The Post-Transcriptional Regulatory Protein CsrA Amplifies Its Targetome through Direct Interactions with Stress-Response Regulatory Hubs: The EvgA and AcnA Cases.

Global rewiring of bacterial gene expressions in response to environmental cues is mediated by regulatory proteins such as the CsrA global regulator from E. coli. Several direct mRNA and sRNA targets of this protein have been identified; however, high-throughput studies suggest an expanded RNA targetome for this protein. In this work, we demonstrate that CsrA can extend its network by directly binding and regulating the evgA and acnA transcripts, encoding for regulatory proteins. CsrA represses EvgA and AcnA expression and disrupting the CsrA binding sites of evgA and acnA, results in broader gene expression changes to stress response networks. Specifically, altering CsrA-evgA binding impacts the genes related to acidic stress adaptation, and disrupting the CsrA-acnA interaction affects the genes involved in metal-induced oxidative stress responses. We show that these interactions are biologically relevant, as evidenced by the improved tolerance of evgA and acnA genomic mutants depleted of CsrA binding sites when challenged with acid and metal ions, respectively. We conclude that EvgA and AcnA are intermediate regulatory hubs through which CsrA can expand its regulatory role. The indirect CsrA regulation of gene networks coordinated by EvgA and AcnA likely contributes to optimizing cellular resources to promote exponential growth in the absence of stress.

Peeling the onion: additional layers of regulation in the acid stress response.

Bacteria are capable of withstanding large changes in osmolality and cytoplasmic pH, unlike eukaryotes that tightly regulate their pH and cellular composition. Previous studies on the bacterial acid stress response described a rapid, brief acidification, followed by immediate recovery. More recent experiments with better pH probes have imaged single living cells, and we now appreciate that following acid stress, bacteria maintain an acidic cytoplasm for as long as the stress remains. This acidification enables pathogens to sense a host environment and turn on their virulence programs, for example, enabling survival and replication within acidic vacuoles. Single-cell analysis identified an intracellular pH threshold of ~6.5. Acid stress reduces the internal pH below this threshold, triggering the assembly of a type III secretion system in Salmonella and the secretion of virulence factors in the host. These pathways are significant because preventing intracellular acidification of Salmonella renders it avirulent, suggesting that acid stress pathways represent a potential therapeutic target. Although we refer to the acid stress response as singular, it is actually a complex response that involves numerous two-component signaling systems, several amino acid decarboxylation systems, as well as cellular buffering systems and electron transport chain components, among others. In a recent paper in the Journal of Bacteriology, M. G. Gorelik, H. Yakhnin, A. Pannuri, A. C. Walker, C. Pourciau, D. Czyz, T. Romeo, and P. Babitzke (J Bacteriol 206:e00354-23, 2024, https://doi.org/10.1128/jb.00354-23) describe a new connection linking the carbon storage regulator CsrA to the acid stress response, highlighting new additional layers of complexity.

Multitier regulation of the E. coli extreme acid stress response by CsrA.

CsrA is an RNA-binding protein that regulates processes critical for growth and survival, including central carbon metabolism, motility, biofilm formation, stress responses, and expression of virulence factors in pathogens. Transcriptomics studies in Escherichia coli suggested that CsrA repressed genes involved in surviving extremely acidic conditions. Here, we examine the effects of disrupting CsrA-dependent regulation on the expression of genes and circuitry for acid stress survival and demonstrate CsrA-mediated repression at multiple levels. We show that this repression is critical for managing the trade-off between growth and survival; overexpression of acid stress genes caused by csrA disruption enhances survival under extreme acidity but is detrimental for growth under mildly acidic conditions. In vitro studies confirmed that CsrA binds specifically to mRNAs of structural and regulatory genes for acid stress survival, causing translational repression. We also found that translation of the top-tier acid stress regulator, evgA, is coupled to that of a small leader peptide, evgL, which is repressed by CsrA. Unlike dedicated acid stress response genes, csrA and its sRNA antagonists, csrB and csrC, did not exhibit a substantial response to acid shock. Furthermore, disruption of CsrA regulation of acid stress genes impacted host-microbe interactions in Caenorhabditis elegans, alleviating GABA deficiencies. This study expands the known regulon of CsrA to genes of the extreme acid stress response of E. coli and highlights a new facet of the global role played by CsrA in balancing the opposing physiological demands of stress resistance with the capacity for growth and modulating host interactions.IMPORTANCETo colonize/infect the mammalian intestinal tract, bacteria must survive exposure to the extreme acidity of the stomach. E. coli does this by expressing proteins that neutralize cytoplasmic acidity and cope with molecular damage caused by low pH. Because of the metabolic cost of these processes, genes for surviving acid stress are tightly regulated. Here, we show that CsrA negatively regulates the cascade of expression responsible for the acid stress response. Increased expression of acid response genes due to csrA disruption improved survival at extremely low pH but inhibited growth under mildly acidic conditions. Our findings define a new layer of regulation in the acid stress response of E. coli and a novel physiological function for CsrA.

