The rpoS mRNA leader recruits Hfq to facilitate annealing with DsrA sRNA

Small noncoding RNAs (sRNAs) regulate the response of bacteria to environmental stress in conjunction with the Sm-like RNA binding protein Hfq. DsrA sRNA stimulates translation of the RpoS stress response factor in Escherichia coli by base-pairing with the 5′ leader of the rpoS mRNA and opening a stem–loop that represses translation initiation. We report that rpoS leader sequences upstream of this stem–loop greatly increase the sensitivity of rpoS mRNA to Hfq and DsrA. Native gel mobility shift assays show that Hfq increases the rate of DsrA binding to the full 576 nt rpoS leader as much as 50-fold. By contrast, base-pairing with a 138-nt RNA containing just the repressor stem–loop is accelerated only twofold. Deletion and mutagenesis experiments showed that sensitivity to Hfq requires an upstream AAYAA sequence. Leaders long enough to contain this sequence bind Hfq tightly and form stable ternary complexes with Hfq and DsrA. A model is proposed in which Hfq recruits DsrA to the rpoS mRNA by binding both RNAs, releasing the self-repressing structure in the mRNA. Once base-pairing between DsrA and rpoS mRNA is established, interactions between Hfq and the mRNA may stabilize the RNA complex by removing Hfq from the sRNA.     

A new Vibrio cholerae sRNA modulates colonization and affects release of outer membrane vesicles

We discovered a new small non-coding RNA (sRNA) gene, vrrA of Vibrio cholerae O1 strain A1552. A vrrA mutant overproduces OmpA porin, and we demonstrate that the 140 nt VrrA RNA represses ompA translation by base-pairing with the 5' region of the mRNA. The RNA chaperone Hfq is not stringently required for VrrA action, but expression of the vrrA gene requires the membrane stress sigma factor, sigma(E), suggesting that VrrA acts on ompA in response to periplasmic protein folding stress. We also observed that OmpA levels inversely correlated with the number of outer membrane vesicles (OMVs), and that VrrA increased OMV production comparable to loss of OmpA. VrrA is the first sRNA known to control OMV formation. Moreover, a vrrA mutant showed a fivefold increased ability to colonize the intestines of infant mice as compared with the wild type. There was increased expression of the main colonization factor of V. cholerae, the toxin co-regulated pili, in the vrrA mutant as monitored by immunoblot detection of the TcpA protein. VrrA overproduction caused a distinct reduction in the TcpA protein level. Our findings suggest that VrrA contributes to bacterial fitness in certain stressful environments, and modulates infection of the host intestinal tract.     

Relationship between structure and antigenicity of O1 Vibrio cholerae lipopolysaccharides

The relationship between the release of fructose from O1 Vibrio cholerae lipopolysaccharides (LPS) by dilute acetic acid hydrolysis and the decrease in their antigenicity was examined. Decrease in the antigenicity of LPS was not parallel with the release of fructose, and occurred very much later than the latter. Periodate oxidation of LPS resulted in the total elimination of the fructose and glucose, and two-thirds of the heptose constituents, but no difference in the antigenicity of LPS was observed before and after oxidation. These findings indicate that the fructose present in O1 V. cholerae LPS is not substantially involved in their specific antigenicity. In the O1 V. cholerae LPS, the fructose is in the branch structure, most probably in the core region.     

A serogroup of non-O1 Vibrio cholerae possessing the Inaba antigen of Vibrio cholerae O1

A serogroup of non-O1 Vibrio cholerae, tentatively named Hakata, possessing the C (Inaba) factor but not the B (Ogawa) and A factors of V. cholerae O1 is described. Strains of this serogroup were isolated from river and estuarine waters and from frozen shrimps.     

A serogroup of non-O1 Vibrio cholerae possessing the Inaba antigen of Vibrio cholerae O1

A serogroup of non-O1 Vibrio cholerae , tentatively named Hakata, possessing the C (Inaba) factor but not the B (Ogawa) and A factors of V. cholerae O1 is described. Strains of this serogroup were isolated from river and estuarine waters and from frozen shrimps.     

The Zymovars of Vibrio cholerae: multilocus enzyme electrophoresis of Vibrio cholerae

Zymovars analysis also known as multilocus enzyme electrophoresis is applied here to investigate the genetic variation of Vibrio cholerae strains and characterise strains or group of strains of medical and epidemiological interest. Fourteen loci were analyzed in 171 strains of non-O1 non-O139, 32 classical and 61 El Tor from America, Africa, Europe and Asia. The mean genetic diversity was 0.339. It is shown that the same O antigen (both O1 and non-O1) may be present in several genetically diverse (different zymovars) strains. Conversely the same zymovar may contain more than one serogroup. It is confirmed that the South American epidemic strain differs from the 7th pandemic El Tor strain in locus LAP (leucyl leucyl aminopeptidase). Here it is shown that this rare allele is present in 1 V. mimicus and 4 non-O1 V. cholerae. Non toxigenic O1 strains from South India epidemic share zymovar 14A with the epidemic El Tor from the 7th pandemic, while another group have diverse zymovars. The sucrose negative epidemic strains isolated in French Guiana and Brazil have the same zymovar of the current American epidemic V. cholerae.     

