserted into bacterial cells.

Review Questions 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

What is the difference between reproduction and gene transfer What are the major genetic differences between bacteria and higher organisms? How does gene transfer in bacteria differ from that in higher organisms? What are the three mechanisms of bacterial gene transfer? Briefly define each type of transfer. What are the three possible fates of incoming DNA fragments during gene transfer? What is required for an incoming piece of DNA to survive without a recombination event? What types of DNA molecules can do this? What type of DNA must be incorporated into the chromosome in order to survive? What are “competent” cells? What are two ways to make cells “competent”? Describe Oswald Avery’s experiment to prove that DNA is the genetic material. Why was his experiment not absolute proof that DNA is the genetic material? What term is used to describe the uptake of naked viral DNA? What is the viral DNA alone able to do? What does the term “transformation” mean to a cancer specialist? What is the purpose of competence pheromones? How and when do these compounds work? Compare and contrast the mechanisms of natural competence and artificially induced competence. How does Vibrio cholerae kill other bacterial cells and steal their DNA? Describe transduction. How is transduction performed in a laboratory? What conditions are required for transduction? Describe the two types of transduction. Give examples of bacteriophage that perform each type of transduction. What is meant by the term “headful packaging”? For which type of transduction is “headful packaging” essential? What is attλ (att-lambda)? Between which genes and in which organism is this site located? What are the two major differences between generalized and specialized transduction with regard to bacterial DNA mistakenly carried by bacteriophages?

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20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43.

Why do specialized transducing particles arise at extremely low frequencies? What are helper phage and defective phage? What is the term used to describe the ability of certain plasmids to move themselves from one bacterial cell to another? Give some examples of families of plasmids that have this ability. What is a sex pilus and a conjugation bridge? How does plasmid DNA move from one cell to another during conjugation? How is the plasmid DNA replicated during transfer? What are “tra genes” and where are they located? Why cannot very small plasmids transfer by conjugation? What does mobilizable mean? How can plasmids mediate the transfer of chromosomal genes by conjugation? What are Hfr-strains? Why were they useful in the early days of bacterial genetics? Why is it possible for gene transfer to be either clockwise or counterclockwise? Why is the presence of insertion sequences on the F plasmid significant? What are Fprime (or F’) plasmids? What are the major structural differences between gram-negative and gram-positive bacteria? What usually promotes the transfer of plasmids between gram-positive bacteria? What are conjugative transposons and how are they transferred? What types of environments do Archaea often thrive in? What are some of the problems associated with the development of genetic systems within Archaea? Compare and contrast the transformation of genetic material in Archaea versus Eubacteria. What are “pathogenicity islands”? What usually flanks these regions? What do these flanking regions indicate? What do differences in GC to AT ratios between “pathogenicity islands” and the rest of the chromosome indicate? What else gives a similar indication? Give an example of an extra segment of DNA that E. coli has that is missing in Salmonella? Why can “pathogenicity islands” best be described as “specialization islands”? What are three properties that describe them? What is horizontal or lateral gene transfer? By which mechanisms may this transfer occur? What is genome transplantation? What was the first organism to have its genome completely synthesized? How was this achieved?

Further Reading Berry, J.L., Pelicic, V., 2015. Exceptionally widespread nanomachines composed of type IV pilins: the prokaryotic Swiss Army knives. FEMS Microbiol Rev 39, 134154. Borgeaud, S., Metzger, L.C., Scrignari, T., Blokesch, M., 2015. The type VI secretion system of Vibrio cholerae fosters horizontal gene transfer. Science. 347, 6367. Cook, L.C., Dunny, G.M., 2014. The Influence of Biofilms in the Biology of Plasmids. Microbiol Spectr 2 (5). Danchin, A., Fang, G., 2016. Unknown unknowns: essential genes in quest for function. Microb Biotechnol. 9, 530540. Hutchison 3rd, C.A., et al., 2016. Design and synthesis of a minimal bacterial genome. Science. 351 (6280), aad6253. Matthey, N., Blokesch, M., 2016. The DNA-Uptake Process of Naturally Competent Vibrio cholerae. Trends Microbiol. 24, 98110. Metzger, L.C., Blokesch, M., 2016. Regulation of competence-mediated horizontal gene transfer in the natural habitat of Vibrio cholerae. Curr Opin Microbiol 30, 17. Pohlschroder, M., Esquivel, R.N., 2015. Archaeal type IV pili and their involvement in biofilm formation. Front Microbiol. 6, 190. Suzuki, Y., et al., 2015. Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling. Genome Res. 25, 435444. van Wolferen, M., Wagner, A., van der Does, C., Albers, S.V., 2016. The archaeal Ced system imports DNA. Proc Natl Acad Sci U S A 113, 24962501.