Vibrio fischeri Biofilm Formation Prevented by a Trio of Regulators.

Biofilms, complex communities of microorganisms surrounded by a self-produced matrix, facilitate attachment and provide protection to bacteria. A natural model used to study biofilm formation is the symbiosis between Vibrio fischeri and its host, the Hawaiian bobtail squid, Euprymna scolopes Host-relevant biofilm formation is a tightly regulated process and is observed in vitro only with strains that have been genetically manipulated to overexpress or disrupt specific regulators, primarily two-component signaling (TCS) regulators. These regulators control biofilm formation by dictating the production of the symbiosis polysaccharide (Syp-PS), the major component of the biofilm matrix. Control occurs both at and below the level of transcription of the syp genes, which are responsible for Syp-PS production. Here, we probed the roles of the two known negative regulators of biofilm formation, BinK and SypE, by generating double mutants. We also mapped and evaluated a point mutation using natural transformation and linkage analysis. We examined traditional biofilm formation phenotypes and established a new assay for evaluating the start of biofilm formation in the form of microscopic aggregates in shaking liquid cultures, in the absence of the known biofilm-inducing signal calcium. We found that wrinkled colony formation is negatively controlled not only by BinK and SypE but also by SypF. SypF is both required for and inhibitory to biofilm formation. Together, these data reveal that these three regulators are sufficient to prevent wild-type V. fischeri from forming biofilms under these conditions.IMPORTANCE Bacterial biofilms promote attachment to a variety of surfaces and protect the constituent bacteria from environmental stresses, including antimicrobials. Understanding the mechanisms by which biofilms form will promote our ability to resolve them when they occur in the context of an infection. In this study, we found that Vibrio fischeri tightly controls biofilm formation using three negative regulators; the presence of a single one of these regulators was sufficient to prevent full biofilm development, while disruption of all three permitted robust biofilm formation. This work increases our understanding of the functions of specific regulators and demonstrates the substantial negative control that one benign microbe exerts over biofilm formation, potentially to ensure that it occurs only under the appropriate conditions.

Genome-wide analysis of small RNAs expressed by Yersinia pestis identifies a regulator of the Yop-Ysc type III secretion system.

Small noncoding RNA (sRNA) molecules are integral components of the regulatory machinery for many bacterial species and are known to posttranscriptionally regulate metabolic and stress-response pathways, quorum sensing, virulence factors, and more. The Yop-Ysc type III secretion system (T3SS) is a critical virulence component for the pathogenic Yersinia species, and the regulation of this system is tightly controlled at each step from transcription to translocation of effectors into host cells. The contribution of sRNAs to the regulation of the T3SS in Yersinia has been largely unstudied, however. Previously, our lab identified a role for the sRNA chaperone protein Hfq in the regulation of components of the T3SS in the gastrointestinal pathogen Yersinia pseudotuberculosis. Here we present data demonstrating a similar requirement for Hfq in the closely related species Yersinia pestis. Through deep sequencing analysis of the Y. pestis sRNA-ome, we found 63 previously unidentified putative sRNAs in this species. We identified a Yersinia-specific sRNA, Ysr141, carried by the T3SS plasmid pCD1 that is required for the production of multiple T3SS proteins. In addition, we show that Ysr141 targets an untranslated region upstream of yopJ to posttranscriptionally activate the synthesis of the YopJ protein. Furthermore, Ysr141 may be an unstable and/or processed sRNA, which could contribute to its function in the regulation of the T3SS. The discovery of an sRNA that influences the synthesis of the T3SS adds an additional layer of regulation to this tightly controlled virulence determinant of Y. pestis.

Global discovery of small RNAs in Yersinia pseudotuberculosis identifies Yersinia-specific small, noncoding RNAs required for virulence.

