Generalized Transduction<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />
Transduction is a phenomena in which bacterial <?xml:namespace prefix = st1 ns = "urn:schemas-microsoft-com:office:smarttags" />DNA is transferred from one bacterial cell to another by a phage particle.Phage particles that contain bacterial DNA are called Transducing Particles.There are two types of transducing particles-generalized and specialized.
Generalized transducing phage produce some particles that contain only DNA obtained from the host bacterium rather than phage DNA& the bacterial DNA fragment can be derived from any part of the bacterial chromosome.
Generalized Transduction by the Salmonella typhimurium phage P22 & E.coli phage P1.P1 packages about twice as much DNA (approx.2 min.or 100 kb) as P22(Approx.1 min.or 50 kb),but certain P22 mutants are much more efficient transducing phage than P1. The mechanism of G.T. by P22 is better understood.
Mechanism: P22 is a temperate phage yhat infects Salmonella typhimurium by binding to the O-antigen,part of the lipopolysaccharide on the outer membrane.After infection,P22 circularize by recombition b/w terminal redundancies at each end of the phage DNA. During lytic growth ,the circular genome of P22 initially undergoes several rounds of replication. Rolling circle replication produce long cocatemeres of double strand P22 DNA.These concatemeres are packaged into phage heads by a heedful mechanism.
Packaging is initiated by a P22 nuclease that cuts P22 DNA initially at a specific sequence called a 'pac-site'.The head is filled with about 44kb of DNA,then the nuclease cuts the DNA &moves onto package a second heedful of DNA,into another phage head,typically about five heedful of DNA are package into another phage heads from each cocatemeres.Because the P22 genome is only 42 kb ,this yields the terminal redundancies at the ends of P22.Thus linear ds DNA is packaged into new phage particles.When the lyses,it released 50-100 new phage. Because G.T.is generally carried out by lytic phage.
There are sequenes on the Salmonella chromosome that are homologus to P22 pack site. When P22 site infects a cell, occasionally the P22 nuclease cuts one of these chromosomal sites DNA into P22 phage heads. These phage heads contain only bacterial Drano phage DNA.The P22 particles carrying bacterial DNA(transducing particles) can inject this DNA into a new host ,and the DNA can the recombine into the chromosome by homologus recombination. Because P22 can transfer DNA fragment from all regions of the chromosome, this is called Generalized Transduction.
Normally the P22 nuclease is quite specific, so only 1٪ of the phage host actually carry bacterial DNA.In addition, all genes are not transduced at the same frequency because cleavage of the chromosome is not truly random.
Generalized Transduction
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Abortive Transduction:
When a generalized transducing particle enters a recipient cell, the DNA can recombine with the recipient chromosome. 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". 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.
For example, when a Salmonella del(proBA) recipient is transduced with phage P22 grown on a prototrophic strain of Salmonella selecting for growth on minimal medium without proline, some large colonies appear and some tiny colonies appear. The large colonies reproduce when restreaked on the same medium, but the tiny colonies fail to reproduce when restreaked. Suggest an explanation for these results.
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.
Imagine what would happen if a phage incorporates host DNA instead of phage DNA inside a fraction of the produced phage heads. The cell would still lyse, and these novel particles, termed transducing particles, could still recognize an appropriate bacteria, attach, and inject their DNA (sometimes called a transducing fragment). The result would be the transfer of DNA from one host to another, since no phage DNA would be found in transducing particles of this type. This DNA transfer mechanism has been enormously useful for E. coli, S. typhimurium and B. subtilis, but such phage have not been identified and developed for most bacteria.
- How is this host DNA "mispackaged"? The most commonly used generalized transducing phages, P22 (infecting S. typhimurium) and P1 (infecting E. coli), package by "headfull packaging", so that they fill the head with a little more than one phage length of DNA. They typically recognize their own DNA as appropriate for packaging because of special sequences termed pac sites. Occasionally, the phage make an error and begin to package a host "concatamer", presumably because a site on the host DNA is reminiscent of the phage pac site. It is also conceivable that insertion sequences in the host might cause a transposition of the host DNA into the phage concatamer, thus allowing packaging of host DNA even when the packaging scheme started correctly on phage concatamers.
- How often does this "mispackaging" occur? For both P22 and P1, about one phage virion in l,000 actually contains host DNA. It is possible to obtain mutants which make errors more frequently. An example of this is the mutant termed P22HT which has completely lost the ability to recognize its pac sites so that it packages DNA randomly. Since an infected cell contains about half host and half phage DNA, the P22HT variant causes the generation of a phage lysate in which half of the phage heads contain host DNA. In the normal case, to the extent that mispackaging is due to pseudo-pac sites in the host DNA, one would expect that not all regions of the host DNA would be transduced at equal frequency. Such seems to be the case with wild-type P22 and this differential transduction is eliminated in mutants like P22HT.
- How is the transduced DNA stably inherited? As always, for the transferred DNA to become stably inherited, it must become associated with a replicon in the recipient cell. Since it is a linear molecule, this requires two homologous recombination events with the net result that a portion of the chromosome in the recipient is replaced by a portion of the incoming DNA. Thus, in contrast with specialized transduction, generalized transduction requires that the recipient possess both a functional rec system and DNA homologous to the transferred fragment. It has been observed in generalized transduction that there is an approximately l0% chance of the incoming chromosomal DNA being recombined into the recipient's chromosome. A recipient cell whose genotype and phenotype have been changed by a transduction event is called a transductant.
- What are the frequencies at which phage can generate transductants? The two phage referred to above carry l-2% of the host DNA and normally package chromosomal DNA at a frequency of 10-3. Thus, about one in l05 phage particles carries a given region of the host DNA. Since a given gene injected into a recipient cell by a transducing particle has a l0% chance of being recombined into the chromosome then one might expect to get approximately one transductant for a given region per l06 phage particles. In the case of the P22HT derivative, since approximately half of the phage particles carry chromosomal DNA, one can achieve the extraordinary frequency of one transductant per l000 phage particles. There is also a remarkable phage in B. subtilis that carries l0% of the host chromosome, though apparently its frequency of mispackaging is sufficiently low that the number of transductants per phage particle is around one in l07. In general, the virtue of generalized transduction is not its efficiency, but its ease of use. For strain construction, it is also an advantage that only a portion of the chromosome is transferred, in contrast to Hfr's.