Dynamic Refolding of OxyS sRNA by the Hfq RNA Chaperone

The Sm protein Hfq chaperones small non-coding RNAs (sRNAs) in bacteria, facilitating sRNA regulation of target mRNAs. Hfq acts in part by remodeling the sRNA and mRNA structures, yet the basis for this remodeling activity is not understood. To understand how Hfq remodels RNA, we used single-molecule Förster resonance energy transfer (smFRET) to monitor conformational changes in OxyS sRNA upon Hfq binding. The results show that E. coli Hfq first compacts OxyS, bringing its 5′ and 3 ends together. Next, Hfq destabilizes an internal stem-loop in OxyS, allowing the RNA to adopt a more open conformation that is stabilized by a conserved arginine on the rim of Hfq. The frequency of transitions between compact and open conformations depend on interactions with Hfqs flexible C-terminal domain (CTD), being more rapid when the CTD is deleted, and slower when OxyS is bound to Caulobacter crescentus Hfq, which has a shorter and more stable CTD than E. coli Hfq. We propose that the CTDs gate transitions between OxyS conformations that are stabilized by interaction with one or more arginines. These results suggest a general model for how basic residues and intrinsically disordered regions of RNA chaperones act together to refold RNA.     

A cis-encoded sRNA,Hfq and mRNA secondary structure act independently to suppress IS200 transposition

IS200 is found throughout Enterobacteriaceae and transposes at a notoriously low frequency. In addition to the transposase protein (TnpA), IS200 encodes an uncharacterized Hfq-binding sRNA that is encoded opposite to the tnpA 5''UTR. In the current work we asked if this sRNA represses tnpA expression. We show here that the IS200 sRNA (named art200 for antisense regulator of transposase IS200) basepairs with tnpA to inhibit translation initiation. Unexpectedly, art200-tnpA pairing is limited to 40 bp, despite 90 nt of perfect complementarity. Additionally, we show that Hfq and RNA secondary structure in the tnpA 5''UTR each repress tnpA expression in an art200-independent manner. Finally, we show that disrupting translational control of tnpA expression leads to increased IS200 transposition in E. coli. The current work provides new mechanistic insight into why IS200 transposition is so strongly suppressed. The possibility of art200 acting in trans to regulate a yet-unidentified target is discussed as well as potential applications of the IS200 system for designing novel riboregulators.     

Crystal structure of Hfq from Bacillus subtilis in complex with SELEX-derived RNA aptamer: insight into RNA-binding properties of bacterial Hfq

Bacterial Hfq is a protein that plays an important role in the regulation of genes in cooperation with sRNAs. Escherichia coli Hfq (EcHfq) has two or more sites that bind RNA(s) including U-rich and/or the poly(A) tail of mRNA. However, functional and structural information about Bacillus subtilis Hfq (BsHfq) including the RNA sequences that specifically bind to it remain unknown. Here, we describe RNA aptamers including fragment (AG)₃A that are recognized by BsHfq and crystal structures of the BsHfq–(AG)₃A complex at 2.2 Å resolution. Mutational and structural studies revealed that the RNA fragment binds to the distal site, one of the two binding sites on Hfq, and identified amino acid residues that are critical for sequence-specific interactions between BsHfq and (AG)₃A. In particular, R32 appears to interact with G bases in (AG)₃A. Poly(A) also binds to the distal site of EcHfq, but the overall RNA structure and protein–RNA interaction patterns engaged in the R32 residues of BsHfq–(AG)₃A differ from those of EcHfq–poly(A). These findings provide novel insight into how the Hfq homologue recognizes RNA.     

