Gene Transfer and Genetic Recombination in Bacteria

The following points highlight the three modes of gene transfer and genetic recombination in bacteria. The modes are: 1. Transformation 2. Transduction 3. Bacterial Conjugation.

Mode # 1. Transformation:

Historically, the discovery of transformation in bacteria preceded the other two modes of gene transfer. The experiments conducted by Frederick Griffith in 1928 indicated for the first time that a gene-controlled character, viz. formation of capsule in pneumococci, could be transferred to a non­-capsulated variety of these bacteria. The transformation experiments with pneumococci eventually led to an equally significant discovery that genes are made of DNA.

In these experiments, Griffith used two strains of pneumococci (Streptococcus pneumoniae): one with a polysaccharide capsule producing ‘smooth’ colonies (S-type) on agar plates which was pathogenic. The other strain was without capsule producing ‘rough’ colonies (R-type) and was non-pathogenic.

When the capsulated living bacteria (S-bacteria) were injected into experimental animals, like laboratory mice, a significant proportion of the mice died of pneumonia and live S-bacteria could be isolated from the autopsied animals.

When the non-capsulated living pneumococci (R-bacteria) were similarly injected into mice, they remained unaffected and healthy. Also, when S-pneumococci or R-pneumococci were killed by heat and injected separately into experimental mice, the animals did not show any disease symptom and remained healthy. But an unexpected result was encountered when a mixture of living R-pneumococci and heat-killed S-pneumococci was injected.

A significant number of injected animals died, and, surprisingly, living capsulated S-pneumococci could be isolated from the dead mice. The experiment produced strong evidence in favour of the conclusion that some substance came out from the heat-killed S-bacteria in the environment and was taken up by some of the living R-bacteria converting them to the S-form. The phenomenon was designated as transformation and the substance whose nature was unknown at that time was called the transforming principle.

With further refinement of transformation experiments carried out subsequently, it was observed that transformation of R-form to S-form in pneumococci could be conducted more directly without involving laboratory animals.

An outline of these experiments is schematically drawn in Fig. 9.96:

At the time when Griffith and others made the transformation experiments, the chemical nature of the transforming principle was unknown. Avery, Mac Leod and McCarty took up this task by stepwise elimination of different components of the cell-free extract of capsulated pneumococci to find out component that possessed the property of transformation.

After several years of painstaking research they found that a highly purified sample of the cell-extract containing not less than 99.9% DNA of S-pneumococci could transform on the average one bacterium of R-form per 10,000 to an S-form. Furthermore, the transforming ability of the purified sample was destroyed by DNase. These findings made in 1944 provided the first conclusive evidence to prove that the genetic material is DNA.

It was shown that a genetic character, like the capacity to synthesise a polysaccharide capsule in pneumococci, could be transmitted to bacteria lacking this property through transfer of DNA. In other words, the gene controlling this ability to synthesise capsular polysaccharide was present in the DNA of the S-pneumococci.

Thus, transformation can be defined as a means of horizontal gene transfer mediated by uptake of free DNA by other bacteria, either spontaneously from the environment or by forced uptake under laboratory conditions.

Accordingly, transformation in bacteria is called:

a. Natural and

b. Artificial.

It may be pointed out to avoid misunderstanding that the term ‘transformation’ carries a different meaning when used in connection with eukaryotic organisms. In eukaryotic cell-biology, this term is used to indicate the ability of a normal differentiated cell to regain the capacity to divide actively and indefinitely. This happens when a normal body cell is transformed into a cancer cell. Such transformation in an animal cell can be due to a mutation, or through uptake of foreign DNA.

(a) Natural Transformation:

In natural transformation of bacteria, free naked fragments of double-stranded DNA become attached to the surface of the recipient cell. Such free DNA molecules become available in the environment by natural decay and lysis of bacteria.

After attachment to the bacterial surface, the double-stranded DNA fragment is nicked and one strand is digested by bacterial nuclease resulting in a single-stranded DNA which is then taken in by the recipient by an energy-requiring transport system.

The ability to take up DNA is developed in bacteria when they are in the late logarithmic phase of growth. This ability is called competence. The single-stranded incoming DNA can then be exchanged with a homologous segment of the chromosome of a recipient cell and integrated as a part of the chromosomal DNA resulting in recombination. If the incoming DNA fails to recombine with the chromosomal DNA, it is digested by the cellular DNase and it is lost.

