Isolation and characterization of host extract-induced transposon mutants of Pectobacterium carotovorum

Pectobacterium carotovorum is a gram-negative phytopathogenic enterobacterium that is responsible for diseases such as soft rot, black leg, and stem rot in many crops. The main pathogenicity factors that allow bacteria to cause disease are exoenzymes that macerate the cell wall of its plant host. The production of exoenzymes is highly induced in the presence of the host extract, but the genetic mechanisms by which this induction takes place is poorly understood. The goal of this research was to identify mutants of P. carotovorum in host extract-inducible genes that may play a role in pathogenesis. The objective of this research therefore is to isolate transposon mutants in genes that are inducible by host extract and to characterize the selected mutants. A promoter-less green fluorescence protein (GFP) transposon, pEVS168 was used to randomly mutagenize P. carotovorum strain KD100. The mutants were subsequently screened in media containing celery extract (CE) for GFP activity. Approximately 17,600 colonies were initially screened, of which 720 colonies showed high GFP activity. These were selected and grown on minimal medium without celery extract to determine which mutant\u27s GFP activity was a result of celery extract (CE) induction. There were 240 mutants that were induced on media containing CE. I Measured the GFP activity of the 240 mutants semi-quantitatively in Minimal Media with and without celery extract and found 30 mutants with high GFP activity. Three mutants were selected as they were deficient in protease (Prt) activity and also showed reduced pectinase activity in the enzyme analysis. These were designated R1, R2 and R3. Pathogenesis was done on these mutants, they all showed reduced virulence