Project description:The plant pathogen Pseudomonas syringae pv. syringae B728a grows and survives on leaf surfaces and in the leaf apoplast of its host, bean. Global transcriptome profiling was used to understand the contribution of distinct regulators to B728a fitness and pathogenicity. We performed a transcriptome analysis of B728a and nine regulator mutants recovered from the surface and interior of leaves and exposed to various environmental stresses in culture. These mutants were nonpolar deletion mutants lacking a single target regulator gene: ahlR, aefR, gacS, retS, salA, rpoS, algU, hrpL and rpoN. RNA was collected from cells of these strains that were exposed to five treatments in vitro, namely a basal medium, sodium chloride to confer an osmotic stress, hydrogen peroxide to confer an oxidative stress, iron limitation, and nitrogen limitation, and were recovered from two bean (Phaseolus vulgaris L.) leaf sites, namely epiphytic sites after leaf surface inoculation and 72 h of growth and apoplastic sites after leaf infiltration and 48 h of growth. The results indicated that the quorum-sensing regulators AhlR and AefR influenced few genes in planta or in vitro. In contrast, GacS and a downstream regulator SalA formed a large regulatory network that included a branch that regulated diverse traits and was independent of plant-specific environmental signals, and a signal-dependent branch that positively regulated secondary metabolite genes and negatively regulated the type III secretion system. RetS functioned almost exclusively to repress secondary metabolite genes in cells in culture but not on leaves. SalA functioned as a central regulator of iron status, based on its reciprocal regulation of pyoverdine and achromobactin genes, and also sulfur uptake, suggesting a role in the iron-sulfur balance. Among the sigma factors examined, AlgU influenced many more genes than RpoS, and most AlgU-regulated genes depended on RpoN. RpoN differentially impacted many AlgU- and GacS-activated genes in cells recovered from apoplastic versus epiphytic sites, suggesting differences in environmental signals or bacterial stress status in these two habitats. Collectively, our findings illustrate a central role for GacS, SalA, RpoN and AlgU in global regulation in B728a in planta and a high level of plasticity in their response to distinct environmental signals.

Project description:The plant pathogen Pseudomonas syringae pv. syringae B728a grows and survives on leaf surfaces and in the leaf apoplast of its host, bean. Global transcriptome profiling was used to understand the contribution of distinct regulators to B728a fitness and pathogenicity. We performed a transcriptome analysis of B728a and nine regulator mutants recovered from the surface and interior of leaves and exposed to various environmental stresses in culture. These mutants were nonpolar deletion mutants lacking a single target regulator gene: ahlR, aefR, gacS, retS, salA, rpoS, algU, hrpL and rpoN. RNA was collected from cells of these strains that were exposed to five treatments in vitro, namely a basal medium, sodium chloride to confer an osmotic stress, hydrogen peroxide to confer an oxidative stress, iron limitation, and nitrogen limitation, and were recovered from two bean (Phaseolus vulgaris L.) leaf sites, namely epiphytic sites after leaf surface inoculation and 72 h of growth and apoplastic sites after leaf infiltration and 48 h of growth. The results indicated that the quorum-sensing regulators AhlR and AefR influenced few genes in planta or in vitro. In contrast, GacS and a downstream regulator SalA formed a large regulatory network that included a branch that regulated diverse traits and was independent of plant-specific environmental signals, and a signal-dependent branch that positively regulated secondary metabolite genes and negatively regulated the type III secretion system. RetS functioned almost exclusively to repress secondary metabolite genes in cells in culture but not on leaves. SalA functioned as a central regulator of iron status, based on its reciprocal regulation of pyoverdine and achromobactin genes, and also sulfur uptake, suggesting a role in the iron-sulfur balance. Among the sigma factors examined, AlgU influenced many more genes than RpoS, and most AlgU-regulated genes depended on RpoN. RpoN differentially impacted many AlgU- and GacS-activated genes in cells recovered from apoplastic versus epiphytic sites, suggesting differences in environmental signals or bacterial stress status in these two habitats. Collectively, our findings illustrate a central role for GacS, SalA, RpoN and AlgU in global regulation in B728a in planta and a high level of plasticity in their response to distinct environmental signals. RNA was collected from cells of B728a and nine regulator mutants following exposure to seven treatments. Each treatment was performed using two biological replicates, with B728a, M-bM-^HM-^FrpoS, M-bM-^HM-^FalgU, M-bM-^HM-^FhrpL and M-bM-^HM-^FrpoN examined in one laboratory, designated lab A, B728a, M-bM-^HM-^FahlR and M-bM-^HM-^FaefR examined in lab B, and B728a, M-bM-^HM-^FretS, M-bM-^HM-^FgacS and M-bM-^HM-^FsalA examined in lab C. Experimental methods were standardized across the three laboratories. For the in vitro treatments, exponential cells from two independent cultures were each exposed to the five treatments. For each treatment, the cells originating from the two cultures were pooled and the RNA was extracted; this was repeated in its entirety and the two RNA pools were combined. The resulting RNA representing four independent cultures served as a single biological replicate. Two biological replicates for each strain in each treatment were generated in this manner, with B728a included as a strain in each of the laboratories. Epiphytic populations of B728a and the nine regulator mutants were established following spray inoculation onto bean leaves and subsequent incubation; the epiphytic cells recovered from 400 to 600 leaves inoculated at one time served as a biological replicate, and the procedure was repeated to provide two biological replicates. Due to the availability of facilities, all of the epiphytic treatments were conducted at a single laboratory, lab B, with cultures provided from each of the other laboratories, and the cell pellets were returned to those laboratories for RNA extraction and analysis. Apoplastic B728a populations were established following vacuum infiltration into bean leaves and subsequent incubation; the apoplastic cells recovered from 40 to 80 leaves inoculated at one time served as a biological replicate, and two biological replicates were generated at each of the three laboratories. The RNA from all treatments was submitted to Roche Nimblegen, Inc where it was labeled and hybridized to an ORF-based microarray that included 5,071 ORFs and 61 putative sRNAs, with each ORF represented by 14 60-mer nucleotide probes. The fluorescence intensity for each probe was measured and subjected to robust multiarray averaging, which included adjustment for the background intensity, log2 transformation, quantile normalization and median polishing, and a robust estimated mean value was determined for each ORF and putative sRNA on the array.