In the process of recombination, Rec A type of protein plays an important role. These proteins bind to the single-stranded DNA as it enters the recipient cell forming a coating around the DNA strand. The coated DNA strand then loosely binds to the chromosomal DNA which is double-stranded. The coated DNA strand and the chromosomal DNA then move relative to each other until homologous sequences are arrived at.

Next, RecA type proteins actively displace one strand of the chromosomal DNA causing a nick. The displacement of one strand of the chromosomal DNA requires hydrolysis of ATP i.e. it is an energy-requiring process.

The incoming DNA strand is integrated by base-pairing with the single-strand of the chromosomal DNA and ligation with DNA-ligase. The displaced strand of the double-helix is nicked and digested by cellular DNase activity. If there is any mismatch between the two strands of DNA, these are corrected. Thereby, transformation is completed.

The sequence of events in natural transformation is shown schematically in Fig. 9.97:

Natural transformation has been reported in several bacterial species, like Streptococcus pneumoniae. Bacillus subtilis, Haemophilus influenzae, Neisseria gonorrhoae etc., though the phenomenon is not common among the bacteria associated with humans and animals. Recent observations indicate that natural transformation among the soil and water-inhabiting bacteria may not be so infrequent. This suggests that transformation may be a significant mode of horizontal gene transfer in nature.

(b) Artificial Transformation:

For a long time, E. coli — a very important organism employed as a model in genetical and molecular biological research — was thought to be not amenable to transformation, because this organism is not naturally transformable.

It has been discovered later that E. coli cells can also be made competent to take up exogenous DNA by subjecting them to special chemical and physical treatments, such as high concentration of CaCl₂ (salt-shock), or exposure to high-voltage electric field. Under such artificial conditions, the cells are forced to take up foreign DNA bypassing the transport system operating in naturally transformable bacteria. The type of transformation occurring in E. coli is called artificial. In this process, the recipient cells are able to take up double-stranded DNA fragments which may be linear or circular.

In case of artificial transformation, physical or chemical stress forces the recipient cells to take up exogenous DNA. The incoming DNA is then integrated into the chromosome by homologous recombination mediated by RecA protein.

The two DNA molecules having homologous sequences exchange parts by crossing over. The RecA protein catalyses the annealing of two DNA segments and exchange of homologous segments. This involves nicking of the DNA strands and resealing of exchanged parts (breakage and reunion).

A generally accepted model explaining homologous recombination is diagrammatically shown in Fig. 9.98:

Co -Transformation:

It is obvious that if the exogenous DNA that enters a recipient bacterial cell, contains known marker genes, say x, y and z, then these genes appear in the trans-formants, provided the segment or segments containing these genes are successfully integrated into the host chromosome. When x and y or x and z or y and z appear in the same trans-formant, the phenomenon is called co-transformation and the particular trans-formant is called a co-trans-formant.

Naturally, the probability of co-transformation and hence the frequency of co-trans-formants depend on the relative distance between the pair of marker genes. For detecting co-transformation, the recipient bacterium must have the corresponding recessive genes, x ,y^– and z^–, because only then the presence of the x, y and z genes can be detected. For convenience, the x, y and z genes may be represented as x⁺, y⁺ and z⁺.

Co-transformation frequency may be used for preparing gene-maps. Thus, if it is observed that x⁺ y⁺ trans-formants appear more frequently than x⁺z⁺, it can be concluded that x⁺ and y⁺ are closer to each oilier than x⁺ and z⁺.

As transforming DNA generally consists of fragments, a particular fragment may or may not contain a marker gene. If the fragment taken up by a cell does not contain any marker gene, there will be no transformation of the marker genes, although other genes not taken into consideration may be present. If the fragment taken up by the recipient contains a marker x⁺ or y⁺, or both markers x⁺ and y⁺, the trans-formants may have the genotypes, x⁺x-, y⁺y- or x⁺x-y⁺y^–. Only the last genotype represents a co-trans-formant.

Now, if the probability of x⁺x^– and y⁺y^– trans-formants in the population is 10^-3 each, then the probability of co-transformation of x⁺x^–y⁺y^– will also be 10^-3 provided x⁺ and y⁺ are present in the same DNA fragment. But if x⁺ and y⁺ are present on different DNA fragments, the probability of taking up the two fragments simultaneously will be 10^-3 x 10^-3 i.e. 10^-6.