A major class of bacterial small, noncoding RNAs (sRNAs) acts by base-pairing with mRNAs to alter the translation from and/or stability of the transcript. Our laboratory has shown that Hfq, the chaperone that mediates the interaction of many sRNAs with their targets, is required for the virulence of the enteropathogen Yersinia pseudotuberculosis. This finding suggests that sRNAs play a critical role in the regulation of virulence in this pathogen, but these sRNAs are not known. Using a deep sequencing approach, we identified the global set of sRNAs expressed in vitro by Y. pseudotuberculosis. Sequencing of RNA libraries from bacteria grown at 26 °C and 37 °C resulted in the identification of 150 unannotated sRNAs. The majority of these sRNAs are Yersinia specific, without orthologs in either Escherichia coli or Salmonella typhimurium. Six sRNAs are Y. pseudotuberculosis specific and are absent from the genome of the closely related species Yersinia pestis. We found that the expression of many sRNAs conserved between Y. pseudotuberculosis and Y. pestis differs in both timing and dependence on Hfq, suggesting evolutionary changes in posttranscriptional regulation between these species. Deletion of multiple sRNAs in Y. pseudotuberculosis leads to attenuation of the pathogen in a mouse model of yersiniosis, as does the inactivation in Y. pestis of a conserved, Yersinia-specific sRNA in a mouse model of pneumonic plague. Finally, we determined the regulon controlled by one of these sRNAs, revealing potential virulence determinants in Y. pseudotuberculosis that are regulated in a posttranscriptional manner.

Correction: Improved Control of Tuberculosis and Activation of Macrophages in Mice Lacking Protein Kinase R.

[This corrects the article DOI: 10.1371/journal.pone.0030512.].

Posttranscriptional regulation of the Yersinia pestis cyclic AMP receptor protein Crp and impact on virulence.

UNLABELLED: The cyclic AMP receptor protein (Crp) is a transcriptional regulator that controls the expression of numerous bacterial genes, usually in response to environmental conditions and particularly by sensing the availability of carbon. In the plague pathogen Yersinia pestis, Crp regulates the expression of multiple virulence factors, including components of the type III secretion system and the plasminogen activator protease Pla. The regulation of Crp itself, however, is distinctly different from that found in the well-studied Escherichia coli system. Here, we show that at physiological temperatures, the synthesis of Crp in Y. pestis is positively regulated at the posttranscriptional level. The loss of the small RNA chaperone Hfq results in decreased Crp protein levels but not in steady-state Crp transcript levels, and this regulatory effect occurs within the 5' untranslated region (UTR) of the Crp mRNA. The posttranscriptional activation of Crp synthesis is required for the expression of pla, and decoupling crp from Hfq through the use of an exogenously controlled promoter and 5' UTR increases Pla protein levels as well as partially rescues the growth defect associated with the loss of Hfq. Finally, we show that both Hfq and the posttranscriptional regulation of Crp contribute to the virulence of Y. pestis during pneumonic plague. The Hfq-dependent, posttranscriptional regulation of Crp may be specific to Yersinia species, and thus our data help explain the dramatic growth and virulence defects associated with the loss of Hfq in Y. pestis. IMPORTANCE: The Crp protein is a major transcriptional regulator in bacteria, and its synthesis is tightly controlled to avoid inappropriate induction of the Crp regulon. In this report, we provide the first evidence of Crp regulation in an Hfq-dependent manner at the posttranscriptional level. Our discovery that the synthesis of Crp in Yersinia pestis is Hfq dependent adds an additional layer of regulation to catabolite repression in this bacterium. Our work provides a mechanism by which the plague pathogen links not just the sensing of glucose or other carbon sources but also other signals that influence Crp abundance via the expression of small RNAs to the induction of the Crp regulon. In turn, this allows Y. pestis to fine-tune Crp levels to optimize virulence gene expression during plague infection and may allow the bacterium to adapt to its unique environmental niches.

Improved control of tuberculosis and activation of macrophages in mice lacking protein kinase R.

