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While large membrane-impermeable antibiotics cannot traverse a lipid barrier, spacious importers such as TonB-dependent receptors (TBDRs) can mistakenly ferry antibiotics across the bacterial outer membrane (OM). We discovered that loss of PerA, an enigmatic β-helix protein in the OM of the oligotrophic α-proteobacteriumCaulobacter crescentus, reprograms the OM TBDR proteome from ChvT that imports the glycopeptide antibiotic vancomycin to an uncharacterized TBDR (BugA) that confers sensitivity to the polypeptide antibiotic bacitracin. Both antibiotics are large zinc-binding molecules that target the peptidoglycan, echoing the zinc stress response induces destabilization of PerA. Inactivation of PerA launches two conserved and interwoven envelope stress programs that remodel the OM with TBDRs, a tripartite multidrug efflux pump and periplasmic proteases. Thus, unanticipated entry routes for antibiotics emerge in stressed diderm bacteria that may be treatable with membrane impermeable antimicrobials owing to an underlying transcriptional stress response pathways coordinated by a novel type of OM regulator.

In the past decades, Caulobacter crescentus has been extensively studied, mostly regarding its dimorphic, asymmetric life cycle. Its distinct mode of reproduction and the need to optimally adapt to ever-changing environmental conditions require tight coordination of gene regulation. Post-transcriptional regulation through non-coding RNAs and RNA-binding proteins constitutes an important layer of expression control in bacteria, but its principles and mechanisms in Caulobacter have only recently been explored. RNA-binding proteins including the RNA chaperone Hfq and ribonuclease RNase E contribute to the activity of regulatory RNAs. Riboswitches and RNA thermometers govern expression of downstream open reading frames, while the small regulatory RNAs CrfA, ChvR and GsrN instead control targets encoded in trans by direct base-pairing interactions.

Significance Bacteria constantly transition between conditions of feast and famine. Colonization of the human intestine by Vibrio cholerae is associated with a surge in host-derived fatty acids, demanding rapid regulation of fatty acid metabolism. Here, we provide evidence for an RNA-based mechanism controlling the expression of central fatty acid metabolism genes in response to changing external fatty acid concentrations. We identified a small regulatory RNA, FarS, which is processed from the 3′UTR of the fabB fatty acid biosynthesis gene and inhibits the production of proteins required for fatty acid degradation. Tight control of fatty acid biosynthesis and degradation is vital for all bacteria, and, in V. cholerae, FarS plays an important role in balancing these processes., 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.

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.

Hfq (host factor for phage Q beta) is key for posttranscriptional generegulation in many bacteria. Hfq’s function is to stabilize sRNAs andto facilitate base-pairing withtrans-encoded target mRNAs. Lossof Hfq typically results in pleiotropic phenotypes, and, in the majorhuman pathogenVibrio cholerae, Hfq inactivation has been linkedto reduced virulence, failure to produce biofilms, and impaired in-tercellular communication. However, the RNA ligands of Hfq inV. choleraeare currently unknown. Here, we used RIP-seq (RNA immu-noprecipitation followed by high-throughput sequencing) analysis toidentify Hfq-bound RNAs inV. cholerae. Our work revealed 603 cod-ing and 85 noncoding transcripts associated with Hfq, including 44sRNAs originating from the 3′end of mRNAs. Detailed investigationof one of these latter transcripts, named FarS (fatty acid regulated sRNA),showed that this sRNA is produced by RNase E-mediated maturationof thefabB3′UTR, and, together with Hfq, inhibits the expression oftwo paralogousfadEmRNAs. ThefabBandfadEgenes are antag-onistically regulated by the major fatty acid transcription factor,FadR, and we show that, together, FadR, FarS, and FadE constitutea mixed feed-forward loop regulating the transition between fattyacid biosynthesis and degradation inV. cholerae. Our results providethe molecular basis for studies on Hfq inV. choleraeand highlight theimportance of a previously unrecognized sRNA for fatty acid metabo-lism in this major human pathogen.

