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Vibrio cholerae, a facultative human pathogen and causative agent of the severe diarrheal disease cholera, transits between the human intestinal tract and aquatic reservoirs. Like other bacterial species, V. cholerae continuously releases bacterial extracellular vesicles (BEVs) from its surface, which have been recently characterised for their role during in vivo colonisation. However, between epidemic outbreaks, V. cholerae persists in the biofilm mode for extended periods in aquatic reservoirs, which enhances environmental fitness and host transition. In this study, we investigated the effect of V. cholerae BEVs on biofilm formation, a critical feature for ex vivo survival. In contrast to BEVs from planktonic cultures, our results show that physiological concentrations of BEVs from dynamic biofilm cultures facilitate V. cholerae biofilm formation, which could be linked to a proteinaceous factor. Comparative proteomic analyses of planktonic‐ and biofilm‐derived BEVs identified a previously uncharacterised outer membrane protein as an abundant component of dynamic biofilm‐derived BEVs, which was found to be responsible for the BEV‐dependent enhancement of biofilm production. Consequently, this protein was named outer membrane‐associated biofilm facilitating protein A (ObfA). Comprehensive molecular studies unravelled ObfA as a negative modulator of HapR activity. HapR is a key transcriptional regulator of the V. cholerae quorum sensing (QS) cascade acting as a potent repressor of biofilm formation and virulence. Consistently, obfA mutants not only exhibited reduced biofilm production but also reduced colonisation fitness. Surprisingly, our results demonstrate that ObfA does not affect HapR through the canonical QS system but via the Csr‐cascade altering the expression of the small regulatory RNAs CsrC and CsrD. In summary, this study elucidates a novel intraspecies BEV‐based communication in V. cholerae that influences biofilm formation and colonisation fitness via a new regulatory pathway involving HapR, Csr‐cascade and the BEV‐associated protein ObfA. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract Vibrio cholerae, a facultative human pathogen and causative agent of the severe diarrheal disease cholera, transits between the human intestinal tract and aquatic reservoirs. Like other bacterial species, V. cholerae continuously releases bacterial extracellular vesicles (BEVs) from its surface, which have been recently characterised for their role during in vivo colonisation. However, between epidemic outbreaks, V. cholerae persists in the biofilm mode for extended periods in aquatic reservoirs, which enhances environmental fitness and host transition. In this study, we investigated the effect of V. cholerae BEVs on biofilm formation, a critical feature for ex vivo survival. In contrast to BEVs from planktonic cultures, our results show that physiological concentrations of BEVs from dynamic biofilm cultures facilitate V. cholerae biofilm formation, which could be linked to a proteinaceous factor. Comparative proteomic analyses of planktonic‐ and biofilm‐derived BEVs identified a previously uncharacterised outer membrane protein as an abundant component of dynamic biofilm‐derived BEVs, which was found to be responsible for the BEV‐dependent enhancement of biofilm production. Consequently, this protein was named outer membrane‐associated biofilm facilitating protein A (ObfA). Comprehensive molecular studies unravelled ObfA as a negative modulator of HapR activity. HapR is a key transcriptional regulator of the V. cholerae quorum sensing (QS) cascade acting as a potent repressor of biofilm formation and virulence. Consistently, obfA mutants not only exhibited reduced biofilm production but also reduced colonisation fitness. Surprisingly, our results demonstrate that ObfA does not affect HapR through the canonical QS system but via the Csr‐cascade altering the expression of the small regulatory RNAs CsrC and CsrD. In summary, this study elucidates a novel intraspecies BEV‐based communication in V. cholerae that influences biofilm formation and colonisation fitness via a new regulatory pathway involving HapR, Csr‐cascade and the BEV‐associated protein ObfA.

ABSTRACTBacterial 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.

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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.

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.

Proteins belonging to the RsmA (regulator of secondary metabolism)/CsrA (carbon storage regulator) family are small RNA-binding proteins that play crucial roles post-transcriptionally regulating gene expression in many Gram-negative and some Gram-positive bacteria. Although most of the bacteria studied have a single RsmA/CsrA gene, Pseudomonas syringae pv. tomato (Pto) DC3000 encodes five Rsm proteins: RsmA/CsrA2, RsmC/CsrA1, RsmD/CsrA4, RsmE/CsrA3, and RsmH/CsrA5. This work aims to provide a comprehensive analysis of the expression of these five rsm protein-encoding genes, elucidate the regulatory mechanisms governing their expression, as well as the physiological relevance of each variant. To achieve this, we examined the expression of rsmA, rsmE, rsmC, rsmD, and rsmH within their genetic contexts, identified their promoter regions, and assessed the impact of both their deletion and overexpression on various Pto DC3000 phenotypes. A novel finding is that rsmA and rsmC are part of an operon with the upstream genes, whereas rsmH seems to be co-transcribed with two downstream genes. We also observed significant variability in expression levels and RpoS dependence among the five rsm paralogs. Thus, despite the extensive repertoire of rsm genes in Pto DC3000, only rsmA , rsmE and rsmH were significantly expressed under all tested conditions (swarming, minimal and T3SS-inducing liquid media). Among these, RsmE and RsmA were corroborated as the most important paralogs at the functional level, whereas RsmH played a minor role in regulating free life and plant-associated phenotypes. Conversely, RsmC and RsmD did not seem to be functional under the conditions tested. [Display omitted] [ABSTRACT FROM AUTHOR]

ABSTRACT 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.

Intestinal colonization and virulence factor production in response to environmental cues is mediated through several regulatory factors in Vibrio cholerae , including the highly conserved RNA-binding global regulatory protein CsrA. We have shown previously that CsrA increases synthesis of the virulence-associated transcription factor ToxR in response to specific amino acids (NRES) and is required for the virulence of V. cholerae in the infant mouse model of cholera. In this study, we mapped the 5' untranslated region (5' UTR) of toxR and showed that CsrA can bind directly to an RNA sequence encompassing the 5' UTR, indicating that the regulation of ToxR levels by CsrA is direct. Consistent with this observation, the 5' UTR of toxR contains multiple putative CsrA binding sequences (GGA motifs), and mutating these motifs disrupted the CsrA-mediated increase in ToxR. Optimal binding of CsrA to a defined RNA oligonucleotide required the bridging of two GGA motifs within a single RNA strand. To determine the mechanism of regulation by CsrA, we assayed toxR transcript levels, stability, and efficiency of translation. Both the amount of toxR mRNA in NRES and the stability of the toxR transcript were increased by CsrA. Using an in vitro translation assay, we further showed that synthesis of ToxR was greatly enhanced in the presence of purified CsrA, suggesting a direct role for CsrA in the translation of toxR mRNA. We propose a model in which CsrA binding to the 5' UTR of the toxR transcript promotes ribosomal access while precluding interactions with RNA-degrading enzymes.IMPORTANCE Vibrio cholerae is uniquely adapted to marine environments as well as the human intestinal tract. Global regulators, such as CsrA, which help translate environmental cues into an appropriate cellular response, are critical for switching between these distinct environments. Understanding the pathways involved in relaying environmental signals is essential for understanding both the environmental persistence and the intestinal pathogenesis of this devastating human pathogen. In this study, we demonstrate that CsrA directly regulates the synthesis of ToxR, a key virulence factor of V. cholerae . Under conditions favoring high levels of active CsrA in the cell, such as in the presence of particular amino acids, CsrA increases ToxR protein levels by binding to the toxR transcript and enhancing both its stability and translation. By responding to nutrient availability, CsrA is perfectly poised to activate the virulence gene regulatory cascade at the preferred site of colonization in the human host, the nutrient-rich small intestinal mucosa.

