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Cloning of binding sequences for the Escherichia coli transcription activators, FNR and CRP: location of bases involved in discrimination between FNR and CRP.

ABSTRACT: Expression from the E.coli melR promoter (pmelR) is normally totally dependent on the transcription activator protein, CRP. We describe experiments with a genetically engineered DNA fragment carrying pmelR in which the wild type CRP binding site was replaced with synthetic oligonucleotides containing either FNR or CRP binding sequences. When the synthetic oligonucleotide contains the 22 bp consensus for FNR binding sites, expression from pmelR is dependent on FNR but not CRP. Single changes at either of two symmetrically-related positions create sites that are recognised by both FNR and CRP. Changes at both positions result in a site that is not recognised by FNR but which binds CRP tightly.

SUBMITTER: Bell AI

PROVIDER: S-EPMC317865 | biostudies-other | 1989 May

REPOSITORIES: biostudies-other

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Cloning of binding sequences for the Escherichia coli transcription activators, FNR and CRP: location of bases involved in discrimination between FNR and CRP.

Bell A I AI Gaston K L KL Cole J A JA Busby S J SJ

Nucleic acids research 19890501 10

Expression from the E.coli melR promoter (pmelR) is normally totally dependent on the transcription activator protein, CRP. We describe experiments with a genetically engineered DNA fragment carrying pmelR in which the wild type CRP binding site was replaced with synthetic oligonucleotides containing either FNR or CRP binding sequences. When the synthetic oligonucleotide contains the 22 bp consensus for FNR binding sites, expression from pmelR is dependent on FNR but not CRP. Single changes at e ...[more]

PMID: 2543955

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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.

Project description:BackgroundNeisseria gonorrhoeae can survive during oxygen starvation by reducing nitrite to nitrous oxide catalysed by the nitrite and nitric oxide reductases, AniA and NorB. The oxygen-sensing transcription factor, FNR, is essential for transcription activation at the aniA promoter, and full activation also requires the two-component regulatory system, NarQ-NarP, and the presence of nitrite. The only other gene known to be activated by the gonococcal FNR is ccp encoding a cytochrome c peroxidase, and no FNR-repressed genes have been reported in the gonococcus. In contrast, FNR acts as both an activator and repressor involved in the control of more than 100 operons in E. coli regulating major changes in the adaptation from aerobic to anaerobic conditions. In this study we have performed a microarray-led investigation of the FNR-mediated responses in N. gonorrhoeae to determine the physiological similarities and differences in the role of FNR in cellular regulation in this species.ResultsMicroarray experiments show that N. gonorrhoeae FNR controls a much smaller regulon than its E. coli counterpart; it activates transcription of aniA and thirteen other genes, and represses transcription of six genes that include dnrN and norB. Having previously shown that a single amino acid substitution is sufficient to enable the gonococcal FNR to complement an E. coli fnr mutation, we investigated whether the gonococcal NarQ-NarP can substitute for E. coli NarX-NarL or NarQ-NarP. A plasmid expressing gonococcal narQ-narP was unable to complement E. coli narQP or narXL mutants, and was insensitive to nitrate or nitrite. Mutations that progressively changed the periplasmic nitrate sensing region, the P box, of E. coli NarQ to the sequence of the corresponding region of gonococcal NarQ resulted in loss of transcription activation in response to the availability of either nitrate or nitrite. However, the previously reported ligand-insensitive ability of gonococcal NarQ, the "locked on" phenotype, to activate either E. coli NarL or NarP was confirmed.ConclusionDespite the sequence similarities between transcription activators of E. coli and N. gonorrhoeae, these results emphasise the fundamental differences in transcription regulation between these two types of pathogenic bacteria.

Dataset Information

Cloning of binding sequences for the Escherichia coli transcription activators, FNR and CRP: location of bases involved in discrimination between FNR and CRP.

Publications

Cloning of binding sequences for the Escherichia coli transcription activators, FNR and CRP: location of bases involved in discrimination between FNR and CRP.

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