RNA-seq of Clostridioides difficile ∆rpoN mutant against wild-type control
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina NovaSeq 6000
ORGANISM(S): Clostridioides difficile
SUBMITTER: Ying Yang
PROVIDER: E-MTAB-14512 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:The experiment intends to reveal the difference in gene expression profiles between the wild-type strain and the ∆cwp66 mutant of Clostridioides difficile. We first constructed the ∆cwp66 mutant, and the phenotypic changes of the ∆cwp66 mutant against the wild-type strain were studied. To further elucidate the mechanism of phenotypic changes of the ∆cwp66 mutant, RNA-sequencing experiments were carried out to reveal the underlying mechanism of phenotypic changes.
2022-03-01 | E-MTAB-11180 | biostudies-arrayexpress
Project description:The phytopathogen Xylella fastidiosa produces two classes of pili, long type IV pili and short type I pili, which are involved in motility and adhesion. In this work, we have investigated the role of Ï54 factor and its involvement in the regulation of fimbrial biogenesis in X. fastidiosa. An rpoN null mutant was constructed from citrus strain J1a12, and analyses of global gene expression profile by microarrays comparing the wild type and rpoN mutant strains showed that few genes exhibited differential expression. At least one gene, pilA1 (XF2542), which encodes the structural pilin protein of type IV fimbriae, showed decreased expression in the rpoN mutant whereas an operon encoding proteins of type I fimbriae were twofold more expressed in the mutant. Quantitative real time RT-PCR (qRT-PCR) analysis confirmed that pilA1 transcript was significantly reduced in the rpoN mutant. The transcriptional start site of pilA1 was determined by primer extension, and a canonical Ï54-dependent promoter was found upstream of the start site. Genome sequence analysis of X. fastidiosa revealed five paralogues of pilA, but microarray and qRT-PCR data demonstrated that only the pilA1 transcript was significantly affected in the rpoN mutant. The rpoN mutant made more biofilm than the wild type strain and presented a cell-cell aggregative phenotype. These results indicate that Ï54 regulates biofilm formation, probably via differential regulation of genes involved in type IV and type I fimbrial biogenesis. Direct comparison between wild type strain J1a12 and mutant strain rpoN-.
2010-06-25 | E-GEOD-8097 | biostudies-arrayexpress
Project description:The protein inventory of the anaerobic pathogen Clostridioides difficile grown in colony or aggregate biofilms was investigated in a comprehensive LC-MS/MS approach. C. difficile has a versatile metabolism and is perfectly equipped to survive and persist inside the mammalian intestine. However, knowledge on how C. difficile exactly resides inside the intestine is still scare. Aggregate or biofilms attached to the epithelium are discussed as possible growth forms and have become a focus in research. However, recent data suggest that the choice of the model strain, timepoint of analysis and the biofilm model significantly impact the outcome of biofilm experiments leading to contradictory results. To address some of these issues, a comprehensive proteomics approach was applied to investigate the metabolism of C. difficile strain 630erm grown in colony or aggregate biofilms. Besides verification of recent findings, a comprehensive overview on the differential expression profile of C. difficile’s cell surface proteins as well as its energy and stress metabolism during the two different forms of biofilm growth could be provided. Additionally, regulatory circuits that were activated in the two different biofilm models were analysed whereby RpoN was determined to be an important regulator of aggregate biofilm formation in C. difficile.
2021-07-12 | PXD022830 | Pride
Project description:The bacterial transcription factor RpoN regulates an extensive network of genes whose products are involved in diverse biological functions. We constructed a small peptide termed the RpoN molecular roadblock, which binds to and blocks transcription from RpoN promoters. This RpoN molecular roadblock can be used in any bacterium to obtain information on the RpoN regulon. We expressed the RpoN molecular roadblock in P. aeruginosa PAO1 and used microarrays to identify genes that were differentially transcribed due to the RpoN molecular roadblock. The RpoN molecular roadblock was expressed in P. aeruginosa PAO1 in mid-exponential phase in rich media. Total RNA was isolated and prepped for Affymetrix GeneChips.
2012-02-09 | E-GEOD-35632 | biostudies-arrayexpress
Project description:Clostridioides difficile interactions with the gut mucosa are crucial for colonisation and establishment of infection, however key infection events during the establishment of disease are still poorly defined. To better understand the initial events that occur during C. difficile colonisation, we employed a dual RNA-sequencing approach to study the host and bacterial transcriptomic profiles during C. difficile infection in a dual-environment in vitro human gut model. Temporal changes in gene expression were analysed over 3-24h post infection and comparisons were made with uninfected controls.