Bacterial small RNAs (sRNAs) are a heterogeneous group of post-transcriptional regulators that often act at the heart of large networks. Hundreds of sRNAs have been discovered by genome-wide screens and most of these sRNAs exert their functions by base-pairing with target mRNAs. However, studies addressing the molecular roles of sRNAs have been largely confined to gamma-proteobacteria, such as Escherichia coli. Here we identify and characterize a novel sRNA, ChvR, from the alpha-proteobacterium Caulobacter crescentus. Transcription of chvR is controlled by the conserved two-component system ChvI-ChvG and it is expressed in response to DNA damage, low pH, and growth in minimal medium. Transient over-expression of ChvR in combination with genome-wide transcriptome profiling identified the mRNA of the TonB-dependent receptor ChvT as the sole target of ChvR. Genetic and biochemical analyses showed that ChvR represses ChvT at the post-transcriptional level through direct base-pairing. Fine-mapping of the ChvR-chvT interaction revealed the requirement of two distinct base-pairing sites for full target regulation. Finally, we show that ChvR-controlled repression of chvT is independent of the ubiquitous RNA-chaperone Hfq, and therefore distinct from previously reported mechanisms employed by prototypical bacterial sRNAs. These findings have implications for the mechanism and evolution of sRNA function across bacterial species.

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 5ʹ 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., Small RNAs (sRNAs) are key players in the post‐transcriptional control of bacterial gene expression. Typically, sRNAs base‐pair to the translation initiation site of their target mRNAs, which blocks access of the ribosome and results in repression of target translation. The SgrS sRNA from Escherichia coli is able to influence translation initiation from a distance: Upstream of the ribosome binding site, SgrS competes with ribosomal protein S1 for binding to an AU‐rich site on the manY transcript. Binding of S1 stimulates manY translation, and binding of SgrS interferes with this process.

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 posttranscriptional 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.

Summary 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.

Small RNAs (sRNAs) in bacteria are abundant and play important roles in posttranscriptional regulation of gene expression, particularly under stress conditions. Some mRNAs are targets for regulation by multiple sRNAs, each responding to different environmental signals. Uncovering the regulatory mechanisms governing sRNA-mRNA interactions and the relevant conditions for these interactions is an ongoing challenge. In this study, we discovered that multiple sRNAs control membrane lipid composition by regulating stability of a single mRNA target. The sRNA-dependent regulation occurred in response to changing pH and was important for cell viability under acid stress conditions. This work reveals yet another aspect of bacterial physiology controlled at the posttranscriptional level by sRNA regulators., Altering membrane protein and lipid composition is an important strategy for maintaining membrane integrity during environmental stress. Many bacterial small RNAs (sRNAs) control membrane protein production, but sRNA-mediated regulation of membrane fatty acid composition is less well understood. The sRNA RydC was previously shown to stabilize cfa (cyclopropane fatty acid synthase) mRNA, resulting in higher levels of cyclopropane fatty acids in the cell membrane. Here, we report that additional sRNAs, ArrS and CpxQ, also directly regulate cfa posttranscriptionally. RydC and ArrS act through masking an RNase E cleavage site in the cfa mRNA 5′ untranslated region (UTR), and both sRNAs posttranscriptionally activate cfa. In contrast, CpxQ binds to a different site in the cfa mRNA 5′ UTR and represses cfa expression. Alteration of membrane lipid composition is a key mechanism for bacteria to survive low-pH environments, and we show that cfa translation increases in an sRNA-dependent manner when cells are subjected to mild acid stress. This work suggests an important role for sRNAs in the acid stress response through regulation of cfa mRNA. IMPORTANCE Small RNAs (sRNAs) in bacteria are abundant and play important roles in posttranscriptional regulation of gene expression, particularly under stress conditions. Some mRNAs are targets for regulation by multiple sRNAs, each responding to different environmental signals. Uncovering the regulatory mechanisms governing sRNA-mRNA interactions and the relevant conditions for these interactions is an ongoing challenge. In this study, we discovered that multiple sRNAs control membrane lipid composition by regulating stability of a single mRNA target. The sRNA-dependent regulation occurred in response to changing pH and was important for cell viability under acid stress conditions. This work reveals yet another aspect of bacterial physiology controlled at the posttranscriptional level by sRNA regulators.

