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The base pairing RNA Spot 42 participates in a multi-output feedforward loop to help enact catabolite repression in Escherichia coli

ABSTRACT: Bacteria selectively consume some carbon sources over others through a regulatory mechanism termed catabolite repression. Here, we show that the base pairing RNA Spot 42 plays a broad role in catabolite repression in Escherichia coli by directly repressing genes involved in central and secondary metabolism, redox balancing, and the consumption of diverse non-preferred carbon sources. Many of the genes repressed by Spot 42 are transcriptionally activated by the global regulator CRP. Since CRP represses Spot 42, these regulators participate in a specific regulatory circuit called a multi-output feedforward loop. We found that this loop can reduce leaky expression of target genes in the presence of glucose and can maintain repression of target genes under changing nutrient conditions. Our results suggest that base pairing RNAs in feedforward loops can help shape the steady-state levels and dynamics of gene expression. MG1655 lacIq cells harboring the control vector pBRplac or the Spot 42-inducible vector pSpot42 were grown in LB to an ABS600 of ~0.4 and treated with 1 mM IPTG. After 7 min, total RNA was isolated using the hot phenol extraction procedure.

ORGANISM(S): Escherichia coli str. K-12 substr. MG1655

SUBMITTER: Chase Beisel

PROVIDER: E-GEOD-24875 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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The base pairing RNA Spot 42 participates in a multi-output feedforward loop to help enact catabolite repression in Escherichia coli

Project description:Bacteria selectively consume some carbon sources over others through a regulatory mechanism termed catabolite repression. Here, we show that the base pairing RNA Spot 42 plays a broad role in catabolite repression in Escherichia coli by directly repressing genes involved in central and secondary metabolism, redox balancing, and the consumption of diverse non-preferred carbon sources. Many of the genes repressed by Spot 42 are transcriptionally activated by the global regulator CRP. Since CRP represses Spot 42, these regulators participate in a specific regulatory circuit called a multi-output feedforward loop. We found that this loop can reduce leaky expression of target genes in the presence of glucose and can maintain repression of target genes under changing nutrient conditions. Our results suggest that base pairing RNAs in feedforward loops can help shape the steady-state levels and dynamics of gene expression.

2011-01-01 | GSE24875 | GEO

Escherichia coli str. K-12 substr. MG1655

Project description:The base pairing RNA Spot 42 participates in a multi-output feedforward loop to help enact catabolite repression in Escherichia coli

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Transcriptional effects of CRP* expression in Escherichia coli

Project description:Escherichia 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*) alleviates diauxic effects in E. coli and enables co-utilization of glucose and other sugars. 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.

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Transcriptional effects of CRP* expression in Escherichia coli

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Expression analysis, A. actinomycetemcomitans wild type (JP2) vs CRP deletion mutant both grown anaerobically.

Project description:The goal of this analysis is to define the anaerobic regulon for the global transcription factor CRP (catabolite repressor protein) in the periodontal pathogen Aggregatibacter actinomycetemcomitans. The objectives were met by comparing the mRNA profile of a wild type strain, JP2, to that of an isogenic mutant deleted of the CRP gene. Both strains were grown anaerobically.

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Expression profiling of a spf deletion mutant suggests biological roles and mRNA targets for Spot 42 in the fish pathogen Aliivibrio salmonicida

