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ChIP-Seq of the Sinorhizobium meliloti CRP-like protein Clr in combination with either cAMP or cGMP under different growth conditions

ABSTRACT: In bacteria, Crp-Fnr superfamily transcription factors mediate 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) signaling. The CRP-like protein Clr of the soil-dwelling and plant-symbiotic α-proteobacterium Sinorhizobium meliloti was previously shown to activate target promoters in both its cAMP- and cGMP-bound states (Krol et al., Microbiology 62:1840–1856, 2016). In order to further characterize the overall regulon of Clr in S. meliloti Chromatin Immuno Precipitation DNA-Sequencing (ChIP-Seq) experiments were performed with C-terminally FLAG tagged Clr under four different growth conditions, namely growth in TY complex medium and MOPS minimal medium, each supplemented with either cAMP or cGMP. For each condition, the respective immunoprecipitated (IP) and non-immunoprecipitated (control) samples were analyzed and compared to locate genomic positions in which Clr-DNA binding occurs. In combining ChIP-Seq with Electrophoretic Mobility Shift Assays and promoter-probe assays we expanded the list of known Clr-regulated target promoters and showed that virtually all of these promoters containing a palindromic Clr binding site (CBS) motif are activated both by Clr•cAMP and Clr•cGMP.

INSTRUMENT(S): Illumina MiSeq

ORGANISM(S): Sinorhizobium meliloti

SUBMITTER: Neda Farmani

PROVIDER: E-MTAB-11788 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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ChIP-Seq of the Sinorhizobium meliloti CRP-like protein Clr in combination with either cAMP or cGMP under different growth conditions

Project description:ChIP-Seq of the Sinorhizobium meliloti CRP-like protein Clr in combination with either cAMP or cGMP under different growth conditions

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A novel conserved transcription factor CLR-4 regulates cellulase genes expression through controlling the intracellular cAMP level in ascomycete fungi

Project description:Fungal degradation of lignocellulosic biomass requires various (hemi-)cellulases and plays key roles in biological carbon cycle. Although cellulases induction recently described in some saprobic filamentous fungi, regulation of cellulase transcription has not been studied thoroughly. Here, we identified and characterized the novel cellulase regulation factors clr-4 in Neurospora crassa and its ortholog Mtclr-4 in Myceliophthora thermophila. Deletion of clr-4 and Mtclr-4 displayed similarly defective phenotypes in cellulolytic enzymes production and activities. Transcriptomics analysis of Δclr-4/ΔMtclr-4 revealed down-regulation of not only encoding genes of (hemi-)cellulases and pivotal regulators (clr-1, clr-2 and xyr-1), but also the key genes of cAMP signaling pathway such as adenylate cyclase cr-1. Consistently, the significant decreased levels of intracellular cAMP were observed in Δclr-4/ΔMtclr-4 compared to wild-type during cellulose utilization. Electrophoretic mobility shift assays (EMSA) verified that CLR-4 could directly bind to the promoter regions of adenylyl cyclase (Nccr-1) and cellulose regulator clr-1, while MtCLR-4 bind to upstream regions of adenylyl cyclase Mtcr-1 and biomass deconstruction regulators Mtclr-2 and Mtxyr-1. Concluded, the novel cellulase expression regulators (CLR-4/MtCLR-4) findings here significantly enrich our understanding of the regulatory network of cellulose degradation and provide new targets for industrial fungi strain engineering for plant biomass deconstruction in biorefinery.

2021-03-18 | GSE111986 | GEO

Time course of the mRNA expression after phage ϕYS40 infection in crp deletion mutant of Thermus thermophilus HB8.

Project description:We observed the expression profile of the total mRNA in crp (TTHA1437) deletion mutant strain of Thermus thermophilus HB8 during infection of bacteriophage ϕYS40. Keywords: time course, bacteriophage, infection, CRP, cAMP receptor protein, deletion mutant

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Lysine 100 acetylation effect of CRP (catabolite activator protein) in gene expression in Escherichia coli

Project description:cAMP receptor protein (CRP, also known as the catabolite activator protein [CAP]) is arguably the best-studied of the global transcription factors of E coli. CRP alone is responsible for regulating at least 283 operons. Upon binding cAMP, the CRP dimer binds DNA and directly interacts with RNA polymerase (RNAP). At Class II promoters, CRP binds near position -41,5 relative to the transcription start site and contacts the amino-terminal domain of the RNAP α subunit (RNAPα-NTD). This interaction requires AR2, a patch of primarily positively charged residues (H19, H21, E96, and K101) that interact with negatively charged residues on RNAPα-NTD. Acetylome analyses consistently detect lysine 100 (K100) of CRP as acetylated. Since K100 is adjacent to the positively charged AR2, we hypothesized that the K100 positive charge may also play a role in CRP function. We further hypothesized that acetylation of K100 would neutralize this positive charge, leading to a potential regulatory mechanism

2017-03-24 | GSE96955 | GEO

Overexpression of cyaJ in Rm2011 to study effects of increased cAMP on the transcriptome of Sinorhizobium meliloti

Project description:Alpha-rhizobia are soil-dwelling alphaproteobacteria existing either in free-living states or in symbiosis with a leguminous plant host. cyaJ is a putative adenylate/guanylate cyclase transmembrane protein. Overexpression of cyaJ leads to increased > levels of cAMP. To investigate the effect of increased cAMP on the transcriptome of Sinorhizobium meliloti we performed microarray analysis of > Sinorhizobium meliloti Rm2011 carrying an empty vector pWBT or cyaJ overexpression construct pWBT-cyaJ.

