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

Abstract

SummaryBacteria that survive under variable conditions possess 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 of PvdS, a group IV sigma factor encoded by Pseudomonas syringae pv. tomato DC3000 (DC3000), a plant pathogen that is likely to encounter variations in nutrient availability as well as plant host defences. The gene encoding PvdS was previously identified by sequence similarity to the Pseudomonas aeruginosa orthologue, which directs transcription of genes encoding the biosynthesis of pyoverdine, a siderophore involved in iron acquisition, and is responsible for the characteristic fluorescence of the pseudomonads. We identified 15 promoters regulated by PvdS in DC3000 and characterized the promoter motif using computational analysis. 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. Hidden Markov models constructed from alignments of sequence motifs extracted from DC3000 and PAO1 were used to query genomes of DC3000 and other fluorescent pseudomonads for similar motifs. We conclude that the role of PvdS as a regulator of pyoverdine synthesis is conserved among the fluorescent pseudomonads, but the promoters recognized by PvdS orthologues may differ subtly from species to species.

Effect of Iron Concentration on the Growth Rate of Pseudomonas syringae and the Expression of Virulence Factors in hrp -Inducing Minimal Medium

“…RNA was treated with Turbo DNase (Ambion, Austin, TX) to remove residual DNA and then cleaned and concentrated using a MinElute kit (Qiagen). Removal of DNA was verified by quantitative real-time PCR (23). Real-time PCR was performed by using the IQ5 sequence detection system (Bio-Rad) and iQ SYBR green Supermix (Bio-Rad) in accordance with the manufacturer's protocols.…”

“…One hundred nanograms of total RNA was reverse transcribed in a thermocycler with an iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer's instructions. One milliliter of the resulting total cDNA population was mixed with 0.4 M concentrations of each primer previously reported (23,25) and 10 l of iQ SYBR green Supermix (Bio-Rad) in a 20-l final volume. The PCR assay was carried out with one cycle at 95°C for 2 minutes and 30 seconds, followed by 32 cycles at 95°C for 15 s and 60°C for 30 s. The amount of double-stranded DNA in each sample was determined at the end of every PCR cycle by measuring fluorescence, which is generated by the incorporation of SYBR green dye.…”

Transcriptome Analysis of Pseudomonas syringae Identifies New Genes, Noncoding RNAs, and Antisense Activity

“…We along with others have employed molecular and computational approaches to identify regulatory factors and genes used by DC3000 to adapt to specific environmental conditions (17,28,29,45,71). Although these studies have provided important information concerning the transcriptional regulation of predicted genes, more studies aimed at determining the location of promoters and regulatory sites, operon membership, and the identification of noncoding RNAs (ncRNAs) are needed to establish more complete and detailed regulatory gene networks for DC3000.…”

Transcription factor function and promoter architecture govern the evolution of bacterial regulons

“…First, it has been suggested that TFs are more likely to evolve if they acquire new target genes (7) and, unlike closely related eukaryotic organisms, closely related bacterial species often exhibit substantial differences in gene content, so that orthologous TFs control largely distinct gene sets (11)(12)(13)(14)(15). Second, the position and orientation of a TF binding site within a bacterial promoter (i.e., the promoter architecture) are critical for gene transcription (16).…”