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1.
mBio ; 15(4): e0315323, 2024 Apr 10.
Article in English | MEDLINE | ID: mdl-38511926

ABSTRACT

The alphaproteobacterium Caulobacter crescentus thrives in oligotrophic environments and is able to optimally exploit minimal resources by entertaining an intricate network of gene expression control mechanisms. Numerous transcriptional activators and repressors have been reported to contribute to these processes, but only few studies have focused on regulation at the post-transcriptional level in C. crescentus. Small RNAs (sRNAs) are a prominent class of regulators of bacterial gene expression, and most sRNAs characterized today engage in direct base-pairing interactions to modulate the translation and/or stability of target mRNAs. In many cases, the ubiquitous RNA chaperone, Hfq, contributes to the establishment of RNA-RNA interactions. Although the deletion of the hfq gene is associated with a severe loss of fitness in C. crescentus, the RNA ligands of the chaperone have remained largely unexplored. Here we report on the identification of coding and non-coding transcripts associated with Hfq in C. crescentus and demonstrate Hfq-dependent post-transcriptional regulation in this organism. We show that the Hfq-bound sRNA RusT is transcriptionally controlled by the NtrYX two-component system and induced in response to iron starvation. By combining RusT pulse expression with whole-genome transcriptome analysis, we determine 16 candidate target transcripts that are deregulated, many of which encode outer membrane transporters. We hence suggest RusT to support remodeling of the C. crescentus cell surface when iron supplies are limited.IMPORTANCEThe conserved RNA-binding protein Hfq contributes significantly to the adaptation of bacteria to different environmental conditions. Hfq not only stabilizes associated sRNAs but also promotes inter-molecular base-pairing interactions with target transcripts. Hfq plays a pivotal role for growth and survival, controlling central metabolism and cell wall synthesis in the oligotroph Caulobacter crescentus. However, direct evidence for Hfq-dependent post-transcriptional regulation and potential oligotrophy in C. crescentus has been lacking. Here, we identified sRNAs and mRNAs associated with Hfq in vivo, and demonstrated the requirement of Hfq for sRNA-mediated regulation, particularly of outer membrane transporters in C. crescentus.


Subject(s)
Caulobacter crescentus , RNA, Small Untranslated , Caulobacter crescentus/genetics , Caulobacter crescentus/metabolism , RNA, Small Untranslated/metabolism , RNA, Bacterial/metabolism , RNA, Messenger/genetics , Membrane Transport Proteins/metabolism , Host Factor 1 Protein/genetics , Host Factor 1 Protein/metabolism , Gene Expression Regulation, Bacterial
2.
Nat Commun ; 11(1): 6067, 2020 11 27.
Article in English | MEDLINE | ID: mdl-33247102

ABSTRACT

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.


Subject(s)
Drug Resistance, Microbial/genetics , RNA, Bacterial/genetics , Vibrio cholerae/cytology , Vibrio cholerae/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Sequence , Biofilms/growth & development , Gene Expression Regulation, Bacterial , Genes, Bacterial , Mutation/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Repressor Proteins/metabolism , Transcription, Genetic , Vibrio cholerae/physiology
3.
EMBO J ; 38(16): e101650, 2019 08 15.
Article in English | MEDLINE | ID: mdl-31313835

ABSTRACT

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.


Subject(s)
RNA, Small Untranslated/chemistry , RNA, Small Untranslated/genetics , Vibrio cholerae/genetics , Bacterial Outer Membrane Proteins/genetics , Conserved Sequence , Gene Expression Regulation, Bacterial , Nucleic Acid Conformation , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , Stress, Physiological
4.
Nucleic Acids Res ; 47(6): 3171-3183, 2019 04 08.
Article in English | MEDLINE | ID: mdl-30649554

ABSTRACT

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 ∼400 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.


Subject(s)
Gene Expression Regulation, Bacterial/genetics , Homoserine/analogs & derivatives , Ketones , Vibrio cholerae/genetics , Virulence/genetics , Biofilms/growth & development , Gene Expression Profiling , Homoserine/genetics , Lactones , Promoter Regions, Genetic , Quorum Sensing/genetics , Signal Transduction/genetics , Vibrio cholerae/pathogenicity
5.
Proc Natl Acad Sci U S A ; 114(40): E8498-E8507, 2017 10 03.
Article in English | MEDLINE | ID: mdl-28923955

ABSTRACT

Successful pathogens use complex signaling mechanisms to monitor their environment and reprogram global gene expression during specific stages of infection. Group A Streptococcus (GAS) is a major human pathogen that causes significant disease burden worldwide. A secreted cysteine protease known as streptococcal pyrogenic exotoxin B (SpeB) is a key virulence factor that is produced abundantly during infection and is critical for GAS pathogenesis. Although identified nearly a century ago, the molecular basis for growth phase control of speB gene expression remains unknown. We have discovered that GAS uses a previously unknown peptide-mediated intercellular signaling system to control SpeB production, alter global gene expression, and enhance virulence. GAS produces an eight-amino acid leaderless peptide [SpeB-inducing peptide (SIP)] during high cell density and uses the secreted peptide for cell-to-cell signaling to induce population-wide speB expression. The SIP signaling pathway includes peptide secretion, reimportation into the cytosol, and interaction with the intracellular global gene regulator Regulator of Protease B (RopB), resulting in SIP-dependent modulation of DNA binding and regulatory activity of RopB. Notably, SIP signaling causes differential expression of ∼14% of GAS core genes. Several genes that encode toxins and other virulence genes that enhance pathogen dissemination and infection are significantly up-regulated. Using three mouse infection models, we show that the SIP signaling pathway is active during infection and contributes significantly to GAS pathogenesis at multiple host anatomic sites. Together, our results delineate the molecular mechanisms involved in a previously undescribed virulence regulatory pathway of an important human pathogen and suggest new therapeutic strategies.


Subject(s)
Bacterial Proteins/metabolism , Exotoxins/metabolism , Gene Expression Regulation, Bacterial/drug effects , Peptide Fragments/pharmacology , Streptococcal Infections/microbiology , Streptococcus pyogenes/pathogenicity , Virulence , Amino Acid Sequence , Animals , Bacterial Proteins/genetics , Base Sequence , Exotoxins/genetics , Gene Expression Profiling , Humans , Mice , Sequence Homology , Streptococcal Infections/genetics , Streptococcal Infections/metabolism , Streptococcus pyogenes/isolation & purification
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