The RNA-binding protein CsrA is a global posttranscriptional regulator and controls many physiological processes and virulence traits. Deletion of csrA caused loss of virulence, reduced motility and production of xanthan gum and substantial increase in glycogen accumulation, as well as enhanced bacterial aggregation and cell adhesion in Xanthomonas spp. How CsrA controls these traits is poorly understood. In this study, an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic analysis was conducted to compare the protein profile of wild-type strain Xanthomonas citri subsp. citri and the isogenic ΔcsrA strain. A total of 2,374 proteins were identified, and 284 were considered to be differentially expressed proteins (DEPS), among which 151 proteins were up-regulated and 133 were down-regulated in the ΔcsrA strain with respect to the wild-type strain. Enrichment analysis and a protein-protein interaction network analysis showed that CsrA regulates bacterial secretion systems, flagella, and xanthan gum biosynthesis. Several proteins encoded by the gumB operon were down-regulated, whereas proteins associated with flagellum assembly and the type IV secretion system were up-regulated in the ΔcsrA strain relative to the Xcc306 strain. These results were confirmed by β-glucuronidase assay or Western blot. RNA secondary structure prediction and a gel-shift assay indicated that CsrA binds to the Shine-Dalgarno sequence of virB5. In addition, the iTRAQ analysis identified 248 DEPs that were not previously identified in transcriptome analyses. Among them, CsrA regulates levels of eight regulatory proteins (ColR, GacA, GlpR, KdgR, MoxR, PilH, RecX, and YgiX), seven TonB-dependent receptors, four outer membrane proteins, and two ferric enterobactin receptors. Taken together, this study greatly expands understanding of the regulatory network of CsrA in X. citri subsp. citri.[Graphic: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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. [ABSTRACT FROM AUTHOR]

We discuss present and developing techniques for studying radiocarbon in marine organic carbon (C). Bulk DOC (dissolved organic C) Δ14C measurements reveal information about the cycling time and sources of DOC in the ocean, yet they are time consuming and need to be streamlined. To further elucidate the cycling of DOC, various fractions have been separated from bulk DOC, through solid phase extraction of DOC, and ultrafiltration of high and low molecular weight DOC. Research using 14C of DOC and particulate organic C separated into organic fractions revealed that the acid insoluble fraction is similar in 14C signature to that of the lipid fraction. Plans for utilizing this methodology are described. Studies using compound specific radiocarbon analyses to study the origin of biomarkers in the marine environment are reviewed and plans for the future are outlined. Development of ramped pyrolysis oxidation methods are discussed and scientific questions addressed. A modified elemental analysis (EA) combustion reactor is described that allows high particulate organic C sample throughput by direct coupling with the MIniCArbonDAtingSystem. [ABSTRACT FROM AUTHOR]

Abstract Background Colorectal cancer is a major public concern, being the second deadliest cancer in the world. Whereas survival is high for localized forms, metastatic colorectal cancer has showed poor prognosis, with a 5-year survival barely surpassing 11%. Conventional chemotherapies against this disease proved their efficiency and remain essential in first-line treatment. However, the large number of authorized protocols complexifies treatment decision. In common practice, such decision is made on an empirical basis, by assessing benefits and risks for the patient. In other words, there is currently no efficient means of predicting the efficacy of any chemotherapy protocol for metastatic colorectal cancer. Methods/design The use of a chemosensitivity assay, the Oncogramme®, should help clinicians administer the best chemotherapy regimen to their patients. We hypothesize it would ultimately improve their survival. In this multicentred, prospective trial (ONCOGRAM), eligible patients with metastatic colorectal cancer are randomized to determine whether they will receive an Oncogramme®. For clinicians whose patients benefited from the assay (arm A), results are used as a decision support tool. Patients not undergoing the Oncogramme® procedure are treated according to current practice, without the assistance of the assay (arm B). Primary outcome is 1-year progression-free survival. Secondary outcomes include response rates, as well as 6-month and 1-year survival rates. Discussion This study aims at investigating the clinical utility of the Oncogramme® as a decision support tool for the treatment of patients with metastatic colorectal cancer. If the Oncogramme® positively influenced patient overall survival and/or progression-free survival, it would be of great value for clinicians to implement this assay within the current landscape of personalized medicine tools, which include genomics and biomarker assays. Trial registration ClinicalTrials.gov identifier NCT03133273 . Registered on April 28, 2017.

ABSTRACT 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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Poly-3-hydroxybutyrate (PHB) can be efficiently produced in recombinant Escherichia coli by the overexpression of an operon (NphaCAB) encoding PHB synthetase. Strain improvement is considered to be one of critical factors to lower the production cost of PHB in recombinant system. In this study, one of key regulators that affect the cell growth and PHB content was confirmed and analyzed. Result: S17-3, a mutant E. coli strain derived from S17-1, was found to be able to achieve high cell density when expressing NphaCAB with the plasmid pBhya-CAB. Whole genome sequencing of S17-3 revealed genetic alternations on the upstream regions of csrA, encoding a global regulator cross-talking between stress response, catabolite repression and other metabolic activities. Deletion of csrA or expression of mutant csrA resulted in improved cell density and PHB content. Conclusion: The impact of gene deletion of csrA was determined, dysfunction of the regulators improved the cell density of recombinant E. coli and PHB production, however, the detail mechanism needs to be further clarified.How to cite: Wu H, Li S, Ji M, et al. Improvement of polyhydroxybutyrate production by deletion of csrA in Escherichia coli. Electron J Biotechnol 2020;46. https://doi.org/10.1016/j.ejbt.2020.04.005.

