Leading regions of enterobacterial plasmids.

Gram-negative bacterial conjugation is a specialised replicative event that increases the population size of a plasmid during its horizontal transfer between organisms. Work reported in this thesis has focused on the genetic structure of the leading regions of enterobacterial plasmids. A leading region is the first segment of a plasmid to enter the recipient cell during conjugation. Some leading regions carry conserved loci. Examples are ssb encoding a single-stranded DNA binding protein; psiB which functions to inhibit induction of the bacterial SOS response; ardA which encodes antirestriction activity, and hok, a gene that contributes to plasmid maintenance. The objective of this work was to determine the distribution of these genes on enterobacterial plasmids and their arrangement relative to the origin of conjugative transfer (oriT). Sequences homologous to ardA were found on plasmid representatives of five (FV, B, I, K and N) out of 23 incompatibility groups tested. Mapping data shows that the ardA homologues are leading region genes, and subcloning experiments coupled with phenotypic assays provide evidence that the IncB and IncFV ardA homologues are functional antirestriction genes. Sequence data reveal that the ardA gene family has diverged by at least 60% at the nucleotide sequence level when compared to the prototype ardA gene of ColIb-F9. A non-homologous antirestriction gene, ardB, was only detected on IncN plasmids. Conserved ssb and psiB genes are known to be present on a number of enterobacterial plasmids representing nine different incompatibility groups (B, com9, I1, FI, FII, FIV, K and Y). It is reported that the leading region of an IncFV plasmid, F0lac, also carries conserved ssb-psiB genes. In common with previous findings, the genes are separated by a spacer of ~2.5kb, indicating their presence in a conserved module. Genes of the hok family of killer genes are found on the leading region of FI and FII plasmids. Southern hybridisation experiments revealed that IncI1 plasmids lack a member of the hok subfamily but contain a member of the pnd subfamily. Functional and hybridisation tests localised pnd to the trailing region, defined as the last segment of a plasmid to enter the recipient during conjugation. Hybridisation data also localised the pnd genes of B and K plasmids to be outside of the leading region. The finding that the killer genes are scattered is consistent with the concept that the ecological role of the hok gene family is in plasmid maintenance rather than in conjugation. The results show that while enterobacterial plasmid leading regions collectively contain some conserved genes, there is considerable heterogeneity in their combination and arrangement on different plasmids with respect to oriT. It is postulated that the difference reflects independent acquisition of modules during the evolution of the different plasmid types. An important finding is that leading region genes are each orientated such that the transcribed strand is the same as the transferred strand. This arrangement holds for plasmids ColIb (IncI1), F (IncFI), pKM101 (IncN) and F0lac (IncFV) and could be important for the expression of these genes. One possibility is that the genes are expressed from the incoming plasmid strand before it is converted into duplex DNA.