|Thete,, Deepti - CORNELL UNIVERSITY|
|Myers,, Christopher - CORNELL UNIVERSITY|
Submitted to: American Society for Microbiology Conference
Publication Type: Abstract Only
Publication Acceptance Date: August 20, 2007
Publication Date: August 26, 2007
Citation: Swingle, B.M., Thete,, D., Moll, M., Myers,, C., Schneider, D.J., Cartinhour, S.W. Pyoverdine and beyond: PvdS dependent gene regulation in Pseudomonas syringae. American Society for Microbiology Conference. 159A:p. 90. Technical Abstract: The extracytoplasmic function (ECF) sigma factor PvdS regulates the expression of genes in Pseudomonas aeruginosa encoding virulence factors and the biosynthesis and transport of pyoverdine, a siderophore involved in iron acquisition. The production of pyoverdine is a distinctive trait of the fluorescent pseudomonads and the regulation of the genes associated with its biosynthesis and uptake is likely to be conserved among this group of bacteria. We have conducted a global search to identify genes regulated by PvdS in Pseudomonas syringae pv. tomato DC3000 (DC3000). To accomplish this goal we constructed a model of the PvdS regulated promoter motif (PvdS-box) by computational analysis of PvdS-regulated promoter regions obtained by screening a DC3000 genomic DNA promoter trap library. Scanning the DC3000 genome with the PvdS-box model identified 22 high-scoring matches to the motif. The validity of these predictions was verified using promoter-reporter fusion assays and/or quantitative real time (qRT)-PCR. We found that the majority of genes linked with the motif were pyoverdine related; however, there was also an interesting subset of genes (~35%) that were not recognizable as being involved with pyoverdine or iron metabolism. In addition to its utility in predicting PvdS-regulated genes, the PvdS-box model also suggests the identity of specific bases involved in the functional interaction of PvdS with its cognate promoters. We tested this idea using two independent methods. First, the location of the transcription start point downstream of the PvdS-box was identified using 5’-RACE. This analysis showed, for 7 regions tested, that the PvdS-dependent transcript initiated between 5 and 11 bp downstream of the motif. This result showed that the conserved domains of the motif are correctly spaced relative to the transcription start point to function as the -10 and -35 promoter elements. Second, we used a PvdS-dependent promoter-reporter fusion construct to examine the effect of nucleotide substitutions within the PvdS-box. This analysis revealed that mutagenesis of conserved nucleotides within the motif interferes with the promoter function. Collectively these analyses provide strong evidence that the PvdS-box motif identified functional elements of DC3000 PvdS-dependent promoters. Finally, comparisons of the DC3000 PvdS-box and the existing motifs associated with PvdS regulation in P. aeruginosa (eg. IS-box-16(n)-CGT) show that they share considerable sequence similarity, consistent with the idea that PvdS regulation is conserved among the fluorescent pseudomonads. However, our data suggests that there is a functional hierarchy between the -10 and -35 domains of the motif at the nucleotide level.