A network of small RNAs regulates sporulation initiation in C. difficile
Ontology highlight
ABSTRACT:
ORGANISM(S): Clostridioides difficile
PROVIDER: GSE213005 | GEO | 2022/12/31
REPOSITORIES: GEO
Similar Datasets
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.
2013-08-10 | GSE45977 | GEO
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.
2013-08-10 | E-GEOD-45977 | biostudies-arrayexpress
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.
2014-12-02 | GSE63777 | GEO
Project description:Clostridium difficile is an important nosocomial pathogen and the leading cause of hospital-acquired diarrhea. Antibiotic use is the primary risk factor for the development of C. difficile-associated disease because it disrupts normal protective gut flora and enables C. difficile to colonize the colon. C. difficile damages host tissue by secreting toxins and disseminates by forming spores. The toxin-encoding genes, tcdA and tcdB are part of a pathogenicity locus, which also encodes the gene tcdR that codes for the toxin genes positive regulator. TcdR is an alternate sigma factor that initiates transcription of tcdA and tcdB at their promoters. Alternative sigma factors are known to regulate virulence and virulence associated genes in many pathogenic bacteria. We created a tcdR mutant in the epidemic-type C. difficile R20291 strain in an attempt to identify the global role of tcdR. A site-directed mutation in tcdR affected both toxin production and sporulation in C. difficile R20291. Spores derived from the tcdR mutant were found to be mildly temperature sensitive. Moreover, nearly two fold more taurocholate was needed to germinate spores from the tcdR mutant than the spores prepared from the wild-type parent strain. Comparison of the tcdR mutant transcriptome with the parent strain revealed many differentially expressed late sporulation genes in the tcdR mutant. These data suggests that gene regulatory networks of toxin production and sporulation in Clostridium difficile are linked with each other.
2017-03-07 | GSE85395 | GEO
Project description:The universally conserved protein Elongation Factor P facilitates translation at amino acids that form peptide bonds with low efficiency, particularly poly-proline tracts. Despite its wide conservation, it is not essential in most bacteria and its physiological role remains unclear. Here, we show that EF-P affects the process of sporulation initiation in the bacterium Bacillus subtilis. We observe that lack of EF-P delays expression of sporulation-specific genes. Using ribosome profiling, we observe that expression of spo0A, encoding a transcription factor that functions as the master regulator of sporulation, is lower in a ∆efp strain as compared to the wildtype. Ectopic expression of Spo0A rescues the sporulation initiation phenotype, indicating that reduced spo0A expression explains the sporulation defect in ∆efp cells. Since Spo0A is the earliest sporulation transcription factor, these data suggest that sporulation initiation can be delayed when protein synthesis is impaired.
2023-01-04 | GSE222121 | GEO
Project description:The pathogen Clostridioides difficile causes toxin-mediated diarrhea and is the leading cause of hospital-acquired infection in the United States. Due to growing antibiotic resistance and recurrent infection, targeting C. difficile metabolism presents a new approach to combat this infection. Genome-scale metabolic network reconstructions (GENREs) have been used to identify therapeutic targets and uncover properties that determine cellular behaviors. Thus, we constructed C. difficile GENREs for a hypervirulent isolate (strain [str.] R20291) and a historic strain (str. 630), validating both with in vitro and in vivo data sets. Growth simulations revealed significant correlations with measured carbon source usage (positive predictive value [PPV] ≥ 92.7%), and single-gene deletion analysis showed >89.0% accuracy. Next, we utilized each GENRE to identify metabolic drivers of both sporulation and biofilm formation. Through contextualization of each model using transcriptomes generated from in vitro and infection conditions, we discovered reliance on the pentose phosphate pathway as well as increased usage of cytidine and N-acetylneuraminate when virulence expression is reduced, which was subsequently supported experimentally. Our results highlight the ability of GENREs to identify novel metabolite signals in higher-order phenotypes like bacterial pathogenesis.
2023-11-27 | MODEL2205060002 | BioModels
Project description:Clostridioides difficile is the leading cause of intestinal nosocomial post-antibiotic infections in adults. During infection, the bacterium must rapidly respond and adapt to the host environment by using survival strategies. Protein phosphorylation is a reversible post-translational modification employed ubiquitously for signal transduction and cellular regulation. Recently, Hanks-type Serine/Threonine kinases (STKs) and phosphatases (STPs), have emerged as important players in bacterial cell signaling and pathogenicity. However, characterization of STKs targets in prokaryotes remained a difficult challenge. Here, we applied and adapted the TiO2 phosphopeptide enrichment approach to determine the phosphoproteome of C. difficile. We have identified and quantified 2497 proteins representing 63,1% of the theoretical proteome. Among these proteins, 649 contained phosphorylated peptides with a localization probability >0,75. This pathogen encodes two STKs (PrkC and CD2148) and one associated phosphatase (STP), PrkC was shown to be crucial in the regulation of cell wall homeostasis, antibiotic resistance and cell division. Comparative phosphoproteomics of wild-type, prkC, CD2148, stp and double prkC CD2148 strains were performed in order to determinate STKs targets. 104 proteins were identified as specific substrates of PrkC, 46 of CD2148 and 94 of both kinases. Using a combination of in vivo and in vitro approaches, FtsK and Spo0A were validated as a direct target of PrkC. This study provides a detailed mapping of kinase-substrate relationships which may aid in the identification of new biomarkers and therapeutic targets.