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.

Pathogenic bacteria express a myriad of systems aimed at subversion and exploitation of a host to promote proliferation and survival. Naturally, host cells have evolved equally complex defensive mechanisms which the bacterial pathogen must overcome in order to successfully establish an infection. Regulatory RNAs can operate at all layers of gene regulation, ranging from transcriptional initiation to protein activity. This chapter selectively details the best-characterized examples of small noncoding regulatory RNAs (sRNAs) and their molecular function in bacterial pathogens of special interest. Salmonella enterica serovar Typhimurium is a gram-negative pathogen causing gastroenteritis in humans. The two most recent additions to the list of Vibrio sRNA regulators affecting pathogenicity are TarA and TarB, both of which are controlled by the master virulence regulator ToxT. Virulence of Pseudomonas aeruginosa is modulated by quorum sensing (QS) systems that control the production of several virulence factors in a cell density-dependent manner. Posttranscriptional control by the RNA-binding protein RsmA (CsrA) regulates many virulence genes of P. aeruginosa. There are a few notable exceptions, including RNAIII in Staphylococcus, IsrM in Salmonella, and several sRNAs in Listeria, deletion of which leads to clear virulence phenotypes. While many functionally related virulence factors are often clustered in horizontally acquired pathogenicity islands, trans-acting sRNAs located in the ancestral genome can be co-opted into regulation of horizontally acquired genes, thus linking expression of virulence factors with regulation of the core genome.

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.

Bacterial small RNAs (sRNAs) are key elements of regulatory networks that modulate gene expression. The sRNA RydC of Salmonella sp. and Escherichia coli is an example of this class of riboregulators. Like many other sRNAs, RydC bears a ‘seed’ region that recognises specific transcripts through base-pairing, and its activities are facilitated by the RNA chaperone Hfq. The crystal structure of RydC in complex with E. coli Hfq at a 3.48 Å resolution illuminates how the protein interacts with and presents the sRNA for target recognition. Consolidating the protein–RNA complex is a host of distributed interactions mediated by the natively unstructured termini of Hfq. Based on the structure and other data, we propose a model for a dynamic effector complex comprising Hfq, small RNA, and the cognate mRNA target. DOI: http://dx.doi.org/10.7554/eLife.05375.001, eLife digest A crucial step in the production of proteins is the translation of messenger RNA molecules. Other RNA molecules called small RNAs are also involved in this process: these small RNAs bind to the messenger RNA molecules to either increase or decrease the production of proteins. Bacteria and other microorganisms use small RNA molecules to help them respond to stress conditions and to changes in their environment, such as fluctuations in temperature or the availability of nutrients. The ability to rapidly adapt to these changes enables bacteria to withstand harmful conditions and to make efficient use of resources available to them. Many small RNA molecules use a protein called Hfq to help them interact with their target messenger RNAs. In some cases this protein protects the small RNA molecules when they are not bound to their targets. Hfq also helps the small RNA to bind to the messenger RNA, and then recruits other enzymes that eventually degrade the complex formed by the different RNA molecules. Previous research has shown that six Hfq subunits combine to form a ring-shaped structure and has also provided some clues about the way in which Hfq can recognise a short stretch of a small RNA molecule, but the precise details of the interaction between them are not fully understood. Now Dimastrogiovanni et al. have used a technique called X-ray crystallography to visualize the interaction between Hfq and a small RNA molecule called RydC. These experiments reveal that a particular region of RydC adopts a structure known as a pseudoknot and that this structure is critical for the interactions between the RydC molecules and the Hfq ring. Dimastrogiovanni et al. find that one RydC molecule interacts with one Hfq ring, and they identify the contact points between the RydC molecule and different regions of the Hfq ring. Based on this information, Dimastrogiovanni et al. propose a model for how the RydC:Hfq complex is likely to interact with a messenger RNA molecule. The next step will be to test this model in experiments. DOI: http://dx.doi.org/10.7554/eLife.05375.002

