In this article we will discuss about:- 1. Transformation in Bacteria 2. Conjugation in Bacteria 3. Transduction.
Transformation in Bacteria:
In 1928, Fred Griffith, an English bacteriologist, made an important observation which initiated a biological revolution. He was working with the bacterium Pneumococcus pneumoniae (then called Diplococcus), the causal organism of ‘pneumonia’. He had both the virulent and the avirulent strains which differed in morphology and colony characters. The virulent strain was capsulated i.e., had a capsule around the cell and formed a smooth colony on growth medium. The avirulent strain was non-capsulated and formed a rough colony.
The virulent strain caused death of mice on being injected, while the avirulent strain was harmless. The heat-killed virulent strain failed to kill the mice. However, when the living avirulent strain and the heat-killed virulent strains were mixed and injected, the mice died. How?
Was the avirulent strain transformed into a virulent strain in association with the dead virulent bacteria? Yes! Griffith could isolate from the dead mice, bacteria, which had the characters of the virulent strain and proved virulent on being injected into other mice. This character was transmitted to the progeny as a stable heritable character.
Griffith called the phenomenon transformation and suggested that the heat-killed bacteria provided the transforming principle. He thought (erroneously), that the transforming principle was the polysaccharide of the capsule. The nucleic acids as bearers of hereditary characters were not known then.
The phenomenon caught the attention of medical bacteriologists and, during the subsequent years, it was found that the transformation could be achieved without involving mice, by growing the avirulent strains and heat-killed virulent strains together in Petri dishes. Transformation thus became a laboratory process.
In 1944, Avery, MacLeod and McCarty showed that DNA is a genetic material. After ten years of vigorous experimentation, these American scientists in 1944 identified the transforming principle—DNA. This was a big achievement which proved for the first time that DNA was the genetic material.
For pinpointing the transforming principle, they extracted DNA, polysaccharides and proteins from the killed bacterial cells in pure form, and added each separately to the avirulent cultures. It was only DNA that brought about the transformation. Protease and RNA degrading enzymes could not prevent the transforming action, whereas DNase inactivated the transforming activity. This confirmed DNA as the transforming principle.
After this, transformation in several other characters was noted, such as the transformation of streptomycin-sensitive cells to streptomycin-resistant cells, and tryptophan-independent strains to tryptophan-requiring strains.
A fragment of DNA, present in the medium, gets adsorbed on the recipient cells and then enters the cells. Inside the cell, it replaces the homologous part of the bacterial genome. This results in the development of new characters which pass on to the progeny cells as stable and heritable character.
To take up the external DNA, the recipient cell should be competent, i.e., must have binding sites for DNA at the surface, and must not have DNase that degrades DNA. Temperature and cations (Ca and Mg ions) enhance the efficiency of transformation. Relatively, few bacterial genera are competent or capable of taking up DNA naturally.
Conjugation in Bacteria:
In 1946, Lederberg and Tatum discovered conjugation in Escherichia coli. Edward Tatum, by inducing mutation, with the help of ultraviolet and X-ray irradiations, developed strains of E. coli (strain K12) which showed deficiency for some growth factors (auxotroph). These strains could be used as genetic markers to identify the introduction of new characters.
Tatum, with his student Joshua Lederberg, set about to look for genetic recombination in E. coli. They succeeded. Lederberg and Tatum reported genetic recombination in E. coli in 1946. In 1958, along with G.W. Beadle, they were awarded the Nobel Prize for explaining the genetic mechanism.
One parental strain (A– B– C+ D+ Sr Ps) requires factors A and B for growth and was resistant to streptomycin and sensitive to phage. The other parent (A+ B+ C–D– Ss Pr) required supply of growth factors C and D and was streptomycin sensitive and phage resistant. Individually, both the strains were unable to grow on minimal media (which lack any growth factor).
But when the two strains were mixed and then spread on the same minimal medium, colonies appeared and grew in the absence of any of the growth factors A, B, C, or D. How did that come about? The growth of the colonies suggests the development of recombinant bacteria which must be A+ B+ C+ D+ genetically.
When tested for streptomycin and phage resistance, these markers also appeared in new combinations. None of the parents were double sensitive or double resistant (Ss Ps or Sr Pr) but, the two colonies (3 and 4) were double resistant Sr Pr, as they grew on both streptomycin and phage-containing media. Three colonies viz., 6, 7, and 8 were doubly sensitive as they did not grow on any plate.
The exact mechanism of the genetic exchange was worked out by three groups of scientists led by Lederberg in Wisconsin, USA, Hayes in London, and Woolman in Paris. That transformation was not involved, was proved by the finding that cell-free extracts of one bacterium failed to transform the other cells.
It was demonstrated that in E. coli K12 strain, physical contact was involved and the DNA passed from one into the other cell through a conjugation tube. This was demonstrated by a method called the ‘interrupted mating experiment’. The process is vastly different from chromosome transfer in higher organisms.
Strains of E. coli show sexual differences, one acting as donor of genes (male) and the other as recipient of genes (the female). The donor strains produce tubular F pili, which establish contact with the recipient cell and serves as a conjugation tube for the passage of DNA. The maleness is bestowed by a fertility factor, called F factor (F plasmid) and not a chromosomal gene.
The F factor is now known to be a small circular DNA molecule (equal in size to the DNA of a bacteriophage) which is autonomous and lies free in the cytoplasm. In a conjugation between F+ and F the bacterial chromosome is not involved. Only a duplicate of the F factor passes into the female cell. The recipient is converted into a male cell. So, the sex in E. coli can be said to be ‘infectious’.
