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The primary transcriptome of Neisseria meningitidis and its interaction with the RNA chaperone Hfq.

ABSTRACT: Neisseria meningitidis is a human commensal that can also cause life-threatening meningitis and septicemia. Despite growing evidence for RNA-based regulation in meningococci, their transcriptome structure and output of regulatory small RNAs (sRNAs) are incompletely understood. Using dRNA-seq, we have mapped at single-nucleotide resolution the primary transcriptome of N. meningitidis strain 8013. Annotation of 1625 transcriptional start sites defines transcription units for most protein-coding genes but also reveals a paucity of classical ?70-type promoters, suggesting the existence of activators that compensate for the lack of -35 consensus sequences in N. meningitidis. The transcriptome maps also reveal 65 candidate sRNAs, a third of which were validated by northern blot analysis. Immunoprecipitation with the RNA chaperone Hfq drafts an unexpectedly large post-transcriptional regulatory network in this organism, comprising 23 sRNAs and hundreds of potential mRNA targets. Based on this data, using a newly developed gfp reporter system we validate an Hfq-dependent mRNA repression of the putative colonization factor PrpB by the two trans-acting sRNAs RcoF1/2. Our genome-wide RNA compendium will allow for a better understanding of meningococcal transcriptome organization and riboregulation with implications for colonization of the human nasopharynx.

SUBMITTER: Heidrich N

PROVIDER: S-EPMC5449619 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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The primary transcriptome of Neisseria meningitidis and its interaction with the RNA chaperone Hfq.

Heidrich Nadja N Bauriedl Saskia S Barquist Lars L Li Lei L Schoen Christoph C Vogel Jörg J

Nucleic acids research 20170601 10

Neisseria meningitidis is a human commensal that can also cause life-threatening meningitis and septicemia. Despite growing evidence for RNA-based regulation in meningococci, their transcriptome structure and output of regulatory small RNAs (sRNAs) are incompletely understood. Using dRNA-seq, we have mapped at single-nucleotide resolution the primary transcriptome of N. meningitidis strain 8013. Annotation of 1625 transcriptional start sites defines transcription units for most protein-coding ge ...[more]

PMID: 28334889

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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:BackgroundThe bacterial Hfq protein is able to interact with diverse RNA molecules, including regulatory small non-coding RNAs (sRNAs), and thus it is recognized as a global post-transcriptional regulator of gene expression. Loss of Hfq has an extensive impact in bacterial physiology which in several animal pathogens influences virulence. Sinorhizobium meliloti is a model soil bacterium known for its ability to establish a beneficial nitrogen-fixing intracellular symbiosis with alfalfa. Despite the predicted general involvement of Hfq in the establishment of successful bacteria-eukaryote interactions, its function in S. meliloti has remained unexplored.ResultsTwo independent S. meliloti mutants, 2011-3.4 and 1021Deltahfq, were obtained by disruption and deletion of the hfq gene in the wild-type strains 2011 and 1021, respectively, both exhibiting similar growth defects as free-living bacteria. Transcriptomic profiling of 1021Deltahfq revealed a general down-regulation of genes of sugar transporters and some enzymes of the central carbon metabolism, whereas transcripts specifying the uptake and metabolism of nitrogen sources (mainly amino acids) were more abundant than in the wild-type strain. Proteomic analysis of the 2011-3.4 mutant independently confirmed these observations. Symbiotic tests showed that lack of Hfq led to a delayed nodulation, severely compromised bacterial competitiveness on alfalfa roots and impaired normal plant growth. Furthermore, a large proportion of nodules (55%-64%) elicited by the 1021Deltahfq mutant were non-fixing, with scarce content in bacteroids and signs of premature senescence of endosymbiotic bacteria. RT-PCR experiments on RNA from bacteria grown under aerobic and microoxic conditions revealed that Hfq contributes to regulation of nifA and fixK1/K2, the genes controlling nitrogen fixation, although the Hfq-mediated regulation of fixK is only aerobiosis dependent. Finally, we found that some of the recently identified S. meliloti sRNAs co-inmunoprecipitate with a FLAG-epitope tagged Hfq protein.ConclusionsOur results support that the S. meliloti RNA chaperone Hfq contributes to the control of central metabolic pathways in free-living bacteria and influences rhizospheric competence, survival of the microsymbiont within the nodule cells and nitrogen fixation during the symbiotic interaction with its legume host alfalfa. The identified S. meliloti Hfq-binding sRNAs are predicted to participate in the Hfq regulatory network.

Dataset Information

The primary transcriptome of Neisseria meningitidis and its interaction with the RNA chaperone Hfq.

Publications

The primary transcriptome of Neisseria meningitidis and its interaction with the RNA chaperone Hfq.

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