2023-03-23 | E-MTAB-12660 | biostudies-arrayexpress
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
2008-09-09 | E-GEOD-11284 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE17834: Transcriptome analysis of Geobacter sulfurreducens grown with different nitrogen sources GSE17837: ChIP-chip of Geobacter sulfurreducens PCA with antibody against RpoN under various conditions. Refer to individual Series
2010-06-28 | E-GEOD-17838 | biostudies-arrayexpress
Project description:Background: Xylella fastidiosa, a Gram-negative fastidious bacterium, grows exclusively in the xylem of several plants, causing diseases such as citrus variegated chlorosis. As the xylem sap contains low concentrations of amino acids and other compounds, X. fastidiosa needs to cope with nitrogen limitation in its natural habitat. Results: In this work, we performed a whole-genome microarray analysis of the X. fastidiosa nitrogen starvation response. A time-course experiment (2, 8 and 12 hours) revealed many differentially expressed genes under nitrogen starvation, such as genes related to transport, nitrogen assimilation, amino acid biosynthesis, transcriptional regulation, and many genes encoding hypothetical proteins. In addition, a decrease in the expression levels of many genes involved in carbon metabolism and energy generation pathways was also observed. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes induced by nitrogen starvation in a ?54-dependent manner. A more complete picture of the ?54 regulon was achieved by combining the transcriptome data with an in silico search for potential ?54-dependent promoters, using a position weight matrix approach. One of these ?54-predicted binding sites, located upstream of the glnA gene (encoding a glutamine synthetase), was validated by primer extension assays, confirming that this gene has a ?54-dependent promoter and contains a predicted NtrC binding site. Conclusions: Together, these results show that nitrogen starvation causes intense changes in the X. fastidiosa transcriptome and some of these differentially expressed genes belong to the ?54 regulon. For time-course studies, cells cultivated at late-exponential phase in PWG medium were used to inoculate a culture in 100 ml XDM2 medium to an optical density at 600 nm (OD600 nm) of 0.1. Cells were grown during 12 days in the XDM2 medium (mid-log phase) and harvested by centrifugation. Then, the culture was divided into two 6 portions and cells were washed with XDM2 medium (zero time) or XDM2 medium lacking all nitrogen sources (XDM0), respectively. The cultivation was continued for 2h, 8h and 12h in XDM0 to establish nitrogen starvation conditions. For each time point, cells in a 25-ml culture were collected by centrifugation and rapidly frozen in dry ice, until RNA isolation. Three RNA samples isolated from independently grown cultures of the cells at each starvation period (2h, 8h and 12h) were examined, and each preparation was subjected to microarray analysis. As the genes were spotted at least in duplicate, we obtained six replicates for each gene from three independent data sets per gene per starvation period. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes induced by nitrogen starvation in a ?54-dependent manner. To determine the effect of rpoN inactivation on gene expression after nitrogen starvation, the transcriptomes of the wild type and the rpoN strains were compared using DNA microarrays, with both strains grown on XDM2 medium and submitted to nitrogen starvation (XDM0) during 2 hours. Three RNA samples isolated from independently grown cultures of the cells were examined. Due to the platform design, each microarray slide was divided into "LEFT" and "RIGHT", allowing the probing of two technical replicates per slide.
2010-12-21 | E-GEOD-21647 | biostudies-arrayexpress
Project description:Experiment conducted to investigate the genetic factors associated with variable and heterogeneous non-planktonic in a mutant lacking rpoN unique to CFT073 bacteria. The aim was to try to understand why RpoN suppresses this heterogeneity in the wild-type during colony growth, but also asses why this phenotype does not occur in other pathogenic strains by also sequencing transcripts from EHEC EDL933. The rpoN mutant strains was complemented with plasmid-borne rpoN containing its native promoter to ensure any differences in gene expression could solely be attributed to the absence of rpoN alone.
2022-01-13 | E-MTAB-11288 | biostudies-arrayexpress
Project description:We applied a ChIP-chip approach to elucidate the binding profiles of sigma factors (RpH, RpoN and RpoS) experimentally under different growth conditions. This technique localizes DNA fragments within DNA-protein complexes enriched by chromatin immunoprecipitation using high-density oligonucleotide tilling arrays. A 10 ChIP-chip study using immunoprecipitated DNA (IP-DNA) from three culture condtions against three alternative sigma factors . The high-density oligonucleotide tiling arrays used were consisted of 381,174 oligonucleotide probes spaced 20 bp apart (30-bp overlap between two probes) across the G.sulfurreducens genome (NimbleGen). Experiments were conducted as three bioliogical replicates (different cultures).
2013-05-01 | E-GEOD-37778 | biostudies-arrayexpress