This work represents a comprehensive analysis of the precise structural relationship and organization existing amongst the gonococcal beta-lactamase-producing plasmids. The second major aspect of this work deals with investigations into the genetic and molecular characterization of the multiple origins of replication of the gonococcal beta-lactamase-producing plasmids. The Asia-plasmid, pJD4, a prototype gonococcal beta-lactamase-producing plasmid, is 7,426 bp and contains two large, direct repeats (DR-30A, 507 bp and DR-30B, 509 bp) which are implicated in the formation of deletion variant plasmids, such as the naturally-occurring Africa-type plasmid. The deletion observed in Africa-type plasmids, represented by pJD5, is 1,827 bp. One of the DR-30 repeats is also missing in the formation of Africa plasmids. The deletion in the Rio-type and several Toronto-type is 2,273 bp and the sequence spanning the deletion was identical irrespective of geographic or temporal origin. Thus, the Rio and Toronto-type plasmids are identical. The Nimes-type plasmid is proposed to be identical to the Africa-type but contains an IS 5 insertion sequence. Since IS5 has not been identified in gonococcal isolates and is not present in the gonococcal genome, it is suggested that this sequence was inserted after the original gonococcal plasmid was transformed into a strain of Escherichia coli. The New Zealand plasmid was shown to be an Asia-type plasmid which contains an endogenous tandem duplication of 1,883 bp. The direct repeat DR-2 is implicated in this duplication. Branch-point analysis by electron microscopy indicated that the Asia-type plasmid contains three origins of replication, named ori1, ori2, and ori3. Although pJD4 belongs to the incompatibility (Inc) group IncW, it also carries a silent IncFII determinant which is expressed when ori2 and ori3 are absent. The Africa-type plasmid was shown to carry only oril, belongs to the IncFII group, and, in contrast to pJD4, requires DNA polymerase I for replication. Plasmid constructs from pJD4 lacking oril but carrying ori2 and ori3 are incompatible with IncW plasmids, suggesting the ori2/ori3 region contains the IncW determinant. A novel replication initiation protein, RepB, was identified and shown to be necessary for ori2 and ori3 to function. The RepB is distinct from RepA, the replication initiation protein required for plasmids carrying ori1. (Abstract shortened by UMI.)
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/9115 |
| Date | January 2001 |
| Creators | Pagotto, Franco Joesph. |
| Contributors | Dillon, Jo-Anne R., |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Detected Language | English |
| Type | Thesis |
| Format | 174 p. |
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