The same argument holds good for all the pairs. The probability of x⁺, y⁺ and z⁺ occurring on different fragments being co-transformed is much less, in the order of 10^-3 x 10^-3 x 10^-3 i.e. 10^-9. On the other hand, if x⁺, y⁺ and z⁺ occur in the same fragment, the probability of the three genes being co-transformed will be 10^-3. Thus, from the probability measurements, it is possible to construct a gene map showing relative distances between the genes, as well as their order in the chromosome.

The principle of using co-transformation as a tool for gene mapping is illustrated in Fig. 9.99:

Mode # 2. Transduction:

Transduction is another mode of horizontal gene transfer in bacteria, mediated by transducing bacteriophages which act as vehicles of DNA transfer from one bacterium to another, generally belonging to the same species, because species, because phages are host-specific.

Two types of transduction are distinguished:

i. Generalized transduction and

ii. Specialized or restrict transuction.

In the first type, bacteriophages infect a host bacterium and progeny phage particles are assembled. Some of these progeny bacteriophages incorporate host DNA in their heads.

When such bacteriophages infect another host cell after being released by lysis, those carrying the host DNA also inject it into the cell, where it can be incorporated into the bacterial chromosome resulting in genetic recombination. In restricted transduction, temperate bacteriophages take part which have two alternative types of life cycles, – a lytic cycle and lysogenic cycle. The temperate phage carry both phage carry both phage DNA and occasionally also some portions of host DNA taken from restricted sites of the host chromosome.

When the released phage particles infect new host cells, they transmit the phage DNA along with the fragments of host DNA. As the bacteriophage enters into a lysogenic state in the infected host, the phage DNA and the bacterial DNA of the previous host are integrated into its chromosome by exchange of parts resulting in genetic recombination.

(i) Generalized Transduction:

Transduction was discovered by Zinder and Lederberg in 1952 when they were looking for an E. coli type conjugation system in Salmonella typhimurium. A mixture of two cultures of auxotrophic mutants of this bacterium differing in contrasting characters was found to produce small number of prototrophic recombinants. For example, two populations of auxotroph’s having phenotypes, A⁺B^– and A^–B⁺ were mixed in growth medium, and, on incubation, they produced some bacteria having A⁺B⁺ phenotype.

To make sure whether the origin of prototrophs was due to conjugation, they took the two cultures in two arms of a U-tube separated by a glass filter which physically prevented cell to cell contact of the two populations which is essential for conjugation. Under this condition also, the prototrophs appeared suggesting that the gene exchange was not due to conjugation.

That it was not due to transformation was also proved by treatment with DNase which destroys free naked DNA. It was finally proved that the gene exchange was mediated by the bacteriophage P22 which infects S. typhimurium and some other species of Salmonella. This new mode of gene exchange leading to genetic recombination was designated as transduction.

In generalized transduction, the transducing phage infects a bacterial host in the usual way by attachment and injection of its DNA. Phage DNA and proteins are synthesized, and the bacterial chromosome is broken down to small fragments. During maturation of progeny phage particles, the host DNA fragments having approximately the same size as that of phage DNA are packaged inadvertently in some of phage heads.

After release by lysis of the infected host cell, such aberrant or defective phage particles may infect new host cells and inject the DNA into the cell. But because the DNA of these phages is of bacterial origin and does not contain phage genes necessary for replication, its life-cycle is not completed. Instead, the DNA can be integrated into the bacterial chromosome by homologous recombination.

In this way, one or more genes can be transferred from one bacterium to another. For example, if a transducing phage carrying a bacterial gene controlling motility infects a non-motile mutant bacterium, the latter may acquire the property of motility.

A characteristic feature of the transducing phages mediating generalized transduction is that any part of the bacterial chromosome can be transferred without any restriction regarding the site. In general, the fragment in a particular phage head is about one-hundredth part of the bacterial chromosome. Also, the frequency of an aberrant phage carrying bacterial DNA in its head in the total phage population is very low, not more than 1 in 10⁵ to 10⁷.

A well-studied example of generalized transduction is by phage PI of E. coli K12. PI is a temperate phage which has also a lytic cycle, like other temperate phages. PI DNA has a molecular weight of 5.9 x 10⁷ Daltons. It encodes a DNase which can cleave bacterial chromosome into fragments having molecular weight of 1 x 10⁷ to 1 x 10⁸ Daltons. As the phage particles are assembled following active replication, one of the host DNA fragments may be taken up by mistake and packaged into the head. Such phage particles become defective and they are the transducing phages.

