Posttranscriptional control is known to contribute to the regulation of secondary metabolism and virulence determinants in certain gram-negative bacteria. Here we report the isolation of a Pseudomonas aeruginosa gene which encodes a global translational regulatory protein, RsmA (regulator of secondary metabolites). Overexpression of rsmA resulted in a substantial reduction in the levels of extracellular products, including protease, elastase, and staphylolytic (LasA protease) activity as well as the PA-IL lectin, hydrogen cyanide (HCN), and the phenazine pigment pyocyanin. While inactivation of rsmA in P. aeruginosa had only minor effects on the extracellular enzymes and the PA-IL lectin, the production of HCN and pyocyanin was enhanced during the exponential phase. The influence of RsmA on N-acylhomoserine lactone-mediated quorum sensing was determined by assaying the levels of N-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL) and N-butanoylhomoserine lactone (C4-HSL) produced by the rsmA mutant and the rsmA-overexpressing strain. RsmA exerted a negative effect on the synthesis of both 3-oxo-C12-HSL and C4-HSL, which was confirmed by using lasI and rhlI translational fusions. These data also highlighted the temporal expression control of the lasI gene, which was induced much earlier and to a higher level during the exponential growth phase in an rsmA mutant. To investigate whether RsmA modulates HCN production solely via quorum-sensing control, hcn translational fusions were employed to monitor the regulation of the cyanide biosynthesis genes (hcnABC). RsmA was shown to exert an additional negative effect on cyanogenesis posttranscriptionally by acting on a region surrounding the hcnA ribosome-binding site. This suggests that, in P. aeruginosa, RsmA functions as a pleiotropic posttranscriptional regulator of secondary metabolites directly and also indirectly by modulating the quorum-sensing circuitry.

The attenuation and immunogenicity of two novel Salmonella vaccine strains, Salmonella enterica serovar Typhi (Ty2 ⌬aroC ⌬ssaV, designated ZH9) and S. enterica serovar Typhimurium (TML ⌬aroC ⌬ssaV, designated WT05), were evaluated after their oral administration to volunteers as single escalating doses of 10 7 , 10 8 , or 10 9 CFU. ZH9 was well tolerated, not detected in blood, nor persistently excreted in stool. Six of nine volunteers elicited anti-serovar Typhi lipopolysaccharide (LPS) immunoglobulin A (IgA) antibody-secreting cell (ASC) responses, with three of three vaccinees receiving 10 8 and two of three receiving 10 9 CFU which elicited high-titer LPS-specific serum IgG. WT05 was also well tolerated with no diarrhea, although the administration of 10 8 and 10 9 CFU resulted in shedding in stools for up to 23 days. Only volunteers immunized with 10 9 CFU of WT05 mounted detectable serovar Typhimurium LPS-specific ASC responses and serum antibody responses were variable. These data indicate that mutations in type III secretion systems may provide a route to the development of live vaccines in humans and highlight significant differences in the potential use of serovars Typhimurium and Typhi.

2A: "(pME3849)" and "(pME6001)" should be deleted from all three graphs. The data are for the plasmid-free PAO1 and PAZH13 strains.

BackgroundTyphoid persists as a major cause of global morbidity. While several licensed vaccines to prevent typhoid are available, they are of only moderate efficacy and unsuitable for use in children less than two years of age. Development of new efficacious vaccines is complicated by the human host-restriction of Salmonella enterica serovar Typhi (S. Typhi) and lack of clear correlates of protection. In this study, we aimed to evaluate the protective efficacy of a single dose of the oral vaccine candidate, M01ZH09, in susceptible volunteers by direct typhoid challenge.Methods and FindingsWe performed a randomised, double-blind, placebo-controlled trial in healthy adult participants at a single centre in Oxford (UK). Participants were allocated to receive one dose of double-blinded M01ZH09 or placebo or 3-doses of open-label Ty21a. Twenty-eight days after vaccination, participants were challenged with 104CFU S. Typhi Quailes strain. The efficacy of M01ZH09 compared with placebo (primary outcome) was assessed as the percentage of participants reaching pre-defined endpoints constituting typhoid diagnosis (fever and/or bacteraemia) during the 14 days after challenge. Ninety-nine participants were randomised to receive M01ZH09 (n = 33), placebo (n = 33) or 3-doses of Ty21a (n = 33). After challenge, typhoid was diagnosed in 18/31 (58.1% [95% CI 39.1 to 75.5]) M01ZH09, 20/30 (66.7% [47.2 to 87.2]) placebo, and 13/30 (43.3% [25.5 to 62.6]) Ty21a vaccine recipients. Vaccine efficacy (VE) for one dose of M01ZH09 was 13% [95% CI -29 to 41] and 35% [-5 to 60] for 3-doses of Ty21a. Retrospective multivariable analyses demonstrated that pre-existing anti-Vi antibody significantly reduced susceptibility to infection after challenge; a 1 log increase in anti-Vi IgG resulting in a 71% decrease in the hazard ratio of typhoid diagnosis ([95% CI 30 to 88%], p = 0.006) during the 14 day challenge period. Limitations to the study included the requirement to limit the challenge period prior to treatment to 2 weeks, the intensity of the study procedures and the high challenge dose used resulting in a stringent model.ConclusionsDespite successfully demonstrating the use of a human challenge study to directly evaluate vaccine efficacy, a single-dose M01ZH09 failed to demonstrate significant protection after challenge with virulent Salmonella Typhi in this model. Anti-Vi antibody detected prior to vaccination played a major role in outcome after challenge.Trial registrationClinicalTrials.gov (NCT01405521) and EudraCT (number 2011-000381-35).

