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Superfolder GFP reporters validate diverse new mRNA targets of the classic porin regulator, MicF RNA

ABSTRACT: MicF is a textbook example of a small regulatory RNA (sRNA) that acts on a trans-encoded target mRNA through imperfect base paring. The discovery of MicF as a post-transcriptional repressor of the major Escherichia coli porin OmpF established the paradigm for a meanwhile common mechanism of translational inhibition, through antisense sequestration of a ribosome binding site. However, whether MicF regulates additional genes has remained unknown for almost three decades. Here, we have harnessed the novel superfolder variant of GFP for reporter-gene fusions to validate newly predicted targets of MicF in Salmonella. We show that the conserved 5’ end of MicF acts by seed pairing to repress the mRNAs of global transcriptional regulator Lrp, periplasmic protein YahO, and lipid A-modifying enzyme LpxR. Whilst MicF binds lrp and yahO in the 5’ UTR, it targets lpxR at both the ribosome binding site and deep within the coding sequence. Repression in the coding sequence of lpxR may be achieved by decreasing mRNA stability through exacerbating the use of a native RNase E site proximal to the short MicF-lpxR duplex. Altogether, this study assigns the classic MicF sRNA to the growing class of Hfqassociated regulators that use diverse mechanisms to impact multiple loci.

ORGANISM(S): Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344 Salmonella enterica

PROVIDER: GSE35848 | GEO | 2012/02/17

SECONDARY ACCESSION(S): PRJNA151865

REPOSITORIES: GEO

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Superfolder GFP reporters validate diverse new mRNA targets of the classic porin regulator, MicF RNA

Project description:MicF is a textbook example of a small regulatory RNA (sRNA) that acts on a trans-encoded target mRNA through imperfect base paring. The discovery of MicF as a post-transcriptional repressor of the major Escherichia coli porin OmpF established the paradigm for a meanwhile common mechanism of translational inhibition, through antisense sequestration of a ribosome binding site. However, whether MicF regulates additional genes has remained unknown for almost three decades. Here, we have harnessed the novel superfolder variant of GFP for reporter-gene fusions to validate newly predicted targets of MicF in Salmonella. We show that the conserved 5’ end of MicF acts by seed pairing to repress the mRNAs of global transcriptional regulator Lrp, periplasmic protein YahO, and lipid A-modifying enzyme LpxR. Whilst MicF binds lrp and yahO in the 5’ UTR, it targets lpxR at both the ribosome binding site and deep within the coding sequence. Repression in the coding sequence of lpxR may be achieved by decreasing mRNA stability through exacerbating the use of a native RNase E site proximal to the short MicF-lpxR duplex. Altogether, this study assigns the classic MicF sRNA to the growing class of Hfqassociated regulators that use diverse mechanisms to impact multiple loci. To determine the targets of the small regulatory RNA MicF in S. Typhimurium, we looked at the effect of a short pulse of MicF over-expression on the Salmonella transcriptome. To achieve over-expression, the micF gene was cloned in the pBAD plasmid and induced with 0.2% L-arabinose for 10 min. We then extracted the total RNA for transcriptional profiling. A strain carrying the pBAD plasmid w/o insert was used as negative control (also induced by L-arabinose). 3 biological replicates were performed. This sRNA target identification strategy has been described in Papenfort et al; Molecular Microbiology (2006) 62(6), 1674?1688.

2012-02-17 | E-GEOD-35848 | biostudies-arrayexpress

Neisseria meningitides small RNA's regulate Citric acid cycle activity under Feast and Famine conditions

Project description:We identified twin small non-coding RNAs regulating tricarboxylic acid (TCA) cycle activity in Neisseria meningitidis. Expression of TCA cycle enzymes was elevated in sRNA deletion mutants. Direct interaction between sRNAs and the ribosomal entry sites on target mRNAs was demonstrated. Expression of the sRNAs was down-regulated in cells grown in poor medium with glucose as the sole carbon source but not without lrp, indicating that sRNA expression is controlled by the stringent response. N. meningitidis, over-expressing the sRNAs replicated in blood, but not in human cerebrospinal fluid. In addition, Lrp synthesis was inhibited by direct interaction between the sRNA and the 5’ UTR of lrp. sRNAs control adaptation of N. meningitidis to variation in nutrient supply in different niches of its host.

