Dataset Information

CRP Interacts Specifically With Sxy to Activate Transcription in Escherichia coli.

ABSTRACT: Horizontal gene transfer through natural competence is an important driving force of bacterial evolution and antibiotic resistance development. In several Gram-negative pathogens natural competence is regulated by the concerted action of cAMP receptor protein (CRP) and the transcriptional co-regulator Sxy through a subset of CRP-binding sites (CRP-S sites) at genes encoding competence factors. Despite the wealth of knowledge on CRP's structure and function it is not known how CRP and Sxy act together to activate transcription. In order to get an insight into the regulatory mechanism by which these two proteins activate gene expression, we performed a series of mutational analyses on CRP and Sxy. We found that CRP contains a previously uncharacterized region necessary for Sxy dependent induction of CRP-S sites, here named "Sxy Interacting Region" (SIR) encompassing residues Q194 and L196. Lost promoter induction in SIR mutants could be restored in the presence of specific complementary Sxy mutants, presenting evidence for a direct interaction of CRP and Sxy proteins in transcriptional activation. Moreover, we identified constitutive mutants of Sxy causing higher levels of CRP-S site promoter activation than wild-type Sxy. Both suppressor and constitutive mutations are located within the same area of Sxy.

SUBMITTER: Sondberg E

PROVIDER: S-EPMC6728893 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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CRP Interacts Specifically With Sxy to Activate Transcription in <i>Escherichia coli</i>.

Søndberg Emilie E Sinha Anurag Kumar AK Sinha Anurag Kumar AK Gerdes Kenn K Semsey Szabolcs S

Frontiers in microbiology 20190830

Horizontal gene transfer through natural competence is an important driving force of bacterial evolution and antibiotic resistance development. In several Gram-negative pathogens natural competence is regulated by the concerted action of cAMP receptor protein (CRP) and the transcriptional co-regulator Sxy through a subset of CRP-binding sites (CRP-S sites) at genes encoding competence factors. Despite the wealth of knowledge on CRP's structure and function it is not known how CRP and Sxy act tog ...[more]

PMID: 31543875

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Project description:BackgroundEscherichia coli exhibits diauxic growth in sugar mixtures due to CRP-mediated catabolite repression and inducer exclusion related to phosphotransferase system enzyme activity. Replacement of the native crp gene with a catabolite repression mutant (referred to as crp*) enables co-utilization of glucose and other sugars in E. coli. While previous studies have examined the effects of expressing CRP* mutants on the expression of specific catabolic genes, little is known about the global transcriptional effects of CRP* expression. In this study, we compare the transcriptome of E. coli W3110 (expressing wild-type CRP) to that of mutant strain PC05 (expressing CRP*) in the presence and absence of glucose.ResultsThe glucose effect is significantly suppressed in strain PC05 relative to strain W3110. The expression levels of glucose-sensitive genes are generally not altered by glucose to the same extent in strain PCO5 as compared to W3110. Only 23 of the 80 genes showing significant differential expression in the presence of glucose for strain PC05 are present among the 418 genes believed to be directly regulated by CRP. Genes involved in central carbon metabolism (including several TCA cycle genes) and amino acid biosynthesis, as well as genes encoding nutrient transport systems are among those whose transcript levels are most significantly affected by CRP* expression.We present a detailed transcription analysis and relate these results to phenotypic differences between strains expressing wild-type CRP and CRP*. Notably, CRP* expression in the presence of glucose results in an elevated intracellular NADPH concentration and reduced NADH concentration relative to wild-type CRP. Meanwhile, a more drastic decrease in the NADPH/NADP+ ratio is observed for the case of CRP* expression in strains engineered to reduce xylose to xylitol via a heterologously expressed, NADPH-dependent xylose reductase. Altered expression levels of transhydrogenase and TCA cycle genes, among others, are consistent with these observations.ConclusionWhile the simplest model of CRP*-mediated gene expression assumes insensitivity to glucose (or cAMP), our results show that gene expression in the context of CRP* is very different from that of wild-type in the absence of glucose, and is influenced by the presence of glucose. Most of the transcription changes in response to CRP* expression are difficult to interpret in terms of possible systematic effects on metabolism. Elevated NADPH availability resulting from CRP* expression suggests potential biocatalytic applications of crp* strains that extend beyond relief of catabolite repression.

Dataset Information

CRP Interacts Specifically With Sxy to Activate Transcription in Escherichia coli.

Publications

CRP Interacts Specifically With Sxy to Activate Transcription in <i>Escherichia coli</i>.

Similar Datasets

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