Clostridioides difficile flagellin FliC is associated with toxin gene expression, bacterial colonization, and virulence, and is also involved in pleiotropic gene regulation during in vivo infection. However, how fliC expression is regulated in C. difficile remains unclear. In Bacillus subtilis , flagellin homeostasis and motility are coregulated by flagellar assembly factor (FliW), flagellin Hag (FliC homolog), and Carbon storage regulator A (CsrA), which is referred to as partner-switching mechanism "FliW-CsrA-Hag." In this study, we characterized FliW and CsrA functions by deleting or overexpressing fliW , csrA , and fliW - csrA in C. difficile R20291. We showed that fliW deletion, csrA overexpression in R20291, and csrA complementation in R20291ΔWA (fliW - csrA codeletion mutant) dramatically decreased FliC production, but not fliC gene transcription. Suppression of fliC translation by csrA overexpression can be relieved mostly when fliW was coexpressed, and no significant difference in FliC production was detected when only fliW was complemented in R20291ΔWA. Further, loss of fliW led to increased biofilm formation, cell adhesion, toxin production, and pathogenicity in a mouse model of C. difficile infection (CDI), while fliW - csrA codeletion decreased toxin production and mortality in vivo. Our data suggest that CsrA negatively modulates fliC expression and FliW indirectly affects fliC expression through inhibition of CsrA post-transcriptional regulation. In light of "FliW-CsrA-Hag" switch coregulation mechanism reported in B. subtilis , our data also suggest that "FliW-CsrA- fliC /FliC" can regulate many facets of C. difficile R20291 pathogenicity. These findings further aid us in understanding the virulence regulation in C. difficile. [ABSTRACT FROM AUTHOR]

BackgroundA previous study identified a Yersinia enterocolitica transposon mutant, GY448, that was unable to export the flagellar type three secretion system (T3SS)-dependent phospholipase, YplA. This strain was also deficient for motility and unable to form colonies on Lauria-Bertani agar medium. Preliminary analysis suggested it carried a mutation in csrA. CsrA in Escherichia coli is an RNA-binding protein that is involved in specific post-transcriptional regulation of a myriad of physiological activities. This study investigated how CsrA affects expression of the flagellar regulatory cascade that controls YplA export and motility. It also explored the effect of csrA mutation on Y. enterocolitica in response to conditions that cue physiological changes important for growth in environments found both in nature and the laboratory.ResultsThe precise location of the transposon insertion in GMY448 was mapped within csrA. Genetic complementation restored disruptions in motility and the YplA export phenotype (Yex), which confirmed this mutation disrupted CsrA function. Mutation of csrA affected expression of yplA and flagellar genes involved in flagellar T3SS dependent export and motility by altering expression of the master regulators flhDC. Mutation of csrA also resulted in increased sensitivity of Y. enterocolitica to various osmolytes, temperatures and antibiotics.ConclusionsThe results of this study reveal unique aspects of how CsrA functions in Y. enterocolitica to control its physiology. This provides perspective on how the Csr system is susceptible to adaptation to particular environments and bacterial lifestyles.

ABSTRACT CsrA is a global regulator widespread in bacteria and known to be involved in different physiological processes, including pathogenicity. Deletion of csrA of Acinetobacter baumannii strain ATCC 19606 resulted in a mutant that was unable to utilize a broad range of carbon and energy sources, including amino acids. This defect in amino acid metabolism was most likely responsible for the growth inhibition of the ΔcsrA mutant in human urine, where amino acids are the most abundant carbon source for A. baumannii. Recent studies revealed that deletion of csrA in the A. baumannii strains AB09-003 and ATCC 17961 resulted in an increase in hyperosmotic stress resistance. However, the molecular basis for this observation remained unknown. This study aimed to investigate the role of CsrA in compatible solute synthesis. We observed striking differences in the ability of different A. baumannii strains to cope with hyperosmotic stress. Strains AB09-003 and ATCC 17961 were strongly impaired in hyperosmotic stress resistance in comparison to strain ATCC 19606. These differences were abolished by deletion of csrA and are in line with the ability to synthesize compatible solutes. In the salt-sensitive strains AB09-003 and ATCC 17961, compatible solute synthesis was repressed by CsrA. This impairment is mediated via CsrA and could be overcome by deletion of csrA from the genome. IMPORTANCE The opportunistic human pathogen Acinetobacter baumannii has become one of the leading causes of nosocomial infections around the world due to the increasing prevalence of multidrug-resistant strains and their optimal adaptation to clinical environments and the human host. Recently, it was found that CsrA, a global mRNA binding posttranscriptional regulator, plays a role in osmotic stress adaptation, virulence, and growth on amino acids of A. baumannii AB09-003 and ATCC 17961. Here, we report that this is also the case for A. baumannii ATCC 19606. However, we observed significant differences in the ΔcsrA mutants with respect to osmostress resistance, such as the AB09-003 and 17961 mutants being enhanced in osmostress resistance whereas the ATCC 19606 mutant was not. This suggests that the role of CsrA in osmotic stress adaptation is strain specific. Furthermore, we provide clear evidence that CsrA is essential for growth in human urine and at high temperatures.

Clostridioides difficile flagellin FliC is associated with toxin gene expression, bacterial colonization, and virulence, and is also involved in pleiotropic gene regulation during in vivo infection. However, how fliC expression is regulated in C. difficile remains unclear. In Bacillus subtilis, flagellin homeostasis and motility are coregulated by flagellar assembly factor (FliW), flagellin Hag (FliC homolog), and Carbon storage regulator A (CsrA), which is referred to as partner-switching mechanism “FliW-CsrA-Hag.” In this study, we characterized FliW and CsrA functions by deleting or overexpressing fliW, csrA, and fliW-csrA in C. difficile R20291. We showed that fliW deletion, csrA overexpression in R20291, and csrA complementation in R20291ΔWA (fliW-csrA codeletion mutant) dramatically decreased FliC production, but not fliC gene transcription. Suppression of fliC translation by csrA overexpression can be relieved mostly when fliW was coexpressed, and no significant difference in FliC production was detected when only fliW was complemented in R20291ΔWA. Further, loss of fliW led to increased biofilm formation, cell adhesion, toxin production, and pathogenicity in a mouse model of C. difficile infection (CDI), while fliW-csrA codeletion decreased toxin production and mortality in vivo. Our data suggest that CsrA negatively modulates fliC expression and FliW indirectly affects fliC expression through inhibition of CsrA post-transcriptional regulation. In light of “FliW-CsrA-Hag” switch coregulation mechanism reported in B. subtilis, our data also suggest that “FliW-CsrA-fliC/FliC” can regulate many facets of C. difficile R20291 pathogenicity. These findings further aid us in understanding the virulence regulation in C. difficile.

