A qrr noncoding RNA deploys four different regulatory mechanisms to optimize quorum-sensing dynamics.

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.

Small RNAs, Large Networks: Posttranscriptional Regulons in Gram-Negative Bacteria.

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.

Small RNA-mediated activation of sugar phosphatase mRNA regulates glucose homeostasis.

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.

The global RNA-RNA interactome of Klebsiella pneumoniae unveils a small RNA regulator of cell division.

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.

A dual-function RNA balances carbon uptake and central metabolism in Vibrio cholerae.

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.

In Vivo Cleavage Map Illuminates the Central Role of RNase E in Coding and Non-coding RNA Pathways.

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.

FlrA-independent production of flagellar proteins is required for proper flagellation in Shewanella putrefaciens.

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 (σ54 ) and FliA (σ28 ) 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.

A conserved RNA seed-pairing domain directs small RNA-mediated stress resistance in enterobacteria.

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.

Switching fatty acid metabolism by an RNA-controlled feed forward loop.

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.

Small Proteins; Big Questions.

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.

mRNA dynamics and alternative conformations adopted under low and high arginine concentrations control polyamine biosynthesis in Salmonella.

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.

Vibrio cholerae filamentation promotes chitin surface attachment at the expense of competition in biofilms.

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.

Regulation outside the box: New mechanisms for small RNAs.

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.

The quorum-sensing systems of Vibrio campbellii DS40M4 and BB120 are genetically and functionally distinct.

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.

Three autoinducer molecules act in concert to control virulence gene expression in Vibrio cholerae.

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.

Targeted decay of a regulatory small RNA by an adaptor protein for RNase E and counteraction by an anti-adaptor RNA.

Bacterial small RNAs (sRNAs) are well established to regulate diverse cellular processes, but how they themselves are regulated is less understood. Recently, we identified a regulatory circuit wherein the GlmY and GlmZ sRNAs of Escherichia coli act hierarchically to activate mRNA glmS, which encodes glucosamine-6-phosphate (GlcN6P) synthase. Although the two sRNAs are highly similar, only GlmZ is a direct activator that base-pairs with the glmS mRNA, aided by protein Hfq. GlmY, however, does not bind Hfq and activates glmS indirectly by protecting GlmZ from RNA cleavage. This complex regulation feedback controls the levels of GlmS protein in response to its product, GlcN6P, a key metabolite in cell wall biosynthesis. Here, we reveal the molecular basis for the regulated turnover of GlmZ, identifying RapZ (RNase adaptor protein for sRNA GlmZ; formerly YhbJ) as a novel type of RNA-binding protein that recruits the major endoribonuclease RNase E to GlmZ. This involves direct interaction of RapZ with the catalytic domain of RNase E. GlmY binds RapZ through a secondary structure shared by both sRNAs and therefore acts by molecular mimicry as a specific decoy for RapZ. Thus, in analogy to regulated proteolysis, RapZ is an adaptor, and GlmY is an anti-adaptor in regulated turnover of a regulatory small RNA.

Leaderless secreted peptide signaling molecule alters global gene expression and increases virulence of a human bacterial pathogen.

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.

Correction: Gifsy-1 Prophage IsrK with Dual Function as Small and Messenger RNA Modulates Vital Bacterial Machineries.

[This corrects the article DOI: 10.1371/journal.pgen.1005975.].

A Vibrio cholerae autoinducer-receptor pair that controls biofilm formation.

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.

Sweet business: Spot42 RNA networks with CRP to modulate catabolite repression.

Spot42 is a paradigm for small RNAs that fine-tune carbon metabolism. In this issue of Molecular Cell, Beisel and Storz (2011) reveal that this conserved RNA acts through a multioutput feedforward loop to modulate the global dynamics of sugar consumption.