Host factors that microbial pathogens exploit for their propagation are potential targets for therapeuic countermeasures. No host enzyme has been identified whose genetic absence benefits the intact mammalian host in vivo during infection with Mycobacterium tuberculosis (Mtb), the leading cause of death from bacterial infection. Here, we report that the dsRNA-dependent protein kinase (PKR) is such an enzyme. PKR-deficient mice contained fewer viable Mtb and showed less pulmonary pathology than wild type mice. We identified two potential mechanisms for the protective effect of PKR deficiency: increased apoptosis of macrophages in response to Mtb and enhanced activation of macrophages in response to IFN-gamma. The restraining effect of PKR on macrophage activation was explained by its mediation of a previously unrecognized ability of IFN-gamma to induce low levels of the macrophage deactivating factor interleukin 10 (IL10). These observations suggest that PKR inhibitors may prove useful as an adjunctive treatment for tuberculosis.

Induction of ER stress in macrophages of tuberculosis granulomas.

BACKGROUND: The endoplasmic reticulum (ER) stress pathway known as the Unfolded Protein Response (UPR) is an adaptive survival pathway that protects cells from the buildup of misfolded proteins, but under certain circumstances it can lead to apoptosis. ER stress has been causally associated with macrophage apoptosis in advanced atherosclerosis of mice and humans. Because atherosclerosis shares certain features with tuberculosis (TB) with regard to lesional macrophage accumulation, foam cell formation, and apoptosis, we investigated if the ER stress pathway is activated during TB infection. PRINCIPAL FINDINGS: Here we show that ER stress markers such as C/EBP homologous protein (CHOP; also known as GADD153), phosphorylated inositol-requiring enzyme 1 alpha (Ire1α) and eukaryotic initiation factor 2 alpha (eIF2α), and activating transcription factor 3 (ATF3) are expressed in macrophage-rich areas of granulomas in lungs of mice infected with virulent Mycobacterium tuberculosis (Mtb). These areas were also positive for numerous apoptotic cells as assayed by TUNEL. Microarray analysis of human caseous TB granulomas isolated by laser capture microdissection reveal that 73% of genes involved in the UPR are upregulated at the mRNA transcript level. The expression of two ER stress markers, ATF3 and CHOP, were also increased in macrophages of human TB granulomas when assayed by immunohistochemistry. CHOP has been causally associated with ER stress-induced macrophage apoptosis. We found that apoptosis was more abundant in granulomas as compared to non-granulomatous tissue isolated from patients with pulmonary TB, and apoptosis correlated with CHOP expression in areas surrounding the centralized areas of caseation. CONCLUSIONS: In summary, ER stress is induced in macrophages of TB granulomas in areas where apoptotic cells accumulate in mice and humans. Although macrophage apoptosis is generally thought to be beneficial in initially protecting the host from Mtb infection, death of infected macrophages in advanced granulomas might favor dissemination of the bacteria. Therefore future work is needed to determine if ER-stress is causative for apoptosis and plays a role in the host response to infection.

HrpA, a DEAH-box RNA helicase, is involved in mRNA processing of a fimbrial operon in Escherichia coli.

Endonucleolytic cleavage of mRNA in the daa operon of Escherichia coli is responsible for co-ordinate regulation of genes involved in F1845 fimbrial biogenesis. Cleavage occurs by an unidentified endoribonuclease, is translation dependent and involves a unique recognition mechanism. Here, we present the results of a genetic strategy used to identify factors involved in daa mRNA processing. We used a reporter construct consisting of the daa mRNA processing region fused to the gene encoding green fluorescent protein (GFP). A mutant defective in daa mRNA processing and expressing high levels of GFP was isolated by flow cytometry. To determine the location of mutations, two different genetic approaches, Hfr crosses and P1 transductions, were used. The mutation responsible for the processing defect was subsequently mapped to the 32 min region of the E. coli chromosome. A putative DEAH-box RNA helicase-encoding gene at this position, hrpA, was able to restore the ability of the mutant to cleave daa mRNA. Site-directed mutagenesis of the hrpA regions predicted to encode nucleotide triphosphate binding and hydrolysis functions abolished the ability of the gene to restore the processing defect in the mutant. We propose that HrpA is a novel enzyme involved in mRNA processing in E. coli.