Date of Award

Winter 1988

Project Type

Dissertation

Program or Major

Microbiology

Degree Name

Doctor of Philosophy

First Advisor

Robert M Zsigray

Abstract

The Vwa plasmid (pYV019::Tn5) of Yersinia pestis was transduced from Escherichia coli LE392 to several F-containing strains of E. coli. Agarose gel electrophoresis of plasmid extracts prepared from the transductants revealed that both plasmids coexisted in these cells, at least to some degree, as autonomous cytoplasmic elements. Transductants demonstrably sensitive to the male specific phage MS-2 and thus carrying the F plasmid were used as donors in matings with several F$\sp-$strains. The results of these matings indicated that the Vwa plasmid was mobilized and cotransferred at a low frequency (0.001%) along with F. Agarose gel electrophoresis of plasmid extracts prepared from the transconjugants demonstrated the presence of both plasmids. Additional matings indicated that mobilization was neither a donor nor a recipient phenomenon and that mobilization was independent of recA function, making cointegrate formation of homologous regions of the plasmids unlikely. The frequency of pYV019::Tn5 transfer to recipient cells was 100-1000 times greater than can be accounted for by transposon-mediated cointegrate formation alone. It appeared likely that the Vwa plasmid of Yersinia pestis contained its own origin of transfer.

The non-mobilizable cloning vector pBR328 was used to create a BamH1 library of the Vwa plasmid. This library was used to clone an origin of transfer of the Vwa by selecting for those clones which mobilized pBR328 in the presence of a second conjugative plasmid (F). The origin of transfer of the Vwa plasmid was found to be contained in the BamH1-5 fragment. This fragment resulted in a ten fold increase in mobilization of pBR328 when compared to transposon-mediated (Tn 1000) cointegrate formation.

Hybridization studies between the Vwa plasmid and known conjugative plasmids from various incompatibility groups revealed that the Vwa plasmid shared sequence homology only to the Sma1-4 fragment of RP4. These results suggest the Vwa plasmid does not share extensive sequence homology with most plasmids of known incompatibility groups.

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