| Summary: | Our understanding of the molecular basis for microbial pathogenicity has increased greatly over the last twenty years due to the application of molecular biology to the host/microbe interaction. This thesis used the same techniques to study the plant pathogen, Erwinia amylovora and its interaction with its rosaceous hosts. The transposon TnphoA was delivered to the E. amylovora chromosome and insertion was shown to be essentially random. Fusions between E. amylovora extracytoplasmic proteins and E. coli alkaline phosphatase were detected in cell envelope fractions of PhoA+ mutants indicating for the first time that sequences encoding signal peptides are present in E. amylovora. The virulence of a number of phenotypically PhoA+ transposon mutants was tested using apple seedlings. This identified a number of mutants altered in virulence. Further analysis of mutants with altered virulence identified four separate classes. The first class were auxotrophs in respect to amino acids or other growth requirements. The second class were altered in their expression of extracytoplasmic polysaccharide (EPS). The third class were altered in cell-shape. Finally the fourth class were identical to the wild-type apart from their altered virulence to apple seedlings. The transposon mutants altered in cell-shape were found to be defective in the expression of the E. amylovora equivalent of an E. coli inner-membrane penicillin binding protein (PBP2), responsible for rod-shape. Wild-type virulence and cell-shape were restored to these cell-shape mutants by complementation with cloned E.coli pbpA/rodA genes. The fourth class of transposon mutant altered in virulence resembled typical dsp mutants, identified by other workers. However, Southern blot analysis of the chromosomal DNA showed that the insertions did not map to the hrp/dsp region of the E. amylovora chromosome. Therefore, another region(s) of the E. amylovora chromosome, encoding functions involved in the virulence of this pathogen, was identified.
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