Indole inhibited the expression of csrA gene in Escherichia coli.

Indole is a very important signal molecule which plays multiple regulatory roles in many physiological and biochemical processes of bacteria, but up to now, the reasons for its wide range of functions have not been revealed. In this study, we found that indole inhibits the motility, promotes glycogen accumulation and enhances starvation resistance of Escherichia coli. However, the regulatory effects of indole became insignificant while the global csrA gene was mutated. To reveal the regulatory relationship between indole and csrA, we studied the effects of indole on the transcription level of csrA, flhDC, glgCAP and cstA, and also the sensing of the promoters of the genes on indole. It was found that indole inhibited the transcription of csrA, and only the promoter of the csrA gene can sense indole. Namely, indole indirectly regulated the translation level of FlhDC, GlgCAP and CstA. These data indicates that indole regulation is related with the regulation of CsrA, which may throw light on the regulation mechanism research of indole.

CsrA selectively modulates sRNA-mRNA regulator outcomes.

Post-transcriptional regulation, by small RNAs (sRNAs) as well as the global Carbon Storage Regulator A (CsrA) protein, play critical roles in bacterial metabolic control and stress responses. The CsrA protein affects selective sRNA-mRNA networks, in addition to regulating transcription factors and sigma factors, providing additional avenues of cross talk between other stress-response regulators. Here, we expand the known set of sRNA-CsrA interactions and study their regulatory effects. In vitro binding assays confirm novel CsrA interactions with ten sRNAs, many of which are previously recognized as key regulatory nodes. Of those 10 sRNA, we identify that McaS, FnrS, SgrS, MicL, and Spot42 interact directly with CsrA in vivo. We find that the presence of CsrA impacts the downstream regulation of mRNA targets of the respective sRNA. In vivo evidence supports enhanced CsrA McaS-csgD mRNA repression and showcases CsrA-dependent repression of the fucP mRNA via the Spot42 sRNA. We additionally identify SgrS and FnrS as potential new sRNA sponges of CsrA. Overall, our results further support the expanding impact of the Csr system on cellular physiology via CsrA impact on the regulatory roles of these sRNAs.

CsrA coordinates the expression of ribosome hibernation and anti-σ factor proteins.

Bacterial growth rate varies due to changing physiological signals and is fundamentally dependent on protein synthesis. Consequently, cells alter their transcription and translation machinery to optimize the capacity for protein production under varying conditions and growth rates. Our findings demonstrate that the post-transcriptional regulator CsrA in Escherichia coli controls the expression of genes that participate in these processes. During exponential growth, CsrA represses the expression of proteins that alter or inhibit RNA polymerase (RNAP) and ribosome activity, including the ribosome hibernation factors RMF, RaiA, YqjD, ElaB, YgaM, and SRA, as well as the anti-σ70 factor, Rsd. Upon entry into the stationary phase, RaiA, YqjD, ElaB, and SRA expression was derepressed and that of RMF, YgaM, and Rsd was activated in the presence of CsrA. This pattern of gene expression likely supports global protein expression during active growth and helps limit protein production to a basal level when nutrients are limited. In addition, we identified genes encoding the paralogous C-tail anchored inner membrane proteins YqjD and ElaB as robust, direct targets of CsrA-mediated translational repression. These proteins bind ribosomes and mediate their localization to the inner cell membrane, impacting a variety of processes including protein expression and membrane integrity. Previous studies found that YqjD overexpression inhibits cell growth, suggesting that appropriate regulation of YqjD expression might play a key role in cell viability. CsrA-mediated regulation of yqjD and ribosome hibernation factors reveals a new role for CsrA in appropriating cellular resources for optimum growth under varying conditions.IMPORTANCEThe Csr/Rsm system (carbon storage regulator or repressor of stationary phase metabolites) is a global post-transcriptional regulatory system that coordinates and responds to environmental cues and signals, facilitating the transition between active growth and stationary phase. Another key determinant of bacterial lifestyle decisions is the management of the cellular gene expression machinery. Here, we investigate the connection between these two processes in Escherichia coli. Disrupted regulation of the transcription and translation machinery impacts many cellular functions, including gene expression, growth, fitness, and stress resistance. Elucidating the role of the Csr system in controlling the activity of RNAP and ribosomes advances our understanding of mechanisms controlling bacterial growth. A more complete understanding of these processes could lead to the improvement of therapeutic strategies for recalcitrant infections.

