ABSTRACT: Comparison of the expression profiles of the 630E JIR8094 strain and sporulation sigma factor mutants (sigE, sigF, sigG, sigK, spoIIID) in MS
Project description:Purpose: The goal of this study was to identify genes whose expression is induced during sporulation in a Spo0A-, σF-, σE-, σG-, and σK-dependent manner. Methods: Whole genome RNA sequencing was performed on wildtype, spo0A-, sigF-, sigE-, sigG-, and sigK- C. difficile strains (strain 630 background; JIR8094 = parent strain), and the transcriptional profiles of the different mutants during growth on 70:30 agar plates were determined using an Illumina HiSeq1000. Results: This analysis identified 185 genes whose expression is collectively activated by sporulation sigma factors: 150 were σF-dependent, 150 were σE-dependent, 30 were σG-dependent, and 31 were σK-dependent. A total of 237 genes were identified as requiring Spo0A for their expression when sporulation was induced on the 70:30 plates. Conclusions: These results provide the first genome-wide transcriptional analysis of genes whose expression is induced by specific sporulation sigma factors in the Clostridia and highlight that diverse mechanisms regulate sporulation sigma factor activity in the Firmicutes. For example, in contrast with the B. subtilis sporulation pathway, C. difficile σE was not required to fully activate σG, and σG was not required to activate σK.
Project description:Purpose: The goal of this study was to identify genes whose expression is induced during sporulation in a Spo0A-, M-OM-^CF-, M-OM-^CE-, M-OM-^CG-, and M-OM-^CK-dependent manner. Methods: Whole genome RNA sequencing was performed on wildtype, spo0A-, sigF-, sigE-, sigG-, and sigK- C. difficile strains (strain 630 background; JIR8094 = parent strain), and the transcriptional profiles of the different mutants during growth on 70:30 agar plates were determined using an Illumina HiSeq1000. Results: This analysis identified 185 genes whose expression is collectively activated by sporulation sigma factors: 150 were M-OM-^CF-dependent, 150 were M-OM-^CE-dependent, 30 were M-OM-^CG-dependent, and 31 were M-OM-^CK-dependent. A total of 237 genes were identified as requiring Spo0A for their expression when sporulation was induced on the 70:30 plates. Conclusions: These results provide the first genome-wide transcriptional analysis of genes whose expression is induced by specific sporulation sigma factors in the Clostridia and highlight that diverse mechanisms regulate sporulation sigma factor activity in the Firmicutes. For example, in contrast with the B. subtilis sporulation pathway, C. difficile M-OM-^CE was not required to fully activate M-OM-^CG, and M-OM-^CG was not required to activate M-OM-^CK. Wildtype, spo0A-, sigF-, sigE-, sigG-, and sigK- were streaked onto 70:30 plates in triplicate, and sporulation was induced. RNA from the three biological replicate samples was harvested, DNAse-I treated, and ribosomally depleted using Ribo-Zero kits. This RNA was transformed into cDNA, and the resulting libraries were subjected to paired end sequencing using an Illumina HiSeq 1000.
Project description:Purpose: A goal of this study was to identify genes induced in the mother cell during Clostridium difficile sporulation (specifically in a σE-, σK-, and SpoIIID-dependent manner). Methods: Whole genome RNA sequencing was performed on wildtype, sigE-, sigK- and spoIIID- C. difficile strains (strain 630 background; JIR8094 = parent strain), and the transcriptional profiles of the different mutants during growth on 70:30 agar plates were determined using an Illumina MiSeq1000. Results: This analysis identified 200 genes whose expression is collectively activated by sporulation sigma factors: 159 were σF-dependent, 162 were σE-dependent, 28 were σG-dependent, and 36 were σK-dependent. A total of 254 genes were identified as requiring Spo0A for their expression when sporulation was induced on the 70:30 plates. Conclusions: These results provide the first genome-wide transcriptional analysis of genes whose expression is induced by specific sporulation sigma factors in the Clostridia and highlight that diverse mechanisms regulate sporulation sigma factor activity in the Firmicutes. For example, in contrast with the B. subtilis sporulation pathway, C. difficile σE was not required to fully activate σG, and σG was not required to activate σK.
Project description:Bacillus subtilis’ sigma factor F is the first forespore specific transcription factor and controls genes required for the early stages of prespore development. The role of sigF is well studied under conditions that induce sporulation. Here, the impact of a sigF disruption on the transcriptome of exponentially growing cultures is studied by micro-array analysis. This is relevant because in various experiments, such as industrial fermentation, prolonged experimental evolution or zero-growth studies, sporulation is an undesirable trait that should be avoided, e.g by a sigF mutation. Under conditions that typically don’t induce sporulation, the transcriptome showed minor signs of sporulation initiation. The number of genes differentially expressed and the magnitude of expression were, as expected, quite small in comparison with sporulation conditions. The genes mildly down-regulated were mostly involved in anabolism and the genes mildly up-regulated were mostly involved in catabolism, in particular fatty acid degradation genes. Probably this is related to the arrest at sporulation stage II occurring in the sigF mutant where additional energy is required for further growth of the polar compartments formed.
