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Gene regulation at the post-transcriptional level is prevalent in all domains of life. In bacteria, ProQ-like proteins have emerged as important RNA chaperones facilitating RNA stability and RNA duplex formation. In the major human pathogen Vibrio cholerae, post-transcriptional gene regulation is key for virulence, biofilm formation, and antibiotic resistance, yet the role of ProQ has not been studied. Here, we show that ProQ interacts with hundreds of transcripts in V. cholerae, including the highly abundant FlaX small RNA (sRNA). Global analyses of RNA duplex formation using RIL-Seq (RNA interaction by ligation and sequencing) revealed a vast network of ProQ-assisted interactions and identified a role for FlaX in motility regulation. Specifically, FlaX base-pairs with multiple sites on the flaB flagellin mRNA, preventing 30S ribosome binding and translation initiation. V. cholerae cells lacking flaX display impaired motility gene expression, altered flagella composition and reduced swimming in liquid environments. Our results provide a global view on ProQ-associated RNA duplex formation and pinpoint the mechanistic and phenotypic consequences associated with ProQ-associated sRNAs in V. cholerae., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The study of small, regulatory RNAs (sRNA) that act by base-pairing with target RNAs in bacteria has been steadily advancing, particularly with the availability of more and more transcriptome and RNA-RNA interactome datasets. While the characterization of multiple sRNAs has helped to elucidate their mechanisms of action, these studies also are providing insights into protein function, control of metabolic flux, and connections between metabolic pathways as we will discuss here. In describing several examples of the metabolic insights gained, we will summarize the different types of base-pairing sRNAs including mRNA-derived sRNAs, sponge RNAs, RNA mimics, and dual-function RNAs as well as suggest how information about sRNAs could be exploited in the future., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Ltd.)

The type III secretion system (T3SS) is a needle-like complex used by numerous bacterial pathogens in host infection by directly injecting exotoxins into the host cell cytoplasm, leading to cell death. The T3SS is a known virulence factor in the shrimp pathogen Vibrio campbellii . The ∼40 genes comprising the V. campbellii T3SS are regulated by a network of transcription factors in response to changes in the cell's environment: cell density (quorum sensing; QS), temperature, calcium, and host cell contact. Under positive environmental stimuli, the master T3SS transcription factor ExsA activates expression of the four structural T3SS operons required for needle formation. Previous studies identified a key role of the master QS transcription factor LuxR: repression of exsA transcription via DNA binding at the exsBA promoter. Here we uncovered a new regulatory role of LuxR: indirect post-translational repression of ExsA activity via direct transcriptional repression of the gene encoding the anti-anti-activator ExsC. In V. campbellii , ExsC is a positive regulator of T3SS transcription: high ExsC expression leads to full ExsA transcription activation of the T3SS structural promoters. LuxR binding at the exsC promoter represses transcription of exsC through disruption of ExsA binding. Our findings collectively show that V. campbellii responds to high cell density signals to shut down the expression of the T3SS. We postulate that this dual regulatory mechanism by LuxR enables both the rapid inactivation of existing ExsA protein and blocks its further synthesis, leading to a rapid shutdown of T3SS activity at high cell density., Importance: Vibrio campbellii utilizes the type III secretion system (T3SS) as a mechanism of pathogenesis, which is a highly studied 'injectisome' complex that delivers exotoxins into host cells during infection. The T3SS pathogenicity island in V. campbellii comprises ∼40 genes that are organized into four structural operons. In this study, we determined that quorum sensing - a method of bacterial communication - regulates T3SS genes both at the transcriptional and post-translational levels to shut down T3SS gene expression at high population densities.

Many, if not all, bacteria use quorum sensing (QS) to control collective behaviors, and more recently, QS has also been discovered in bacteriophages (phages). Phages can produce communication molecules of their own, or "listen in" on the host's communication processes, to switch between lytic and lysogenic modes of infection. Here, we study the interaction of Vibrio cholerae with the lysogenic phage VP882, which is activated by the QS molecule DPO. We discover that induction of VP882 results in the binding of phage transcripts to the major RNA chaperone Hfq, which in turn outcompetes and downregulates host-encoded small RNAs (sRNAs). VP882 itself also encodes Hfq-binding sRNAs, and we demonstrate that one of these sRNAs, named VpdS, promotes phage replication by regulating host and phage mRNA levels. We further show that host-encoded sRNAs can antagonize phage replication by downregulating phage mRNA expression and thus might be part of the host's phage defense arsenal., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

RNA-RNA interactions are a key feature of post-transcriptional gene regulation in all domains of life. While ever more experimental protocols are being developed to study RNA duplex formation on a genome-wide scale, computational methods for the analysis and interpretation of the underlying data are lagging behind. Here, we present ChimericFragments, an analysis framework for RNA-seq experiments that produce chimeric RNA molecules. ChimericFragments implements a novel statistical method based on the complementarity of the base-pairing RNAs around their ligation site and provides an interactive graph-based visualization for data exploration and interpretation. ChimericFragments detects true RNA-RNA interactions with high precision and is compatible with several widely used experimental procedures such as RIL-seq, LIGR-seq or CLASH. We further demonstrate that ChimericFragments enables the systematic detection of novel RNA regulators and RNA-target pairs with crucial roles in microbial physiology and virulence. ChimericFragments is written in Julia and available at: https://github.com/maltesie/ChimericFragments., (© The Author(s) 2024. Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics.)