The F plasmid can live in two states—either free in the cytoplasm or integrated with a bacterial ‘chromosome’. When inserted in the ‘chromosome’, the F+ male becomes a Hfr male, ‘high fertility male’. It is a new type of male, which shows enormous increase in its frequency of recombinations. Super males of James Bond! When such a Hfr male conjugates with a female F–, the genetic material is transferred.
The female is not converted into a male as the F+ factor is not passed on. The genetic material may replace homologous portions of the female genome. This brings about genetic recombination. The bacterial chromosome breaks at the site of attachment and becomes a linear DNA molecule having the F factor always at the rear end.
With various Hfr strains, the site of the breakage varies. Chromosomal replication starts at that end which is directed towards the conjugation tube. According to Jacob and Brenner, the F replicator (replicator of the F factor DNA) serves as an initiation point for replication of the integrated DNA. One of the daughter chromosomes enters the female cell.
For the transfer of the complete replicate of the DNA, about 2 hours are needed, but in nature the mating never lasts so long. Due to interruption in the mating, only a portion of the chromosome enters the female cell; the F factor being always at the rear end never goes to the recipient cell.
By artificially interrupting the mating, the sequence in which the genes enter the female cell can be known. This sequence suggests that the bacterial chromosome is a circular DNA molecule. The sequence of genes remains the same though the site of entry varied. This would result by the breaking of the circular chromosome at different places. The direction of entry of chromosome can also be reversed. This is possible only with a circular molecule.
The F factor can spontaneously dissociate and go back to the cytoplasm again. The Hfr males are then reverted to F+ males. Now conjugation is reported in several bacteria.
Sexduction:
During the dissociation of the F factor from the ‘chromosome’, it may carry some of the bacterial genes. Such a mutant fertility factor is designated as F’ (F prime). When such F’ factors are transferred, the recipient bacterial cells become heterozygous for that part of the DNA which the F’ had obtained from the male bacterium. This phenomenon is called sexduction and is employed to obtain partial diploids.
Transduction in Bacteria:
Lederberg and his student Zinder in 1951 started looking for recombination in Salmonella typhimurium—the mouse typhoid bacterium. They used the same techniques which Lederberg and Tatum had used with E. coli. They obtained nutritionally-deficient mutants (auxotrophs) which failed to grow on the minimal medium.
When a mixture of the two mutants was plated together, recombinants appeared in a few cases but not with other strains. When they analysed the cause they discovered a new type of gene exchange, which involved the mediation of bacteriophages. Zinder and Lederberg described this new method of gene acquisition in 1952 as Transduction.
In their experiments, conjugation was ruled out by the following experiment. A U tube was taken which had a sintered glass filter between its two arms, through which bacteria could not pass. Two auxotrophs (=nutritionally deficient mutants) were grown in the two arms (strain A in one arm and strain B in the other). The medium from one could freely go into the other arm. Recombinants appeared in the bacterial population of one of the arms A only. Transformation was also ruled out as DNase (the DNA degrading enzyme) had no effect on the gene transfer.
It was found that the strain A was a lysogenic strain harbouring a temperate phage. A small amount of the phage was always present in the medium as, occasionally, some cells lysed and released phage particles. The phage particles are small enough to pass through the filter and attack the bacterial strain B in the other arm, which were sensitive to the phage.
During lytic cycle of the strain B, the viruses during maturation formed particles that accidentally contained a piece of bacterial DNA, in place of the normal viral DNA. These phages, called transducing phages, when attacking strain A, behaved as a temperate phage. The DNA of strain B, brought by the virus, integrates with the DNA of strain A. This integration results in recombination between the homologous portions of the recipient and acquired DNA. The genome of the recipient is modified.
Generalized and Specialized Transduction:
Generalized Transduction:
During lytic cycle of virulent phage, the DNA of the bacterial host is degraded into fragments. When the virions are formed during maturation the bacterial genes rather than viral genes get encapsulated. When these phages containing only bacterial genes infect new cells, they inject the bacterial DNA, which may integrate into the bacterial chromosome. Such phages which contain bacterial DNA instead of viral DNA are called defective phages as they do not cause death of cells.
Generalized transduction is also brought about by temperate phages during their lytic phase. They contain DNA fragment of lysed bacterium on their own DNA and transmit it to recipient lysogenic cell. The bacterial DNA brings about recombination by integration with the bacterial chromosome.
Specialized (=Restricted) Transduction:
This is caused only by temperate phages which integrate in the host chromosome. Bacterial genes close to the prophage can be transduced (transferred). Lambda phage in E. coli has been studied extensively for the specialized transduction. During the lysogenic cycle the phage genome is integrated with the bacterial chromosome.
When the lysogenic cycle enters the lytic cycle, the phage genome is excised from the bacterial chromosome. Usually the mechanism of excision is highly precise but occasionally (once in million excisions) one or more bacterial genes that were adjacent to the viral genome go with the viral DNA. After lytic cycle, the new progeny of temperate phages containing the bacterial genes enter the lysogenic cell and bring about transduction.
Abortive Transduction:
Sometimes the bacterial DNA, brought by the phage, does not integrate with the genome of the recipient bacterium and expresses itself independently. It does not replicate and so it passes on during binary fission to only one of the two daughter cells. For example the motile character transduced in a non-motile strain, at the time of cell division passes on only one of the daughter cells, as it is neither integrated with the chromosome nor does it replicate itself.
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