2022-12-08 | PXD029827 | Pride
Project description:Clostridium acetobutylicum, the endospore-forming anaerobe best known for its ABE (acetone-butanol-ethanol) fermentation, has received renewed attention recently for the biological production of butanol, both for bulk chemical production and as a potential biofuel. With butanol production in mind, most of the recent research on C. acetobutylicum has focused on increasing butanol production, tolerance to butanol, and optimizing it for various substrates. However, an equally important trait, though less understood, is its sporulation program, which it coupled to solvent formation. The model organism for endospore formation is Bacillus subtilis, but significant physiological, metabolic, and genomic differences exist between the two organisms. Despite these differences, the major sporulation-related transcription/sigma factors are conserved between the two species. These transcription/sigma factors are activated in a cascade manner such that Spo0A becomes active first, followed by σF, then σE, then σG, and finally σK. The goal of this study is to determine the regulons of 4 of these transcription/sigma factors (Spo0A, σF, σE, and σG) and compare them to those in B. subtilis. To accomplish this goal, individual mutant strains were created for Spo0A, σF, σE, and σG, in which the transcription/sigma factor is silenced. These mutants were then compared transcriptionally using microarrays to determine the regulon of each transcription/sigma factor. To help avoid false positives, comparisons were made between strains in which the downstream transcription/sigma factor is silenced (e.g., for the Spo0A regulon, the Spo0A mutant was compared against the σF mutant and the σE mutant, since they are both upregulated by Spo0A) rather than just the WT. For each regulon, 4 timepoints were taken, since it is very difficult to synchronize sporulation in C. acetobutylicum cultures, and dye-swaps were prepared for each timepoint.
2011-02-28 | GSE24103 | GEO
Project description:Clostridium acetobutylicum, the endospore-forming anaerobe best known for its ABE (acetone-butanol-ethanol) fermentation, has received renewed attention recently for the biological production of butanol, both for bulk chemical production and as a potential biofuel. With butanol production in mind, most of the recent research on C. acetobutylicum has focused on increasing butanol production, tolerance to butanol, and optimizing it for various substrates. However, an equally important trait, though less understood, is its sporulation program, which it coupled to solvent formation. The model organism for endospore formation is Bacillus subtilis, but significant physiological, metabolic, and genomic differences exist between the two organisms. Despite these differences, the major sporulation-related transcription/sigma factors are conserved between the two species. These transcription/sigma factors are activated in a cascade manner such that Spo0A becomes active first, followed by M-OM-^CF, then M-OM-^CE, then M-OM-^CG, and finally M-OM-^CK. The goal of this study is to determine the regulons of 4 of these transcription/sigma factors (Spo0A, M-OM-^CF, M-OM-^CE, and M-OM-^CG) and compare them to those in B. subtilis. To accomplish this goal, individual mutant strains were created for Spo0A, M-OM-^CF, M-OM-^CE, and M-OM-^CG, in which the transcription/sigma factor is silenced. These mutants were then compared transcriptionally using microarrays to determine the regulon of each transcription/sigma factor. To help avoid false positives, comparisons were made between strains in which the downstream transcription/sigma factor is silenced (e.g., for the Spo0A regulon, the Spo0A mutant was compared against the M-OM-^CF mutant and the M-OM-^CE mutant, since they are both upregulated by Spo0A) rather than just the WT. For each regulon, 4 timepoints were taken, since it is very difficult to synchronize sporulation in C. acetobutylicum cultures, and dye-swaps were prepared for each timepoint. Four transcription/sigma factor regulons were investigated: Spo0A, M-OM-^CF, M-OM-^CE, and M-OM-^CG. For Spo0A, two comparison were made: Spo0A mutant vs M-OM-^CF mutant and Spo0A mutant vs M-OM-^CE mutant. For M-OM-^CF, two comparison were also made: M-OM-^CF mutant vs M-OM-^CE mutant and M-OM-^CF mutant vs M-OM-^CG mutant. Finally for M-OM-^CE and M-OM-^CG, only one comparison for each was made: M-OM-^CE mutant vs M-OM-^CG mutant and M-OM-^CG mutant vs wild-type, respectively. For each comparison, 4 timepoints were analyzed, and dye-swaps were prepared for each timepoint comparison. Timepoints were chosen based on when each transcription/sigma factor was expected to be active, based on a previous study [Jones, SW, et al. Genome Biol. 2008;9(7):R114.].
2011-02-28 | E-GEOD-24103 | biostudies-arrayexpress
Project description:Clostridioides difficile is the leading cause of antibiotic-associated infections worldwide. Within the host, C. difficile can transition from a sessile to a motile state by secretion of PPEP-1, which releases the cells from the intestinal epithelium by cleaving adhesion proteins. PPEP-1 belongs to the group of Pro-Pro endopeptidases, which are characterized by their unique ability to cleave proline-proline bonds. Interestingly, another putative member of this group, CD1597, is present in C. difficile. Although it possesses a domain similar to other PPEPs, CD1597 displays several distinct features that suggest a markedly different role for this protein. We investigated the proteolytic activity of CD1597 by testing various potential substrates. In addition, we investigated the effect of the absence of CD1597 by generating an insertional mutant of the cd1597 gene. Using the cd1597 mutant, we sought to identify phenotypic changes through a series of in vitro experiments and quantitative proteomic analyses. Furthermore, we aimed to study the localization of this protein using a fluorogenic fusion protein. Despite its similarities to PPEP-1, CD1597 did not show proteolytic activity. In addition, the absence of CD1597 caused an increase in various sporulation proteins during the stationary phase, yet we did not observe any alterations in the sporulation frequency of the cd1597 mutant. Furthermore, a promoter activity assay indicated a very low expression level of cd1597 in vegetative cells that was independent of the culture medium and growth stage. The low expression was corroborated by our comprehensive proteomics analysis of whole cell cultures, which failed to identify CD1597. However, an analysis of purified C. difficile spores identified CD1597 as part of the spore proteome. Hence, we predict that the protein is involved in sporulation, although we were unable to define a precise role for CD1597 in C. difficile
2024-05-27 | PXD052347 | Pride