Virulence gene expression of Yersinia pseudotuberculosis changes during the different stages of infection and this is tightly controlled by environmental cues. In this study, we show that the small protein YmoA, a member of the Hha family, is part of this process. It controls temperature- and nutrient-dependent early and later stage virulence genes in an opposing manner and co-regulates bacterial stress responses and metabolic functions. Our analysis further revealed that YmoA exerts this function by modulating the global post-transcriptional regulatory Csr system. YmoA pre-dominantly enhances the stability of the regulatory RNA CsrC. This involves a stabilizing stem-loop structure within the 5′-region of CsrC. YmoA-mediated CsrC stabilization depends on H-NS, but not on the RNA chaperone Hfq. YmoA-promoted reprogramming of the Csr system has severe consequences for the cell: we found that a mutant deficient of ymoA is strongly reduced in its ability to enter host cells and to disseminate to the Peyer’s patches, mesenteric lymph nodes, liver and spleen in mice. We propose a model in which YmoA controls transition from the initial colonization phase in the intestine toward the host defense phase important for the long-term establishment of the infection in underlying tissues.

Background: Colorectal cancer is a major public concern, being the second deadliest cancer in the world. Whereas survival is high for localized forms, metastatic colorectal cancer has showed poor prognosis, with a 5-year survival barely surpassing 11%. Conventional chemotherapies against this disease proved their efficiency and remain essential in first-line treatment. However, the large number of authorized protocols complexifies treatment decision. In common practice, such decision is made on an empirical basis, by assessing benefits and risks for the patient. In other words, there is currently no efficient means of predicting the efficacy of any chemotherapy protocol for metastatic colorectal cancer. Methods/design: The use of a chemosensitivity assay, the Oncogramme®, should help clinicians administer the best chemotherapy regimen to their patients. We hypothesize it would ultimately improve their survival. In this multicentred, prospective trial (ONCOGRAM), eligible patients with metastatic colorectal cancer are randomized to determine whether they will receive an Oncogramme®. For clinicians whose patients benefited from the assay (arm A), results are used as a decision support tool. Patients not undergoing the Oncogramme® procedure are treated according to current practice, without the assistance of the assay (arm B). Primary outcome is 1-year progression-free survival. Secondary outcomes include response rates, as well as 6-month and 1-year survival rates. Discussion: This study aims at investigating the clinical utility of the Oncogramme® as a decision support tool for the treatment of patients with metastatic colorectal cancer. If the Oncogramme® positively influenced patient overall survival and/or progression-free survival, it would be of great value for clinicians to implement this assay within the current landscape of personalized medicine tools, which include genomics and biomarker assays. Trial registration: ClinicalTrials.gov identifier NCT03133273. Registered on April 28, 2017. [ABSTRACT FROM AUTHOR]

Virulence gene expression of Yersinia pseudotuberculosis changes during the different stages of infection and this is tightly controlled by environmental cues. In this study, we show that the small protein YmoA, a member of the Hha family, is part of this process. It controls temperature- and nutrient-dependent early and later stage virulence genes in an opposing manner and co-regulates bacterial stress responses and metabolic functions. Our analysis further revealed that YmoA exerts this function by modulating the global post-transcriptional regulatory Csr system. YmoA pre-dominantly enhances the stability of the regulatory RNA CsrC. This involves a stabilizing stem-loop structure within the 5′-region of CsrC. YmoA-mediated CsrC stabilization depends on H-NS, but not on the RNA chaperone Hfq. YmoA-promoted reprogramming of the Csr system has severe consequences for the cell: we found that a mutant deficient of ymoA is strongly reduced in its ability to enter host cells and to disseminate to the Peyer's patches, mesenteric lymph nodes, liver and spleen in mice. We propose a model in which YmoA controls transition from the initial colonization phase in the intestine toward the host defense phase important for the long-term establishment of the infection in underlying tissues. [ABSTRACT FROM AUTHOR]

The formation of biofilms is a survival strategy employed by bacteria to help protect them from changing or unfavourable environments. In this research, 319 genes which govern biofilm formation in V. parahaemolyticus, as reported in 1,625 publications, were analysed using protein-protein-interaction (PPI) network analysis. CsrA was identified as a motility-sessility switch and biofilm formation regulator. Through robust rank aggregation (RRA) analysis of GSE65340, the generation of viable but non-culturable (VBNC) cells that may enhance cell tolerance to stress, was found to be associated with the TCA cycle and carbon metabolism biological pathways. The finding that CsrA is likely to play a role in the development of VBNC cells improves understanding of the molecular mechanisms of VBNC formation in V. parahaemolyticus and contributes to on-going efforts to reduce the hazard posed by this foodborne pathogen. [ABSTRACT FROM AUTHOR]

Three out of the seven ribosomal RNA operons in Escherichia coli end in dual terminator structures. Between the two terminators of each operon is a short sequence that we report here to be an sRNA gene, transcribed as part of the ribosomal RNA primary transcript by read-through of the first terminator. The sRNA genes (rrA, rrB and rrF) from the three operons (rrnA, rrnB and rrnD) are more than 98% identical, and pull-down experiments show that their transcripts interact with Hfq and CsrA. Deletion of rrA, B, F, as well as overexpression of rrB, only modestly affect known CsrA-regulated phenotypes like biofilm formation, pgaA translation and glgC translation, and the role of the sRNAs in vivo may not yet be fully understood. Since RrA, B, F are short-lived and transcribed along with the ribosomal RNA components, their concentration reflect growth-rate regulation at the ribosomal RNA promoters and they could function to fine-tune other growth-phase-dependent processes in the cell. The primary and secondary structure of these small RNAs are conserved among species belonging to different genera of Enterobacteriales.

Three out of the seven ribosomal RNA operons in Escherichia coli end in dual terminator structures. Between the two terminators of each operon is a short sequence that we report here to be an sRNA gene, transcribed as part of the ribosomal RNA primary transcript by read-through of the first terminator. The sRNA genes (rrA , rrB and rrF) from the three operons (rrnA , rrnB and rrnD) are more than 98% identical, and pull-down experiments show that their transcripts interact with Hfq and CsrA. Deletion of rrA, B, F , as well as overexpression of rrB , only modestly affect known CsrA-regulated phenotypes like biofilm formation, pgaA translation and glgC translation, and the role of the sRNAs in vivo may not yet be fully understood. Since RrA, B, F are short-lived and transcribed along with the ribosomal RNA components, their concentration reflect growth-rate regulation at the ribosomal RNA promoters and they could function to fine-tune other growth-phase-dependent processes in the cell. The primary and secondary structure of these small RNAs are conserved among species belonging to different genera of Enterobacteriales. [ABSTRACT FROM AUTHOR]