Project description:Compartment-specific control of gene expression during Bacillus subtilis sporulation is governed by a cascade of four sigma factors, sigmaF and sigmaG in the forespore and sigmaE and sigmaK in the mother cell. In this work, we combined transcriptional analyses and transcriptional start site mapping to define the sigmaF, sigmaE, sigmaG and sigmaK regulons in Clostridium difficile. A total of about 225 genes were under the control of these sigma factors: 25 in the sigmaF regulon, 97 sigmaE-dependent genes, 50 sigmaG-governed genes and 56 genes specifically controlled by sigmaK. A significant fraction of genes in each regulon are of unknown function and we can propose new candidates for spore coat proteins synthesized under the control of sigmaE and sigmaK among proteins previously detected in the spore proteome (Lawley et al., 2009 (PMID 19542279)). Global analysis of developmental gene expression under the control of these sigma factors indicate deviations from the B. subtilis model regarding the communication between mother cell and forespore in C. difficile. We show that the expression of the sigmaE regulon in the mother cell is not strictly under the control of sigmaF despite the fact that the forespore product SpoIIR is required for the processing of pro-sigmaE. In addition, the sigmaK regulon is not controlled by sigmaG in C. difficile in connection with the lack of pro-sigmaK processing in this bacterium. However, a control of the forespore on sigmaK targets is maintained through a sigmaF-dependent regulation of sigmaK-controlled genes, a process that bypasses sigmaG. Here, transcriptional profiling of the Clostridium difficile 630E strain vs. a sigE mutant after 14h of growth in MS is performed. Two-condition experiment: 630E strain 14h vs. sigF mutant 14h. 4 biological replicates for each condition, including dye-swaps.
Project description:This SuperSeries is composed of the following subset Series: GSE35070: Comparison of the expression profiles of 630E JIR8094 strain and a ccpA mutant after 10h of growth in TY with 0.5% glucose. GSE35071: Comparison of the expression profiles of 630E JIR8094 strain and a ccpA mutant after 10h of growth in TY. GSE35072: Clostridium difficile CD630E JIR8094: growth 10h with 0.5% glucose in TY vs growth 10h in TY GSE35073: Clostridium difficile mutant ccpA CD630E JIR8094: growth 10h with 0.5% glucose in TY vs growth 10h in TY Refer to individual Series
Project description:Compartment-specific control of gene expression during Bacillus subtilis sporulation is governed by a cascade of four sigma factors, sigmaF and sigmaG in the forespore and sigmaE and sigmaK in the mother cell. In this work, we combined transcriptional analyses and transcriptional start site mapping to define the sigmaF, sigmaE, sigmaG and sigmaK regulons in Clostridium difficile. A total of about 225 genes were under the control of these sigma factors: 25 in the sigmaF regulon, 97 sigmaE-dependent genes, 50 sigmaG-governed genes and 56 genes specifically controlled by sigmaK. A significant fraction of genes in each regulon are of unknown function and we can propose new candidates for spore coat proteins synthesized under the control of sigmaE and sigmaK among proteins previously detected in the spore proteome (Lawley et al., 2009 (PMID 19542279)). Global analysis of developmental gene expression under the control of these sigma factors indicate deviations from the B. subtilis model regarding the communication between mother cell and forespore in C. difficile. We show that the expression of the sigmaE regulon in the mother cell is not strictly under the control of sigmaF despite the fact that the forespore product SpoIIR is required for the processing of pro-sigmaE. In addition, the sigmaK regulon is not controlled by sigmaG in C. difficile in connection with the lack of pro-sigmaK processing in this bacterium. However, a control of the forespore on sigmaK targets is maintained through a sigmaF-dependent regulation of sigmaK-controlled genes, a process that bypasses sigmaG. Here, transcriptional profiling of the Clostridium difficile 630E strain vs. a sigE mutant after 14h of growth in MS is performed. Two-condition experiment: 630E strain 14h vs. sigE mutant 14h. 4 biological replicates for each condition, including dye-swaps.
Project description:Sigma factor E (SigE) controls the expression of genes that are essential for Mtb virulence. In this work, we have identified the SigE regulon during infection of macrophages Our results indicate that SigE regulates the expression of genes involved in the maintenance of Mtb cell envelope integrity and function (i.e., detoxification and secretion). Keywords: strains comparison
Project description:To understand which gene(s) are affected by 2-methylcitrate to lead to the delayed sporulation in ∆prpD, we extracted the total RNA from BMB171 and ∆prpD at 12 h for whole genome transcription analysis by RNA sequencing (RNA-Seq). Our RNA-seq data showed that transcriptions of almost all of the SigF regulon genes, as concluded from the B. subtilis regulon, were significantly down-regulated in ∆prpD compared with the wild type BMB171. However, almost all the regulon genes of SigH, a sigma factor that functions upstream of SigF, were significantly up-regulated in ∆prpD. Those genes included spo0A, the early-expressed operon spoIIA and sigF itself. These results indicated that dysregulation of genes occurred on various levels. In particular, though, post-transcriptional inhibition of sigF functionality might subsequently lead to down-regulation of SigF regulon genes in the ∆prpD mutant.