Because the DNase coded by the phage cleaves bacterial DNA at random, the fragment packaged into a defective phage head may originate from any part of the bacterial chromosome. These defective particles are released along with large number of normal PI phages which are not transducing. For reasons, the DNA carried by the transducing phage particles can be injected into new host bacteria, but without replication and production of new progeny phages.

However, as the DNA of the transducing PI phages is of bacterial origin, it can be integrated into the chromosome of E. coli. For example, when phage PI is allowed to infect an ampicillin resistant strain of E. coli (amp^r), some of the transducing phages will package the fragment containing the amp^r gene. Such transducing phages on infecting an ampicillin sensitive strain of E. coli (amp^S) will transfer the amp’ gene, making the sensitive strain resistant.

A schematic representation of generalized transduction is shown in Fig. 9.100:

Like co-transformation, generalized transduction can also be used as a tool for gene mapping. In case a single fragment of bacterial chromosome contains two closely linked marker genes, both can be transduced into a host cell together resulting in co-transduction.

In the E. coli-Pl phage system, a number of pairs of genes have been found to be co-transduced quite often indicating their close linkage. For example, the genes controlling threonine (thr) and leucine (leu) synthesis, or leucine synthesis and azide resistance (azi^y), or streptomycin resistance (sir^r) and malate utilization (mal) are co-transduced quite frequently. On the other hand, markers which are located so far apart from each other that they cannot be included in a fragment of the size that can be packaged into a single phage head are never co-transduced together.

Thus, in E. coli-PI phage system, thr and leu pair or leu-azi^y pair are separately co-transduced, but thr-azi^y pair is never co-transduced. This means that thr-azi^y pair of genes are too far apart on E. coli chromosome to be included in a fragment that fits in the phage head. It is also evident that the order of genes is thr-leu-azi^y in the bacterial chromosome.

From the frequency of cotransductants, the relative distances of the marker genes can be determined. For example, if leu-azi^y  pair is more frequent among cotransductants than thr-leu, it would mean that leu is nearer to azi^y than to thr. Thus, by analysis of a large number contransductions, it is possible to prepare a gene-map of the organism concerned (Fig. 9.101).

Besides being used as an instrument for gene mapping, generalized transduction has been utilized widely for transferring genes in genetical research. The development of modern recombinant DNA technology has largely replaced the need of transduction being used for gene transfer. But in natural environmental conditions, transduction probably plays a significant mechanism for horizontal gene transfer.

(ii) Restricted Transduction:

Restricted transduction is mediated by certain temperate bacteriophages, like the λ-phage (lambda phage) of E. coli, which enters into a lysogenic relationship with the host bacterium. This type of phages normally does not cause a lysis of the host, instead the phage DNA is integrated into the host chromosome where it is replicated as a part of the chromosome for many generations in the prophage state.

Occasionally, the phage DNA can be released from the chromosome and can actively multiply to form many copies and synthesise phage proteins to form new progeny phage particles carrying out a lytic cycle. This changeover from lysogenic state to lytic cycle is known as induction.

The release of the phage DNA from the host chromosome — known as excision — is, on rare occasions, imperfect, so that a region of host DNA situated on either side of the prophage DNA may be included into the excised phage DNA. When such a defective phage DNA is packaged into a phage head, the progeny phage on being released and on infecting another host cell transmits the bacterial DNA along with its own DNA into the infected host cell.

On integration with the host DNA, the phage DNA along with the portion of bacterial DNA carried by it becomes part of the host chromosome, thereby causing transduction. Since this type of bacteriophages can be integrated at only specific sites of the host chromosome, they can cause transfer only of specific segments of host DNA from one cell to another.

Hence, this type of transduction is designated as restricted or specialized to distinguish from generalized transduction. Restricted transduction occurs only at a low frequency, generally in the order of 10^-6 to 10^-7 which indicates that incorrect excision is a rare phenomenon.

The best known agent mediating restricted transduction is the λ -phage of E. coli. This phage contains a double-stranded linear DNA molecule as its genome in the virion and has 4,650 base-pairs with 12 base-pair long single-stranded cohesive ends. When the phage infects an E. coli cell, the injected linear DNA circularizes with the complimentary single stranded cohesive segments and this circular DNA is integrated into the E. coli chromosome having about 4700 x 10³ base-pairs. Integration results in extending the length of the bacterial chromosome by about 1%.