The post-transcriptional regulator RsmA globally controls gene expression in bacteria. Previous studies showed that RsmA2 and RsmA3 played critical roles in regulating type III secretion system (T3SS), motility, syringafactin, and alginate productions in Pseudomonas syringae pv. tomato strain DC3000 (PstDC3000). In this study, we investigated global gene expression profiles of the wild-type PstDC3000, the rsmA3 mutant, and the rsmA2/A3 double mutant in the hrp-inducing minimum medium (HMM) and King’s B (KB) medium. By comparing the rsmA2/A3 and rsmA3 mutants to PstDC3000, a total of 1358 and 1074 differentially expressed genes (DEGs) in HMM, and 870 and 1463 DEGs in KB were uncovered, respectively. When comparing the rsmA2/A3 mutant with the rsmA3 mutant, 277 and 741 DEGs in HMM and KB, respectively, were revealed. Transcriptomic analysis revealed that the rsmY, rsmZ, and rsmX1-5 non-coding small RNAs (ncsRNAs) were positively affected by RsmA2 and RsmA3, while RsmA3 positively regulates the expression of the rsmA2 gene and negatively regulates both rsmA1 and rsmA5 gene expression. Comparative transcriptomic analysis showed that RsmA2 and RsmA3 synergistically influenced the expression of genes involved in T3SS and alginate biosynthesis in HMM and chemotaxis in KB. RsmA2 and RsmA3 inversely affected genes involved in syringafactin production in HMM and ribosomal protein biosynthesis in KB. In addition, RsmA2 played a major role in influencing genes involved in sarcosine and thiamine biosynthesis in HMM and in mannitol and phosphate metabolism in KB. On the other hand, genes involved in fatty acid metabolism, cellulose biosynthesis, signal transduction, and stress responses were mainly impacted by RsmA3 in both HMM and KB; whereas RsmA3 played a major role in controlling genes involved in c-di-GMP, phosphate metabolism, chemotaxis, and capsular polysaccharide in HMM. Furthermore, regulation of syringafactin production and oxidative stress by RsmA2 and RsmA3 was experimentally verified. Our results suggested the potential interplay among the RsmA proteins, which exhibit distinct and overlapping roles in modulating virulence and survival in P. syringae under different nutritional conditions.

The post-transcriptional regulator RsmA globally controls gene expression in bacteria. Previous studies showed that RsmA2 and RsmA3 played critical roles in regulating type III secretion system (T3SS), motility, syringafactin, and alginate productions in Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000). In this study, we investigated global gene expression profiles of the wild-type Pst DC3000, the rsmA3 mutant, and the rsmA2/A3 double mutant in the hrp -inducing minimum medium (HMM) and King's B (KB) medium. By comparing the rsmA2/A3 and rsmA3 mutants to Pst DC3000, a total of 1358 and 1074 differentially expressed genes (DEGs) in HMM, and 870 and 1463 DEGs in KB were uncovered, respectively. When comparing the rsmA2/A3 mutant with the rsmA3 mutant, 277 and 741 DEGs in HMM and KB, respectively, were revealed. Transcriptomic analysis revealed that the rsmY , rsmZ , and rsmX1-5 non-coding small RNAs (ncsRNAs) were positively affected by RsmA2 and RsmA3, while RsmA3 positively regulates the expression of the rsmA2 gene and negatively regulates both rsmA1 and rsmA5 gene expression. Comparative transcriptomic analysis showed that RsmA2 and RsmA3 synergistically influenced the expression of genes involved in T3SS and alginate biosynthesis in HMM and chemotaxis in KB. RsmA2 and RsmA3 inversely affected genes involved in syringafactin production in HMM and ribosomal protein biosynthesis in KB. In addition, RsmA2 played a major role in influencing genes involved in sarcosine and thiamine biosynthesis in HMM and in mannitol and phosphate metabolism in KB. On the other hand, genes involved in fatty acid metabolism, cellulose biosynthesis, signal transduction, and stress responses were mainly impacted by RsmA3 in both HMM and KB; whereas RsmA3 played a major role in controlling genes involved in c-di-GMP, phosphate metabolism, chemotaxis, and capsular polysaccharide in HMM. Furthermore, regulation of syringafactin production and oxidative stress by RsmA2 and RsmA3 was experimentally verified. Our results suggested the potential interplay among the RsmA proteins, which exhibit distinct and overlapping roles in modulating virulence and survival in P. syringae under different nutritional conditions. [ABSTRACT FROM AUTHOR]

The RNA-binding protein CsrA is a posttranscriptional regulator encoded by genomes throughout the bacterial phylogeny. In the gammaproteobacteria, the activity of CsrA is inhibited by small RNAs that competitively sequester CsrA binding. In contrast, the firmicute Bacillus subtilis encodes a protein inhibitor of CsrA called FliW, which noncompetitively inhibits CsrA activity but for which the precise mechanism of antagonism is unclear. Here, we take an unbiased genetic approach to identify residues of FliW important for CsrA inhibition and these residues fall into two distinct spatial and functional classes. Most loss-of-function alleles mutated FliW residues surrounding the critical regulatory CsrA residue N55 and abolished interaction between the two proteins. Two loss-of-function alleles, however, mutated FliW residues near the CsrA core dimerization domain and maintained interaction with CsrA. One of the FliW alleles reversed a residue charge to disrupt a salt bridge with the CsrA core, and a compensatory charge reversal in the CsrA partner residue restored both the salt bridge and antagonism. We propose a model in which the initial interaction between FliW and CsrA is necessary but not sufficient for antagonism, and for which salt bridge formation with, and deformation of, the CsrA core domain is likely required to allosterically abolish RNA-binding activity. [ABSTRACT FROM AUTHOR]

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

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., Competing Interests: The author declares no conflict of interest.

