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Title: Characterization of the PvdS-regulated promoter motif in Pseudomonas syringae pv. tomato DC3000 reveals regulon members and insights regarding PvdS function in other pseudomonads

Author
item Swingle, Bryan
item THETE, DEEPTI - CORNELL UNIVERSITY
item Moll, Monica
item MYERS, CHRISTOPHER - CORNELL UNIVERSITY
item Schneider, David
item Cartinhour, Samuel

Submitted to: Molecular Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/7/2008
Publication Date: 5/1/2008
Citation: Swingle, B.M., Thete, D., Moll, M., Myers, C., Schneider, D.J., Cartinhour, S.W. 2008. Characterization of the PvdS-regulated promoter motif in Pseudomonas syringae pv. tomato DC3000 reveals regulon members and insights regarding PvdS function in other pseudomonads. Molecular Microbiology. 68(4):871-889.

Interpretive Summary: The ability of bacteria to sense their environment and regulate gene expression is critical for their survival. In the case of bacterial plant pathogens, cues from the environment may be used to signal the induction of factors necessary for virulence in a suitable host. In this work we have focused on the regulation of systems required for the uptake of iron in the plant pathogen Pseudomonas syringae pathovar tomato DC3000. Iron is an essential nutrient for virtually all forms of life, however it is available in extremely low concentrations. This is especially true in plant fluids where iron is sequestered in plant cells for use in metabolism and to prevent oxidative damage. P. syringae encodes several systems to obtain iron in extremely limiting conditions, likely successfully competing with plant iron-sequestration systems. P. syringae grows in the plant apoplast fluid where the iron concentrations are sufficiently low to induce these iron sequestration systems. This indicates that iron concentration is a possible environmental cue used by P. syringae to conditionally induce the iron sequestration systems and possibly other virulence factors necessary for survival in the plant. In this work we have characterized PvdS, a transcriptional regulator responsible for activating the expression of genes encoding pyoverdine, the principal iron uptake system in P. syringae. We characterized the DNA sequence that PvdS binds to, and used this to predict which genes are regulated by PvdS. Furthermore, we show that different regions of the DNA sequence bound by PvdS have different importance for the binding interaction and we draw comparisons with other pseudomonads (including the human pathogen P. aeruginosa) that suggest that this phenomenon is broadly applicable to the entire group of bacteria.

Technical Abstract: Species of bacteria with lifestyles that require adapting to changing environmental conditions to survive are equipped with an assortment of genetic regulators to meet these challenges. The group IV or extracytoplasmic function (ECF) sigma factors regulate gene expression in response to specific environmental signals by altering the promoter specificity of RNA polymerase. We have undertaken a study PvdS, a group IV sigma factor encoded by Pseudomonas syringae, which is widespread in the environment where individual strains are capable of causing host-specific disease on plants. The gene encoding PvdS in P. syringae pv. tomato DC3000 (DC3000) was identified by sequence similarity to the P. aeruginosa ortholog, which directs transcription of genes encoding the biosynthesis of pyoverdine, a siderophore involved in iron acquisition and responsible for the characteristic fluorescence of the pseudomonads. We have identified the conserved elements of promoters regulated by PvdS in DC3000 using conventional molecular genetic approaches in conjunction with computational analysis. Using this approach we identified and confirmed 16 transcription units that were regulated by PvdS in DC3000. A hidden Markov model was constructed from alignments of conserved sequence motifs (PvdS-box) and was used to predict genes regulated by PvdS in DC3000 and other related pseudomonads. Mutagenesis of conserved nucleotides within the motif interfered with promoter function and the degree of the effect was different depending on which region of the motif was mutated. Collectively these analyses provide strong evidence that the PvdS-box motif represents the functional elements of DC3000 PvdS-dependent promoters.