The integration of the two circular DNA molecules, viz. λ-DNA and circular E. coli chromosome, occurs at specific sites of both DNAs and results in linear insertion of the λ-DNA into the circular chromosome. The attachment sites of λ-DNA and E. coli chromosome are known as POP’ and BOB’, respectively. During integration crossing-over takes place between these sites and the gene order of the λ-DNA is reversed (Fig. 9.102).

After integration, the λ-DNA remains as a prophage in the host cell which is then called a lysogen. In the lysogen, the λ-genes are kept in a repressed state by a repressor produced by a λ-gene which is the only viral gene which remains functional in the lysogenic condition.

The lysogenic host can grow indefinitely without the expression of the other λ-genes. However, the phage genes can be occasionally activated either spontaneously or by external agents, like UV-light — through a process known as induction. On induction, the phage DNA is excised from the bacterial chromosome again in a circular form by reversing the process of integration. While integration is catalysed by the enzyme integrase, excision is catalysed by excisionase. Excisionase binds to the integrase making it to reverse the process.

As a consequence, the original attachment sequences, POP’ and BOB’ are recreated and the λ-DNA is released from the chromosome in its circular form. Although excision is by and large accurate, on rare occasions in one cell per million or ten million (10^-6 to 10^-7) it may be inaccurate.

When such an event takes place, either the gal locus or the bio locus of E. coli chromosome may be included into the λ-DNA. Many of these inaccurate excisions produce DNA segments which are either too large or too small to be packaged into λ-head and they are lost. But aberrant excisions sometimes produce a DNA segment of correct length to fit in the phage head. The phage, progeny particles in such case contain either gal or bio loci in their heads along with λ-DNA.

However, these phage particles are often defective, because some parts of phage DNA may not be included into the DNA segment packaged into their heads. These gal or bio transducing λ-phages are unable to complete their life-cycle by themselves, because they are unable to perform certain functions controlled by the missing phage genes.

With the help of normal λ-phages, the specialized (restricted) defective gal or bio transducing phages, can grow in a host bacterium and integrate the gal or bio loci into its chromosome causing transduction of these genes. Aberrant excision producing λ-gal and λ-bio transducing phages and restricted transduction are diagrammatically shown in Fig. 9.103.

Mode # 3. Bacterial Conjugation:

The third mechanism of gene transfer in bacteria is conjugation. In this mechanism, DNA is transferred from a donor cell to a recipient by cell to cell contact. Conjugation has been found to occur in a number of bacteria. It was first discovered in 1946 by Lederberg and Tatum in E. coli. Other organisms capable of gene transfer through conjugation include Shigella, Salmonella, Pseudomonas etc. The ability to conjugate is conferred by genes present in plasmids. In E. coli this plasmid is known as the fertility factor or F-plasmid. The bacteria possessing the F-plasmid are considered as ‘male’ because they can act as donor in conjugation.

The recipient cell which is without the F-plasmid (F^–) is considered as ‘female’. In conjugation involving F⁺ and F cells, a copy of the fertility factor is transferred through a mating bridge from the donor to the recipient. As a result, the recipient also becomes an F⁺ cell and can act as a donor, while the original F⁺ cell continues to remain as F^– because a copy of the F-plasmid is retained. The ability to act as a donor, that is maleness, is due to genes in the F-plasmid.

The F-plasmid can either remain free in the cell i.e. independent of the chromosome, or it can be integrated as an episome in the chromosomal DNA. When an E. coli F⁺ cell with the free F-plasmid conjugates with an F^– cell, the plasmid alone is transferred without any transfer of chromosomal genes. Chromosomal gene transfer from the donor to the recipient occurs only when the F-plasmid is integrated into the chromosome resulting in the formation of an Hfr male (high frequency of recombination).

(i) F-Plasmid:

The F-plasmid of E. coli is a self-transmissible, low copy-number extra-chromosomal genetic element mediating its own transfer. The transfer process requires products of a good number of genes (about 40) which are located in the plasmid itself. Among the more important functions of these genes are those relating to the replication of the plasmid, production of sex or F-pili and the formation of a mating bridge between the conjugating F⁺ and F^– cells.

The F-plasmid is a comparatively large double- stranded circular DNA molecule having about 100,000 base pairs and a molecular weight of about 63 x 10⁶ Daltons. The plasmid replicates once in each cell cycle and the daughter molecules are segregated to daughter cells. Number of copies per cell is one or two.