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 ., Competing Interests: The authors declare no conflict of interest.

Research on microbial pathogens has traditionally relied on animal and cell culture models to mimic infection processes in the host. Over recent years, developments in microfluidics and bioengineering have led to organ-on-chip (OoC) technologies. These microfluidic systems create conditions that are more physiologically relevant and can be considered humanized in vitro models. Here we review various OoC models and how they have been applied for infectious disease research. We outline the properties that make them valuable tools in microbiology, such as dynamic microenvironments, vascularization, near-physiological tissue constitutions and partial integration of functional immune cells, as well as their limitations. Finally, we discuss the prospects for OoCs and their potential role in future infectious disease research., (© 2024. Springer Nature Limited.)

The ubiquitous RNA chaperone Hfq is involved in the regulation of key biological processes in many species across the bacterial kingdom. In the opportunistic human pathogen Klebsiella pneumoniae , deletion of the hfq gene affects the global transcriptome, virulence, and stress resistance; however, the ligands of the major RNA-binding protein in this species have remained elusive. In this study, we have combined transcriptomic, co-immunoprecipitation, and global RNA interactome analyses to compile an inventory of conserved and species-specific RNAs bound by Hfq and to monitor Hfq-mediated RNA-RNA interactions. In addition to dozens of RNA-RNA pairs, our study revealed an Hfq-dependent small regulatory RNA (sRNA), DinR, which is processed from the 3' terminal portion of dinI mRNA. Transcription of dinI is controlled by the master regulator of the SOS response, LexA. As DinR accumulates in K. pneumoniae in response to DNA damage, the sRNA represses translation of the ftsZ transcript by occupation of the ribosome binding site. Ectopic overexpression of DinR causes depletion of ftsZ mRNA and inhibition of cell division, while deletion of dinR antagonizes cell elongation in the presence of DNA damage. Collectively, our work highlights the important role of RNA-based gene regulation in K. pneumoniae and uncovers the central role of DinR in LexA-controlled division inhibition during the SOS response., Competing Interests: Competing interests statement:The authors declare no competing interest.

Small regulatory RNA (sRNAs) are key mediators of posttranscriptional gene control in bacteria. Assisted by RNA-binding proteins, a single sRNA often modulates the expression of dozens of genes, and thus sRNAs frequently adopt central roles in regulatory networks. Posttranscriptional regulation by sRNAs comes with several unique features that cannot be achieved by transcriptional regulators. However, for optimal network performance, transcriptional and posttranscriptional control mechanisms typically go hand-in-hand. This view is reflected by the ever-growing class of mixed network motifs involving sRNAs and transcription factors, which are ubiquitous in biology and whose regulatory properties we are beginning to understand. In addition, sRNA activity can be antagonized by base-pairing with sponge RNAs, adding yet another layer of complexity to these networks. In this article, we summarize the regulatory concepts underlying sRNA-mediated gene control in bacteria and discuss how sRNAs shape the output of a network, focusing on several key examples.

Vibrio cholerae is a major human pathogen causing the diarrheal disease, cholera. Regulation of virulence in V. cholerae is a multifaceted process involving gene expression changes at the transcriptional and post-transcriptional level. Whereas various transcription factors have been reported to modulate virulence in V. cholerae , small regulatory RNAs (sRNAs) have now been established to also participate in virulence control and the regulation of virulence-associated processes, such as biofilm formation, quorum sensing, stress response, and metabolism. In most cases, these sRNAs act by base-pairing with multiple target transcripts and this process typically requires the aid of an RNA-binding protein, such as the widely conserved Hfq protein. This review article summarizes the functional roles of sRNAs in V. cholerae , their underlying mechanisms of gene expression control, and how sRNAs partner with transcription factors to modulate complex regulatory programs. In addition, we will discuss regulatory principles discovered in V. cholerae that not only apply to other Vibrio species, but further extend into the large field of RNA-mediated gene expression control in bacteria., Competing Interests: None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

Small regulatory RNAs (sRNAs) acting in concert with the RNA chaperone Hfq are prevalent in many bacteria and typically act by base-pairing with multiple target transcripts. In the human pathogen Vibrio cholerae, sRNAs play roles in various processes including antibiotic tolerance, competence, and quorum sensing (QS). Here, we use RIL-seq (RNA-interaction-by-ligation-and-sequencing) to identify Hfq-interacting sRNAs and their targets in V. cholerae. We find hundreds of sRNA-mRNA interactions, as well as RNA duplexes formed between two sRNA regulators. Further analysis of these duplexes identifies an RNA sponge, termed QrrX, that base-pairs with and inactivates the Qrr1-4 sRNAs, which are known to modulate the QS pathway. Transcription of qrrX is activated by QrrT, a previously uncharacterized LysR-type transcriptional regulator. Our results indicate that QrrX and QrrT are required for rapid conversion from individual to community behaviours in V. cholerae., (© 2022. The Author(s).)