ABSTRACT The sequence-specific RNA-binding protein CsrA is the central component of the conserved global regulatory Csr system. In Escherichia coli, CsrA regulates many cellular processes, including biofilm formation, motility, carbon metabolism, iron homeostasis, and stress responses. Such regulation often involves translational repression by CsrA binding to an mRNA target, thereby inhibiting ribosome binding. While CsrA also extensively activates gene expression, no detailed mechanism for CsrA-mediated translational activation has been demonstrated. An integrated transcriptomic study identified ymdA as having the strongest CsrA-mediated activation across the E. coli transcriptome. Here, we determined that CsrA activates ymdA expression posttranscriptionally. Gel mobility shift, footprint, toeprint, and in vitro coupled transcription-translation assays identified two CsrA binding sites in the leader region of the ymdA transcript that are critical for translational activation. Reporter fusion assays confirmed that CsrA activates ymdA expression at the posttranscriptional level in vivo. Furthermore, loss of binding at either of the two CsrA binding sites abolished CsrA-dependent activation. mRNA half-life studies revealed that CsrA also contributes to stabilization of ymdA mRNA. RNA structure prediction revealed an RNA hairpin upstream of the ymdA start codon that sequesters the Shine-Dalgarno (SD) sequence, which would inhibit ribosome binding. This hairpin also contains one of the two critical CsrA binding sites, with the other site located just upstream. Our results demonstrate that bound CsrA destabilizes the SD-sequestering hairpin such that the ribosome can bind and initiate translation. Since YmdA represses biofilm formation, CsrA-mediated activation of ymdA expression may repress biofilm formation under certain conditions. IMPORTANCE The Csr system of E. coli controls gene expression and physiology on a global scale. CsrA protein, the central component of this system, represses translation initiation of numerous genes by binding to target transcripts, thereby competing with ribosome binding. Variations of this mechanism are so common that CsrA is sometimes called a translational repressor. Although CsrA-mediated activation mechanisms have been elucidated in which bound CsrA inhibits RNA degradation, no translation activation mechanism has been defined. Here, we demonstrate that CsrA binding to two sites in the 5′ untranslated leader of ymdA mRNA activates translation by destabilizing a structure that otherwise prevents ribosome binding. The extensive role of CsrA in activating gene expression suggests the common occurrence of similar activation mechanisms.

Gene expression is adjusted according to cellular needs through a combination of mechanisms acting at different layers of the flow of genetic information. At the posttranscriptional level, RNA-binding proteins are key factors controlling the fate of nascent and mature mRNAs. Among them, the members of the CsrA family are small dimeric proteins with heterogeneous distribution across the bacterial tree of life, that act as global regulators of gene expression because they recognize characteristic sequence/structural motifs (short hairpins with GGA triplets in the loop) present in hundreds of mRNAs. The regulatory output of CsrA binding to mRNAs is counteracted in most cases by molecular mimic, non-protein coding RNAs that titrate the CsrA dimers away from the target mRNAs. In γ-proteobacteria, the regulatory modules composed by CsrA homologs and the corresponding antagonistic sRNAs, are mastered by two-component systems of the GacS-GacA type, which control the transcription and the abundance of the sRNAs, thus constituting the rather linear cascade Gac-Rsm that responds to environmental or cellular signals to adjust and coordinate the expression of a set of target genes posttranscriptionally. Within the γ-proteobacteria, the genus Pseudomonas has been shown to contain species with different number of active CsrA (RsmA) homologs and of molecular mimic sRNAs. Here, with the help of the increasing availability of genomic data we provide a comprehensive state-of-the-art picture of the remarkable multiplicity of CsrA lineages, including novel yet uncharacterized paralogues, and discuss evolutionary aspects of the CsrA subfamilies of the genus Pseudomonas, and implications of the striking presence of csrA alleles in natural mobile genetic elements (phages and plasmids).

Small non-coding RNAs (sRNAs) are critical post-transcriptional regulators of gene expression. Distinct RNA-binding proteins (RBPs) influence the processing, stability and activity of bacterial small RNAs. The vast majority of bacterial sRNAs interact with mRNA targets, affecting mRNA stability and/or its translation rate. The assistance of RNA-binding proteins facilitates and brings accuracy to sRNA-mRNA basepairing and the RNA chaperones Hfq and ProQ are now recognized as the most prominent RNA matchmakers in bacteria. These RBPs exhibit distinct high affinity RNA-binding surfaces, promoting RNA strand interaction between a trans-encoding sRNA and its mRNA target. Nevertheless, some organisms lack ProQ and/or Hfq homologs, suggesting the existence of other RBPs involved in sRNA function. Along this line of thought, the global regulator CsrA was recently shown to facilitate the access of an sRNA to its target mRNA and may represent an additional factor involved in sRNA function. Ribonucleases (RNases) can be considered a class of RNA-binding proteins with nucleolytic activity that are responsible for RNA maturation and/or degradation. Presently RNase E, RNase III, and PNPase appear to be the main players not only in sRNA turnover but also in sRNA processing. Here we review the current knowledge on the most important bacterial RNA-binding proteins affecting sRNA activity and sRNA-mediated networks.

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.

Legionella pneumophila, an environmental bacterium that parasitizes protozoa, is the causative pathogen of Legionnaires' disease. L. pneumophila adopts a distinct biphasic life cycle that allows it to adapt to environmental conditions for survival, replication, and transmission. This cycle consists of a non-virulent replicative phase (RP) and a virulent transmissive phase (TP). Timely and fine-tuned expression of growth and virulence factors in a life cycle-dependent manner is crucial. Herein, we report evidence that CsrA, a key regulator of the switch between the RP and the TP, is dually regulated in a ClpP-dependent manner during the biphasic life cycle of L. pneumophila. First, we show that the protein level of CsrA is temporal during the life cycle and is degraded by ClpP during the TP. The ectopic expression of CsrA in a ΔclpP mutant, but not in the wild type, inhibits both the initiation of the RP in vitro and the invasiveness to Acanthamoeba castellanii, indicating that the ClpP-mediated proteolytic pathway regulates the CsrA protein level. We further show that the temporally expressed IHFB is the transcriptional inhibitor of csrA and is degraded via a ClpP-dependent manner during the RP. During the RP, the level of CsrA is increased by promoting the degradation of IHFB and reducing the degradation of the accumulated CsrA via a ClpP-dependent manner. During the TP, the level of CsrA is decreased by inhibiting the degradation of IHFB and promoting the degradation of the accumulated CsrA via a ClpP-dependent manner as well. In conclusion, our results show that the growth-stage-specific expression level of CsrA is dually regulated by ClpP-dependent proteolysis at both the transcription and protein levels during the biphasic life cycle of L. pneumophila.