Transfer of F-plasmid from a donor to a recipient is accompanied by replication of the plasmid. When the two conjugating cells come in contact and a mating-bridge has been formed, the plasmid replicates by the rolling circle model and a single-stranded copy is transferred to the recipient.

New DNA is synthesized in both the donor and the recipient, so that the plasmids in both cells become double stranded and circular by sealing the open ends through ligase. When a small number F⁺ cells is mixed with a large number of F^– cells, all cells become F⁺ after some time, because the F^– cells become F⁺ and can act as donors.

(ii) The Conjugation Process:

An F-plasmid gene confers the ability to produce long, hair-like appendages, known as sex pili (F-pili). These appendages establish contact with F-cells which are without sex-pili. The sex pilus is then retracted within the P cell, bringing the F cell to come in contact with the F cell. The enzymes coded by the F-plasmid are then used to build a mating bridge which establishes a direct contact between the two conjugating cells.

Next, replication of the F-plasmid begins by producing a nick in a single strand of the double- stranded plasmid DNA at a specific site, called the transfer origin (OriT). A protein (probably the nicking enzyme) itself remains bound to the 5′-end of the nicked DNA strand and effects transfer of the nicked strand into the recipient (F) cell through the mating bridge.

The replication of the plasmid DNA occurs by the rolling circle model in which the strand which is being transferred is regenerated by new DNA synthesis in the 5′ —> 3′ direction. Simultaneously, a DNA strand complimentary to the transferred strand is synthesized in the F cell. After completion of the transfer, the two regenerated strands are sealed by ligaso. The two cells separate and each of them now has a copy of the complete F-plasmid. Thus, both the donor and the recipient become F⁺.

The sequences of events in the conjugation process are shown in Fig. 9.104.:

(iii) Hfr-Cells and Transfer of Chromosomal Genes:

In F⁺ x F^– conjugation system, only the F-plasmid is transferred to the recipient and the chromosome plays no role. But in the pioneering experiments conducted by Lederberg and Tatum, they observed recombination of chromosomal genes.

Chromosomal gene transfer does occur, but only when the F-plasmid is integrated into the host cell’s chromosome leading to the formation of an Hfr-male. An Hfr-male can conjugate with an F^– recipient in a similar manner as does an F cell. Before going into the details of Hfr x F^– conjugation, the original experiments of Lederberg and Tatum which proved that gene exchange takes place by conjugation in E. coli may be briefly reviewed.

Lederberg and Tatum employed two double-auxotroph’s of E. coli K 12, each unable to synthesize two essential metabolites. One double-auxotroph was unable to synthesize biotin (B^–) and methionine (M^–) and the other was unable to synthesize threonine (T^–) and leucine (L^–). The genotypes of the two auxotroph’s may be written as B^–M^–T⁺L⁺ and B⁺M⁺FL^–. The first one required addition of biotin and methionine in the growth medium and in the absence of one or both of these growth factors, the double- auxotroph could not grow.

Similarly, the second auxotroph obligately required threonine and leucine in growth medium. Obviously, in a minimal medium which supports growth of wild-type E. coli K 12 containing none of the four growth factors, neither of the two double-auxotroph’s can grow. The justification behind the use of double-auxotroph’s was to rule out the possibility of reverse mutations arising spontaneously. If a reverse mutation — say from B^– to B⁺ or from M^– to M⁺ — occurs at a frequency of 10^-6, the chance of two mutations occurring simultaneously is only 10^-12 which is highly improbable.

When a mixture of the two double-auxotroph’s were allowed to grow in a medium containing all the four growth factors, viz. biotin, methionine, threonine and leucine and an aliquote was plated in a. minimal agar containing none of the growth factors, a small number of bacterial colonies were found to appear. These colonies evidently possessed the genotype B⁺M⁺T⁺L⁺ like that of the wild type E. coli K 12.

The results indicated that B⁺M⁺ genes of the B⁺M⁺T^–L^– double auxotroph have entered into the B^–M^–T⁺L⁺ auxotroph making, the B⁺M⁺T⁺L⁺, or, alternatively, TL⁺ genes of the B^–M^–T⁺L⁺ auxotroph have been transferred to the B⁺M⁺T^–L^– auxotroph. In either case, genetic exchange between the two double-auxotroph’s must have taken place.

