Genetics of bacteria and phages

Bacteria and phages in genetic research:

Advantages: They are haploid, therefore recessiveness or dominance dont come into play except in the case of partial diploids. New generations are produced within one or two days. They are easy to grow in enormous numbers, this allows for analysis of rare genetic events. The offspring is clonal and genetically identical.
Recombination is quite different in prokaryotes: A bacterial cell contains one circular chromosome and rarely encounters another complete chromosome. Recombination is usually between a chromosomal fragment from a donor cell and an intact chromosome of the recipient cell. Incorporation of part of the transferred donor DNA requires at least two or any even number of exchange events because the recipient DNA is circular. Furthermore, recombination is not reciprocal and produces only one recombinant molecule, the circular recipient molecule, containing an integrated piece of donor DNA. A single bacterial cell placed on solid medium will grow exponentially and form a clonally derived colony. The appearance of colonies and the growth requirements of bacteria can be used to identify the genotype of the colonies.

Genetic analysis of bacteria by mutation analysis: Different types of mutations can be used: Antibiotic resistance: these mutants are able to grow in the presence of an antibiotic, such as streptomycin. Nutritional mutants: Wild-type bacteria can synthesize most of the complex nutrients they require from simple molecules present in the minimal growth medium and are hence called prototrophs. This ability to grow on simple or minimal media can be lost due to mutation of enzymes involved in the synthesis of nutrients. Mutants unable to synthesize an essential nutrient cannot grow unless that nutrient is supplied in the growth medium, and are called auxotrophs. Carbon-source mutants cannot utilize particular substances as energy sources, such as lactose and are unable to form colonies on medium containing lactose as the sole energy source. A medium in which wild-type cells form colonies is called nonselective medium. Mutant cells an wild-type cells are not distinguishable on nonselective medium. If the medium allows only one type of cell to grow it is termed selective. Eg. Medium containing streptomycin is selective for Strp-r (streptomycin resistant) phenotyptes. In bacteria phenotypes are designated with 3 letters (first letter capitalized) , a superscript denoting absence or presence of a character and s and r denoting resistance or sensitivity. For example Strp-r or Leu-, a genotype is lowercase italics: leu-.

Criterion Recombination process Transformation Conjugation Cell contact required? no yes

Sensitive to DNase?

yes no

Transduction

no

no

U-tube experiment: Bacteria of different genotypes are in different arms of the tube, separated by sintered glass filter that prevents cell-cell contact. This experiment tests the transfer of genetic material. DNase in the culture medium degrades free DNA, providing a test for transformation. If recombination occurs, it is likely taking place by means of transduction. Why transduction? Bacteria are too large to make it through the filter, that excludes conjugation. Transformation is excluded because the DNA in the media would get degraded by DNA. In a phage, however, the DNA is packaged in protein and protected from digestion.

Transformation involves the uptake of DNA from donor cells and integration into the host recipient genome. In nature DNA becomes available from breakage of donor cells. Transformation efficiencies can be increased significantly by chemical treatment that makes the cells competent. The uptake efficiency is rather poor, even if cells are made competent it is 1 in 1000 cells.

Transformation can be detected by selection for inheritance of a phenotype from the donor DNA by the recipient cells. For example, purified DNA from an erythromycin resistant strain of S. pneumoniae (Eryr) is mixed with cells from a culture of a sensitive strain (erys). After a period of incubation (time for DNA uptake and expression by the donor) cells are plated on erythromycin. The formation of eryr colonies is significantly above the mutation rate, this indicates that transformation has occurred.
Transformation can be useful for gene mapping. If two genes are separated far enough that most of the times the DNA fragment is broken before transformation, then the probabilities of cotransformation is the same as the product of probability for the individual genes i.e. 103 x103. If they are close enough the frequency of cotransformation is substantially greater