ABSTRACT The transition from a planktonic lifestyle to a host-attached state is often critical for bacterial virulence. Upon attachment to host cells, enteropathogenic Escherichia coli (EPEC) employs a type III secretion system (T3SS) to inject into the host cells ∼20 effector proteins, including Tir. CesT, which is encoded from the same operon downstream of tir, is a Tir-bound chaperone that facilitates Tir translocation. Upon Tir translocation, the liberated CesT remains in the bacterial cytoplasm and antagonizes the posttranscriptional regulator CsrA, thus eliciting global regulation in the infecting pathogen. Importantly, tight control of the Tir/CesT ratio is vital, since an uncontrolled surge in free CesT levels may repress CsrA in an untimely manner, thus abrogating colonization. We investigated how fluctuations in Tir translation affect the regulation of this ratio. By creating mutations that cause the premature termination of Tir translation, we revealed that the untranslated tir mRNA becomes highly unstable, resulting in a rapid drop in cesT mRNA levels and, thus, CesT levels. This mechanism couples Tir and CesT levels to ensure a stable Tir/CesT ratio. Our results expose an additional level of regulation that enhances the efficacy of the initial interaction of EPEC with the host cell, providing a better understanding of the bacterial switch from the planktonic to the cell-adherent lifestyle. IMPORTANCE Host colonization by extracellular pathogens often entails the transition from a planktonic lifestyle to a host-attached state. Enteropathogenic E. coli (EPEC), a Gram-negative pathogen, attaches to the intestinal epithelium of the host and employs a type III secretion system (T3SS) to inject effector proteins into the cytoplasm of infected cells. The most abundant effector protein injected is Tir, whose translocation is dependent on the Tir-bound chaperon CesT. Upon Tir injection, the liberated CesT binds to and inhibits the posttranscriptional regulator CsrA, resulting in reprogramming of gene expression in the host-attached bacteria. Thus, adaptation to the host-attached state involves dynamic remodeling of EPEC gene expression, which is mediated by the relative levels of Tir and CesT. Fluctuating from the optimal Tir/CesT ratio results in a decrease in EPEC virulence. Here we elucidate a posttranscriptional circuit that prevents sharp variations from this ratio, thus improving host colonization.

ABSTRACT The global regulatory protein CsrA coordinates gene expression in response to physiological cues reflecting cellular stress and nutrition. CsrA binding to the 5′ segments of mRNA targets affects their translation, RNA stability, and/or transcript elongation. Recent studies identified probable mRNA targets of CsrA that are involved in iron uptake and storage in Escherichia coli, suggesting an unexplored role for CsrA in regulating iron homeostasis. Here, we assessed the impact of CsrA on iron-related gene expression, cellular iron, and growth under various iron levels. We investigated five new targets of CsrA regulation, including the genes for 4 ferritin or ferritin-like iron storage proteins (ISPs) and the stress-inducible Fe-S repair protein, SufA. CsrA bound with high affinity and specificity to ftnB, bfr, and dps mRNAs and inhibited their translation, while it modestly activated ftnA expression. Furthermore, CsrA was found to regulate cellular iron levels and support growth by repressing the expression of genes for ISPs, most importantly, ferritin B (FtnB) and bacterioferritin (Bfr). Iron starvation did not substantially affect cellular levels of CsrA or its small RNA (sRNA) antagonists, CsrB and CsrC. csrA disruption led to increased resistance to the lethal effects of H2O2 during exponential growth, consistent with a regulatory role in oxidative stress resistance. We propose that during exponential growth and under minimal stress, CsrA represses the deleterious expression of the ISPs that function under oxidative stress and stationary-phase conditions (FtnB, Bfr, and Dps), thus ensuring that cellular iron is available to processes that are required for growth. IMPORTANCE Iron is an essential micronutrient for nearly all living organisms but is toxic in excess. Consequently, the maintenance of iron homeostasis is a critical biological process, and the genes involved in this function are tightly regulated. Here, we explored a new role for the bacterial RNA binding protein CsrA in the regulation of iron homeostasis. CsrA was shown to be a key regulator of iron storage genes in Escherichia coli, with consequential effects on cellular iron levels and growth. Our findings establish a model in which robust CsrA activity during the exponential phase of growth leads to repression of genes whose products sequester iron or divert it to unnecessary stress response processes. In so doing, CsrA supports E. coli growth under iron-limiting laboratory conditions and may promote fitness in the competitive iron-limited environment of the host large intestine.

ABSTRACT CsrA, an RNA-binding global regulator, is an essential protein in Vibrio cholerae. V. cholerae CsrA is regulated by three small RNAs (sRNAs), namely, CsrB, CsrC, and CsrD, which act to sequester and antagonize the activity of CsrA. Although the sRNAs were considered to be largely redundant, we found that they differ in expression, half-life, and the ability to regulate CsrA. Further, we identified a feedback loop in the Csr system in which CsrA increases the synthesis of these antagonistic sRNAs. Because the Csr sRNAs are positively regulated by VarA, we determined the effects of CsrA on VarA levels. The level of VarA was reduced in a csrA mutant, and we found that CsrA directly bound to varA mRNA in an electrophoretic mobility shift assay in vitro and in an CsrA-RNA immunoprecipitation assay in vivo. Thus, varA mRNA is an in vivo-verified direct target of CsrA in V. cholerae, and this is the first demonstration of CsrA directly binding to a varA/uvrY/gacA homolog. Additionally, we demonstrated that a varA translational fusion was less active in a csrA mutant than in wild-type V. cholerae, suggesting that CsrA enhances varA translation. We propose that this autoregulatory feedback loop, in which CsrA increases the production of the nonredundant Csr sRNAs by regulating the amount of VarA, provides a mechanism for fine-tuning the availability of CsrA and, thus, of its downstream targets. IMPORTANCE Vibrio cholerae is a major human pathogen, causing epidemics and pandemics of cholera. V. cholerae persists in the aquatic environment, providing a constant source for human infection. Success in transitioning from the environment to the human host and back requires the bacterium to rapidly respond and to adjust its gene expression and metabolism to these two very different habitats. Our findings show that CsrA, an RNA-binding regulatory protein, plays a central role in regulating these transitions. CsrA activity is controlled by the antagonistic sRNAs CsrB, CsrC, and CsrD, and these sRNAs respond to changes in the availability of nutrients. CsrA autoregulates its own activity by controlling these sRNAs via their primary regulator VarA. Thus, the change in CsrA availability in response to nutrient availability allows V. cholerae to alter gene expression in response to environmental cues.

Accurate and efficient transcription termination is an important step for cells to generate functional RNA transcripts. In bacteria, two mechanisms are responsible for terminating transcription: intrinsic (Rho-independent) termination and Rho-dependent termination. Growing examples suggest that neither type of transcription termination is static, but instead are highly dynamic and regulated. Regulatory small RNAs (sRNAs) are key players in bacterial stress responses, are frequently expressed under specific growth conditions, and are predominantly terminated through the intrinsic termination mechanism. Once made, sRNAs can base-pair with mRNA targets and regulate mRNA translation and stability. Recent findings suggest that alterations in the efficiency of intrinsic termination for sRNAs under various growth conditions may affect the availability of a given sRNA and the ability of the sRNA to function properly. Moreover, alterations of mRNA structure, translation, and accessibility by sRNAs have the potential to impact the access of Rho factor to mRNAs and thus termination of the mRNA. Indeed, recent studies have revealed that some sRNAs can modulate target gene expression by stimulating or inhibiting Rho-dependent termination, thus expanding the regulatory power of bacterial sRNAs. Here we review the current knowledge on intrinsic termination of sRNAs and sRNA-mediated Rho-dependent termination of protein coding genes in bacteria.