That cell to cell contact was essential for such gene exchange was proved by growing the two strains (double-auxotroph’s) in two arms of a U-tube separated by a glass filter which prevented cell contact. No recombinants could be recovered under these conditions.

The principle of these experiments is diagrammatically represented in Fig. 9.105.:

An Hfr-cell originates when the F-plasmid is attached to its chromosome and the cell functions as a donor during conjugation with an F^– cell. The attachment of F-plasmid to the chromosome is a rare event, so origin of an Hfr-male in the population occurs only occasionally with a very low frequency.

The attachment sites of the E. coli chromosome contains insertion sequences which are capable of recognizing the F-plasmid DNA. There are several such attachment sites on the chromosome, each giving rise to a different Hfr-strain.

Integration of the F-DNA into the chromosome occurs by reciprocal recombination of a homologous sequence. The circular F-DNA becomes linear after integration and it accounts for about 2% of the total DNA (Fig. 9.106).

When an Hfr male conjugates with an F^– cell, the integrated F mediates transfer of chromosomal DNA into the recipient where recombination occurs between the transferred DNA and the recipient chromosome by crossing over. The initial events in an Hfr x P mating are similar to those of F⁺ x F^– mating. The mating cells, Hfr-male and the P recipient, are brought in contact with the help of sex-pili and a mating bridge is formed.

Next, the F-integrated chromosome begins to replicate by rolling-circle mechanism. Replication starts from OriT which is situated in the F-DNA. The free 3′-end of the elongating strand is transferred through the mating bridge into the recipient Pcell. Because the replication originates within the F-DNA, a small part of the F-DNA is transferred first followed by the chromosomal DNA strand, while the rest of the F-DNA remains attached at the rear end of the F-integrated chromosome.

It takes about 100 minutes for transfer of the entire F-integrated single stranded DNA into the recipient. However, a characteristic feature of Hfr x F^– conjugation is that the mating cells most often break apart before 100 minutes, so that only a part of the F-integrated DNA is transferred into the recipient. As a result, a part of F-DNA is left behind in the Hfr-donor.

Thus, in most of the Hfr x F^– mattings, the recipient remains P and does not become P. Another distinguishing feature between Hfr x F^– and F⁺ x F^– mattings is that the transferred F-DNA does not circularize due to the absence of the complete F-DNA. Also, the transferred F-DNA cannot replicate in the recipient, because all the genes necessary for independent replication of the F-plasmid are not present.

Only rarely the conjugation proceeds to completion when the recipient becomes P. Under such a circumstance, the F-DNA gets detached from the chromosomal DNA strand, generates a complimentary strand and circularizes to produce an F-plasmid, making the recipient P male.

After the mating partners break apart, the part of DNA transferred is replicated in the usual way. This double-stranded DNA segment containing genes of the Hfr-chromosome are recombined by crossing over with the homologous segment of the chromosome of the F^– recipient effecting genetic recombination. Such re-combinations were observed by Lederberg and Tatum in their experiments.

The events occurring in Hfr x F^– mattings are schematically shown in Fig. 9.107:

The major differences between F⁺ x F^– and Hfr x F^– conjugation systems are summarized in Table 9.7.:

(iv) Interrupted Mating and Gene-Mapping:

The time taken for transfer of the F-integrated Hfr chromosome of E. coli is 100 minutes under optimal conditions, though the transfer of the entire chromosome is of rare occurrence. The mating pair usually breaks apart before 100 minutes. The separation of mating partners can also be effected artificially by vigorous agitation in a blender.

This is called interrupted mating technique. By adopting such technique at different time intervals after the two mating partners i.e. Hfr and F^– cells are mixed, it is possible to detect the genes that have been transferred from the donor to the recipient at any particular point of time. The identification of the genes transferred at different intervals in case of a particular Hfr-donox makes it possible to determine the gene order and to prepare a gene map.

In such a map the distance between genes is expressed in terms of time, taking the whole E. coli chromosome to be 100 minutes long. By employing different Hfr-strains in which the F-DNA is integrated at different sites of the circular chromosome, it has become possible to construct the entire gene-map of E. coli containing about 2,000 genes. The relative distances between genes are shown in minutes. A simplified representation of the map of E. coli is shown in Fig. 9.108 with a few selected genes.

The important features of interrupted mating between Hfr x F^– cells of E. coli are briefly mentioned below:

(a) Transfer of Hfr chromosome to P cell begins at a particular point on chromosome determined by the site of integration of the F-plasmid. This means that in different Hfr-strains, transfer begins at different joints of Hfr-chromosome.