Transformation: Griffith discovered transformation in 1928, 17 years later Avery, MacLeod and McCarty demonstrated that DNA was the transforming principle. What is its importance besides these discoveries? It is important in genetic analysis of yeast, and bacteria, and it is central to most cloning strategies. Although not necessarily the most efficient, it is the simplest form of DNA transfer. Griffiths discoveries could be made in Pneumococcus because these cells can become naturally competent to take up DNA. Natural competence for transformation is observed in Bacillus subtilis, Haemophilus influenzae, Str. Pneumoniae etc. and people thought for some time it is limited to these species, but not so. It contributes to antigenic variation in gonococcus (Neisseria gonorrhoeae) where it involves pil genes, which are also involve in attachment to epithelial cells. General features: Competence occurs usually in the late log phase of growth, possibly as a response to increasing cell density, nutrient depletion and accumulation of secreted competence factors. (Natural competence may only last for a short time period). These factors stimulate gene expression of other genes required for competence through a 2 component regulatory system (transmembrane signal transduction). It could be considered a form of quorum sensing. In B. subtilis some genes involved in transformation also are important in early sporulation.

In naturally competent S.pneumoniae (gram-positive) cells, double-stranded DNA fragments bind to cell surface receptors, however, only one strand is taken up. In some species this can be specific because it depends on the presence of species-specific DNA on the fragments. Eg. In Neisseria menengitidis DNA uptake is dependent on a 10bp sequence, of which there are 2000 copies through out the N, menengitidis genome. H. influenzae transformation requires a 29bp sequence which occurs 1500 times in the genome. In other species, such as B. subtilis, and Str. pneumoniae the uptake is non-specific and occurs with almost any linear piece of DNA. H.influenzae takes up double stranded DNA. However, transformation requires integration into the host genome in the case of linear (non-plasmid) sequences and is facilitated by sequence homology. Therefore, the more similar the sequences are to the host genome, the more likely is a recombination event with with the host genome. That is, the recombinant events lead to substitution rather than addition of DNA sequences. Str. Pneumoniae has apparently become penicillin resistant as a result of penicillin target gene replacement with the resistant gene from resistant streptococci.

Following uptake of DNA, the transforming donor DNA replaces or substitutes the recipient DNA through homologous recombination events involving RecA like proteins. DNA is not just added or inserted into the host genome.

Mapping by transformation: When transformation is used to transfer genes to measure recombination frequencies between markers several difficulties arise. The probability of recovering the donor marker in the recipient depends on the molecular weight (or number of bp) of the donor fragment, and on the marker itself. There are low efficiency and high efficiency markers. Low efficiency markers are recovered at low frequencies and high efficiency markers at high frequencies. The efficiency is a result of different mismatch repair probabilities (in strains where transformation takes up only single stranded DNA). Furthermore reciprocal transformation do not yield the same transformation frequencies, eg if there are two alleles Z and z, transformation and recovery is not the same if Z is the donor and z the recipient as with z the donor and Z the recipient allele because of differences in mismatch repair. Therefore, in transformation recombination frequencies between donor and recipient dont depend on, or correlate with distance between markers. But the probability of two markers to be transformed (and detected) together is increased if they are located closely together on the same DNA fragment. Hence cotransformation frequencies go up the closer two markers are.

When DNA from donor bacteria is isolated, most of it is also broken into hundreds of random size smaller fragments. If such DNA is used to transform highly competent recipient cells, the probability or frequency of transformation of most genes is about one cell per 1000. If two genes x and y are separated and the distance between them is larger than the length of most of the transformed fragments, that is, if the two genes are rarely observed on the same DNA fragment , then the transformation frequency of both together is the square of the individual frequences (10-3)2 or 10-6. However, if the two genes are located so closely to each other such that they often end up on the same donor DNA fragment, then the frequency of cotransformation is much closer to 10-3, the frequency of transformation of a single gene alone. Thus cotransformation of two genes at a high frequency implies that they are located closely together. Example: if genes y and z and genes z and x can be cotransformed but not y and x, then the order of genes must by y z x. In general, if the size of the DNA fragments is controlled within a reasonably narrow range, then one can relate the cotransformation frequency with the distance between the genes. I.e. if one measures the cotransformation frequency as a function of the molecular weight (or length of the DNA) one can determine the distance between the genes roughly.

If a and b, and b and c can be cotransformed but not and c, that means the order of the genes must be a,b,c. However, cotransformation frequencies (CF) are not the same as recombination frequencies. The CF is determined mostly by the size of the fragment and the likelyhood of strand breakage of bacterial DNA, rather then than chiasma formation of homologous chromosomes.

