Elucidation of the osmoregulatory locus, ompRZ, in Erwinia chrysanthemi

Master Thesis


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University of Cape Town

Bacteria are constantly faced with harsh environmental conditions to which they have to adapt. These adaptive mechanism generally involve the use of two-component sensory systems, comprising of sensor proteins interacting with their cognate response regulator proteins. To survive fluctuating environments such as osmotic conditions, certain bacterial species employ the ompR-envZ (ompB) two-component system to monitor and respond to the osmotic cue. The EnvZ protein functions as the sensor and relays information regarding changes to the external environment, to the response regulator, OmpR. OmpR, in turn, regulates the porins, OmpF and OmpC in a reciprocal manner, so that one porin predominates over the other, depending on osmotic conditions. Erwinia chrysanthemi, which causes "soft rot" in a wide range of economically important crops, has been demonstrated to contain porin-like proteins similar to OmpF and OmpC. The expression of these porins was regulated in a similar manner to OmpF and OmpC with respect to medium osmolarity. Furthermore, preliminary studies have shown that changes in osmolarity affect the expression of pathogenecity genes. Evidence for an osmoregulatory system analagous to the ompB system of Escherichia coli was, therefore, sought. Primers specific for conserved regions in ompR were designed and used to PCR amplify a 631 bp fragment from E. coli. This fragment was cloned into the vector, pBluescriptSk, and end-sequenced to confirm its authenticity. The same strategy was followed, using envZ-specific primers to generate an E. coli envZ clone. Southern hybridisation analyses, using an ompR probe, confirmed the presence of an ompR homologue in E. chrysanthemi. An E. chrysanthemi genomic library was thus constructed and screened and a clone homologous to the ompR probe was isolated. The resulting plasmid, pRZ69, was partially characterised and determined to have both envZ and ompR homologues resident. Southern hybridisation analyses were employed to localise the ompR and envZ genes on the plasmid. A 1200 bp EcoRV-Pst1 fragment containing the ompR homologue and a 2000 bp EcoRV-EcoRV fragment containing the envZ homologue, were subcloned into pBluescriptSk, generating the plasmids, pRS1 and pZS2 respectively.

Bibliography: pages 148-171.