(b) The integrated F-DNA nicks at the OriT locus and a part of the F-DNA forms the leading end of the Hfr-chromosome being transferred (see Fig. 9.107). The chromosomal genes enter into the recipient in a linear order. The first gene is the one just behind the part of F-DNA forming the leading end. Other genes follow successively.

(c) The time of entry of any specific gene is the time taken by it to enter the recipient calculated from the time of mixing the Hfr and F^– cells. The time of entry, expressed in minutes, is determined by interrupted mating technique.

(d) The number of F^– cells showing the presence of a specific gene transferred from Hfr-donor increases with time till a maximum is reached. This happens because all donor cells of an Hfr-strain do not start transferring genes at the same time. The maximum is reached because all do not cells present in the population have transferred the particular gene to the recipients. A graphical representation of entry of two hypothetical genes x and y is shown in Fig. 9.109.

(e) Though under appropriate selective conditions, a gene entering into a recipient cell can be expressed as soon as it enters, permanent genetic recombination occurs only after the gene has been incorporated into the F^–-chromosome by homologous recombination.

The incoming genes, transferred from Hfr strain, replace homologous genes of F^–. The replaced segments of DNA containing these genes, as well as the segments of Hfr-chromosome which have not been incorporated into the F^– chromosome, are eventually hydrolysed by cellular nucleases.

(f) In different Hfr-strains of E. coli, the F-plasmid is integrated at different loci of the chromosome. There are numerous sites where the F can be integrated. As a result, the linear order of genes during entry into recipient is different in each strain of Hfr. Moreover, the F-plasmid can be integrated in two different orientations. This also changes the linear order of genes.

The gene orders in three hypothetical Hfr-strains are diagrammatically illustrated in Fig. 9.110:

A composite gene-map of the circular chromosome can be produced by combining the gene sequences of the transferred chromosomal segments of different Hfr-strains, even if the transferred portions are in fragments as shown in Fig. 9.111:

(g) In order to detect the entry of a gene in the recipient cell, it becomes necessary to identify such cells in a mixed population of both Hfr cells and F^– cells. Such identification needs the use of marker genes present hi both the donor and the recipient. Auxotrophy for various amino acids or other metabolites and antibiotic resistance are often used as useful markers. For example, an F^– strain resistant to streptomycin (Str^r) can grow in a medium containing an inhibitory concentration of the antibiotic, while a streptomycin-sensitive Hfr-strain (Str^s) fails to grow in such a medium.

Thus, in a mixed population of P and Hfr cells, the latter can be eliminated. Again, if the P strain is an auxotrophic mutant, requiring an essential metabolite, like leucine (leu^–), it fails to grow in a minimal medium unless it receives a gene for leucine synthesis (leu⁺) from the Hfr-strain. Thus, when a mixture of Str^r leu^– F^– cells and Str^s leu⁺ Hfr cells is plated on a minimal agar containing streptomycin, neither the leu^– F^– cells nor Str^s Hfr cells can grow. Only the Str^r leu⁺ F^– cells will form colonies on such plates.

Thus, by adopting the interrupted mating technique and using different selected markers, it is possible to determine the time of entry of specific genes into the recipients. Many markers had to be used and large number of interrupted mating experiments had to be done for determining the gene map of E. coli.

(v) F-Plasmids and Sexduction:

Just as F-plasmids can be integrated into the chromosome, so they can also be occasionally excised from the Hfr-chromosome to produce a free plasmid. Sometimes, the excision does not occur in a precise manner by exact reversal of the integration. Instead, some portion of the chromosomal DNA bordering the integrated F-DNA is included in the excised F-plasmid. Such aberrant F-plasmids containing a fragment of the chromosome are designated as F’-plasmids. 

Different Hfr-strains in which F-plasmid has been integrated at different sites of chromosome may give rise to different F’-plasmids each having a different chromosomal fragment attached to it. The F’-plasmids can be transferred to F^– cells by the usual conjugation process. Thereby, the recipients not only acquire the F-plasmid DNA, but also the chromosomal fragment carried by the F’-plasmid. This process of chromosomal gene transfer via F’-plasmids from a donor to a recipient is known as sexduction or F-duction. The recipient cell becomes partial diploid (or merodiploid) for the genes carried by the chromosomal fragment attached to the F-plasmid (Fig. 9.112).