Obligate micro-parasites must enter and compromise a living host cell in order to reproduce. Distinct from symbiosis and commensalism, parasitism results in host mortality or morbidity; the severity of which may be expressed in terms of virulence. Bacteriophage and viruses are ubiquitous in the environment, and localised co-existence of host with parasite is a priori evidence for mechanisms which act to mitigate virulence. When physical separation of host-parasite is not possible, molecular immunity to infection may be selected. The phage growth limitation (Pgl) system of soil bacterium Streptomyces coelicolor confers resistance to temperate phage ΦC31 and clear plaque (virulent) derivatives. A key component of the Pgl mechanism (pglX) is a putative methyltransferase, which resembles a type I restriction modification protein. Phase variable regulation represents an elegant mechanism for generating bi-directional genetic polymorphism; reversible phenotypic diversity. At ca. 10-3 – 10-4 DNA replications, translational phase variation of pglX results from slipped strand mispairing within a polyG-tract. Potential costs associated with the phage-resistant allele were considered. It was hypothesised that the Pgl phenotypes would be distinct in the absence of ΦC31. Growth differences were observed between phase variant S. coelicolor populations in the absence of ΦC31, indicative of Pgl-derived pleiotropy. Liquid infection studies were executed using virulent phage ΦC31cΔ25, designed to exert various degrees of selective pressure on independent cultures of each host phase variant. Under conditions designed to maximise phage-host encounter, the population and evolutionary dynamics of phage and host were interrogated. It was hypothesised that selection for the Pgl-positive allele would be rapid when host seeds were predominantly Pgl-negative. Shaken liquid microcosms were seeded with host, and exposed to ΦC31cΔ25 at various densities (MOI 10, 0.1, 0.001 and 0). Surviving host were purified of exogenous phage, processed, and progeny spores used to inoculate fresh microcosms. Phage selection was maintained at each respective MOI using ancestral (stock) ΦC31cΔ25. Phage adaptation was predictably constrained, however it was hypothesised that pglX phase variation would prevent ancestral ΦC31cΔ25 extinction at an intermediate MOI. At selected microcosm cycles, host populations were screened for phenotypic resistance to ΦC31cΔ25, and a novel adaptation of dHPLC (Transgenomic Inc) was used to resolve and make semi-quantitative the N+/-1 sized pglX alleles within a mixed PCR product. Using host total community DNA, the contribution of Pgl to supporting free ΦC31cΔ25 in addition to host phage-resistance was ascertained. ΦC31cΔ25 mutants bearing lysogenic characteristics emerged during the first infection cycle; a phenomenon reproduced in soil microcosms. While temperance often gives rise to lytic phage mutants in vitro, the converse was most unexpected, particularly during experimental regimes designed to direct evolution in favour of the host. Mutant ΦC31cΔ25 microcosm isolates produced turbid plaques when assayed using S. lividans TK24, and subsequent S. lividans lysogenic spores were superimmune to infection when screened on ΦC31cΔ25-impregnated agar.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:549503 |
| Date | January 2009 |
| Creators | Griffiths, Scott Andrew |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/3151/ |
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