2016-08-17 | E-MTAB-4932 | biostudies-arrayexpress

Mixed Culture Gene Expression of E. coli and Pseudomonas aeruginosa grown on defined media with N-acetyl glucosamine

Project description:Transcriptional profiles of Escherichia coli MG1655 in mixed culture with Pseudomonas aeruginosa PAO1 showed a number of E. coli genes to be upregulated including purA-F and other genes associated with purine synthesis. In contrast, genes associated with pyrimidine synthesis were unaffected. Competition experiments in both planktonic and biofilm cultures, using three purine synthesis mutants, purD, purH, and purT showed little difference in E. coli survival from the parent strain. As purines are components of the cell signals, cAMP and c-di-GMP, we conducted competition experiments with E. coli mutants lacking adenylate cyclase (cyaA), cAMP phosphodiesterase (cpdA), and the catabolite receptor protein (crp), as well as ydeH, an uncharacterized gene that has been associated with c-di-GMP synthesis. Survival of the cyaA and crp mutants during co-culture were significantly less than the parent strain. Supplementation of the media with 1mM cAMP could restore survival of the cyaA mutant but not the crp mutant. In contrast, survival of the cpdA mutant was similar to the parent strain. Survival of the ydeH mutant was moderately less than the parent, suggesting that cAMP has more impact on E. coli mixed culture growth than c-di-GMP. Addition of 1 mM indole restored the survival of both the cyaA and crp mutations. Mutants in genes for tryptophan synthesis (trpE) and indole production (tnaA) showed a loss of competition and recovery through indole supplementation, comparable to the cyaA and crp mutants. Overall, these results suggest indole and cAMP as major contributing factors to E. coli growth in mixed culture.

2011-11-29 | GSE26932 | GEO

Mixed Culture Gene Expression of E. coli and Pseudomonas aeruginosa

2011-11-09 | GSE26931 | GEO

Cross-talk between PKA and PKG controls pH-dependent host cell egress of Toxoplasma gondii

Project description:Toxoplasma gondii encodes three Protein Kinase A catalytic (PKAc1-3) and one regulatory (PKAr) subunits to integrate cAMP-dependent signals. Here, we show that inactive PKAc1 is maintained at the parasite pellicle by interacting with dually acylated PKAr. Either a conditional knockdown of PKAr or the overexpression of PKAc1 results in a block in parasite division. In contrast, conditional expression of a dominant negative PKAr isoform unable to bind cAMP, triggers premature egress of parasites from infected cells. This untimely egress critically depends on parasite density and host cell acidification. A comparative phosphoproteome analysis reveals that PKA genetic inhibition significantly changed the phosphorylation profile of a putative cGMP-phosphodiesterase, PDE2. Consistently, the phenotype of PKA genetic inhibition is alleviated by chemical inhibition of the cGMP-dependent protein kinase G (PKG). A phosphodiesterase inhibitor is able to circumvent egress repression by PKA or pH neutralisation, indicating that environmental acidification and PKA signalling act as balancing regulators of cGMP degradation to control PKG-mediated egress. Collectively, these results reveal a cross-talk between PKA and PKG pathways to govern egress in T. gondii.

2017-10-12 | PXD006045 | Pride

Transcription profiling by array of overexpression of the Rv0805 phosphodiesterase elicits a cAMP-independent transcriptional response

Project description:The Rv0805 gene in Mycobacterium tuberculosis encodes a metallophosphoesterase which shows cAMP-hydrolytic activity. Overexpression of Rv0805 has been used as a tool to lower intracellular cAMP levels and thereby elucidate the roles of cAMP in mycobacteria. Here we show that levels of cAMP in M. tuberculosis were lowered by only .30% following overexpression of Rv0805, and transcript levels of a number of genes, which include those associated with virulence and the methyl citrate cycle, were altered. The genes that showed altered expression were distinct from those differentially regulated in a strain deleted for the cAMP-receptor protein (CRP(Mt)), consistent with the relatively low dependence on cAMP of CRP(Mt) binding to DNA. Using mutants of Rv0805 we show that the transcriptional signature of Rv0805 overexpression is a combination of catalysis-dependent and independent effects, and that the structurally flexible C-terminus of Rv0805 is crucial for the catalysis-independent effects of the protein. Our study demonstrates the dissociation of Rv0805 and cAMP-regulated gene expression, and reveals alternate functions for this phosphodiesterase from M. tuberculosis. [Data is also available from http://bugs.sgul.ac.uk/E-BUGS-146]

2013-11-02 | E-BUGS-146 | biostudies-arrayexpress

Gene expression in undifferentiated or cyclic adenosine monophosphate-exposed BeWo trophoblast cells

Project description:BeWo trophoblast cells differentiate in response to expsure to cyclic adenosine monophosphate (cAMP) analogs. Differentiation includes syncytialization (fusion) and hormonogenesis. The goal of this study was to globally determine transcripts differentially expressed in BeWo trophoblast cells following a 24-h exposure to 250 uM 8-bromo-cAMP.

2015-08-01 | GSE66840 | GEO

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