Florida-California Cancer Health Equity Center (CaRE2) Community Scientist Research Advocacy Program.

The Community Scientist Program (CSP), a model connecting researchers with community members, is effective to inform and involve the general population in health-related clinical research. Given the existing cancer disparities among Black/African American and Hispanic/Latino/a populations, more models describing how cancer-related CSPs are designed, implemented, and evaluated are needed. The Florida-California Cancer Research, Education and Engagement (CaRE2) Health Equity Center is a tri-institutional, bicoastal center created to eliminate cancer health disparities among Black/African American and Hispanic/Latino/a populations living in California and in Florida. The CaRE2 Center created a Community Scientist Research Advocacy (CSRA) training program for community members to become cancer research advocates. The CSRA program is currently a 13-week program conducted 100% virtually with all materials provided in English and Spanish for participants to learn more about prostate, lung, and pancreas cancers, ongoing research at CaRE2, and ways to share cancer research throughout their communities. Participants attend didactic lectures on cancer research during weeks 1-5. In week 4, participants join CSRA self-selected groups based on cancer-related topics of interest. Each group presents their cancer-related advocacy project developed during weeks 5-12 at the final session. In this paper, we describe the CaRE2 Health Equity Center's CSRA program, share results, and discuss opportunities for improvement in future program evaluation as well as replication of this model in other communities.

Little reason to call them small noncoding RNAs.

Hundreds of different species of small RNAs can populate a bacterial cell. This small transcriptome contains important information for the adaptation of cellular physiology to environmental changes. Underlying cellular networks involving small RNAs are RNA-RNA and RNA-protein interactions, which are often intertwined. In addition, small RNAs can function as mRNAs. In general, small RNAs are referred to as noncoding because very few are known to contain translated open reading frames. In this article, we intend to highlight that the number of small RNAs that fall within the set of translated RNAs is bound to increase. In addition, we aim to emphasize that the dynamics of the small transcriptome involve different functional codes, not just the genetic code. Therefore, since the role of small RNAs is always code-driven, we believe that there is little reason to continue calling them small noncoding RNAs.

CsrA-Mediated Translational Activation of the hmsE mRNA Enhances HmsD-Dependent C-di-GMP-Enabled Biofilm Production in Yersinia pestis.

The plague bacterium, Yersinia pestis, forms a biofilm-mediated blockage in the flea foregut that enhances its transmission by fleabite. Biofilm formation is positively controlled by cyclic di-GMP (c-di-GMP), which is synthesized by the diguanylate cyclases (DGC), HmsD and HmsT. While HmsD primarily promotes biofilm-mediated blockage of fleas, HmsT plays a more minor role in this process. HmsD is a component of the HmsCDE tripartite signaling system. HmsC and HmsE posttranslationally inhibit or activate HmsD, respectively. HmsT-dependent c-di-GMP levels and biofilm formation are positively regulated by the RNA-binding protein CsrA. In this study we determined whether CsrA positively regulates HmsD-dependent biofilm formation through interactions with the hmsE mRNA. Gel mobility shift assays determined that CsrA binds specifically to the hmsE transcript. RNase T1 footprint assays identified a single CsrA binding site and CsrA-induced structural changes in the hmsE leader region. Translational activation of the hmsE mRNA was confirmed in vivo using plasmid-encoded inducible translational fusion reporters and by HmsE protein expression studies. Furthermore, mutation of the CsrA binding site in the hmsE transcript significantly reduced HmsD-dependent biofilm formation. These results suggest that CsrA binding leads to structural changes in the hmsE mRNA that enhance its translation to enable increased HmsD-dependent biofilm formation. Given the requisite function of HmsD in biofilm-mediated flea blockage, this CsrA-dependent increase in HmsD activity underscores that complex and conditionally defined modulation of c-di-GMP synthesis within the flea gut is required for Y. pestis transmission. IMPORTANCE Mutations enhancing c-di-GMP biosynthesis drove the evolution of Y. pestis to flea-borne transmissibility. c-di-GMP-dependent biofilm-mediated blockage of the flea foregut enables regurgitative transmission of Y. pestis by fleabite. The Y. pestis diguanylate cyclases (DGC), HmsT and HmsD, which synthesize c-di-GMP, play significant roles in transmission. Several regulatory proteins involved in environmental sensing, as well as signal transduction and response regulation, tightly control DGC function. An example is CsrA, a global posttranscriptional regulator that modulates carbon metabolism and biofilm formation. CsrA integrates alternative carbon usage metabolism cues to activate c-di-GMP biosynthesis through HmsT. Here, we demonstrated that CsrA additionally activates hmsE translation to promote c-di-GMP biosynthesis through HmsD. This emphasizes that a highly evolved regulatory network controls c-di-GMP synthesis and Y. pestis transmission.