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.

Escherichia coli C is a strong biofilm producer in comparison to E. coli K-12 laboratory strains due to higher expression of the pgaABCD operon encoding the enzymes for the biosynthesis of the extracellular polysaccharide poly-β-1,6-N-acetylglucosamine (PNAG). The pgaABCD operon is negatively regulated at the post-transcriptional level by two factors, namely CsrA, a conserved RNA-binding protein controlling multiple pathways, and the RNA exonuclease polynucleotide phosphorylase (PNPase). In this work, we investigated the molecular bases of different PNAG production in C-1a and MG1655 strains taken as representative of E. coli C and K-12 strains, respectively. We found that pgaABCD operon expression is significantly lower in MG1655 than in C-1a; consistently, CsrA protein levels were much higher in MG1655. In contrast, we show that the negative effect exerted by PNPase on pgaABCD expression is much stronger in C-1a than in MG1655. The amount of CsrA and of the small RNAs CsrB, CsrC, and McaS sRNAs regulating CsrA activity is dramatically different in the two strains, whereas PNPase level is similar. Finally, the compensatory regulation acting between CsrB and CsrC in MG1655 does not occur in E. coli C. Our results suggest that PNPase preserves CsrA-dependent regulation by indirectly modulating csrA expression.

Increasing demands for small-scale radiocarbon (14C) analyses required the installation of a "SO-110 B" type ion source (HVE Europa B.V.) at our 6 MV Tandetron AMS (HVE) dedicated for the direct injection of CO2 using either the gas injection system (GIS) from Ionplus AG or a EuroVector EA 3000 elemental analyzer (EA). We tested both systems with multiple series of 14C-free and modern standards (2.5–50 µg C) combusted in quartz ampoules or EA containers and were able to quantify exogenous C introduced. In EA-GIS-AMS analysis exogenous C is mainly derived from the EA sample containers. Blank values for 50 µg C combusted in solvent-cleaned tin (Sn) vessels were 0.0127 ± 0.0012 F14C (boats) and 0.0090 ± 0.0010 F14C (capsules), while they were much higher for thermally cleaned silver (Ag) capsules. The processing of gas samples for GIS-AMS yields similar blank values corresponding to 0.30 ± 0.08 µg exogenous C with 0.93 ± 0.23 F14C consisting of 0.28 µg C modern and 0.02 µg C fossil C. The combustion of larger amounts of blank material (1 mg C) in a single quartz tube split into aliquots gives lower blanks (0.0064 ± 0.0008 F14C; 50 µg C). Thus, 14C analysis of small, gaseous samples is now possible at CologneAMS. [ABSTRACT FROM AUTHOR]

Legionella pneumophila , an environmental bacterium that parasitizes protozoa, is the causative pathogen of Legionnaires' disease. L. pneumophila adopts a distinct biphasic life cycle that allows it to adapt to environmental conditions for survival, replication, and transmission. This cycle consists of a non-virulent replicative phase (RP) and a virulent transmissive phase (TP). Timely and fine-tuned expression of growth and virulence factors in a life cycle-dependent manner is crucial. Herein, we report evidence that CsrA, a key regulator of the switch between the RP and the TP, is dually regulated in a ClpP-dependent manner during the biphasic life cycle of L. pneumophila. First, we show that the protein level of CsrA is temporal during the life cycle and is degraded by ClpP during the TP. The ectopic expression of CsrA in a Δ clpP mutant, but not in the wild type, inhibits both the initiation of the RP in vitro and the invasiveness to Acanthamoeba castellanii , indicating that the ClpP-mediated proteolytic pathway regulates the CsrA protein level. We further show that the temporally expressed IHFB is the transcriptional inhibitor of csrA and is degraded via a ClpP-dependent manner during the RP. During the RP, the level of CsrA is increased by promoting the degradation of IHFB and reducing the degradation of the accumulated CsrA via a ClpP-dependent manner. During the TP, the level of CsrA is decreased by inhibiting the degradation of IHFB and promoting the degradation of the accumulated CsrA via a ClpP-dependent manner as well. In conclusion, our results show that the growth-stage-specific expression level of CsrA is dually regulated by ClpP-dependent proteolysis at both the transcription and protein levels during the biphasic life cycle of L. pneumophila. [ABSTRACT FROM AUTHOR]

Summary: Pseudomonas syringae pv. tomato DC3000 (PstDC3000) contains five RsmA protein homologues. In this study, four were functionally characterized, with a focus on RsmA2, RsmA3 and RsmA4. RNA electrophoretic mobility shift assays demonstrated that RsmA1 and RsmA4 exhibited similar low binding affinities to non‐coding small RNAs (ncsRNAs), whereas RsmA2 and RsmA3 exhibited similar, but much higher, binding affinities to ncsRNAs. Our results showed that both RsmA2 and RsmA3 were required for disease symptom development and bacterial growth in planta by significantly affecting virulence gene expression. All four RsmA proteins, especially RsmA2 and RsmA3, influenced γ‐amino butyric acid utilization and pyoverdine production to some degree, whereas RsmA2, RsmA3 and RsmA4 influenced protease activities. A single RsmA, RsmA3, played a dominant role in regulating motility. Furthermore, reverse transcription quantitative real‐time PCR and western blot results showed that RsmA proteins, especially RsmA2 and RsmA3, regulated target genes and possibly other RsmA proteins at both transcriptional and translational levels. These results indicate that RsmA proteins in PstDC3000 exhibit distinct binding affinities to ncsRNAs and have distinct roles in virulence. Our results also suggest that RsmA proteins in PstDC3000 interact with each other, where RsmA2 and RsmA3 play a major role in regulating various functions in a complex manner. [ABSTRACT FROM AUTHOR]

This paper presents Base Cube One (BCO), an architecture for smart environments that organizes devices and functionality in a location-addressable service-oriented schema so that the complex interplay between sensors, actuators and intermediary computing modules can be organized and set up in a structured and effective manner. We show how this can be used by smart components to adapt their generic behavior in order to control individual smart environments such as smart homes with heterogeneous hard- and software components. Additionally, a common ground between smart components and occupants is addressed by sharing the same spatial organization of smart things within the environment. This acknowledges the fast pace at which smart environments are evolving and our approach aims to provide a solid basis for later iterative refinements as new devices are included or removed, or the inter-connectivity is structurally changed. We will present the new architecture in detail and exemplify it using our Cognitive Service-Robotics Apartment project in the context of which this architecture has been developed. The discussion will touch in particular on the requirements for an architecture to both provide a stable foundation for the smart environment yet offer a plasticity for ongoing iterative refinement and supporting the overall system adaptation to the resulting changes. [ABSTRACT FROM AUTHOR]