Ecology of Non-O 1 Vibrio cholerae in Toyama Prefecture

The ecology of non-O 1 Vibrio cholerae and Vibrio mimicus as causes of cholera-like diarrhea or seafood-associated gastroenteritis has been investigated in Toyama Prefecture since 1980. The relationship between biological or serological characteristics of the isolates and their enteropathogenicity is discussed. Overall isolation rates from river water, sea water, and fish were 24.0, 59.5, and 33.7%, respectively, the isolation frequency being, in general, extremely high in the summer season, although the organisms were detected all year around in the case of sea water. Most isolates from river water were unable to grow on plates of TCBS agar to which colistin was added at a concentration of 1 μg/ml (CL-TCBS). These strains quickly fermented cellobiose. O-51 and O-70 were the two most frequently detected serogroups among them and they did not show enteropathogenicity in the rabbit ileal loop (RIL) test. On the other hand, almost all isolates from sea water and fish as well as those from human diarrhea cases were able to grow on CL-TCBS, but were unable to ferment cellobiose quickly. O-36, O-10, O-6, O-8, O-39, and O-26 were the dominant serogroups of these isolates, and some of them showed enteropathogenicity in the RIL test. Six out of 98 isolates from river water, 14 out of 116 from sea water, and 19 out of 112 from fish were classified as Vibrio mimicus. All of these strains were able to grow on CL-TCBS and quickly fermented mannose but not cellobiose. I-41 was the most common serogroup among them and some of these strains showed enteropathogenicity in the RIL test. Production of a cholera-like enterotoxin among the isolates in Toyama Prefecture, if any, seemed to be poor.     

Multiplex polymerase chain reaction to detect toxigenic Vibrio cholerae and to biotype Vibrio cholerae O1

A multiplex polymerase chain reaction (PCR) was developed to identify cholera toxin-producing Vibrio cholerae and to biotype V. cholerae O1. Enterotoxin-producing V. cholerae strains were identified with a primer pair that amplified a fragment of the ctxA2-B gene. Vibrio cholerae O1 strains were simultaneously differentiated into biotypes with three primers specified for the hlyA gene in the same reaction. The hlyA amplicon in the multiplex PCR serves as an internal control when testing toxin-producing strains, as hlyA gene sequences exist in all tested V. cholerae strains. Enrichment of V. cholerae present on oysters for 6 h in alkaline peptone water before detection by a nested PCR with internal primers for ctxA2-B gene yielded a detection limit lower than 3 colony-forming units (cfu) per gram of food.     

Clinical Manifestations of Non-O1 Vibrio cholerae Infections

Background

Infections caused by non-O1 Vibrio cholera are uncommon. The aim of our study was to investigate the clinical and microbiological characteristics of patients with non-O1 V. cholera infections.

Methods

The clinical charts of all patients with non-O1 V. cholera infections and who were treated in two hospitals in Taiwan were retrospectively reviewed.

Results

From July 2009 to June 2014, a total of 83 patients with non-O1 V. cholera infections were identified based on the databank of the bacteriology laboratories of two hospitals. The overall mean age was 53.3 years, and men comprised 53 (63.9%) of the patients. Liver cirrhosis and diabetes mellitus were the two most common underlying diseases, followed by malignancy. The most common type of infection was acute gastroenteritis (n = 45, 54.2%), followed by biliary tract infection (n = 12, 14.5%) and primary bacteremia (n = 11, 13.3%). Other types of infection, such as peritonitis (n = 5, 6.0%), skin and soft tissue infection (SSTI) (n = 5, 6.0%), urinary tract infection (n = 3, 3.6%) and pneumonia (2, 2.4%), were rare. July and June were the most common months of occurrence of V. cholera infections. The overall in-hospital mortality of 83 patients with V. cholera infections was 7.2%, but it was significantly higher for patients with primary bacteremia, hemorrhage bullae, acute kidney injury, acute respiratory failure, or admission to an ICU. Furthermore, multivariate analysis showed that in-hospital mortality was significantly associated with acute respiratory failure (odds ratio, 60.47; 95% CI, 4.79-763.90, P = 0.002).

Conclusions

Non-O1 V. cholera infections can cause protean disease, especially in patients with risk factors and during warm-weather months. The overall mortality of 83 patients with non-O1 V. cholera infections was only 7.2%; however, this value varied among different types of infection.     

Distribution of Vibrio cholerae O1 antigen biosynthesis genes among O139 and other non-O1 serogroups of Vibrio cholerae

The organization and distribution of the genes responsible for O antigen biosynthesis in various serogroups of Vibrio cholerae were investigated using several DNA probes derived from various regions of the genes responsible for O1 antigen biosynthesis. Based on the reactivity pattern of the probes against the various serogroups, the cluster of genes responsible for the O1 antigen biosynthesis could be broadly divided into six groups, designated as class 1-6. The class 3 cluster of genes corresponding to gmd to wbeO, wbeT and a part of wbeU was specific for only the O1 serogroup. The other cluster of genes (class 1, 2, 4-6) reacted with other serogroups of V. cholerae. These data indicate that serotype conversion in V. cholerae does not depend on a simple mutational event but may involve horizontal gene transfer not only between V. cholerae strains but also between V. cholerae and species other than V. cholerae.