The work presented in this thesis involves the characterization of a small plasmid, pHD2, of approximately 2 kb in size from Bacillus thuringiansis subspecies kurstaki HDl-Dipel. The plasmid was cloned and sequenced and compared to other plasmids from Gram-positive bacteria for which sequence information was available and for which replication functions had been assigned. Homology between the predicted amino acid sequence of an open reading frame within pHD2 and the rep gene products of the pT181 group of staphylococcal plasmids suggested a common method of plasmid replication. The further identification of possible plus and minus origins indicated that pHD2 was a member of a family of plasmids replicating via a single-stranded DNA intermediate. Plasmid replication control in the staphylococcal plasmids pT181, pC221 and pS194 involves a negative control circuit using countertranscripts. Replication control in the case of pHD2 may utilize an alternative system involving the gene product of a second small open reading frame with homology to RepA of pLSl in which replication control is achieved by the binding of the repA gene product to the promoter region of the replication protein repB. pHD2 is the first plasmid from a Bacillus thuringiensis isolate to which replication functions have been assigned. The work presented here suggests pHD2 to be a member of the ssDNA family of plasmids and extends the range of such plasmids which have been characterized with the suggestion that this group contains, in addition to the highly related staphylococcal plasmids, a number of more distantly related members. Additionally, chimeric plasmids containing pHD2 and pBR322 have been demonstrated to show structural instability, although not segregational instability, in an alternative Bacillus thuringiensis host. Consequently, the use of such constructs in the cloning of heterologous genes in Bacillus thuringiensis in such a system may prove impractical at this stage with further work being required in order to overcome these problems and extend the exploitation of this industrially important family of entomopathogenic bacteria.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:303360 |
| Date | January 1990 |
| Creators | McDowell, David Gordon |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/108622/ |
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