## Doctoral Dissertations

Spring 1993

Dissertation

Microbiology

#### Degree Name

Doctor of Philosophy

Robert M Zsigray

#### Abstract

Virulence plasmid expression in the Yersinia has been shown to vary depending upon which species harbors the virulence plasmid suggesting that chromosomal regulation may in some way be involved. In this study, fertility plasmid expression in Y. pestis was investigated as a means of evaluating mechanisms of virulence plasmid regulation in this bacterium. The F plasmid was found to be minimally expressed in Y. pestis transferring at a rate of 8.8 $\times$ 10$\sp{-5\%}.$ In addition, Y. pestis failed to express F pili as determined by infection with the male specific phage MS2. When F plasmids containing at least 2 E. coli F regulatory genes were transferred to Y. pestis and monitored for MS2 sensitivity, they were found to confer MS2 sensitivity to Y. pestis.

In an attempt to determine if these known plasmid regulatory genes could influence virulence plasmid expression, three clones arcA, rfaH, and cpxA were observed in Y. pestis. All of these genes were found to increase the frequency of F transfer from Y. pestis, but none were able to alter virulence plasmid expression. The effects of anaerobic growth on plasmid expression were investigated. While anaerobicity was observed to have no effect on F plasmid expression, surprisingly virulence plasmid expression was found to be repressed in strains with and without the arcA gene.

Further analysis of two $\beta$-galactosidase insertion mutants of Y. pestis revealed that both yopJ and yopE genes failed to be induced by the normal stimuli when the mutants were grown anaerobically. However, it was noted that when these strains were returned to an aerobic environment plasmid expression returned to normal. Varying gas mixtures were studied for their ability to repress plasmid expression, but only the original mix of 85%N$\sb2,$ 5%CO$\sb2,$ and 10%H$\sb2$ was able to repress plasmid expression. NaHCO$\sb3$ at a final concentration of 10mM was found to relieve growth restriction aerobically suggesting that the anaerobic repression was due to the formation of HCO$\sb{3\sp-}$ in the presence of CO$\sb2$ and H$\sb2.$.

These results represent a new means of relieving Yersinia plasmid-mediated growth restriction in vitro which may be related to the physiological environment encountered in vivo by this bacterium.

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