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Transcriptional organization and regulation of expression of region 1 of the Escherichia coli K5 capsule gene cluster.

ABSTRACT: The transcriptional organization and regulation of region 1 expression of the Escherichia coli K5 capsule gene cluster were studied. Region 1 was transcribed as an 8.0-kb polycistronic mRNA which was processed to form a separate 1.3-kb transcript encoding the 3'-most gene kpsS. Transcription of region 1 of the E. coli K5 capsule gene cluster was directed from a single promoter 225 bp upstream of a previously unidentified gene, kpsF. The promoter had -35 and -10 consensus sequences typical of an E. coli sigma 70 promoter, with no similarities to binding sites for other sigma factors. Two integration host factor (IHF) binding site consensus sequences were identified 110 bp upstream and 130 bp downstream of the transcription start site. In addition, two AT-rich regions separated by 16 bp identified upstream of the region 1 promoter were conserved upstream of the region 3 promoter. The kpsF gene was 98.8% identical with the kpsF gene identified in the E. coli K1 antigen gene cluster and confirms that the kpsF gene is conserved among group II capsule gene clusters. An intragenic Rho-dependent transcriptional terminator was discovered within the kpsF gene. No essential role for KpsF in the expression of the K5 antigen could be established. The temperature regulation of region 1 expression was at the level of transcription, with no transcription detectable in cells grown at 18 degrees C. Mutations in regulatory genes known to control temperature-dependent expression of a number of virulence genes had no effect on the temperature regulation of region 1 expression. Likewise, RfaH, which is known to regulate expression of E. coli group II capsules had no effect on the expression of region 1. Mutations in the himA and himD genes which encode the subunits of the IHF led to a fivefold reduction in the expression of KpsE at 37 degrees C, confirming a regulatory role for IHF in the expression of region 1 genes.

SUBMITTER: Simpson DA

PROVIDER: S-EPMC178532 | biostudies-other | 1996 Nov

REPOSITORIES: biostudies-other

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Transcriptional organization and regulation of expression of region 1 of the Escherichia coli K5 capsule gene cluster.

Simpson D A DA Hammarton T C TC Roberts I S IS

Journal of bacteriology 19961101 22

The transcriptional organization and regulation of region 1 expression of the Escherichia coli K5 capsule gene cluster were studied. Region 1 was transcribed as an 8.0-kb polycistronic mRNA which was processed to form a separate 1.3-kb transcript encoding the 3'-most gene kpsS. Transcription of region 1 of the E. coli K5 capsule gene cluster was directed from a single promoter 225 bp upstream of a previously unidentified gene, kpsF. The promoter had -35 and -10 consensus sequences typical of an ...[more]

PMID: 8932302

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Project description:Post-transcriptional regulation of gene expression plays a crucial role in many bacterial pathways. In particular, the translation of mRNA can be regulated by trans-acting, small, non-coding RNAs (sRNAs) or mRNA-binding proteins, each of which has been successfully treated theoretically using two-component models. An important system that includes a combination of these modes of post-transcriptional regulation is the Colicin E2 system. DNA damage, by triggering the SOS response, leads to the heterogeneous expression of the Colicin E2 operon including the cea gene encoding the toxin colicin E2, and the cel gene that codes for the induction of cell lysis and release of colicin. Although previous studies have uncovered the system's basic regulatory interactions, its dynamical behavior is still unknown. Here, we develop a simple, yet comprehensive, mathematical model of the colicin E2 regulatory network, and study its dynamics. Its post-transcriptional regulation can be reduced to three hierarchically ordered components: the mRNA including the cel gene, the mRNA-binding protein CsrA, and an effective sRNA that regulates CsrA. We demonstrate that the stationary state of this system exhibits a pronounced threshold in the abundance of free mRNA. As post-transcriptional regulation is known to be noisy, we performed a detailed stochastic analysis, and found fluctuations to be largest at production rates close to the threshold. The magnitude of fluctuations can be tuned by the rate of production of the sRNA. To study the dynamics in response to an SOS signal, we incorporated the LexA-RecA SOS response network into our model. We found that CsrA regulation filtered out short-lived activation peaks and caused a delay in lysis gene expression for prolonged SOS signals, which is also seen in experiments. Moreover, we showed that a stochastic SOS signal creates a broad lysis time distribution. Our model thus theoretically describes Colicin E2 expression dynamics in detail and reveals the importance of the specific regulatory components for the timing of toxin release.

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

Transcriptional organization and regulation of expression of region 1 of the Escherichia coli K5 capsule gene cluster.

Publications

Transcriptional organization and regulation of expression of region 1 of the Escherichia coli K5 capsule gene cluster.

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