Project description:Spot 42 was discovered in Escherichia coli nearly 40 years ago as an abundant, small and unstable RNA. Its biological role remained obscure until it was shown to cause discoordinate expression of the galactose operon (gal operon). Recently Spot 42 has also been implicated in having broader roles in the central and secondary metabolism. Spot 42 is encoded by the spf gene. The gene is ubiquitous in the Vibrionaceae family of gamma-proteobacteria, which includes a number of serious pathogens of humans and animals, including the infamous Vibrio cholerae. One member of this family, Aliivibrio salmonicida, causes cold-water vibriosis in farmed Atlantic salmon. Its genome encodes Spot 42 with 84 % identity to E. coli Spot 42 (spf). We generated a A. salmonicida spf deletion mutant. We then and used microarray and Northern blot analyses to monitor global effects on the transcriptome in order to provide insights into the biological roles of Spot 42 in this bacterium. In the presence of glucose we found a surprisingly large number of ≥2× differentially expressed genes, and several major cellular processes were affected, such as carbohydrate metabolism and transport, motility and chemotaxis, iron homeostasis and quorum sensing. A gene encoding a pirin-like protein (VSAL_I1200) showed an on/off expression pattern in the presence/absence of Spot 42, which suggests that Spot 42 plays a key regulatory role in the central metabolism by regulating the switch between fermentation and respiration. Interestingly, in a global search we discovered a sRNA, named VSsrna24, which is encoded immediately downstream of spf. This new sRNA has an expression pattern opposite to that of Spot 42, and its expression is highly dependent on glucose. Our hypothesis is that this novel sRNA works in concert with Spot 42 to regulate carbohydrate metabolism and uptake.

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Project description:As advances are made toward the industrial feasibility of mass-producing biofuels and commodity chemicals with sugar-fermenting microbes, high feedstock costs continue to inhibit commercial application. Hydrolyzed lignocellulosic biomass represents an ideal feedstock for these purposes as it is cheap and prevalent. However, many microbes, including Escherichia coli, struggle to efficiently utilize this mixture of hexose and pentose sugars due to the regulation by the carbon catabolite repression (CCR) system. CCR causes a sequential utilization of sugars, rather than simultaneous utilization, resulting in reduced carbon yield and complex process implications in fed-batch fermentation. A mutation in the gene encoding the cyclic AMP receptor protein, Crp*, has been shown to disable CCR and improve co-utilization of mixed sugar substrates. Here, we present the strain construction and characterization of a site specific crp* chromosomal mutant in E. coli BL21 starTM (DE3). The crp* mutant strain demonstrates simultaneous consumption of glucose and xylose, suggesting a deregulated CCR system. The proteomic analysis further showed that cells link xylose consumption to energy production through the de novo nucleotide synthesis pathway, explaining the relatively slower growth of the crp* mutant strain. This highly characterized strain can be particularly beneficial for chemical production by simultaneously utilizing both C5 and C6 substrates from lignocellulosic biomass.

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Project description:In filamentous ascomycete fungi, the utilization of alternate carbon sources is influenced by the zinc finger transcription factor CreA/CRE-1, which encodes a carbon catabolite repressor protein homologous to Mig1 from Saccharomyces cerevisiae. In Neurospora crassa, deletion of cre-1 results in increased secretion of amylase and β-galactosidase. Here, we determined the CRE-1 regulon by investigating the transcriptome of a Δcre-1 strain compared to wild type when grown on Avicel versus minimal medium (MM). Our data provide comprehensive information on the CRE-1 regulon in N. crassa and contribute to deciphering the global role of carbon catabolite repression in filamentous ascomycete fungi during plant cell wall deconstruction.

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Project description:Carbon catabolite repression (CCR) occurs in the presence of sufficient concentrations of easy metabolizable carbon sources (e.g. glucose), down-regulating the expression of genes encoding enzymes involved in the breakdown of complex carbon sources. The extent of CCR at a global level is unknown in wood-rotting fungi – and was investigated in Dichomitus squalens with transcriptomics and exo-proteomics. Approximately 10% of expressed genes had lower expression in presence of glucose compared to Avicel or xylan alone. The glucose repressed genes included key components for utilization of plant biomass – CAZymes, sugar transporters and carbon catabolic genes. The majority of polysaccharide degrading CAZymes were repressed and included activities towards all major polymers. The repression found in the transcriptome was strongly supported by exo-proteomics – there was repression of almost all of the CAZymes whose transcripts were repressed on Avicel. The clear CCR-mediated effects indicate biotech production of CAZymes by D. squalens would benefit from de-repressed or constitutively expressing strains. The extent of CCR is surprising given that D. squalens rarely experiences high sugar concentrations in its woody environment.

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