Flagella are multiprotein complexes whose assembly and positioning require complex spatiotemporal control. Flagellar assembly is thought to be controlled by several transcriptional tiers, which are mediated through various master regulators. Here, we revisited the regulation of flagellar genes in polarly flagellated gammaproteobacteria by the regulators FlrA, RpoN (σ 54 ) and FliA (σ 28 ) in Shewanella putrefaciens CN-32 at the transcript and protein level. We found that a number of regulatory and structural proteins were present in the absence of the main regulators, suggesting that initiation of flagella assembly and motor activation relies on the abundance control of only a few structural key components that are required for the formation of the MS- and C-ring and the flagellar type III secretion system. We identified FlrA-independent promoters driving expression of the regulators of flagellar number and positioning, FlhF and FlhG. Reduction of the gene expression levels from these promoters resulted in the emergence of hyperflagellation. This finding indicates that basal expression is required to adjust the flagellar counter in Shewanella. This is adding a deeper layer to the regulation of flagellar synthesis and assembly., (© 2022 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

In recent years, there has been increased appreciation that a whole category of proteins, small proteins of around 50 amino acids or fewer in length, has been missed by annotation as well as by genetic and biochemical assays. With the increased recognition that small proteins are stable within cells and have regulatory functions, there has been intensified study of these proteins. As a result, important questions about small proteins in bacteria and archaea are coming to the fore. Here, we give an overview of these questions, the initial answers, and the approaches needed to address these questions more fully. More detailed discussions of how small proteins can be identified by ribosome profiling and mass spectrometry approaches are provided by two accompanying reviews (N. Vazquez-Laslop, C. M. Sharma, A. S. Mankin, and A. R. Buskirk, J Bacteriol 204:e00294-21, 2022, https://doi.org/10.1128/JB.00294-21; C. H. Ahrens, J. T. Wade, M. M. Champion, and J. D. Langer, J Bacteriol 204:e00353-21, 2022, https://doi.org/10.1128/JB.00353-21). We are excited by the prospects of new insights and possible therapeutic approaches coming from this emerging field.

Bacterial small RNAs (sRNAs) are well known to modulate gene expression by base pairing with trans-encoded transcripts and are typically non-coding. However, several sRNAs have been reported to also contain an open reading frame and thus are considered dual-function RNAs. In this study, we discovered a dual-function RNA from Vibrio cholerae, called VcdRP, harboring a 29 amino acid small protein (VcdP), as well as a base-pairing sequence. Using a forward genetic screen, we identified VcdRP as a repressor of cholera toxin production and link this phenotype to the inhibition of carbon transport by the base-pairing segment of the regulator. By contrast, we demonstrate that the VcdP small protein acts downstream of carbon transport by binding to citrate synthase (GltA), the first enzyme of the citric acid cycle. Interaction of VcdP with GltA results in increased enzyme activity and together VcdR and VcdP reroute carbon metabolism. We further show that transcription of vcdRP is repressed by CRP allowing us to provide a model in which VcdRP employs two different molecular mechanisms to synchronize central metabolism in V. cholerae., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Vibrio campbellii BB120 (previously classified as Vibrio harveyi) is a fundamental model strain for studying quorum sensing in vibrios. A phylogenetic evaluation of sequenced Vibrio strains in Genbank revealed that BB120 is closely related to the environmental isolate V. campbellii DS40M4. We exploited DS40M4's competence for exogenous DNA uptake to rapidly generate greater than 30 isogenic strains with deletions of genes encoding BB120 quorum-sensing system homologues. Our results show that the quorum-sensing circuit of DS40M4 is distinct from BB120 in three ways: (i) DS40M4 does not produce an acyl homoserine lactone autoinducer but encodes an active orphan LuxN receptor, (ii) the quorum regulatory small RNAs (Qrrs) are not solely regulated by autoinducer signalling through the response regulator LuxO and (iii) the DS40M4 quorum-sensing regulon is much smaller than BB120 (~100 genes vs. ~400 genes, respectively). Using comparative genomics to expand our understanding of quorum-sensing circuit diversity, we observe that conservation of LuxM/LuxN proteins differs widely both between and within Vibrio species. These strains are also phenotypically distinct: DS40M4 exhibits stronger interbacterial cell killing, whereas BB120 forms more robust biofilms and is bioluminescent. These results underscore the need to examine wild isolates for a broader view of bacterial diversity in the marine ecosystem., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Photorhabdus and Xenorhabdus species have mutualistic associations with nematodes and an entomopathogenic stage 1,2 in their life cycles. In both stages, numerous specialized metabolites are produced that have roles in symbiosis and virulence 3,4 . Although regulators have been implicated in the regulation of these specialized metabolites 3,4 , how small regulatory RNAs (sRNAs) are involved in this process is not clear. Here, we show that the Hfq-dependent sRNA, ArcZ, is required for specialized metabolite production in Photorhabdus and Xenorhabdus. We discovered that ArcZ directly base-pairs with the mRNA encoding HexA, which represses the expression of specialized metabolite gene clusters. In addition to specialized metabolite genes, we show that the ArcZ regulon affects approximately 15% of all transcripts in Photorhabdus and Xenorhabdus. Thus, the ArcZ sRNA is crucial for specialized metabolite production in Photorhabdus and Xenorhabdus species and could become a useful tool for metabolic engineering and identification of commercially relevant natural products.