The pathway for glycerol catabolism in Streptomyces coelicolor is determined by the gylCABX operon, which is transcribed from two closely spaced glycerol-inducible, glucose-repressible promoters. Glucose (or catabolite) repression of gyl is known to be exerted by a general catabolite repression system in which the soluble glucose kinase plays a central role. The gylR gene is contained in a separate glycerol-inducible, weakly glucose-repressible transcription unit immediately upstream from the gyl operon. The role of gylR in the regulation of gyl transcription was assessed by introducing specific null mutations into the chromosomal gylR gene. Direct quantification of gyl transcripts from the gylR null mutants grown on different carbon sources demonstrated that GylR is the repressor of the gylCABX operon and also revealed that GylR functions as a negative autoregulator. Moreover, the transcriptional analysis revealed that the gylR null mutants were relieved of glucose repression of both gylCABX and gylR. We conclude that both substrate induction and catabolite repression of gyl are mediated through the GylR protein. This is the first direct evidence that catabolite repression in Streptomyces is not exerted at the transcriptional level by a general 'catabolite repressor protein'. Models for catabolite repression are discussed.

The dnaK operon of Streptomyces coelicolor contains four genes (5-dnaK-grpE-dnaJ-hspR). The fourth gene encodes a novel heat shock protein, HspR, which appears so far to be unique to the high-G؉C actinomycete group of bacteria. HspR binds with high specificity to three inverted repeat sequences in the promoter region of the S. coelicolor dnaK operon, strongly suggesting a direct role for HspR in heat shock gene regulation. Here we present genetic and biochemical evidence that HspR is the repressor of the dnaK operon. Disruption of hspR leads to high-level constitutive transcription of the dnaK operon. Parallel transcriptional analyses of groESL1 and groEL2 expression demonstrated that heat shock regulation of the groE genes was essentially unaffected in an hspR null mutant, although the basal (uninduced) level of groEL2 transcription was slightly elevated compared with the wild type. The results of HspR titration experiments, where the dnaK operon promoter region was cloned at ca. 50 copies per chromosome, were consistent with the prediction that HspR functions as a negative autoregulator. His-tagged HspR, overproduced and purified from Escherichia coli, was shown to repress transcription from the dnaK operon promoter in vitro, providing additional evidence for the proposal that HspR directly regulates transcription of the dnaK operon. These studies indicate that there are at least two transcriptional mechanisms for controlling heat shock genes in S. coelicolor-one controlling the dnaK operon and another controlling the groE genes.Free-living organisms generally respond to an injury caused by exposure to a physicochemical or metabolically stressful agent by inducing the synthesis of a set of proteins, collectively called heat shock proteins (Hsps), which help to prevent or repair damage caused by partially denatured proteins that may or may not have formed insoluble aggregates (15,19). Some Hsps are molecular chaperones which prevent the aggregation of partially unfolded proteins and help them to fold into their native structures (reviewed in reference 18), while others are proteases, such as Lon and Clp, which degrade unfolded proteins that have formed insoluble aggregates (see, e.g., reference 16). Some Hsps, such as the molecular chaperones Hsp70 (DnaK) and Hsp60 (GroEL), also fulfill crucial roles under normal physiological conditions by assisting in the folding of newly synthesized polypeptides (see, e.g., reference 18). Although these chaperones rank as perhaps the most highly conserved proteins in nature, diverse regulatory mechanisms have evolved in different organisms for controlling their synthesis. In Escherichia coli the coordinated induction of the heat shock regulon is achieved by increasing the cellular concentration of two alternative factors: 32 activates heat shock genes following a mild heat shock (9, 14, 45), while E activates transcription under conditions of severe heat shock (31, 33). This positive mode of regulation is moderately conserved in gramnegative bacteria, particularly in the ␥ s...

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The oral typhoid vaccine M01ZH09 is well tolerated and highly immunogenic in a single oral dose, with and without prevaccination with a bicarbonate buffer. Field studies to demonstrate protective efficacy are planned.