SigB modulates expression of novel SigB regulon members via Bc1009 in non-stressed and heat-stressed cells revealing its alternative roles in Bacillus cereus
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PROVIDER: PRJNA883372 | ENA |
REPOSITORIES: ENA
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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.
2023-03-02 | GSE213958 | GEO
Project description:To investigate whether salt-stress dependent activation of SigB is mitigated when cells are pre-loaded with the compatible solute glycine betaine, we analyzed salt induction of the SigB regulon in the presence of the precursor choline at a genome-wide level. The median expression value of SigB-regulated genes was 1.7-fold higher in cells that received choline immediately before salt stress compared to cells where choline was added 60 minutes prior to the salt stress.
2020-03-01 | GSE141882 | GEO
Project description:In several gram-positive bacterial genera including Bacillus, Staphylococcus, and Listeria, sigma B (σB) has been identified as a stress-responsive alternative sigma factor responsible for initiating transcription of genes (the σB regulon) involved in response to stress-inducing environmental conditions. In L. monocytogenes, a foodborne pathogen of considerable threat to public health and the food industry, σB is involved in regulation of stress response and virulence gene expression. We have defined the σB regulon in L. monocytogenes during early stationary phase and under salt stress (0.3M NaCl) conditions using whole-genome microarrays, identifying 168 genes that generated ≥2.0-fold higher signals in the parental strain 10403S than in an isogenic sigB null mutant (ΔsigB), categorized into nine functional groups including stress-response genes (12), virulence genes (5), and genes related to transport (26) and metabolism (45). To gain a broader biological perspective of the σB stress response system, we applied these microarrays to Listeria innocua under the same environmental conditions. Our studies revealed 64 candidates in the L. innocua σB regulon with ≥2.0-fold higher signals in the parent than in a ΔsigB mutant; 49 of the 64 genes overlap with the L. monocytogenes σB regulon, indicating extensive overlap in σB-controlled genes between the two species. Further transcriptional analysis using TaqMan quantitative real time RT-PCR was performed for selected genes that displayed contrasting fold changes among the four microarray data sets (two stress conditions per species). We report novel members of the L. monocytogenes σB regulon, as well as the initial definition of the L. innocua σB regulon. Our comparative studies of the σB stress response systems in L. monocytogenes and L. innocua revealed features of the σB regulon that are conserved and unique to the two species. Keywords: Listeria monocytogenes, Listeria innocua, SigB regulon, salt stress, stationary phase
2007-11-29 | GSE7492 | GEO
Project description:Listeria monocytogenes SigB and PrfA are pleiotropic regulators of stress response and virulence gene expression, which have been shown to co-regulate genes in L. monocytogenes. We performed whole genome transcriptional profiling in the presence of PrfA* and active SigB, to identify the overlaps between the PrfA virulence regulon and the SigB stress response regulon. In L. monocytogenes, the PrfA* allele contributes to the activation of virulence genes to a level comparable to that of intracellular growing L. monocytogenes. Our results showed that the core PrfA regulon consists of 12 genes previously described as PrfA regulated. Furthermore, we found that the role of SigB during virulence gene regulation changes, dependent on the presence or absence of PrfA*. In the absence of PrfA*, SigB activated the transcription of virulence genes such as inlA and inlB. In the presence of PrfA*, SigB negatively influenced the transcription of genes in the PrfA core regulon. The observed effect of SigB on the transcript level of PrfA regulated genes was shown to reduce the cytotoxic effect of the PrfA* allele in HepG-2 cells. Our results indicate that the SigB-PrfA regulatory network is important for the adjustment of virulence gene transcription to ensure L. monocytogenes success as an intracellular pathogen. Keywords: comparison of gene expression of regulatory mutants
2008-10-01 | GSE11347 | GEO
Project description:Global expression analysis comparing gene expression of wild-type and sigB mutant in the SH1000 background
2016-11-14 | GSE87036 | GEO
Project description:Listeria monocytogenes SigB and PrfA are pleiotropic regulators of stress response and virulence gene expression, which have been shown to co-regulate genes in L. monocytogenes. We performed whole genome transcriptional profiling in the presence of PrfA* and active SigB, to identify the overlaps between the PrfA virulence regulon and the SigB stress response regulon. In L. monocytogenes, the PrfA* allele contributes to the activation of virulence genes to a level comparable to that of intracellular growing L. monocytogenes. Our results showed that the core PrfA regulon consists of 12 genes previously described as PrfA regulated. Furthermore, we found that the role of SigB during virulence gene regulation changes, dependent on the presence or absence of PrfA*. In the absence of PrfA*, SigB activated the transcription of virulence genes such as inlA and inlB. In the presence of PrfA*, SigB negatively influenced the transcription of genes in the PrfA core regulon. The observed effect of SigB on the transcript level of PrfA regulated genes was shown to reduce the cytotoxic effect of the PrfA* allele in HepG-2 cells. Our results indicate that the SigB-PrfA regulatory network is important for the adjustment of virulence gene transcription to ensure L. monocytogenes success as an intracellular pathogen. Keywords: comparison of gene expression of regulatory mutants The experimental design included 4 mutant strains of L. monocytogenes 10403S (PrfA*, delta prfA, delta prfA delta sigB, and PrfA* delta sigB), of which cDNA generated from 4 biological replicates were hybridized in all possible pairwise comparisons. Data were analyzed using a one way ANOVA in R/MAANOVA to determine significant differences in gene expression among the different strains. A two way ANOVA implemented in R/MAANOVA determined significant differences in gene expression due to the presence or absence of SigB and PrfA.