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.

Regulation at the post-transcriptional level is an important mode of gene expression control in bacteria. Small RNA regulators (sRNAs) that act via intramolecular base-pairing with target mRNAs are key players in this process and most often sequester the target's ribosome binding site (RBS) to down-regulate translation initiation. Over the past few years, several exceptions from this mechanism have been reported, revealing that sRNAs are able to influence translation initiation from a distance. In this issue of Molecular Microbiology, Azam and Vanderpool show that repression of the manY mRNA by the sRNA SgrS relies on an unconventional mechanism involving a translational enhancer element and ribosomal protein S1. Binding of S1 to an AU-rich sequence within the 5' untranslated region of the manY transcript promotes translation of the mRNA, and base-pairing of SgrS to the same site can interfere with this process. Therefore, instead of blocking translation initiation by occluding the manY RBS, SgrS reduces ManY synthesis by inhibiting S1-dependent translation activation. These findings increase the base-pairing window for sRNA-mediated gene expression control in bacteria and highlight the role of ribosomal protein S1 for translation initiation., (© 2020 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Negative feedback regulation, that is the ability of a gene to repress its own synthesis, is the most abundant regulatory motif known to biology. Frequently reported for transcriptional regulators, negative feedback control relies on binding of a transcription factor to its own promoter. Here, we report a novel mechanism for gene autoregulation in bacteria relying on small regulatory RNA (sRNA) and the major endoribonuclease, RNase E. TIER-seq analysis (transiently-inactivating-an-endoribonuclease-followed-by-RNA-seq) revealed ~25,000 RNase E-dependent cleavage sites in Vibrio cholerae , several of which resulted in the accumulation of stable sRNAs. Focusing on two examples, OppZ and CarZ, we discovered that these sRNAs are processed from the 3' untranslated region (3' UTR) of the oppABCDF and carAB operons, respectively, and base-pair with their own transcripts to inhibit translation. For OppZ, this process also triggers Rho-dependent transcription termination. Our data show that sRNAs from 3' UTRs serve as autoregulatory elements allowing negative feedback control at the post-transcriptional level., Competing Interests: MH, MH, KF, KP No competing interests declared, (© 2020, Hoyos et al.)

Hfq (host factor for phage Q beta) is key for posttranscriptional gene regulation in many bacteria. Hfq's function is to stabilize sRNAs and to facilitate base-pairing with trans -encoded target mRNAs. Loss of Hfq typically results in pleiotropic phenotypes, and, in the major human pathogen Vibrio cholerae , Hfq inactivation has been linked to reduced virulence, failure to produce biofilms, and impaired intercellular communication. However, the RNA ligands of Hfq in V. cholerae are currently unknown. Here, we used RIP-seq (RNA immunoprecipitation followed by high-throughput sequencing) analysis to identify Hfq-bound RNAs in V. cholerae Our work revealed 603 coding and 85 noncoding transcripts associated with Hfq, including 44 sRNAs originating from the 3' end of mRNAs. Detailed investigation of one of these latter transcripts, named FarS (fatty acid regulated sRNA), showed that this sRNA is produced by RNase E-mediated maturation of the fabB 3'UTR, and, together with Hfq, inhibits the expression of two paralogous fadE mRNAs. The fabB and fadE genes are antagonistically regulated by the major fatty acid transcription factor, FadR, and we show that, together, FadR, FarS, and FadE constitute a mixed feed-forward loop regulating the transition between fatty acid biosynthesis and degradation in V. cholerae Our results provide the molecular basis for studies on Hfq in V. cholerae and highlight the importance of a previously unrecognized sRNA for fatty acid metabolism in this major human pathogen., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

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 (σ E ) and one or more sRNAs. In this study, we identified the MicV sRNA as a new member of the σ E 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 σ E 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 σ E -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., (© 2019 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Collective behavior in spatially structured groups, or biofilms, is the norm among microbes in their natural environments. Though biofilm formation has been studied for decades, tracing the mechanistic and ecological links between individual cell morphologies and the emergent features of cell groups is still in its infancy. Here we use single-cell-resolution confocal microscopy to explore biofilms of the human pathogen Vibrio cholerae in conditions mimicking its marine habitat. Prior reports have noted the occurrence of cellular filamentation in V. cholerae , with variable propensity to filament among both toxigenic and nontoxigenic strains. Using a filamenting strain of V. cholerae O139, we show that cells with this morphotype gain a profound competitive advantage in colonizing and spreading on particles of chitin, the material many marine Vibrio species depend on for growth in seawater. Furthermore, filamentous cells can produce biofilms that are independent of primary secreted components of the V. cholerae biofilm matrix; instead, filamentous biofilm architectural strength appears to derive at least in part from the entangled mesh of cells themselves. The advantage gained by filamentous cells in early chitin colonization and growth is countered in long-term competition experiments with matrix-secreting V. cholerae variants, whose densely packed biofilm structures displace competitors from surfaces. Overall, our results reveal an alternative mode of biofilm architecture that is dependent on filamentous cell morphology and advantageous in environments with rapid chitin particle turnover. This insight provides an environmentally relevant example of how cell morphology can impact bacterial fitness., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