P-bodies are dynamic aggregates of RNA and proteins involved in several post-transcriptional regulation processes. P-bodies have been shown to play important roles in regulating viral infection, whereas their interplay with bacterial pathogens, specifically intracellular bacteria that extensively manipulate host cell pathways, remains unknown. Here, we report that Salmonella infection induces P-body disassembly in a cell type-specific manner, and independently of previously characterized pathways such as inhibition of host cell RNA synthesis or microRNA-mediated gene silencing. We show that the Salmonella-induced P-body disassembly depends on the activation of the SPI-2 encoded type 3 secretion system, and that the secreted effector protein SpvB plays a major role in this process. P-body disruption is also induced by the related pathogen, Shigella flexneri, arguing that this might be a new mechanism by which intracellular bacterial pathogens subvert host cell function.

Small regulatory RNAs (sRNAs) commonly act to downregulate gene expression. In bacteria, however, sRNAs have also been shown to activate genes by a variety of direct or indirect mechanisms. Several sRNAs (DsrA, GlmZ, RNAIII, RprA, RyhB, and Qrr) act as direct translational activators by an 'anti-antisense mechanism' in the 5' mRNA region to liberate a sequestered ribosome binding site, while pairing of GadY sRNA to the 3'-end alters processing and increases stability of its target mRNA. Indirect activation includes cases of RNA mimicry in which degradation of the activating GlmZ sRNA is suppressed by the highly similar GlmY sRNA, or where a pseudo-target mRNA traps MicM sRNA to derepress porin synthesis.

International audience; The human malaria parasite Plasmodium falciparum exports determinants of virulence and pathology to destinations within the host erythrocyte, including the erythrocyte cytoplasm, plasma membrane and membrane profiles of parasite origin termed Maurer's clefts. Most of the exported proteins contain a conserved pentameric motif termed plasmodial export element (PEXEL)/vacuolar transfer signal (VTS) that functions as a cleavable sorting signal permitting export to the host erythrocyte. However, there are some exported proteins, such as the skeleton-binding protein 1 (PfSBP1) that lack the PEXEL/VTS motif and that are not N-terminally processed, suggesting the presence of alternative sorting signals and/or mechanisms. In this study, we have investigated trafficking of PfSBP1 to the Maurer's clefts. Our data show that the transmembrane domain of PfSBP1 functions as an internal signal sequence for entry into the parasite's secretory pathway and for transport to the parasite plasma membrane. Trafficking beyond the parasite's plasma membrane required additional N-terminal domains, which are characterized by a high negative net charge. Biochemical data indicate that these domains affect the solubility and extraction profile, the orientation of the protein within the membrane and the subcellular localization. Our findings suggest new principles of protein export in P. falciparum-infected erythrocytes.

The small GTPase ADP-ribosylation factor-1 (Arf1) plays a key role in the formation of coat protein I (COP I)-coated vesicles. Upon recruitment to the donor Golgi membrane by interaction with dimeric p24 proteins, Arf1's GDP is exchanged for GTP. Arf1-GTP then dissociates from p24, and together with other Golgi membrane proteins, it recruits coatomer, the heptameric coat protein complex of COP I vesicles, from the cytosol. In this process, Arf1 was shown to specifically interact with the coatomer beta and gamma-COP subunits through its switch I region, and with epsilon-COP. Here, we mapped the interaction of the Arf1-GTP switch I region to the trunk domains of beta and gamma-COP. Site-directed photolabeling at position 167 in the C-terminal helix of Arf1 revealed a novel interaction with coatomer via a putative longin domain of delta-COP. Thus, coatomer is linked to the Golgi through multiple interfaces with membrane-bound Arf1-GTP. These interactions are located within the core, adaptor-like domain of coatomer, indicating an organizational similarity between the COP I coat and clathrin adaptor complexes.