Regulation of OmpA Translation and Shigella dysenteriae Virulence by an RNA Thermometer.

RNA thermometers are cis-acting riboregulators that mediate the posttranscriptional regulation of gene expression in response to environmental temperature. Such regulation is conferred by temperature-responsive structural changes within the RNA thermometer that directly result in differential ribosomal binding to the regulated transcript. The significance of RNA thermometers in controlling bacterial physiology and pathogenesis is becoming increasingly clear. This study combines in silico, molecular genetics, and biochemical analyses to characterize both the structure and function of a newly identified RNA thermometer within the ompA transcript of Shigella dysenteriae First identified by in silico structural predictions, genetic analyses have demonstrated that the ompA RNA thermometer is a functional riboregulator sufficient to confer posttranscriptional temperature-dependent regulation, with optimal expression observed at the host-associated temperature of 37°C. Structural studies and ribosomal binding analyses have revealed both increased exposure of the ribosomal binding site and increased ribosomal binding to the ompA transcript at permissive temperatures. The introduction of site-specific mutations predicted to alter the temperature responsiveness of the ompA RNA thermometer has predictable consequences for both the structure and function of the regulatory element. Finally, in vitro tissue culture-based analyses implicate the ompA RNA thermometer as a bona fide S. dysenteriae virulence factor in this bacterial pathogen. Given that ompA is highly conserved among Gram-negative pathogens, these studies not only provide insight into the significance of riboregulation in controlling Shigella virulence, but they also have the potential to facilitate further understanding of the physiology and/or pathogenesis of a wide range of bacterial species.

Subunit structure of benzylsuccinate synthase.

Benzylsuccinate synthase is a member of the glycyl radical family of enzymes. It catalyzes the addition of toluene to fumarate to form benzylsuccinate as the first step in the anaerobic pathway of toluene fermentation. The enzyme comprises three subunits, alpha, beta, and gamma, that in Thauera aromatica strain T1 are encoded by the tutD, tutG, and tutF genes, respectively. The large alpha-subunit contains the essential glycine and cysteine residues that are conserved in all glycyl radical enzymes. However, the function of the small beta- and gamma-subunits has remained unclear. We have overexpressed all three subunits of benzylsuccinate synthase in Escherichia coli, both individually and in combination. Coexpression of the gamma-subunit (but not the beta-subunit) is essential for efficient expression of the alpha-subunit. The benzylsuccinate synthase complex lacking the glycyl radical could be purified as an alpha(2)beta(2)gamma(2) hexamer by nickel affinity chromatography through a "His(6)" affinity tag engineered onto the C-terminus of the alpha-subunit. Unexpectedly, BSS was found to contain two iron-sulfur clusters, one associated with the beta-subunit and the other with the gamma-subunit that appear to be necessary for the structural integrity of the complex. The spectroscopic properties of these clusters suggest that they are most likely [4Fe-4S] clusters. Removal of iron with chelating agents results in dissociation of the complex; similarly, a mutant gamma-subunit lacking the [4Fe-4S] cluster is unable to stabilize the alpha-subunit when the proteins are coexpressed.

Sibling sRNA RyfA1 Influences Shigella dysenteriae Pathogenesis.

Small regulatory RNAs (sRNAs) of Shigella dysenteriae and other pathogens are vital for the regulation of virulence-associated genes and processes. Here, we characterize RyfA1, one member of a sibling pair of sRNAs produced by S. dysenteriae. Unlike its nearly identical sibling molecule, RyfA2, predicted to be encoded almost exclusively by non-pathogenic species, the presence of a gene encoding RyfA1, or a RyfA1-like molecule, is strongly correlated with virulence in a variety of enteropathogens. In S. dysenteriae, the overproduction of RyfA1 negatively impacts the virulence-associated process of cell-to-cell spread as well as the expression of ompC, a gene encoding a major outer membrane protein important for the pathogenesis of Shigella. Interestingly, the production of RyfA1 is controlled by a second sRNA, here termed RyfB1, the first incidence of one regulatory small RNA controlling another in S. dysenteriae or any Shigella species.

Construction and characterization of insertion/deletion mutations of the tutF, tutD, and tutG genes of Thauera aromatica strain T1.

Thauera aromatica T1 was isolated for its ability to use toluene as a sole carbon source under denitrifying conditions. A genetic approach was used to examine the roles of the tutF, tutD, and tutG gene products (part of a single operon) in the metabolism of toluene. The genes were individually deleted from the chromosome and each resulting mutant strain was unable to metabolize toluene. Plasmids carrying individual in-frame gene deletions failed to complement the corresponding chromosomal deletions but did complement chromosomal deletions downstream of the in-frame deletion. Hence, the tutF, tutD, and tutG genes are each essential for toluene metabolism in T. aromatica T1.

Role of benzylsuccinate in the induction of the tutE tutFDGH gene complex of T. aromatica strain T1.

Expression of the tutE tutFDGH gene cluster of Thauera aromatica strain T1 was examined by Northern and Western analysis in a wild-type strain and chromosomally deleted strains with or without in-frame deletion plasmids. While expression was observed when the wild-type strain was induced with toluene, various chromosomally deleted strains exhibited little or no expression of the tut genes. In contrast, both wild-type and chromosomally deleted strains expressed the tut genes when induced with benzylsuccinate. We conclude that benzylsuccinate is required for the full induction of the tutE tutFDGH gene cluster of T. aromatica strain T1.

Site-directed mutagenesis of the Thauera aromatica strain T1 tutE tutFDGH gene cluster.

Benzylsuccinate synthase, encoded by the tutF, tutD, and tutG genes of Thauera aromatica strain T1, is responsible for the first step of anaerobic toluene metabolism. Previous work has shown that these genes are part of the tutE tutFDGH gene cluster and strains carrying a mutation in the tutE, tutF, tutD, or tutG genes are unable to metabolize toluene. In this study, we performed site-directed mutagenesis of the tutE, tutF, and tutG genes and determined that the cysteines at position 72 and 79 of TutE are likely to be critical for the radical activation of benzylsuccinate synthase, while the cysteine alanine at positions 9 and 10 of TutF, and the cysteine at position 29 of TutG are also essential for toluene metabolism. Additionally, we report that the tutH gene is necessary for toluene metabolism and the glycine lysine serine (part of the putative ATP/GTP binding domain) at positions 52-54 of the TutH protein is essential for toluene metabolism.