2010-05-19 | E-GEOD-11347 | biostudies-arrayexpress
Project description:Acclimation of cyanobacterium Synechocystis sp. PCC6803 to suboptimal conditions is largely dependent on adjustments of gene expression, which is highly controlled by the σ factor subunits of RNA polymerase (RNAP). The SigB and SigD σ factors are close homologues. Here we show that sigB and sigD genes are both induced in bright light and high temperature stresses. Comparison of transcriptomes of the control strain (CS), ΔsigB, ΔsigD, ΔsigBCE (SigD is an only functional group 2 σ factor), and ΔsigCDE (SigD is an only functional group 2 σ factor) strains in standard, bright light and high temperature conditions revealed that the SigB and SigD factors regulate different set of genes, and that SigB and SigD regulons are highly dependent on stress conditions. The SigB regulon is bigger than the SigD regulon at high temperature, whereas in bright light the SigD regulon is bigger the SigB regulon. Furthermore, our results show that favoring the SigB or SigD factor by deleting other group 2 σ factors do not lead to superior acclimation to bright light or high temperature conditions, indicating that all group 2 σ factors play roles in acclimation processes.
2022-01-19 | GSE192357 | GEO
Project description:Protein samples were measured using the LC-MS/MS platform containing reversed-phase nano liquid chromatography (nano Acquity M-class UPLC) (Waters Corporation, Massachusetts, USA), which was coupled to nanospray ionization tandem mass spectrometry with traveling wave ion mobility using high-definition data-independent (HD-MSE) acquisition and enabled with hybrid quadrupole orthogonal acceleration time of flight mass spectrometer (Synapt G2Si, Waters Corporation). The peptide mixture was separated on ACQUITY UPLC M-Class HSS T3 1.8 um, 75 um x 200 mm column (Waters Corporation) using a mixture of Buffers A (0.1% v/v Acetic acid in water) and B (0.1% v/v acetic acid in acetonitrile) through a linear concentration gradient of Buffer B in 170 min, with a flow rate of 300 nl/min from 5-26% (v/v). The eluents were sprayed at a voltage of 1.85-1.90 kV using PicoTip emitters (Waters Corporation), and other source parameters were set (sampling cone = 40V, source offset = 0 V, source temperature = 80C, gas flows, cone gas = 50 l/h, nano gas flow = 0.4 bar, purge gas = unchanged, IMS = optimized for wave velocity with a start velocity of 870 m/s to end at 564 m/s that corresponds to the separation of GluFib fragments in the drift time range of 0-200 bins). The data was acquired using the MassLynx software program version V4.1 (Waters Corporation) that automatically switches between MS and MS/MS (HDMSE) scans in resolution mode at 20000 with 1 second (sec) scan time and the scan range of 50-2000 m/z. Calibration was done by injecting GluFib at an interval of 1 min. The acquired data were analyzed for protein identifications against the Uniprot fasta database of B. cereus ATCC14579 strain that contained 5240 sequences via the PLGS v3.3 program (Water Corporation). Spectra were processed using low and high-energy thresholds of 135, 20 counts, and lock mass calibration 785.8456 m/z for GluFib. The workflow search parameters were set as: (protease = trypsin, one missed cleavage, carbamidomethyl for cysteine as fixed, variable modifier = oxidation of methionine). The protein was quantified when 1) the top 3 peptides had no modifications; 2) pass one match had peptide fragment one; and 3) ranked the first three highest peptides. The PLGS independent identification output was imported to ISOQuant 1.8 (Distler et al., 2014) for sample analyses. [doi:10.25345/C5Z892K4R] [dataset license: CC0 1.0 Universal (CC0 1.0)]
| MSV000090315 | MassIVE
Project description:The numerous sigma factors present in Mycobacterium tuberculosis (MTB) are indicative of adaptability to different environmental conditions. In this report we describe the sigma factor B (sigB) regulon and the phenotypes of a MTB sigB mutant strain exposed to different stresses like SDS and Diamide. This experiment set compares expression profiles between H37Rv wild type and H37Rv sigB null mutant as well as under different stress conditions. Both H37Rv wild type and H37Rv sigB null mutants were treated with either 0.05% SDS or 5mM Diamide for 60 min and their expression profiles were compared with untreated wild type or mutant controls.
2009-06-23 | GSE15976 | GEO
Project description:The numerous sigma factors present in Mycobacterium tuberculosis (MTB) are indicative of adaptability to different environmental conditions. In this report we describe the sigma factor B (sigB) regulon and the phenotypes of a MTB sigB mutant strain exposed to different stresses like SDS and Diamide. This experiment set compares expression profiles between H37Rv wild type and H37Rv sigB null mutant as well as under different stress conditions. Both H37Rv wild type and H37Rv sigB null mutants were treated with either 0.05% SDS or 5mM Diamide for 60 min and their expression profiles were compared with untreated wild type or mutant controls. Biological Replicate
2010-06-25 | E-GEOD-15976 | biostudies-arrayexpress