RESUMO
Small regulatory RNAs (sRNAs) are crucial components of many stress response systems. The envelope stress response (ESR) of Gram-negative bacteria is a paradigm for sRNA-mediated stress management and involves, among other factors, the alternative sigma factor E (σE ) and one or more sRNAs. In this study, we identified the MicV sRNA as a new member of the σE regulon in Vibrio cholerae. We show that MicV acts redundantly with another sRNA, VrrA, and that both sRNAs share a conserved seed-pairing domain allowing them to regulate multiple target mRNAs. V. cholerae lacking σE displayed increased sensitivity toward antimicrobials, and over-expression of either of the sRNAs suppressed this phenotype. Laboratory selection experiments using a library of synthetic sRNA regulators revealed that the seed-pairing domain of σE -dependent sRNAs is strongly enriched among sRNAs identified under membrane-damaging conditions and that repression of OmpA is crucial for sRNA-mediated stress relief. Together, our work shows that MicV and VrrA act as global regulators in the ESR of V. cholerae and provides evidence that bacterial sRNAs can be functionally annotated by their seed-pairing sequences.
Assuntos
Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/genética , Vibrio cholerae/genética , Proteínas da Membrana Bacteriana Externa/genética , Sequência Conservada , Regulação Bacteriana da Expressão Gênica , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/genética , Estresse FisiológicoRESUMO
Bacteria use quorum sensing to monitor cell density and coordinate group behaviours. In Vibrio cholerae, the causative agent of the diarrheal disease cholera, quorum sensing is connected to virulence gene expression via the two autoinducer molecules, AI-2 and CAI-1. Both autoinducers share one signal transduction pathway to control the production of AphA, a key transcriptional activator of biofilm formation and virulence genes. In this study, we demonstrate that the recently identified autoinducer, DPO, also controls AphA production in V. cholerae. DPO, functioning through the transcription factor VqmA and the VqmR small RNA, reduces AphA levels at the post-transcriptional level and consequently inhibits virulence gene expression. VqmR-mediated repression of AphA provides an important link between the AI-2/CAI-1 and DPO-dependent quorum sensing pathways in V. cholerae. Transcriptome analyses comparing the effect of single autoinducers versus autoinducer combinations show that quorum sensing controls the expression of â¼400 genes in V. cholerae and that all three autoinducers are required for a full quorum sensing response. Together, our data provide a global view on autoinducer interplay in V. cholerae and highlight the importance of RNA-based gene control for collective functions in this major human pathogen.
Assuntos
Regulação Bacteriana da Expressão Gênica/genética , Homosserina/análogos & derivados , Cetonas , Vibrio cholerae/genética , Virulência/genética , Biofilmes/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Homosserina/genética , Lactonas , Regiões Promotoras Genéticas , Percepção de Quorum/genética , Transdução de Sinais/genética , Vibrio cholerae/patogenicidadeRESUMO
Vibrio cholerae, the cause of cholera disease, exhibits a characteristic curved rod morphology, which promotes infectivity and motility in dense hydrogels. Periplasmic protein CrvA determines cell curvature in V. cholerae, yet the regulatory factors controlling CrvA are unknown. Here, we discover the VadR small RNA (sRNA) as a post-transcriptional inhibitor of the crvA mRNA. Mutation of vadR increases cell curvature, whereas overexpression has the inverse effect. We show that vadR transcription is activated by the VxrAB two-component system and triggered by cell-wall-targeting antibiotics. V. cholerae cells failing to repress crvA by VadR display decreased survival upon challenge with penicillin G indicating that cell shape maintenance by the sRNA is critical for antibiotic resistance. VadR also blocks the expression of various key biofilm genes and thereby inhibits biofilm formation in V. cholerae. Thus, VadR is an important regulator for synchronizing peptidoglycan integrity, cell shape, and biofilm formation in V. cholerae.
Assuntos
Resistência Microbiana a Medicamentos/genética , RNA Bacteriano/genética , Vibrio cholerae/citologia , Vibrio cholerae/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Mutação/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica , Vibrio cholerae/fisiologiaRESUMO
Transcriptome analysis using RNA-sequencing (RNA-seq) has now become the standard approach to determine the transcriptional output of an organism. Various modifications to this technology have been developed over the years, usually aiming to improve the annotation of transcript borders, or to identify novel classes of RNAs, such as small regulatory RNAs (sRNAs) and antisense transcripts. RNA-seq has also led to the identification of dozens of new sRNAs in the major human pathogen, Vibrio cholerae. Several of these sRNAs function in the context of a cell-to-cell communication process, called quorum sensing (QS). QS is key for pathogenicity and biofilm formation of V. cholerae and the sRNAs involved typically act by base pairing with multiple target mRNAs to control gene expression at the posttranscriptional level. In this chapter, we describe the use of RNA-seq technologies for the discovery and characterization of regulatory RNAs in V. cholerae and discuss their relevance to QS and collective functions, such as biofilm formation. We further outline possible methods for the identification and validation of sRNA target genes, which can provide crucial information as to the physiological roles of an sRNA.