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., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Putrescine belongs to the large group of polyamines, an essential class of metabolites that exists throughout all kingdoms of life. The Salmonella speF gene encodes an inducible ornithine decarboxylase that produces putrescine from ornithine. Putrescine can be also synthesized from arginine in a parallel metabolic pathway. Here, we show that speF expression is controlled at multiple levels through regulatory elements contained in a long leader sequence. At the heart of this regulation is a short open reading frame, orf34, which is required for speF production. Translation of orf34 interferes with Rho-dependent transcription termination and helps to unfold an inhibitory RNA structure sequestering speF ribosome-binding site. Two consecutive arginine codons in the conserved domain of orf34 provide a third level of speF regulation. Uninterrupted translation of orf34 under conditions of high arginine allows the formation of a speF mRNA structure that is degraded by RNase G, whereas ribosome pausing at the consecutive arginine codons in the absence of arginine enables the formation of an alternative structure that is resistant to RNase G. Thus, the rate of ribosome progression during translation of the upstream ORF influences the dynamics of speF mRNA folding and putrescine production. The identification of orf34 and its regulatory functions provides evidence for the evolutionary conservation of ornithine decarboxylase regulatory elements and putrescine production., Competing Interests: The authors have declared that no competing interests exist.

The study of bacteriophages (phages) and prophages has provided key insights into almost every cellular process as well as led to the discovery of unexpected new mechanisms and the development of valuable tools. This is exemplified for RNA-based regulation. For instance, the characterization and exploitation of the antiphage CRISPR (clustered regularly interspaced short palindromic repeat) systems is revolutionizing molecular biology. Phage-encoded proteins such as the RNA-binding MS2 protein, which is broadly used to isolate tagged RNAs, also have been developed as valuable tools. Hfq, the RNA chaperone protein central to the function of many base-pairing small RNAs (sRNAs), was first characterized as a bacterial host factor required for Qβ phage replication. The ongoing studies of RNAs are continuing to reveal regulatory connections between infecting phages, prophages, and bacteria and to provide novel insights. There are bacterial and prophage sRNAs that regulate prophage genes, which impact bacterial virulence as well as bacterial cell killing. Conversely, phage- and prophage-encoded sRNAs modulate the expression of bacterial genes modifying metabolism. An interesting subcategory of the prophage-encoded sRNAs are sponge RNAs that inhibit the activities of bacterial-encoded sRNAs. Phages also affect posttranscriptional regulation in bacteria through proteins that inhibit or alter the activities of key bacterial proteins involved in posttranscriptional regulation. However, what is most exciting about phage and prophage research, given the millions of phage-encoded genes that have not yet been characterized, is the vast potential for discovering new RNA regulators and novel mechanisms and for gaining insight into the evolution of regulatory RNAs.

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., (© 2018 Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare no conflict of interest.

Quorum sensing (QS) is a cell-cell communication process that enables bacteria to track cell population density and orchestrate collective behaviors. QS relies on the production and detection of, and the response to, extracellular signal molecules called autoinducers. In Vibrio cholerae, multiple QS circuits control pathogenesis and biofilm formation. Here, we identify and characterize a new QS autoinducer-receptor pair. The autoinducer is 3,5-dimethylpyrazin-2-ol (DPO). DPO is made from threonine and alanine, and its synthesis depends on threonine dehydrogenase (Tdh). DPO binds to and activates a transcription factor, VqmA. The VqmA-DPO complex activates expression of vqmR, which encodes a small regulatory RNA. VqmR represses genes required for biofilm formation and toxin production. We propose that DPO allows V. cholerae to regulate collective behaviors to, among other possible roles, diversify its QS output during colonization of the human host.