Distance is not the only variable in cotransformation frequency: At high DNA concentrations relative to the number of competent cells, more than one DNA fragments binds each cell and the cell may take up several fragments, this may appear as if two genes are cotransformed and lead to the wrong interpretation of the data. Therefore, one may have to perform a dilution test. If following dilution, the second gene B is cotransformed with the other gene A, at a similar frequency as gene A or B alone then the two genes are closely linked. Alternatively, if the cotransformation frequency drops severely following dilution, then the two genes are not (closely) linked. If the genes are not closely linked then a tenfold decrease in DNA concentration should lead to a hundred fold decrease in cotransformation of both genes, while the drop off should be by a factor of 10 for the individual genes. If the two genes are closely linked, then a 10-fold dilution should lead to only a 10 fold drop in cotransformation.

Problem: Because transformation is performed with DNA fragments and because the genetic markers used for gene mapping may be far apart, there will be problems in the determination of neighboring genes in such areas (where there are only few or no markers) because one may not be able to observe cotransformation. As a result, mapping a circular bacterial genome through cotransformation produces areas where the linkage between genes is determined (linkage groups) and gaps between those linkage groups where there are not enough closely spaced markers. (It is possible to close those gaps by taking cultures in which DNA replication is synchronized and determining the sequence of gene replication from an origin of replication, this involves separating newly synthsized DNA from old DNA by 15N labeling. )

Linkage group 1

linkage group2

No cotransformation between genes from these two linkage groups because the distance between genes/markers is too large.

In vitro plasmid transfer: How does one transfer a non-transmissible plasmid to a specific host cell? One purifies the plasmid DNA and transforms the strain of interest with it, applying a genetic selection, usually antibiotic resistance. Transformation means spontaneous uptake of the DNA by the recipient bacterial strain. Some bacterial strains are naturally transformable, however, the strains most commonly used in molecular biology need to be prepared to be come transformation competent. A common method of making E. coli chemically competent is through hypotonic shock in the present of divalent ions, such as Ca2+, Mn2+ and Mg2+. Early log phase cultures are centrifuged, and resuspended in hypotonic solution. When DNA is added it forms a complex with the Calcium that adsorbs to the cell surface. Cells are then warmed/heat shocked, this transport into the cell. Alternatively cells can be made permeable with electroporation. The cells are exposed to an electric field and and electric discharge that polarizes the membrane. The voltage potential across the membrane forms transiently small pores, making the cell permeable, allowing macromolecules such as DNA to enter. In vitro transformation uses double stranded DNA, and because it uses selfreplicating plasmids it does not require recombination. However the size of DNA that can be taken up by bacteria in that way is limited.

Conjugation Conjugation is the direct unidirectional transfer of DNA from on bacterial cell to another, in most cases plasmid DNA, although in some species chromosomal transfer can also occur. Conjugation can easily be demonstrated among Enterobacteria and other Gram-negative bacteria. But gram positives like Streptomyces also possess conjugation systems. Conjugation is not necessarily confined to members of the same species, therefore, it is another avenue for horizontal gene transfer across taxonomic boundaries. As a result plasmids that are present in the normal gut flora can be transmitted to infecting pathogens, which then can become resistant to a range of antibiotics. Mechanism: As already mentioned, it requires a donor strain containing a plasmid that carries the genes required for promoting DNA transfer. In E.coli and other Gram-negative species, the donor cells carry pili, which vary in structure length, flexibility etc., depending on the plasmid. The pili bring the cells into contact and then a channel or pore is made through which the DNA is transferred from donor to recipient. Apparently this mechanism has many things in common with protein secretion systems used to deliver bacteria toxins directly into host cells.

Some large plasmids contain genes that enable plasmids to be transferred between cells. In E. coli For example there is a large plasmid called F factor (F like fertility). Cells containing F factor are F+, those lacking it are F-. Transfer of the F plasmid is mediated through a tube like structure, called the pilus, during conjugation. The F plasmids or conjugative plasmids contain about 20 genes that are required for pilus assembly and gene transfer. Most smaller plasmids dont contain those genes, however, by means of recombination with the F plasmids, they can tag along with the conjugative plasmids.