Identification and Regulatory Roles of a New Csr Small RNA from Arctic Pseudoalteromonas fuliginea BSW20308 in Temperature Responses.

Small RNAs (sRNAs) play a very important role in gene regulation at the posttranscriptional level. However, sRNAs from nonmodel microorganisms, extremophiles in particular, have been rarely explored. We discovered a putative sRNA, termed Pf1 sRNA, in Pseudoalteromonas fuliginea BSW20308 isolated from the polar regions in our previous work. In this study, we identified the sRNA and investigated its regulatory role in gene expression under different temperatures. Pf1 sRNA was confirmed to be a new member of the CsrB family but has little sequence similarity with Escherichia coli CsrB. However, Pf1 sRNA was able to bind to CsrA from E. coli and P. fuliginea BSW20308 to regulate glycogen synthesis. The Pf1 sRNA knockout strain (ΔPf1) affected motility, fitness, and global gene expression in transcriptomes and proteomes at 4°C and 32°C. Genes related to carbon metabolism, amino acid metabolism, salinity tolerance, antibiotic resistance, oxidative stress, motility, chemotaxis, biofilm, and secretion systems were differentially expressed in the wild-type strain and the ΔPf1 mutant. Our study suggested that Pf1 sRNA might play an important role in response to environmental changes by regulating global gene expression. Specific targets of the Pf1 sRNA-CsrA system were tentatively proposed, such as genes involved in the type VI secretion system, TonB-dependent receptors, and response regulators, but most of them have an unknown function. Since this is the first study of CsrB family sRNA in Pseudoalteromonas and microbes from the polar regions, it provides a novel insight at the posttranscriptional level into the responses and adaptation to temperature changes in bacteria from extreme environments. This study also sheds light on the evolution of sRNA in extreme environments and expands the bacterial sRNA database. IMPORTANCE Previous research on microbial temperature adaptation has focused primarily on functional genes, with little attention paid to posttranscriptional regulation. Small RNAs, the major posttranscriptional modulators of gene expression, are greatly underexplored, especially in nonpathogenic and nonmodel microorganisms. In this study, we verified the first Csr sRNA, named Pf1 sRNA, from Pseudoalteromonas, a model genus for studying cold adaptation. We revealed that Pf1 sRNA played an important role in global regulation and was indispensable in improving fitness. This study provided us a comprehensive view of sRNAs from Pseudoalteromonas and expanded our understanding of bacterial sRNAs from extreme environments.

Global profiling of the RNA and protein complexes of Escherichia coli by size exclusion chromatography followed by RNA sequencing and mass spectrometry (SEC-seq).

New methods for the global identification of RNA-protein interactions have led to greater recognition of the abundance and importance of RNA-binding proteins (RBPs) in bacteria. Here, we expand this tool kit by developing SEC-seq, a method based on a similar concept as the established Grad-seq approach. In Grad-seq, cellular RNA and protein complexes of a bacterium of interest are separated in a glycerol gradient, followed by high-throughput RNA-sequencing and mass spectrometry analyses of individual gradient fractions. New RNA-protein complexes are predicted based on the similarity of their elution profiles. In SEC-seq, we have replaced the glycerol gradient with separation by size exclusion chromatography, which shortens operation times and offers greater potential for automation. Applying SEC-seq to Escherichia coli, we find that the method provides a higher resolution than Grad-seq in the lower molecular weight range up to ~500 kDa. This is illustrated by the ability of SEC-seq to resolve two distinct, but similarly sized complexes of the global translational repressor CsrA with either of its antagonistic small RNAs, CsrB and CsrC. We also characterized changes in the SEC-seq profiles of the small RNA MicA upon deletion of its RNA chaperones Hfq and ProQ and investigated the redistribution of these two proteins upon RNase treatment. Overall, we demonstrate that SEC-seq is a tractable and reproducible method for the global profiling of bacterial RNA-protein complexes that offers the potential to discover yet-unrecognized associations between bacterial RNAs and proteins.