2017-03-22 | PXD000891 | Pride

Phenotypic and transcriptional analysis of the osmotic regulator OmpR in Yersinia pestis

Project description:Background The osmotic regulator OmpR in Escherichia coli regulates differentially the expression of major porin proteins OmpF and OmpC. In Yersinia enterocolitica and Y. pseudotuberculosis, OmpR is required for both virulence and survival within macrophages. However, the phenotypic and regulatory roles of OmpR in Y. pestis are not yet fully understood. Results Y. pestis OmpR is involved in building resistance against phagocytosis and controls the adaptation to various stressful conditions met in macrophages. The ompR mutation likely did not affect the virulence of Y. pestis strain 201 that was a human-avirulent enzootic strain. The microarray-based comparative transcriptome analysis disclosed a set of 224 genes whose expressions were affected by the ompR mutation, indicating the global regulatory role of OmpR in Y. pestis. Real-time RT-PCR or lacZ fusion reporter assay further validated 16 OmpR-dependent genes, for which OmpR consensus-like sequences were found within their upstream DNA regions. ompC, F, X, and R were up-regulated dramatically with the increase of medium osmolarity, which was mediated by OmpR occupying the target promoter regions in a tandem manner. Conclusion OmpR contributes to the resistance against phagocytosis or survival within macrophages, which is conserved in the pathogenic yersiniae. Y. pestis OmpR regulates ompC, F, X, and R directly through OmpR-promoter DNA association. There is an inducible expressions of the pore-forming proteins OmpF, C, and X at high osmolarity in Y. pestis, in contrast to the reciprocal regulation of them in E. coli. The main difference is that ompF expression is not repressed at high osmolarity in Y. pestis, which is likely due to the absence of a promoter-distal OmpR-binding site for ompF.

2011-10-24 | GSE26601 | GEO

Fosfomycin uptake in Escherichia coli is mediated by the outer membrane porins OmpF, OmpC and LamB

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2024-01-24 | GSE236554 | GEO

Escherichia coli Lrp regulates one-third of the genome via direct, cooperative, and indirect paths

Project description:The global regulator Lrp plays a crucial role in regulating metabolism, virulence and motility in response to environmental conditions. Lrp has previously been shown to activate or repress approximately 10% of genes in Escherichia coli. However, the full spectrum of targets, and how Lrp acts to regulate them, has stymied earlier study. We have combined matched ChIP-seq and RNA sequencing under nine physiological conditions to map the binding and regulatory activity of Lrp as it directs responses to nutrient abundance. In addition to identifying hundreds of novel Lrp targets, we observe two new global trends: first, that Lrp will often bind to promoters in a poised position under conditions when it has no regulatory activity, and second, that nutrient levels induce a global shift in the equilibrium between non-specific and sequence-specific DNA binding. The overall regulatory behavior of Lrp, which as we now show regulates 35% of E. coli genes directly or indirectly under at least one condition, thus arises from the interaction between changes in Lrp binding specificity and cooperative action with other regulators.

2018-11-28 | GSE111874 | GEO

Bi-directional ribosome scanning controls the stringency of start codon selection

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2021-11-24 | GSE176058 | GEO

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Project description:We present HDX-MS data of apo-SurA, SurA in complex with OmpX, OmpF and a peptide with sequence WEYIPNV to define (1) interdomain dynamics in the periplasmic chaperone SurA, and (2) the binding site of OmpX, OmpF and WEYIPNV on SurA.

2020-05-22 | PXD017010 | Pride

New RoxS sRNA targets identified in B. subtilis by pulsed SILAC

Project description:Non-coding RNA (sRNA) are playing a key role in gene expression in bacteria. In general, sRNA regulates gene expression by base-pairing with ribosome binding sites to block translation. The modification of the ribosome trafficking generally affects mRNA stability. However, few examples have been described in bacteria where sRNA binding are mainly affected translation without modifying mRNA stability. In order to identify such targets in B. subtilis, we developed a pulsed-SILAC method allowing to label new protein synthesis after a short expression of the sRNA. We applied it to the best characterized sRNA in this bacteria, namely RoxS. RoxS sRNA was previously shown to interfere with the expression of genes encoding enzymes of the central metabolism to control the NAD/NADH ratio in B. subtilis.

2023-01-18 | MSV000091084 | MassIVE

Improving the purification of membrane proteins from Escherichia coli C41(DE3)

Project description:The overexpression and purification of membrane proteins to high purity and homogeneity is a challenging task. Over time, several strains have been developed that decrease the toxic side-effects and thus result in higher bio mass and protein yield. However, two major contaminants have been identified in membrane protein preparations from E. coli: the outer membrane porin OmpF and AcrB, which is part of a tripartite efflux pump. Both proteins crystallise from low concentrations and diverse conditions, which make them a major problem, especially in membrane protein crystallography. In this study, we present a C41(DE3)-derived expression strains that is depleted of these two proteins.

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