Conjugation is the process in which DNA is transferred from a donor cell to a recipient cell, by cell to cell contact and DNA transfer through a conjugation tube (or F- pilus). It occurs in many species, originally discovered by J. Lederberg. The process is controlled by and is dependent on a set of genes that is encoded in an independent circular DNA molecule (F plasmid, autonomous state) in F+ cells, or it can be integrated in the bacterial chromosome in hfr cells (high frequency recombination). When an F+ donor cell conjugates with and F- recipient cell, only the F factor is transferred, both the donor and recipient cells end up F+, since the plasmid is replicated by a rolling circle mechanism during transfer. If a few F+ cells are mixed into a majority of F-cells, most of them end up being F+. (F stands for fertility.)

The transfer of plasmid DNA is initiated by a protein (Mob) which makes a single-strand break (nick) at a specific site, the origin of transfer (oriT). A plasmid-encoded helicase unwinds the plasmid DNA and the 5 end of the nicked strand is transferred to the recipient, while the 3 end is extended by a DNA polymerase during rolling circle replication.

Nicking proteins remains attached to the 5 end of the transferred DNA, creating a second nick and closing the circle upon completion of replication

Conjugation is a replicative process that transfers a copy into the recipient, but also leaves another copy in the donor cell. Thus both cells are donors after the mating, hence this process can lead to an epidemic spread of the plasmid throughout a bacterial population. Plasmids that can mediate the complete DNA transfer process are called conjugative plasmids. In some cases the conjugative plasmids can also promote (mobilize) the transfer of a second otherwise non-conjugative plasmids from the donor cell (donation). ColE1 is an example of a plasmid that has the genes needed for DNA transfer, but not the genes required for mating-pair formation. Mobilization involves the mob gene which encodes site specific nuclease that nicks DNA at the bom site (=oriT, the origin of transfer). Not all mobilizable plasmids have the mob gene. In that case, the Mob nuclease has to be provided by the conjugative plasmid for mobilization. However, no mobilization happens without a bom site on the second mobilizable plasmid. Removal of the bom site from plasmid makes it nonmobilizable, and non-transferrable through conjugation. The primary goal of conjugation is transfer of the conjugative plasmid to the recipient, converting the recipient into a male donor cell, spreading the plasmid through the whole population of bacteria. However in some cases some types of plasmids can also promote transfer of chromosomal DNA.

bom=oriT

In the case of the F plasmid, the transfer of chromosomal sequences involves prior integration of the (conjugative) F plasmid into the host chromosome. As a result a part of the host chromosome is transferred, as part of the attempt of the F plasmid to transfer itself. (In other cases chromosomal transfer occurs without stable association between the plasmid and the host chromosome). It is however impossible for the complete copy of the chromosome to be transferred because DNA transfer during conjugation is a slow process that would take about 100 min for the whole chromosome. Matings rarely last that long, and therefore, the process tends to get disrupted before the transfer of the genome is complete. The mating lasts long enough for a plasmid of 40-100kb which requires 1 min. to be transferred, but not for the 4 mb of a chromosome which would require 100 min. Hfr strains (high frequency of recombination). Hfr strains arise by integration of the F plasmid into the bacterial chromosome. As in the conjugative plasmid the starts at an origin of transfer of the plasmid and is determined by the site of insertion of the plasmid within the genome. The direction of transfer is determined by the orientation of the plasmid inserted. Hence different Hfr strains of the same species have different origins of transfer and different directions, depending on the particulars of the site and direction of insertion into the host chromosome.

The F factor can integrate into the bacterial chromosome by site specific recombination to create Hfr cells (High frequency recombination.) Recombination occurs by site specific homologous recombination between insertion sequences (IS) present on the plasmid and the chromosome. IS sequences originate from transposons.

After conjugation between Hfr cells and F- cells the transferred DNA will not become circular, and is not capable of further replication, because most of the time the transferred DNA does not contain all the genes necessary for conjugation and autonomous replication, as transfer usually gets interrupted prior to completion of transfer. The DNA transfer is
Bact. Chromosome in Hfr cell

B-chromosome

controlled by the integrated F factor, and is initiated on the Hfr chromosome at the same site as in the F plasmid. A part of F DNA is transferred first, followed by chromosomal genes and the remainder of F last, however, the latter rarely makes it into the recipient cell, hence the recipient remains F-.