Project description:The RpoN-RpoS alternative sigma factor pathway is essential for key adaptive responses by Borrelia burgdorferi, particularly those involved in the infection of a mammalian host. A putative response regulator, Rrp2, ostensibly is required for activation of the RpoN-dependent transcription of rpoS. However, questions remain regarding the extent to which the three major constituents of this pathway (Rrp2, RpoN, and RpoS) act interdependently. To assess the functional interplay between Rrp2, RpoN, and RpoS, we employed microarray analyses to compare gene expression levels in rrp2, rpoN, or rpoS mutants of parental strain 297. We identified 98 genes that were similarly regulated by Rrp2, RpoN and RpoS, and an additional 47 genes were determined to be likely regulated by this pathway. The substantial overlap between genes regulated by RpoS and RpoN provides compelling evidence that these two alternative sigma factors form a congruous pathway and that RpoN regulates B. burgdorferi gene expression through RpoS. Although several known B. burgdorferi virulence determinants were regulated by the RpoN-RpoS pathway, a defined function has yet to be ascribed to most of the genes substantially regulated by Rrp2, RpoN and RpoS, therefore providing additional avenues for future research into the genetic determinants contributing to virulence expression by B. burgdorferi. Keywords: gene profiling 297 (wt) vs rpoS mutant: 6 slides, 12 hybridizations (including dyeswaps), three biological replicates 297 (wt) vs rpoN mutant: 5 slides, 10 hybridizations (including dyeswaps), three biological replicates 297 (wt) vs rrp2 mutant: 6 slides, 12 hybridizations (including dyeswaps), three biological replicates 5A18 (wt) vs XY424.3 (rrp2 mutant): 5 slides, 10 hybridizations (including dyeswaps), three biological replicates 297 (wt) vs rpoS mutant: 6 slides, 12 hybridizations (including dyeswaps), three biological replicates 297 (wt) vs rpoN mutant: 5 slides, 10 hybridizations (including dyeswaps), three biological replicates 297 (wt) vs rrp2 mutant: 6 slides, 12 hybridizations (including dyeswaps), three biological replicates 5A18 (wt) vs XY424.3 (rrp2 mutant): 5 slides, 10 hybridizations (including dyeswaps), three biological replicates

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Pseudomonas syringae

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