Transduction

Transduction is the heritable transfer of bacterial DNA from one cell (the donor) to another (the recipient) by a bacteriophage.

Transduction was discovered by Zinder and Lederberg in 1952 (217, 218) during a search for genetic recombination in salmonellae. Expecting to find conjugative transfer, they grew two mutant strains together. Recombinants were indeed produced, but to the authors’ surprise, cell-to-cell contact was not required. Rather, recombination proved to be mediated by a DNase-resistant filterable agent which was later demonstrated to be identical to the bacteriophage P22. In 1955, Lennox (103) reported that, similarly, the temperate coliphage P1 could also act as a vector to carry out what has come to be termed generalized transduction.

Transducing bacteriophage particles are formed in donor bacterial cells during phage development. They are of one of two types, distinguished by the nature of the DNA molecule which they carry.

Generalized transducing particles, competent to mediate generalized transduction, carry a fragment of host DNA; specialized transducing particles, able to carry out specialized transduction, contain both host and viral DNA sequences as part of a single molecule. Such hybrid molecules are formed, in vivo, by aberrant excision of a prophage (or other recombinational event) to yield DNA molecules which can be both
replicated and packaged. Example; P1 and P22.

Specialized transducing particles arise at low frequencies, although, once identified, they can be propagated, with the aid of a helper phage if required, to yield high frequency transducing lysates. Specialized transductants are likely to be lysogens which are diploid for the transduced
host markers. The transduced DNA will have been added to the recipient genome and the transductants are able to produce transducing particles in their turn. (Example: Lambda) The range of host markers which can be transduced in this fashion is limited to those near prophage insertion sites or other sites of recombination between phage and host sequences unless the transducing particles have been engineered in vitro to contain other DNA. 

The products of generalized transduction are quite different from those of specialized transduction. Generalized transducing lysates, although composed primarily of infectious phages, contain, in addition, a small proportion of transducing which, on subsequent infection of a recipient culture, deliver a fragment of host DNA to a minority of cells. Generalized transducing particles completely lack DNA originating from the viral vector, containing instead only sequences of host origin.They arises when the viral genome sized fragments of donor DNA are packaged into phage heads in place of viral DNA. The process is called generalized transduction because any part of the host genome can be packaged and transferred in this way. Provided that transduced cells have not been coinfected with normal viral particles, they will have received only nonviral sequences. Stable transductants result from RecA-dependent replacement of recipient by transduced DNA, and progeny cells will thus be haploid for the transduced region and nonlysogenic. Generalized transduction need leave no evidence, other than a possible exchange of alleles, that it has occurred. 

In specialized transduction in case of Lambda, integration will always occur between gal (galactosidase) and bio (biotin) gene. In this case some portion of lambda remain and rest part of excession occurs with gal/bio genes. So in case of Lambda mixture of host and phage DNA is packed into the head of phage. This phenomenon is called specialized transduction. 

 

Events in the Donor Cell

Packaging mechanism uses only host DNA instead of concatemeric phage DNA. Transducing particles are formed when the phage packaging mechanism seizes upon host DNA, instead of concatemeric phage DNA, as a packaging substrate. The elegant experimental work which first permitted this conclusion was completed by Ikeda and Tomizawa in 1965. They studied the formation of P1 transducing phages by density labeling the DNA of donor cells with 5-bromouracil (BU) before infection and replacing the BU with thymine at the time of infection. Lysates were centrifuged through CsCl density gradients, and infective and transducing particles were assayed in each fraction (78). In control experiments in which either thymine or BU labeling was used throughout, transducing and infective particles had similar but distinguishable densities which reflected the differing GC/AT ratios of the two types of DNA. When, however, the medium was altered at the time of infection, density measurements clearly showed that transducing particles contained only DNA which had been present before infection; conversely, the infective particles contained only DNA made postinfection.

Litter or no specificity.The earliest evidence suggesting lack of specificity in packaging P1 transducing DNA was the observation by Ikeda and Tomizawa that all transducing particles, irrespective of the markers assayed (five separate markers were tested), had identical density profiles. If DNA density differs among chromosome segments of the size encapsidated in a P1 particle (and it does; analysis of the 0.5 Mb of available continuous sequence from the 80- to 90-min region reveals that P1-sized fragments vary in GC content from 51 to 53% [M. Masters and J. F. Collins, unpublished results]), it can be inferred that the particles carrying a particular marker are not likely to contain DNA identical in sequence, but rather a set of overlapping segments which have the selected marker in common. Consistent with this is the fact that no closely linked pairs of markers have been identified with cotransduction frequencies very much lower than anticipated, as would be found if they were seldom within the same transducing fragment. Lack of packaging specificity can also be inferred from the observation that the frequencies with which individual markers can be transduced by P1 do not vary over a very wide range; in several studies, a 10- to 20-fold range in frequencies, from 1 ´ 10–6 to 2 ´ 10–5 per infectious particle, was typically found. Masters , in a more extensive study, measured transduction frequencies for 26 markers and found that except for markers close to oriC (to be discussed further below), transduction frequencies varied over only a 10-fold range. Part of this variation originates in the gradient in gene dosage, from replication origin to terminus, characteristic of the exponentially growing donor cells used to prepare lysates, and much of the remainder is due to recombinational selectivity.

About 30% of the phage particles in a lysate contain host DNA rather than phage DNA. In a lysate approximately 1 in 1500 phage particles are present.

Unlike regular virions, transducing virions has a single molecule of a specific phage protein attached to each end. 

Events in the Recipient Cell

When a generalized transducing particle enters a recipient cell, 2% of the DNA can recombine  and integrated with the recipient chromosome. And 8% fail to transfer gene and these DNA are degraded. Alternatively, sometimes a phage protein binds to the ends of the transducing DNA, causing it to circularize and protecting it from nucleases. When this happens the transducing DNA is not a substrate for the RecBCD recombination pathway and thus remains in the cytoplasm as an “abortive transductant“. About 90% of donor DNA under go abortive transduction. Abortive transductants typically form tiny colonies that never grow to full size and usually fail to form colonies when picked and restreaked on a fresh plate.

The large colonies are “true transductants” due to recombination between the pro+ alleles on the linear transducing DNA and the chromosomal DNA, resulting in repair of the auxotrophic mutation. The tiny colonies are due to abortive transduction. The DNA on an abortive transducing particle can be transcribed and translated, allowing complementation of the chromosomal mutation. However, because an abortive transducing particle does not have an origin of replication it cannot be replicated. Everytime a cell divides only one of the two daughter cells will get a copy of the abortive transducing particle that complements the chromosomal auxotrophy. The other daughter cell will retain a proportion of the complementing proteins made before cell division, but it will only be able to continue growing until the gene products are degraded or too dilute to satifify the auxotrophic requirement. Hence, at each cell division only one of the two cells will be able to continue to divide and produce daughter cells, so instead of the cells reproduces exponentally (i.e. # cells = 2[# generations]) the abortive transductant only reproduces geometrically (i.e. # cells = 1 + [2 x # generations]) and most of the cells in the colony cannot reproduce when restreaked.

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