Molecular characterization of novel pathogenicity genes of soft rot Erwiniae
Abstract
The bacterial pathogens Erwinia carotova subsp. carotovora (Ecc), Erwinia carotovora subsp. astroseptica (Eca), and Erwinia chrysanthemi (Echr) cause soft-rot disease in many plant species. Our laboratory had previously isolated prototrophic mutants of Ecc which display reduced virulence but are able to synthesize and secrete pectolytic enzymes in a normal fashion. I have constructed several genomic clone banks of these mutants' DNAs and isolated cosmid clones which complement these mutations. I have cloned and characterized adjacent DNA fragments (a 0.6 kb PstI-EcoRI fragment and a 5.0 kb PstI-SalI fragment) from a recombinant cosmid that complements the mutation in Ecc AH2552. Each of these fragments pleiotrophically inhibits production of pectate lyases, polygalaturonase, pectin methyl esterase, cellulase and protease in Ecc, Eca and Echr, and thereby imparts an nonpathogenic phenotype to these phytopathogenic bacteria. The effects of these two DNA fragments were observed only when they were present on a high copy number plasmid (pBR322) and not on a low copy number plasmid (pLAFR3). DNA mobility retardation experiments showed that a 174 base pair PstI-NcoI fragment from the 0.6 kb PstI-EcoRI fragment binds a factor(s) from Ecc protein extracts. The binding can be competed effectively by both the 0.6 kb PstI-EcoRI and 5.0 kb PstI-SalI fragments, but not by pBR322 DNA. I have determined the complete DNA sequence of the 0.6 kb PstI-EcoRI fragment. Comparison of the determined DNA sequence with those available in various gene data banks has indicated that the promoters of some cellulase, protease and pectate lyase genes have regions which are 60 to 100% homologous with various sequences on the 174 bp PstI-NcoI fragment. Based on these data, I hypothesize that the 0.6 kb PstI-EcoRI fragment competes for a common trans-acting factor required for activation of various pathogenicity genes, including those specifying hydrolytic enzymes, in soft-rot Erwinia species. This suggests that these Erwinia species specify a common transcriptional regulator which controls the expression of many virulence genes and the pathogenicity process.
Degree
Ph.D.
Advisors
Handa, Purdue University.
Subject Area
Molecular biology|Plant pathology
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