Because it involves transfer of a bacterial chromosome (4600kb) conjugation between Hfr cells and F- cells would take 100 min, vs. 2 min with F+ cells (100kb). Usually only a fraction of several 100 genes of the chromosome is transferred before conjugation is disrupted and the cells separate. The recipient usually remains F- because separation takes place before the entire chromosome is transferred. Some regions of the transferred DNA becomes integrated into the donor cells by homologous recombination. Where integration occurs, cells become recombinant, however the donor genomes remain unchanged. Eg. Donors are Hfr Leu+, recipients F- leu-, recombinant recipients will be F- Leu+. Because recombinants occurs in only a small fraction of all the bacteria, selectable markers need to be employed to identify recombinants, and counterselection is used to get rid of donor cells. Eg. The the transferred selectable marker for recombinants could be Leu+, while the counterselection marker employed could be Str-s (streptomycin resistance) which would be only present in recipients. Hfr matings can be used for bacterial chromosome mapping. In this case, the genetic map is based on transfer order, not on meiotic recombination. The genetic map is obtained by interrupting the DNA transfer during the mating (i.e. after Hfr and F- cells have been mixed) with a blender like device. The earliest time at which a particular gene is transferred (time of entry) , and at which mating disruption no longer interferes with the appearance of recombinants, that time denotes the relative position of that gene on the chromosome, since transfer is unidirectional. Many selectable marker genes have been mapped in that way.

Interrupted mating technique

The number of recombinants increases with length of time of mating. Each marker has time of entry before which no recombinants are detected. Each curve has a linear region that can be extrapolated back to the time axis, defining the time of entry. The period of increasing recombination is due to a variation in the initiation of conjugation. The number of recombinants of each type reaches a maximum or plateau, the value of which decreases with successive times of entry.

The genes that can be mapped with the interrupted mating technique, depend on the location and the direction in which the F-plasmid integrated into the host genome.
Different F- strains contain different alleles, thus adding more information to the map (C). Different Hfr strains differ in their location of F and hence in origins and direction of transfer, since F can integrate at multiple sites. Combining the overlapping maps obtained with different Hfr strains yields a composite map, that is circular because the E. coli chromosome is circular.

Circular map of E.coli. Map distances are given in minutes. The total map length is 100 min. For some loci that encode related gene products, the map order of the clustered genes is shown along with the direction of the transcription and length of transcript, (black arrows). The purple arrow heads show the origin and of transfer of a number of HFR strains. For example, Hfr transfers the thr early, followed by leu and other genes in a clockwise direction.

Complete F plasmids are occasionally excised. Aberrant excision creates an Fplasmid that contains a fragment of chromosomal DNA. By the use of different Hfr strains with different origins of transfer, Fplasmids carrying segments from many regions of the chromosome have been isolated. F plasmids dont require packaging, and their size is less restricted than that of phage. Any recipient cell is partially diploid (meroploid) for the chromosome segment carried on the plasmid. This is useful for testing dominance, and gene dosage, as well as complementation. F particles may carry selectable markers such as thr+ or leu+ that can recombine with the recipient genomewhile the rest of the F particle may be lost. Recombination mediated by F particles is called sexduction or F-duction.

Mapping closely linked genes in bacteria: Interrupted mating experiments provide approximate map distances for genes that are several map units apart. The resolution is , however too coarse for genes that are one or two minutes apart. In order to resolve the relationships between closely linked genes, three-point crosses are necessary. With one major difference the logic of three-point crosses is mostly the same as the logic observed in diploids. With rare exceptions, recombination in bacteria takes place between fragments of a donor chromosome carried on a transferred (exogenote) piece of DNA (by transduction, transformation or sexduction,) and a recipient chromosome, the endogenote. Recombination requires a double crossover since bacterial chromosomes are circular. Therefore, with merozygotes or partially diploid cells, a three factor cross can be performed in two ways: (i) double mutant donor X single mutant recipient, (ii) single mutant donor X double mutant recipient. The result of such a reciprocal cross can be used to order the markers involved.

