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Genome-wide transcriptional start site mapping and sRNA identification in the pathogen Leptospira interrogans

ABSTRACT: Leptospira are emerging zoonotic pathogens transmitted from animals to humans typically through contaminated environmental sources of water and soil. Transcriptional regulation of pathogenic Leptospira spp. underlying the adaptive response to different hosts and environmental conditions remains elusive. In this study, we provide the first global Transcriptional Start Site (TSS) map of a Leptospira species. RNA was obtained from the pathogen Leptospira interrogans grown at 30° (optimal in vitro temperature) and 37°C (host temperature) and selectively enriched for 5' ends of native transcripts. Primary TSS (pTSS) was identified for 2,865 genes, accounting for 67% of the total genome. The majority of the TSSs were located between 0 to 10 nucleotides from the translational start site. Comparative dRNA-seq analysis revealed conservation of most pTSS at 30° and 37°C. Promoter prediction algorithms allow the identification of the binding sites of the alternative sigma factor sigma 54. However, other motifs were not identified indicating that Leptospira consensus promoter sequences are inherently different from the E. coli model. RNA sequencing also identified 277 and 226 putative small regulatory RNAs (sRNAs) at 30°C and 37°C, respectively, including 8 validated sRNAs by Northern blots. These results provide the first global view of transcriptional start sites and the repertoire of sRNAs in L. interrogans, and will establish a foundation for future experimental work on gene regulation under various environmental conditions including those in the host.

ORGANISM(S): Leptospira interrogans serovar Manilae

PROVIDER: GSE92976 | GEO | 2017/01/23

SECONDARY ACCESSION(S): PRJNA359198

REPOSITORIES: GEO

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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.

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Genome-wide transcriptional start site mapping and sRNA identification in the pathogen Leptospira interrogans

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