Understanding RNA processing and turnover requires knowledge of cleavages by major endoribonucleases within a living cell. We have employed TIER-seq (transiently inactivating an endoribonuclease followed by RNA-seq) to profile cleavage products of the essential endoribonuclease RNase E in Salmonella enterica. A dominating cleavage signature is the location of a uridine two nucleotides downstream in a single-stranded segment, which we rationalize structurally as a key recognition determinant that may favor RNase E catalysis. Our results suggest a prominent biogenesis pathway for bacterial regulatory small RNAs whereby RNase E acts together with the RNA chaperone Hfq to liberate stable 3' fragments from various precursor RNAs. Recapitulating this process in vitro, Hfq guides RNase E cleavage of a representative small-RNA precursor for interaction with a mRNA target. In vivo, the processing is required for target regulation. Our findings reveal a general maturation mechanism for a major class of post-transcriptional regulators., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Model enteric bacteria such as Escherichia coli and Salmonella enterica express hundreds of small non-coding RNAs (sRNAs), targets for most of which are yet unknown. Some sRNAs are remarkably well conserved, indicating that they serve cellular functions that go beyond the necessities of a single species. One of these 'core sRNAs' of largely unknown function is the abundant ∼100-nucleotide SdsR sRNA which is transcribed by the general stress σ-factor, σ S and accumulates in stationary phase. In Salmonella, SdsR was known to inhibit the synthesis of the species-specific porin, OmpD. However, sdsR genes are present in almost all enterobacterial genomes, suggesting that additional, conserved targets of this sRNA must exist. Here, we have combined SdsR pulse-expression with whole genome transcriptomics to discover 20 previously unknown candidate targets of SdsR which include mRNAs coding for physiologically important regulators such as the carbon utilization regulator, CRP, the nucleoid-associated chaperone, StpA and the antibiotic resistance transporter, TolC. Processing of SdsR by RNase E results in two cellular SdsR variants with distinct target spectra. While the overall physiological role of this orphan core sRNA remains to be fully understood, the new SdsR targets present valuable leads to determine sRNA functions in resting bacteria., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Horizontal transfer of genetic information is a major driving force of evolution. In bacteria, genome plasticity is intimately linked to the ability of the bacterium to integrate novel material into existing gene expression circuits. Small RNAs (sRNAs) are a versatile class of regulatory molecules, and have recently been discovered to perform important tasks in the interplay between core genomic elements and horizontally-acquired DNA. Together with auxiliary proteins such as the RNA-chaperone Hfq and cellular ribonucleases, sRNAs typically act post-transcriptionally to either promote or restrict the expression of multiple target genes. Bacterial sRNAs have been identified in core and peripheral (acquired) genome sequences, and their target suites may likewise include genes from both locations. In this review, we discuss how sRNAs influence the expression of foreign genetic material in enterobacterial pathogens, and outline the processes that foster the integration of horizontally-acquired RNAs into existing regulatory networks. We also consider potential benefits and risks of horizontal gene transfer for RNA-based gene regulation., (© 2016 John Wiley & Sons Ltd.)

Bacteria use quorum sensing to orchestrate gene expression programmes that underlie collective behaviours. Quorum sensing relies on the production, release, detection and group-level response to extracellular signalling molecules, which are called autoinducers. Recent work has discovered new autoinducers in Gram-negative bacteria, shown how these molecules are recognized by cognate receptors, revealed new regulatory components that are embedded in canonical signalling circuits and identified novel regulatory network designs. In this Review we examine how, together, these features of quorum sensing signal-response systems combine to control collective behaviours in Gram-negative bacteria and we discuss the implications for host-microbial associations and antibacterial therapy.

While an increasing number of conserved small regulatory RNAs (sRNAs) are known to function in general bacterial physiology, the roles and modes of action of sRNAs from horizontally acquired genomic regions remain little understood. The IsrK sRNA of Gifsy-1 prophage of Salmonella belongs to the latter class. This regulatory RNA exists in two isoforms. The first forms, when a portion of transcripts originating from isrK promoter reads-through the IsrK transcription-terminator producing a translationally inactive mRNA target. Acting in trans, the second isoform, short IsrK RNA, binds the inactive transcript rendering it translationally active. By switching on translation of the first isoform, short IsrK indirectly activates the production of AntQ, an antiterminator protein located upstream of isrK. Expression of antQ globally interferes with transcription termination resulting in bacterial growth arrest and ultimately cell death. Escherichia coli and Salmonella cells expressing AntQ display condensed chromatin morphology and localization of UvrD to the nucleoid. The toxic phenotype of AntQ can be rescued by co-expression of the transcription termination factor, Rho, or RNase H, which protects genomic DNA from breaks by resolving R-loops. We propose that AntQ causes conflicts between transcription and replication machineries and thus promotes DNA damage. The isrK locus represents a unique example of an island-encoded sRNA that exerts a highly complex regulatory mechanism to tune the expression of a toxic protein.

Horizontal gene transfer via plasmid conjugation is a major driving force in microbial evolution but constitutes a complex process that requires synchronization with the physiological state of the host bacteria. Although several host transcription factors are known to regulate plasmid-borne transfer genes, RNA-based regulatory circuits for host-plasmid communication remain unknown. We describe a posttranscriptional mechanism whereby the Hfq-dependent small RNA, RprA, inhibits transfer of pSLT, the virulence plasmid of Salmonella enterica. RprA employs two separate seed-pairing domains to activate the mRNAs of both the sigma-factor σ(S) and the RicI protein, a previously uncharacterized membrane protein here shown to inhibit conjugation. Transcription of ricI requires σ(S) and, together, RprA and σ(S) orchestrate a coherent feedforward loop with AND-gate logic to tightly control the activation of RicI synthesis. RicI interacts with the conjugation apparatus protein TraV and limits plasmid transfer under membrane-damaging conditions. To our knowledge, this study reports the first small RNA-controlled feedforward loop relying on posttranscriptional activation of two independent targets and an unexpected role of the conserved RprA small RNA in controlling extrachromosomal DNA transfer.