Consider:

a+b1+ b2 donor X a b1 b2+ recipient a b1 b2+ donor X a+b1+ b2 recipient

If the order is a b1 b2 then a+b1+b2+ recombinants will occur with similar frequency because only one double cross over is required for both crosses to get a+b1+b2+ recombinant, assuming that all markers are conditionally lethal:

cross1
a+ a b1+ b1 b2 b2
+

cross2
a a+ b1 b1+ b2+ b2

Donor/ exogenote recipient/ endogenote

If the order is a b2b1 then the requirements are different: a+ b2

+

b1+

Donor/ exogenote recipient/ endogenote

b2+

b1 b1+

b2

b1

a+

b2

Two double crossovers low frequency.

One exchange/double crossover, higher frequency.

donor

recipient

If the order is a-b-c then the frequency of a+b+c+ in cross 1 will be approx. equal to the frequency of a+b+c+ in cross2. If the order is a-c-b, then the frequencey of a+b+c+ in cross 1 will be much lower than the frequency of a+b+c+ in cross 2.

Nuclease catalyzed fragmentation of b. chromosomes

Transduction Generalized Transduction: Transfer of bacterial DNA from one bacterial cell to another by phage particles is called transduction. General transduction is the result of accidental packaging of (nuclease fragmented) bacterial DNA (from any part of the bacterial chromosome) into phage particles. In specialized transduction the phage particles contain both phage DNA and bacterial DNA in one molecule, however, the bacterial DNA is derived from a particular chromosomal region close to the phage integration site.

1in 106 Transducing particle contains about 50 genes

After recombination leu+, will survive on selective growth medium

Demonstration of linkage by cotransduction. The transduced Fragment contains about 50 genes. Whether 2 genes are cotransduced or not depends on the distance. Cotransduction of bio and leu markers can be detected by growth on selective medium. Gal+ and bio+ are cotransduced about 12% of the time. Leu and gal cotransduction does usually not occur because the distance is too far. Cotransduction frequencies make it possible to construct linkage maps. Three factor crosses can be applied as described above.

Temperate phage cycle. Transduction requires a lysogenic, rather than a lytic phage.

Loss of prophage (curing) Nonlysogenic cell lysogenic response lysogenic cell

lytic response Vegetative phase

induction

Mapping in phage.

Life cycle of a (lytic) bacteriophage

If two phage particles with different genotypes infect the same bacterium, recombination can and does occur. When crossing r-h+ X r+h- all possible recombinants are observed. Furthermore, reciprocal recombinants occur in similar numbers.

Recombination frequency: number of recomb.phage RF= total number of phage X100

Phagemaps can be constructed from phage recombination experiments. Early mutation mapping experiments suggested T4 phage mapped to 3 different clusters. All clusters showed linkage. Three point crosses indicated that the map could be circular. In T4 genes are also clustered according to function.

rII mutants proliferate in E. coli B cells but not in K12(l). WT T4 phage can grow in both strains of E.coli.

The wildtype phage T4 can be grown in either E.coli strain B or K12(l). T4 with mutations in the rII gene can be grown as large round plagues in B but not in K12(l) which is a lambda lysogen. If the B strain is infected with two different phageT4 rII mutant recombination occurs (somewhat rare) from which rII+ phage results that can grow on K12(l). Mutagenesis yields a large number of rII mutations. Mutations that fail to recombine with several known point mutants (that were known to recombine with other point mutants) these are taken to be deletion mutants. The deletion mutants eliminates a part of the phage genome. The deletions can be used to order maps obtained by point mutations. If a cross between a point mutant and a deletion mutant produces a wildtype recombinant, that means the point mutant was outside the region that was missing in the deletion (eg. Mutants 1,2,3. If a deletion and a point mutant overlaps with the deletion, then wildtype recombinant progeny cannot be produced by crossing the two.. 1 2 3 4 5 6 7

Deletion mutant

No recombinants in this interval between the deletion mutant and point mutants 5 and 6

These studies provided important experimental support for the following concepts: Genetic exchange can take place within the coding sequence for a gene, at almost any nucleotide. Mutations are not distributed evenly across a gene. Mutational hotspots exist, i.e. sites that are much more likely to be mutated. Also this mutation analysis helped distinguish between three different definitions of gene as a unit of function or cistron: a stretch of DNA encoding a functional protein. Complementation test : When mutants in rIIA and rIIB were combined to infect E. coli simultaneously, normal numbers of phage were produced without recombination. I.e. rIIA and B function independently of each other and can therefore complement each other. Different mutants within the same gene eg. rIIA cannot complement. ( A functional protein may be encoded by two subunits locaten on two different genes.) Other meanings of gene: (1) unit of genetic transmission that participates in recombination. (2) unit of genetic change or mutation. Both can correspond to individual nucleotides in a gene.

