Project description:In Neisseria meningitidis iron responsive gene regulation is mediated primarily by the Ferric Uptake Regulator (Fur) protein. When complexed with iron, Fur represses gene expression by preventing transcription initiation. Fur can also indirectly activate gene expression via the repression of regulatory small RNAs (sRNA). One such Fur-and iron-regulated sRNA, NrrF, was previously identified in N. meningitidis and shown to repress expression of the sdhA and sdhC genes encoding subunits of the succinate dehydrogenase complex. In the majority of Gram-negative bacteria sRNA-mediated regulation requires a cofactor RNA-binding protein (Hfq) for proper gene regulation and stabilization. In this study we examined the role of Hfq in NrrF-mediated regulation of the succinate dehydrogenase genes in N. meningitidis and the effect of an hfq- mutation on iron-responsive gene regulation more broadly. We first demonstrated that the stability of Nrrf as well as the regulation of sdhC and sdhA in vivo was unaltered in the hfq- mutant. Secondly, we established that iron responsive gene regulation of the Fur-regulated sodB gene was dependent on Hfq. Finally, we demonstrate that in N. meningitidis Hfq functions to control expression of both ORFs and intergenic regions via iron independent mechanisms. Collectively these studies demonstrate that in N. meningitidis iron and NrrF mediated regulation of sdhC and sdhA can occur independently of Hfq, although Hfq functions more globally to control regulation of other N. meningitidis genes primarily by iron-independent mechanisms.
Project description:In Neisseria meningitidis iron responsive gene regulation is mediated primarily by the Ferric Uptake Regulator (Fur) protein. When complexed with iron, Fur represses gene expression by preventing transcription initiation. Fur can also indirectly activate gene expression via the repression of regulatory small RNAs (sRNA). One such Fur-and iron-regulated sRNA, NrrF, was previously identified in N. meningitidis and shown to repress expression of the sdhA and sdhC genes encoding subunits of the succinate dehydrogenase complex. In the majority of Gram-negative bacteria sRNA-mediated regulation requires a cofactor RNA-binding protein (Hfq) for proper gene regulation and stabilization. In this study we examined the role of Hfq in NrrF-mediated regulation of the succinate dehydrogenase genes in N. meningitidis and the effect of an hfq- mutation on iron-responsive gene regulation more broadly. We first demonstrated that the stability of Nrrf as well as the regulation of sdhC and sdhA in vivo was unaltered in the hfq- mutant. Secondly, we established that iron responsive gene regulation of the Fur-regulated sodB gene was dependent on Hfq. Finally, we demonstrate that in N. meningitidis Hfq functions to control expression of both ORFs and intergenic regions via iron independent mechanisms. Collectively these studies demonstrate that in N. meningitidis iron and NrrF mediated regulation of sdhC and sdhA can occur independently of Hfq, although Hfq functions more globally to control regulation of other N. meningitidis genes primarily by iron-independent mechanisms. RNA was isolated from wild-type MC58 Neisseria meningitidis, from an hfq- mutant, and from a complemented hfq- mutant under both iron-replete and iron-deplete conditions. Three biological replicates were analyzed for each strain and condition were analyzed.
Project description:Neisseria meningitidis is a human commensal that occasionally causes life-threatening infections such as bacterial meningitis and septicemia. Despite experimental evidence that gene regulation as well as the expression of small non-coding RNAs (sRNAs) affect meningococcal virulence, the organization of its transcriptome, including in particular the biogenesis of sRNAs and their mode of action, is only poorly understood. Here, we addressed these issues using a combination of high-throughput technologies. We applied differential RNA-seq (dRNA-seq) to produce a single-nucleotide resolution map of the primary transcriptome of N. meningitidis strain 8103. Our dRNA-seq analysis predicted 1,625 transcriptional start sites (TSS) including 65 non-coding RNA transcripts, of which 20 were further validated by Northern analysis. This allowed for the discovery of a novel CRISPR-associated sRNA with a Cas9-independent biogenesis. Genome-wide mapping of σ 70-dependent and independent promoters revealed that classical Escherichia coli-like σ70 promoter are absent in most of the protein coding genes in meningococci. The majority of the 706 primary TSSs (pTSSs) were associated with coding sequences, including 382 pTSS obtained for single genes and 240 pTSSs obtained for genes located in operons. By Hfq RNA immunoprecipitation sequencing (RIP-seq) we identified a large Hfq-centered post-transcriptional regulatory network comprising 24 sRNAs and 407 potential mRNA targets, and rifampicin stability assays demonstrated that Hfq binding confers enhanced stability on sRNAs. We finally confirmed the interactions of two sRNAs and their cognate target mRNA in vivo. Both directly repress prpB encoding a methylisocitrate lyse which was previously shown to be involved in meningococcal colonization of the human nasopharynx.The combination of both high-throughput approaches thus creates a compendium that not only provides a valuable data resource, but also allows for a better understanding of meningococcal transcriptome organization and riboregulation with implications for colonization of the human nasopharynx.
Project description:The zur regulon in Neisseria meningitidis was elucidated in the strain MC58 using a zur knockout strain and conditions which activate Zur ( zinc supplementation in the medium)
Project description:Transcriptome analysis of Neisseria meningitidis exposed to physiologically relevant stress signals (e.g. heat shock, oxidative stress, iron and carbon source limitation) and identification of sRNAs resulting differentially expressed in vitro.
Project description:Microarray comparative genome hybridization (mCGH) data was collected from one Neisseria cinerea, two Neisseria lactamica, two Neisseria gonorrhoeae, and 48 Neisseria meningitidis isolates. For N. meningitidis, these isolates are from diverse clonal complexes, invasive and carriage strains, and all major serogroups. The microarray platform represented N. meningitidis strains MC58, Z2491, and FAM18 and N. gonorrhoeae FA1090.
Project description:Transcriptome analysis of Neisseria meningitidis exposed to physiologically relevant stress signals (e.g. heat shock, oxidative stress, iron and carbon source limitation) and identification of sRNAs resulting differentially expressed in vitro.
Project description:Neisseria meningitidis is an obligate commensal colonising the human nasopharynx and occasionally invades the bloodstream causing life-threatening meningitis and septicaemia. The gene NMB0419 on the genome of N. meningitidis MC58 encodes a putative Sel1-like repeat (SLR) containing protein, which has been implicated in mediating meningococcal invasion of epithelial cells. We prepared RNA samples from N. meningitidis MC58 (WT) and its isogenic mutant of NMB0419 grown to log phase in in-vitro culture. The RNA samples were subjected to RNA sequencing. The resulting transcriptomes were compared to determine the genes that differentially expressed in NMB0419 mutant.
Project description:Neisseria meningitidis is a major cause of bacterial meningitis and septicemia worldwide. Seven new serogroup C meningococci were isolated from two provinces of China in January, 2006. Their PorA VR types were P1.20, 9. Multilocus sequence typing results indicated that they all belonged to ST-7. It is a new serogroup C N. meningitidis sequence type clone identified in China. Here we also present the results of a genomic comparison of these isolates with other 15 N. meningitidis serogroup A and B isolates, which belonged to ST-7, based on comparative genomic hybridization analysis. The data described here would be helpful to monitor the spread of this new serogroup C meningococci sequence type clone in China and worldwide. Keywords: comparative genomic hybridization