Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Probing the regulatory effects of specific mutations in three major binding domains of the pleiotropic regulator CcpA of Bacillus subtilis

ABSTRACT: Study the effect of single amino acid point mutations in CcpA at the transcriptome level. Cells were grown in LB with or without 1% glucose and harvested at an OD600 of 0.3 (early exponential phase). Bacillus subtilis strain ccpA::spec carrying pHT304 (empty plasmid), pWH_ccpA_M17R, pWH_ccpA_T62H, or pWH_ccpA_R304W were grown in LB with or without 1% glucose. The control in the microarray study was the strain Bacillus subtilis ccpA::spec carrying pWH_ccpA-wt. All Bacillus subtilis cultures were harvested in the early exponential growth phase at an OD600 of 0.3 from 100 ml culture. Two independent cultures of each strain per growth condition (+/- glucose) and a technical replicate (dye-swap) were used. Please note that '[A}' and '{B}' channels (in the raw data *slide.txt files) correspond to Cy3 (Ch1) and Cy5 (Ch2) in each sample record.

ORGANISM(S): Bacillus subtilis

SUBMITTER: Ruud Detert Oude Weme

PROVIDER: E-GEOD-69575 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Probing the regulatory effects of specific mutations in three major binding domains of the pleiotropic regulator CcpA of Bacillus subtilis.

Detert Oude Weme Ruud R Seidel Gerald G Kuipers Oscar P OP

Frontiers in microbiology 20151002

Carbon catabolite control is required for efficient use of available carbon sources to ensure rapid growth of bacteria. CcpA is a global regulator of carbon metabolism in Gram-positive bacteria like Bacillus subtilis. In this study the genome-wide gene regulation of a CcpA knockout and three specific CcpA mutants were studied by transcriptome analysis, to further elucidate the function of specific binding sites in CcpA. The following three amino acids were mutated to characterize their function: ...[more]

PMID: 26483775

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Project description:In Bacillus subtilis and its relatives carbon catabolite control, a mechanism enabling to reach maximal efficiency of carbon and energy sources metabolism, is achieved by the global regulator CcpA (carbon catabolite protein A). CcpA in a complex with HPr-Ser-P (seryl-phosphorylated form of histidine-containing protein, HPr) binds to operator sites called catabolite responsive elements, cre. Depending on the cre box position relative to the promoter, the CcpA/HPr-Ser-P complex can either act as a positive or a negative regulator. The cre boxes are highly degenerate semi-palindromes with a lowly conserved consensus sequence. So far, studies aimed at revealing how CcpA can bind such diverse sites were focused on the analysis of single cre boxes. In this study, a genome-wide analysis of cre sites was performed in order to identify differences in cre sequence and position, which determine their binding affinity. The transcriptomes of B. subtilis cultures with three different CcpA expression levels were compared. The higher the amount of CcpA in the cells, the more operons possessing cre sites were differentially regulated. The cre boxes that mediated regulation at low CcpA levels were designated as strong (high affinity) and those which responded only to high amounts of CcpA, as weak (low affinity). Differences in the sequence and position in relation to the transcription start site between strong and weak cre boxes were revealed. Certain residues at specific positions in the cre box as well as, to a certain extent, a more palindromic nature of cre sequences and the location of cre in close vicinity to the transcription start site contribute to the strength of CcpA-dependent regulation. The main factors contributing to cre regulatory efficiencies, enabling subtle differential control of various subregulons of the CcpA regulon, are identified. Bacillus subtilis strain MP902 [strain MP901 (Ptet-ccpA, KmR) carrying pWH119 (Pxyl-tetR, EmR)] was grown in presence of three different concentrations of Ptet inducer, anhydrotetracycline (ATc), leading to three levels of ccpA expression induction. The control culture was grown in absence of ATc. The total RNA for transcriptome analyses was isolated at OD600 = 0.8 from 16 ml culture. Three independent cultures of each strain (target strains and controls) were used, and cells were sampled for microarray experiment.

Dataset Information

Probing the regulatory effects of specific mutations in three major binding domains of the pleiotropic regulator CcpA of Bacillus subtilis

Publications

Probing the regulatory effects of specific mutations in three major binding domains of the pleiotropic regulator CcpA of Bacillus subtilis.

Similar Datasets

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