Dual role of CsrA in regulating the hemolytic activity of Escherichia coli O157:H7.

Post-transcriptional global carbon storage regulator A (CsrA) is a sequence-specific RNA-binding protein involved in the regulation of multiple bacterial processes. Hemolysin is an important virulence factor in the enterohemorrhagic Escherichia coli O157:H7 (EHEC). Here, we show that CsrA plays a dual role in the regulation of hemolysis in EHEC. CsrA significantly represses plasmid-borne enterohemolysin (EhxA)-mediated hemolysis and activates chromosome-borne hemolysin E (HlyE)-mediated hemolysis through different mechanisms. RNA structure prediction revealed a well-matched stem-loop structure with two potential CsrA binding sites located on the 5' untranslated region (UTR) of ehxB, which encodes a translocator required for EhxA secretion. CsrA inhibits EhxA secretion by directly binding to the RNA leader sequence of ehxB to repress its expression in two different ways: CsrA either binds to the Shine-Dalgarno sequence of ehxB to block ribosome access or to ehxB transcript to promote its mRNA decay. The predicted CsrA-binding site 1 of ehxB is essential for its regulation. There is a single potential CsrA-binding site at the 5'-end of the hlyE transcript, and its mutation completely abolishes CsrA-dependent activation. CsrA can also stabilize hlyE mRNA by directly binding to its 5' UTR. Overall, our results indicate that CsrA acts as a hemolysis modulator to regulate pathogenicity under certain conditions.

Activation of the Type III Secretion System of Enteropathogenic Escherichia coli Leads to Remodeling of Its Membrane Composition and Function.

The cell envelope of Gram-negative bacteria is a complex structure, essential for bacterial survival and for resistance to many antibiotics. Channels that cross the bacterial envelope and the host cell membrane form secretion systems that are activated upon attachment to host, enabling bacteria to inject effector molecules into the host cell, required for bacterium-host interaction. The type III secretion system (T3SS) is critical for the virulence of several pathogenic bacteria, including enteropathogenic Escherichia coli (EPEC). EPEC T3SS activation is associated with repression of carbon storage regulator (CsrA), resulting in gene expression remodeling, which is known to affect EPEC central carbon metabolism and contributes to the adaptation to a cell-adherent lifestyle in a poorly understood manner. We reasoned that the changes in the bacterial envelope upon attachment to the host and the activation of a secretion system may involve a modification of the lipid composition of bacterial envelope. Accordingly, we performed a lipidomics analysis on mutant strains that simulate T3SS activation. We saw a shift in glycerophospholipid metabolism toward the formation of lysophospholipids, attributed to corresponding upregulation of the phospholipase gene pldA and the acyltransferase gene ygiH upon T3SS activation in EPEC. We also detected a shift from menaquinones and ubiquinones to undecaprenyl lipids, concomitant with abnormal synthesis of O antigen. The remodeling of lipid metabolism is mediated by CsrA and associated with increased bacterial cell size and zeta potential and a corresponding alteration in EPEC permeability to vancomycin, increasing the sensitivity of T3SS-activated strains and of adherent wild-type EPEC to the antibiotic. IMPORTANCE The characterization of EPEC membrane lipid metabolism upon attachment to the host is an important step toward a better understanding the shift of EPEC, a notable human pathogen, from a planktonic to adherent lifestyle. It may also apply to other pathogenic bacteria that use this secretion system. We predict that upon attachment to host cells, the lipid remodeling upon T3SS activation contributes to bacterial fitness and promotes host colonization, and we show that it is associated with increased cell permeability and higher sensitivity to vancomycin. To the best of our knowledge, this is the first demonstration of a bacterial lipid remodeling due to activation of a secretion system.

Bacterial RNA chaperones and chaperone-like riboregulators: behind the scenes of RNA-mediated regulation of cellular metabolism.

