ARS | Publication request: Characterization of two small RNAs of Pseudomonas syringae DC3000

Submitted to: American Society for Microbiology Meeting
Publication Type: Abstract Only
Publication Acceptance Date: February 20, 2008
Publication Date: June 1, 2008
Citation: Filiatrault, M.J., Bronstein, P., Stodghill, P., Cartinhour, S.W. 2008. Characterization of two small RNAs of Pseudomonas syringae DC3000. American Society for Microbiology Meeting.

Technical Abstract: Small RNAs (sRNAs) are important components of many regulatory pathways and have been shown to have key roles in regulation of factors important for virulence. To date, only a few sRNAs have been described for the bacterial plant pathogen Pseudomonas syringae. One approach to identify novel candidate sRNAs is to globally search intergenic regions for promoter motifs and transcriptional terminator sites. A RpoN promoter motif was developed for P. syringae DC3000 and applied to the genome in combination with terminator predictions. This study focuses on two intergenic regions in the genome, which do not contain called ORFs and found to contain RpoN promoter motifs and predicted terminators. To investigate these candidate sRNAs, Northern blot analyses were performed using RNA isolated from DC3000 grown under iron-limited conditions. The determined sizes of the sRNAs were found to be ~400 nt each which were consistent with our predictions. This also provided evidence that these sRNAs are transcribed independently of downstream neighboring genes. 5’ RACE was used to determine the transcriptional start sites of the sRNAs. These results were consistent with the location of the predicted RpoN promoter region. Transcriptional start sites were also determined for neighboring genes and confirmed that the neighboring genes posses independent transcriptional start sites. For phytopathogenic bacteria such as P. syringae, RpoN is an important virulence regulator and is required for the elicitation of the hypersensitive response and modulating pathogenicity-related genes. To determine if the sRNAs play a role in RpoN-dependent phenotypes, strains over-expressing each of these sRNAs were constructed. Results demonstrated that one sRNA is involved in swarming motility. We are continuing studies to elucidate the mechanism of regulation of these sRNAs in P. syringae and to determine their role in pathogenesis. These sRNAs are conserved among the Pseudomonads therefore analysis of the regulation of these sRNA and their role in pathogenesis can provide information about global gene regulation in other Pseudomonads.