Publications by Year: 2012

Escherichia coli alkyl hydroperoxide reductase subunit C (AhpC) is a peroxiredoxin that detoxifies peroxides. Here we show an additional role for AhpC in cellular iron metabolism of E. coli. Deletion of ahpC resulted in reduced growth and reduced accumulation of iron by cells grown in low-iron media. Liquid chromatography-mass spectroscopy (LC-MS) analysis of culture supernatants showed that the ahpC mutant secreted much less enterobactin, the siderophore that chelates and transports ferric iron under iron-limiting conditions, than wild-type E. coli did. The ahpC mutant produced less 2,3-dihydroxybenzoate, the intermediate in the enterobactin biosynthesis pathway, and providing 2,3-dihydroxybenzoate restored wild-type growth of the ahpC mutant. These data indicated that the defect was in an early step in enterobactin biosynthesis. Providing additional copies of entC, which functions in the first dedicated step of enterobactin biosynthesis, but not of other enterobactin biosynthesis genes, suppressed the mutant phenotype. Additionally, providing either shikimate or a mixture of para-aminobenzoate, tryptophan, tyrosine, and phenylalanine, which, like enterobactin, are synthesized from the precursor chorismate, also suppressed the mutant phenotype. These data suggested that AhpC affected the activity of EntC or the availability of the chorismate substrate.

Vibrio cholerae responds to environmental changes by altering the protein composition of its outer membrane. In rich medium, V. cholerae expresses almost exclusively the outer membrane porin OmpU, whereas in minimal medium, OmpT is the dominant porin. The supplementation of a minimal medium with a mixture of asparagine, arginine, glutamic acid, and serine (NRES) promotes OmpU production and OmpT repression at levels similar to those seen with rich media. Here we show that the altered Omp profile is not due to an increase in the growth rate in the presence of supplemental amino acids but requires the addition of specific amino acids. The effects of the NRES mix on Omp production were mediated by ToxR, a known regulator of omp gene expression. No changes in the Omp profile were detected in a toxR mutant. Supplementation with the NRES mix resulted in significantly higher levels of ToxR, and the elevated ToxR levels were sufficient to cause a switch in Omp synthesis. The increase in the level of the ToxR protein correlated with an increase in toxR mRNA levels and was observed only when toxR was expressed from its native promoter. ToxS, which is required for ToxR activity, was necessary for NRES-mediated omp gene regulation but not for the increase in ToxR levels. The growth of V. cholerae in the presence of bile acids also resulted in Omp switching, and this required ToxR. However, unlike the NRES mix, bile acids did not increase either ToxR protein or toxR mRNA levels, suggesting a different mechanism of omp gene regulation by bile than that by amino acids.

BACKGROUND: Glutamyl queuosine-tRNA(Asp) synthetase (GluQ-RS) is a paralog of the catalytic domain of glutamyl-tRNA synthetase and catalyzes the formation of glutamyl-queuosine on the wobble position of tRNA(Asp). Here we analyze the transcription of its gene in Shigella flexneri, where it is found downstream of dksA, which encodes a transcriptional regulator involved in stress responses. RESULTS: The genomic organization, dksA-gluQ-rs, is conserved in more than 40 bacterial species. RT-PCR assays show co-transcription of both genes without a significant change in transcript levels during growth of S. flexneri. However, mRNA levels of the intergenic region changed during growth, increasing at stationary phase, indicating an additional level of control over the expression of gluQ-rs gene. Transcriptional fusions with lacZ as a reporter gene only produced β-galactosidase activity when the constructs included the dksA promoter, indicating that gluQ-rs do not have a separate promoter. Using bioinformatics, we identified a putative transcriptional terminator between dksA and gluQ-rs. Deletion or alteration of the predicted terminator resulted in increased expression of the lacZ reporter compared with cells containing the wild type terminator sequence. Analysis of the phenotype of a gluQ-rs mutant suggested that it may play a role in some stress responses, since growth of the mutant was impaired in the presence of osmolytes. CONCLUSIONS: The results presented here, show that the expression of gluQ-rs depends on the dksA promoter, and strongly suggest the presence and the functionality of a transcriptional terminator regulating its expression. Also, the results indicate a link between glutamyl-queuosine synthesis and stress response in Shigella flexneri.

Infection of the human host by Shigella species requires the coordinated production of specific Shigella virulence factors, a process mediated largely by the VirF/VirB regulatory cascade. VirF promotes the transcription of virB, a gene encoding the transcriptional activator of several virulence-associated genes. This study reveals that transcription of virB is also regulated by the small RNA RyhB, and importantly, that this regulation is not achieved indirectly via modulation of VirF activity. These data are the first to demonstrate that the regulation of virB transcription can be uncoupled from the master regulator VirF. It is also established that efficient RyhB-dependent regulation of transcription is facilitated by specific nucleic acid sequences within virB. This study not only reveals RyhB-dependent regulation of virB transcription as a novel point of control in the central regulatory circuit modulating Shigella virulence, but also highlights the versatility of RyhB in controlling bacterial gene expression.