In all domains of life, RNA chaperones safeguard and guide the fate of the cellular RNA pool. RNA chaperones comprise structurally diverse proteins that ensure proper folding, stability, and ribonuclease resistance of RNA, and they support regulatory activities mediated by RNA. RNA chaperones constitute a topologically diverse group of proteins that often present an unstructured region and bind RNA with limited nucleotide sequence preferences. In bacteria, three main proteins - Hfq, ProQ, and CsrA - have been shown to regulate numerous complex processes, including bacterial growth, stress response and virulence. Hfq and ProQ have well-studied activities as global chaperones with pleiotropic impact, while CsrA has a chaperone-like role with more defined riboregulatory function. Here, we describe relevant novel insights into their common features, including RNA binding properties, unstructured domains, and interplay with other proteins important to RNA metabolism.

CsrD regulates amylovoran biosynthesis and virulence in Erwinia amylovora in a novel cyclic-di-GMP dependent manner.

Erwinia amylovora is an economically devastating plant pathogen that causes fire blight disease in members of the Rosaceae family, most notably in apple and pear. The exopolysaccharide amylovoran is a pathogenicity determinant in E. amylovora and a major component of the extracellular matrix of biofilms formed within the xylem vasculature of the host plant. The second messenger cyclic-di-GMP (c-di-GMP) has been reported to positively regulate the transcription of amsG (the first gene in the 12-gene amylovoran [ams] biosynthetic operon), thus impacting amylovoran production. However, the regulatory mechanism by which this interaction occurs is largely unknown. Here, we report that c-di-GMP can bind to specific residues in the EAL domain of the E. amylovora protein CsrD. CsrD and RNase E regulate the degradation of the sRNA CsrB in E. amylovora. When CsrD is bound to c-di-GMP, there is an enhancement in the level of RNase E-mediated degradation of CsrB, which then alters amsG transcription. Additionally, csrD was also found to positively contribute to virulence and biofilm formation. We thus present a pathway of conditional regulation of amylovoran production mediated by changing intracellular levels of c-di-GMP, which impacts disease progression.

CsrA-Controlled Proteins Reveal New Dimensions of Acinetobacter baumannii Desiccation Tolerance.

Hospital environments are excellent reservoirs for the opportunistic pathogen Acinetobacter baumannii in part because it is exceptionally tolerant to desiccation. We found that relative to other A. baumannii strains, the virulent strain AB5075 was strikingly desiccation resistant at 2% relative humidity (RH), suggesting that it is a good model for studies of the functional basis of this trait. Consistent with results from other A. baumannii strains at 40% RH, we found the global posttranscriptional regulator CsrA to be critically important for desiccation tolerance of AB5075 at 2% RH. Proteomics experiments identified proteins that were differentially present in wild-type and csrA mutant cells. Subsequent analysis of mutants in genes encoding some of these proteins revealed six genes that were required for wild-type levels of desiccation tolerance. These include genes for catalase, a universal stress protein, a hypothetical protein, and a biofilm-associated protein. Two genes of unknown function had very strong desiccation phenotypes, with one of the two genes predicting an intrinsically disordered protein (IDP) that binds to DNA. Intrinsically disordered proteins are widespread in eukaryotes but less so in prokaryotes. Our results suggest there are new mechanisms underlying desiccation tolerance in bacteria and identify several key functions involved. IMPORTANCE Acinetobacter baumannii is found in terrestrial environments but can cause nosocomial infections in very sick patients. A factor that contributes to the prevalence of A. baumannii in hospital settings is that it is intrinsically resistant to dry conditions. Here, we established the virulent strain A. baumannii AB5075 as a model for studies of desiccation tolerance at very low relative humidity. Our results show that this trait depends on two proteins of unknown function, one of which is predicted to be an intrinsically disordered protein. This category of protein is critical for the small animals named tardigrades to survive desiccation. Our results suggest that A. baumannii may have novel strategies to survive desiccation that have not previously been seen in bacteria.

Phage display-based discovery of cyclic peptides against the broad spectrum bacterial anti-virulence target CsrA.