A virulent phage undergoes only the lytic cycle, in which the phage takes over the cell to produce only phage protein and replicate phage DNA. In the lysogenic cycle the phage genome is integrated into the bacterial genome, where it replicates along with the bacterial genome as prophage. The bacterial cell is now called lysogen, eg. K12(l). The phage DNA can be activated and excised under certain conditions that damage DNA, such as UV radiation. Lysis follows.

At this point the genome of l phage has been analysed extensively, both in therms of sequence and functionally. l genes exhibit extensive clustering by function, eg head proteins, tail proteins, DNA replication, recombination , lysis immunity, etc. Clustering also occurs in terms of the timing of product synthesis, eg early and late genes. Interrupted mating of E.coli cells nonlysogenic and lysogenic for l phage, revealed that the l genome is inserted between the gal and bio genes of E. coli chromosomes. I.e. the presence of prophage increases the physical distance between gal and bio, since gal and bio can be cotransduced by P1 phage on a nonlysogen, but not on a lysogen for l. Genetic mapping of the prophage yields a permutation of the genetic phage map derived from standard phage crosses.

The DNA of phage l is a linear molecule with complementary cohesive ends (cos) each 12b, such that they can recombine and form a closed circle upon ligation. Circularization is required for both the lytic and lysogenic cycle.

The site of breakage and rejoining in bacterial and phage DNA are called attachment sites. They have three segments each. The central segments are identical in their nucleotide sequence for both sites. POP is located near the middle of the linear form of the phage. A phage protein, integrase, recognizes the attachment sites and catalyzes site specific recombination between them. Hence the phage map is a permutation of the prophage map. Upon integration the often only one phage protein is expressed in the lysogen, the phage repressor, which prevents other phages of the same kind from infecting the lysogen, conferring immunity from lytic infection.

The geometry of integration and excision of phage l. The phage attachment site is POP. The bacterial attachment site is BOB. The prophage si flanked by two hybrid attachment sites denoted BOPand POB.

The prophage can become activated to excise and undergo a lytic cycle upon induction by DNA damaging UV light or environmental agents. Excision requires two enzymes, excisionase and integrase, the latter for site recognition. In about 1out of 106 or 107 excision events errors occur, in which the sites of breakage and rejoining are displaced. Sometimes such an aberrant molecule can replicate and gets packaged. The aberration occurs either to the right or the left of the molecule, including either bio or gal genes. The resulting phage particles are called ldgal or ldbio. They are called specialized transducing phages because they can transduce only a limited number of genes, such as the gal or bio genes.

Because specialized transduction particle are deficient in some genes, they require wildtype l helper phage to integrate and replicate. Wildtype l phage is required for integration because its integration provides two hybrid attachment sites homologous to the one present in ld phage. Also wildtype l phage provides genes that are missing within the ld phage. The segment of donor DNA and the phage chromosome are added to the recipients chromosome, producing a partially dipoid transductant. Usually when lysogenic phage is excised, few defective transducing particles are formed by aberrant excision, hence the resulting lysates are called low frequency transducing lysates. If enough wildtype phage is present the recipient cells are also infected with wildtype phage that integrates at the normal attachment site, producing double lysogens, (carrying l+ and ldgal). The resulting transductants are thus gal-/gal+ partial diploids [and are also called gal+/galheterogenotes, containing gal+ exogenote (the donor DNA fragment) and gal- endogenote (recipient chromosome)]. The partial diploids are unstable transductants because can exit the chromosome and be lost. In addition, ldgal-l+ double lysogens can be induced to lyse with UV light , producing 50% ldgal and 50% l+ HFT (high frequency transduction ) lysates. HFT lysates facilitate genetic analysis by via specialized transduction by dramatically increasing the frequency of transduction events. Gal can be used as a selection marker, since only gal+ cells can utilize galactose as a carbohydrate substrate. In general specialized transduction can be used to map phage attachment sites and the genes that are close to the attachment site. In general specialized transduction is not the most useful technique encountered in prokaryotic genetics.