Bacterial small regulatory RNAs (sRNAs) are commonly known to repress gene expression by base pairing to target mRNAs. In many cases, sRNAs base pair with and sequester mRNA ribosome-binding sites, resulting in translational repression and accelerated transcript decay. In contrast, a growing number of examples of translational activation and mRNA stabilization by sRNAs have now been documented. A given sRNA often employs a conserved region to interact with and regulate both repressed and activated targets. However, the mechanisms underlying activation differ substantially from repression. Base pairing resulting in target activation can involve sRNA interactions with the 5(') untranslated region (UTR), the coding sequence or the 3(') UTR of the target mRNAs. Frequently, the activities of protein factors such as cellular ribonucleases and the RNA chaperone Hfq are required for activation. Bacterial sRNAs, including those that function as activators, frequently control stress response pathways or virulence-associated functions required for immediate responses to changing environments. This review aims to summarize recent advances in knowledge regarding target mRNA activation by bacterial sRNAs, highlighting the molecular mechanisms and biological relevance of regulation., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Quorum sensing (QS) is a process of cell-to-cell communication that enables bacteria to transition between individual and collective lifestyles. QS controls virulence and biofilm formation in Vibrio cholerae, the causative agent of cholera disease. Differential RNA sequencing (RNA-seq) of wild-type V. cholerae and a locked low-cell-density QS-mutant strain identified 7,240 transcriptional start sites with ∼ 47% initiated in the antisense direction. A total of 107 of the transcripts do not appear to encode proteins, suggesting they specify regulatory RNAs. We focused on one such transcript that we name VqmR. vqmR is located upstream of the vqmA gene encoding a DNA-binding transcription factor. Mutagenesis and microarray analyses demonstrate that VqmA activates vqmR transcription, that vqmR encodes a regulatory RNA, and VqmR directly controls at least eight mRNA targets including the rtx (repeats in toxin) toxin genes and the vpsT transcriptional regulator of biofilm production. We show that VqmR inhibits biofilm formation through repression of vpsT. Together, these data provide to our knowledege the first global annotation of the transcriptional start sites in V. cholerae and highlight the importance of posttranscriptional regulation for collective behaviors in this human pathogen.

Quorum sensing is a cell-cell communication process that bacteria use to transition between individual and social lifestyles. In vibrios, homologous small RNAs called the Qrr sRNAs function at the center of quorum-sensing pathways. The Qrr sRNAs regulate multiple mRNA targets including those encoding the quorum-sensing regulatory components luxR, luxO, luxM, and aphA. We show that a representative Qrr, Qrr3, uses four distinct mechanisms to control its particular targets: the Qrr3 sRNA represses luxR through catalytic degradation, represses luxM through coupled degradation, represses luxO through sequestration, and activates aphA by revealing the ribosome binding site while the sRNA itself is degraded. Qrr3 forms different base-pairing interactions with each mRNA target, and the particular pairing strategy determines which regulatory mechanism occurs. Combined mathematical modeling and experiments show that the specific Qrr regulatory mechanism employed governs the potency, dynamics, and competition of target mRNA regulation, which in turn, defines the overall quorum-sensing response., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Small non-coding RNAs (sRNAs) are emerging regulatory elements in bacteria. The Vibrio cholerae sRNA VrrA has previously been shown to down-regulate outer membrane proteins (OmpA and OmpT) and biofilm matrix protein (RbmC) by base-pairing with the 5' region of the corresponding mRNAs. In this study, we present an additional target of VrrA in V. cholerae, the mRNA coding for the ribosome binding protein Vrp. Vrp is homologous to ribosome-associated inhibitor A (RaiA) of Escherichia coli which facilitates stationary phase survival through ribosome hibernation. We show that VrrA down-regulates Vrp protein synthesis by base-pairing to the 5' region of vrp mRNA and that the regulation requires the RNA chaperone protein, Hfq. We further demonstrate that Vrp is highly expressed during stationary phase growth and associates with the ribosome of V. cholerae. The effect of the Vrp protein in starvation survival is synergistic with that of the VC2530 protein, a homolog of the E. coli hibernation promoting factor HPF, suggesting a combined role for these proteins in ribosome hibernation in V. cholerae. Vrp and VC2530 are important for V. cholerae starvation survival under nutrient deficient conditions. While VC2530 is down-regulated in cells lacking vrrA, mutation of vrp results in VC2530 activation. This is the first report indicating a regulatory role for an sRNA, modulating stationary factors involved in bacterial ribosome hibernation.