Small macrocyclic peptides are promising candidates for new anti-infective drugs. To date, such peptides have been poorly studied in the context of anti-virulence targets. Using phage display and a self-designed peptide library, we identified a cyclic heptapeptide that can bind the carbon storage regulator A (CsrA) from Yersinia pseudotuberculosis and displace bound RNA. This disulfide-bridged peptide, showed an IC50 value in the low micromolar range. Upon further characterization, cyclisation was found to be essential for its activity. To increase metabolic stability, a series of disulfide mimetics were designed and a redox-stable 1,4-disubstituted 1,2,3-triazole analogue displayed activity in the double-digit micromolar range. Further experiments revealed that this triazole peptidomimetic is also active against CsrA from Escherichia coli and RsmA from Pseudomonas aeruginosa. This study provides an ideal starting point for medicinal chemistry optimization of this macrocyclic peptide and might pave the way towards broad-acting virulence modulators.

CsrA regulation via binding to the base-pairing small RNA Spot 42.

The carbon storage regulator system and base-pairing small RNAs (sRNAs) represent two predominant modes of bacterial post-transcriptional regulation, which globally influence gene expression. Binding of CsrA protein to the 5' UTR or initial mRNA coding sequences can affect translation, RNA stability, and/or transcript elongation. Base-pairing sRNAs also regulate these processes, often requiring assistance from the RNA chaperone Hfq. Transcriptomics studies in Escherichia coli have identified many new CsrA targets, including Spot 42 and other base-pairing sRNAs. Spot 42 synthesis is repressed by cAMP-CRP, induced by the presence of glucose, and Spot 42 post-transcriptionally represses operons that facilitate metabolism of nonpreferred carbon sources. CsrA activity is also increased by glucose via effects on CsrA sRNA antagonists, CsrB/C. Here, we elucidate a mechanism wherein CsrA binds to and protects Spot 42 sRNA from RNase E-mediated cleavage. This protection leads to enhanced repression of srlA by Spot 42, a gene required for sorbitol uptake. A second, independent mechanism by which CsrA represses srlA is by binding to and inhibiting translation of srlM mRNA, encoding a transcriptional activator of srlA. Our findings demonstrate a novel means of regulation, by CsrA binding to a sRNA, and indicate that such interactions can help to shape complex bacterial regulatory circuitry.

CsrA enters Hfq's territory: Regulation of a base-pairing small RNA.

Post-transcriptional regulatory networks in Gammaproteobacteria are to a large extent built around the two globally acting RNA-binding proteins (RBPs) CsrA and Hfq. Both RBPs interact with small regulatory RNAs (sRNAs), but the functional outcomes of these interactions are generally distinct. Whereas Hfq both stabilizes sRNAs and promotes their base-pairing to target mRNAs, the sRNAs bound by CsrA act as sequestering molecules that titrate the RBP away from its mRNA targets. In this issue of Molecular Microbiology, Lai et al. reveal that CsrA interacts with the Hfq-associated and base-pairing sRNA Spot 42. In this case, CsrA increases Spot 42 stability by masking a cleavage site for endoribonuclease RNase E, thereby promoting Spot 42-dependent regulation of srlA mRNA. Interestingly, the effect of CsrA on srlA expression is two-fold. In addition to affecting Spot 42-dependent regulation, CsrA directly inhibits translation of SrlM, an activator of srlA transcription. Together, this study reveals a new function for CsrA and indicates more intricate connections between the CsrA and Hfq networks than previously anticipated. Several recent studies have identified additional RBPs that interact with sRNAs. With new RBP identification methods at hand, it will be intriguing to see how many more sRNA-binding proteins will be uncovered.

CsrA Regulates Swarming Motility and Carbohydrate and Amino Acid Metabolism in Vibrio alginolyticus.

Vibrio alginolyticus, like other vibrio species, is a widely distributed marine bacterium that is able to outcompete other species in variable niches where diverse organic matters are supplied. However, it remains unclear how these cells sense and adjust metabolic flux in response to the changing environment. CsrA is a conserved RNA-binding protein that modulates critical cellular processes such as growth ability, central metabolism, virulence, and the stress response in gamma-proteobacteria. Here, we first characterize the csrA homolog in V. alginolyticus. The results show that CsrA activates swarming but not swimming motility, possibly by enhancing the expression of lateral flagellar associated genes. It is also revealed that CsrA modulates the carbon and nitrogen metabolism of V. alginolyticus, as evidenced by a change in the growth kinetics of various carbon and nitrogen sources when CsrA is altered. Quantitative RT-PCR shows that the transcripts of the genes encoding key enzymes involved in the TCA cycle and amino acid metabolism change significantly, which is probably due to the variation in mRNA stability given by CsrA binding. This may suggest that CsrA plays an important role in sensing and responding to environmental changes.