|Thete,, Deepti - CORNELL UNIVERSITY|
|Myers,, Christopher - CORNELL UNIVERSITY|
Submitted to: Molecular Genetics of Bacteria and Phage
Publication Type: Abstract Only
Publication Acceptance Date: August 26, 2007
Publication Date: August 7, 2007
Citation: Swingle, B.M., Thete,, D., Moll, M., Myers,, C., Schneider, D.J., Cartinhour, S.W. 2007. Extracytoplasmic Function (ECF) Sigma Factor Gene Regulation in Pseudomonas syringae: Integrated Molecular and Computational Characterization of PvdS-Regulated Promoters. Molecular Genetics of Bacteria and Phage. p. 262. Technical Abstract: The extracytoplasmic function (ECF) sigma factor PvdS regulates the expression of genes required for the biosynthesis and transport of pyoverdine, a siderophore that functions 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. In this work, we sought to determine the genes regulated by PvdS in the plant pathogen Pseudomonas syringae pv. tomato DC3000 (DC3000), including genes related to pyoverdine as well as novel genes elsewhere in the genome. To accomplish this we identified the conserved features 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 functional interactions with the PvdS-holoenzyme. 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 spaced relative to the transcription start point to function as canonical -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. Additionally, our results provide new information that suggests a functional hierarchy between the -10 and -35 domains of the motif. Finally, comparisons of the DC3000 PvdS-box and the existing motifs associated with PvdS regulation in P. aeruginosa show that they share considerable sequence similarity, consistent with the idea that PvdS regulation is conserved among the fluorescent pseudomonads. This has prompted us to evaluate the utility of the DC3000 PvdS-box model for identification of potentially PvdS-regulated promoters in other pseudomonads.