Enteric pathogens often cycle between virulent and saprophytic lifestyles. To endure these frequent changes in nutrient availability and composition bacteria possess an arsenal of regulatory and metabolic genes allowing rapid adaptation and high flexibility. While numerous proteins have been characterized with regard to metabolic control in pathogenic bacteria, small non-coding RNAs have emerged as additional regulators of metabolism. Recent advances in sequencing technology have vastly increased the number of candidate regulatory RNAs and several of them have been found to act at the interface of bacterial metabolism and virulence factor expression. Importantly, studying these riboregulators has not only provided insight into their metabolic control functions but also revealed new mechanisms of post-transcriptional gene control. This review will focus on the recent advances in this area of host-microbe interaction and discuss how regulatory small RNAs may help coordinate metabolism and virulence of enteric pathogens.

Small RNAs use a diversity of well-characterized mechanisms to repress mRNAs, but how they activate gene expression at the mRNA level remains not well understood. The predominant activation mechanism of Hfq-associated small RNAs has been translational control whereby base pairing with the target prevents the formation of an intrinsic inhibitory structure in the mRNA and promotes translation initiation. Here, we report a translation-independent mechanism whereby the small RNA RydC selectively activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) in Salmonella enterica. Target activation is achieved through seed pairing of the pseudoknot-exposed, conserved 5' end of RydC to an upstream region of the cfa mRNA. The seed pairing stabilizes the messenger, likely by interfering directly with RNase E-mediated decay in the 5' untranslated region. Intriguingly, this mechanism is generic such that the activation is equally achieved by seed pairing of unrelated small RNAs, suggesting that this mechanism may be utilized in the design of RNA-controlled synthetic circuits. Physiologically, RydC is the first small RNA known to regulate membrane stability.

Small RNAs (sRNAs) constitute a large and heterogeneous class of bacterial gene expression regulators. Much like eukaryotic microRNAs, these sRNAs typically target multiple mRNAs through short seed pairing, thereby acting as global posttranscriptional regulators. In some bacteria, evidence for hundreds to possibly more than 1,000 different sRNAs has been obtained by transcriptome sequencing. However, the experimental identification of possible targets and, therefore, their confirmation as functional regulators of gene expression has remained laborious. Here, we present a strategy that integrates phylogenetic information to predict sRNA targets at the genomic scale and reconstructs regulatory networks upon functional enrichment and network analysis (CopraRNA, for Comparative Prediction Algorithm for sRNA Targets). Furthermore, CopraRNA precisely predicts the sRNA domains for target recognition and interaction. When applied to several model sRNAs, CopraRNA revealed additional targets and functions for the sRNAs CyaR, FnrS, RybB, RyhB, SgrS, and Spot42. Moreover, the mRNAs gdhA, lrp, marA, nagZ, ptsI, sdhA, and yobF-cspC were suggested as regulatory hubs targeted by up to seven different sRNAs. The verification of many previously undetected targets by CopraRNA, even for extensively investigated sRNAs, demonstrates its advantages and shows that CopraRNA-based analyses can compete with experimental target prediction approaches. A Web interface allows high-confidence target prediction and efficient classification of bacterial sRNAs.

Quorum sensing is a chemical communication process that bacteria use to control collective behaviours including bioluminescence, biofilm formation, and virulence factor production. In Vibrio harveyi, five homologous small RNAs (sRNAs) called Qrr1-5, control quorum-sensing transitions. Here, we identify 16 new targets of the Qrr sRNAs. Mutagenesis reveals that particular sequence differences among the Qrr sRNAs determine their target specificities. Modelling coupled with biochemical and genetic analyses show that all five of the Qrr sRNAs possess four stem-loops: the first stem-loop is crucial for base pairing with a subset of targets. This stem-loop also protects the Qrr sRNAs from RNase E-mediated degradation. The second stem-loop contains conserved sequences required for base pairing with the majority of the target mRNAs. The third stem-loop plays an accessory role in base pairing and stability. The fourth stem-loop functions as a rho-independent terminator. In the quorum-sensing regulon, Qrr sRNAs-controlled genes are the most rapid to respond to quorum-sensing autoinducers. The Qrr sRNAs are conserved throughout vibrios, thus insights from this work could apply generally to Vibrio quorum sensing.

Glucose homeostasis is strictly controlled in all domains of life. Bacteria that are unable to balance intracellular sugar levels and deal with potentially toxic phosphosugars cease growth and risk being outcompeted. Here, we identify the conserved haloacid dehalogenase (HAD)-like enzyme YigL as the previously hypothesized phosphatase for detoxification of phosphosugars and reveal that its synthesis is activated by an Hfq-dependent small RNA in Salmonella typhimurium. We show that the glucose-6-P-responsive small RNA SgrS activates YigL synthesis in a translation-independent fashion by the selective stabilization of a decay intermediate of the dicistronic pldB-yigL messenger RNA (mRNA). Intriguingly, the major endoribonuclease RNase E, previously known to function together with small RNAs to degrade mRNA targets, is also essential for this process of mRNA activation. The exploitation of and targeted interference with regular RNA turnover described here may constitute a general route for small RNAs to rapidly activate both coding and noncoding genes., (Copyright © 2013 Elsevier Inc. All rights reserved.)