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Genome-wide transcriptional analysis of the phosphate starvation stimulon of Bacillus subtilis

ABSTRACT: Bacillus subtilis responds to phosphate starvation stress by inducing the PhoP and SigB regulons. While the PhoP regulon provides a specific response to phosphate starvation stress, maximizing the acquisition of phosphate (Pi) from the environment and reducing the cellular requirement for this essential nutrient, the SigB regulon provide non-specific resistance to stress by protecting essential cellular components such as DNA and membranes. We have characterized the phosphate starvation stress response of B. subtilis at a genome-wide level using DNA macroarrays. A combination of outlier and cluster analyses identified putatively new members of the PhoP regulon, namely yfkN (2',3'-cyclic-nucleotide 2'-phosphodiesterase), yurI (ribonuclease), yjdB (unknown) and vpr (extracellular serine protease). YurI is thought to be responsible for the non-specific degradation of RNA, whilst the activity of YfkN on various nucleotide phosphates suggests that it could act on substrates liberated by YurI which produces 3` or 5` phosphoribonucleotides. The putative new PhoP regulon members are either known or predicted to be secreted and are likely to be important for the recovery of inorganic phosphate from a variety of organic sources of phosphate in the environment. Keywords: other

ORGANISM(S): Bacillus subtilis

PROVIDER: GSE2667 | GEO | 2005/11/16

SECONDARY ACCESSION(S): PRJNA92101

REPOSITORIES: GEO

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Project description:Background: The Bacillus cereus Sigma B (SigB) dependent general stress response is activated via the two-component RsbKY system, which involves a phosphate transfer from RsbK to RsbY. It has been hypothesized that the Hpr-like phosphocarrier protein (Bc1009) encoded by bc1009 in the SigB gene cluster may play a role in this transfer, thereby acting as a regulator of SigB activation. Alternatively, Bc1009 may be involved in the activation of a subset of SigB regulon members. Results: We first investigated the potential role of bc1009 to act as a SigB regulator but ruled out this possibility as the deletion of bc1009 did not affect the expression of sigB and other SigB gene cluster members. The SigB-dependent functions of Bc1009 were further examined in B. cereus ATCC14579 via comparative proteome profiling (backed up by transcriptomics) of wt, Δbc1009 and ΔsigB deletion mutants under heat stress at 42°C. This revealed 284 proteins displaying SigB-dependent alterations in protein expression levels in heat-stressed cells, including a subgroup of 138 proteins for which alterations were also Bc1009-dependent. Next to proteins with roles in stress defense, newly identified SigB and Bc1009-dependent proteins have roles in cell motility, signal transduction, transcription, cell wall biogenesis, and amino acid transport and metabolism. Analysis of lethal stress survival at 50°C after pre-adaptation at 42°C showed intermediate survival efficacy of Δbc1009 cells, highest survival of wt, and lowest survival of ΔsigB cells, respectively. Additional comparative proteome analysis of non-stressed wt and mutant cells at 30°C revealed 96 proteins with SigB and Bc1009-dependent differences in levels, 51 were also identified under heat stress, and 45 showed significant differential expression at 30°C, including proteins with roles in carbohydrate/ion transport and metabolism. Overlapping functions at 30°C and 42°C included proteins involved in motility, and ΔsigB and Δbc1009 cells showed reduced motility compared to wt cells in swimming assays at both temperatures. Conclusion: our results extend the B. cereus SigB regulon to >300 members, with a novel role of SigB-dependent Bc1009 in the activation of a subregulon of >180 members, conceivably via phosphotransfer-based interactions with other transcriptional regulatory networks.

Project description:Inorganic phosphate is an essential nutrient required by organisms for growth. During phosphate starvation, Saccharomyces cerevisiae activates the phosphate signal transduction (PHO) pathway leading to the expression of the secreted acid phosphatase, PHO5. The fission yeast, Schizosaccharomyces pombe, regulates expression of the ScPHO5 homolog (pho1+) via a non-orthologous PHO pathway. The genes induced by phosphate limitation and the molecular mechanism by which the genetically identified positive (pho7+) and negative (csk1+) regulators function are not known. Here we use a combination of molecular biology, expression microarrays, and chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) to characterize the role of pho7+ and csk1+ in the PHO response. We define the set of genes that comprise the initial response to phosphate starvation in S. pombe. We identify a conserved PHO response for the ScPHO5 (pho1+), ScPHO84 (spbc8e4.01c+), and ScGIT1 (spbc1271.09+) orthologs. We use ChIP-Seq to identify members of the Pho7 regulon and characterize Pho7 binding in response to phosphate-limitation and Csk1 activity. We demonstrate that activation of pho1+ requires Pho7 binding to a UAS in the pho1+ promoter and that Csk1 repression does not regulate Pho7 enrichment. Further, we find that Pho7-dependent activation is not limited to phosphate-starvation, as additional environmental stress response pathways require pho7+ for maximal induction. We provide a global analysis of the PHO pathway in S. pombe. Our results elucidate the conserved core regulon required for responding to phosphate starvation between distantly related ascomycetes and a better understanding of flexibility in environmental stress response networks. Schizosaccharomyces pombe 972 h- cells were starved for inorganic phosphate for 0, 30, 60, 120, or 240 minutes pior to microarray preparation to determine the extent and temporal resolution of the phosphate starvation response. At 120 minutes post-starvation we define a set of genes that are directly and specifically induced by phosphate starvation, providing a time-point at which all other experiments were performed. We characterize the pho7+- and csk1+- dependency of this PHO response at 120 minutes post-starvation in pho7+csk1+, pho7M-NM-^T, csk1M-NM-^T, and pho7M-NM-^Tcsk1M-NM-^T cells. In a seperate set of experiments we characterized the S. pombe stress response to copper limitation, iron limitation, and carbon switching at 120 minutes post-stress and osmotic shift at 20 minutes post-stress in both pho7+ and pho7M-NM-^T cells.

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Genome-wide transcriptional analysis of the phosphate starvation stimulon of Bacillus subtilis

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