Recombinant Salmonella Expressing Burkholderia mallei LPS O Antigen Provides Protection in a Murine Model of Melioidosis and Glanders.

Burkholderia pseudomallei and Burkholderia mallei are the etiologic agents of melioidosis and glanders, respectively. These bacteria are highly infectious via the respiratory route and can cause severe and often fatal diseases in humans and animals. Both species are considered potential agents of biological warfare; they are classified as category B priority pathogens. Currently there are no human or veterinary vaccines available against these pathogens. Consequently efforts are directed towards the development of an efficacious and safe vaccine. Lipopolysaccharide (LPS) is an immunodominant antigen and potent stimulator of host immune responses. B. mallei express LPS that is structurally similar to that expressed by B. pseudomallei, suggesting the possibility of constructing a single protective vaccine against melioidosis and glanders. Previous studies of others have shown that antibodies against B. mallei or B. pseudomallei LPS partially protect mice against subsequent lethal virulent Burkholderia challenge. In this study, we evaluated the protective efficacy of recombinant Salmonella enterica serovar Typhimurium SL3261 expressing B. mallei O antigen against lethal intranasal infection with Burkholderia thailandensis, a surrogate for biothreat Burkholderia spp. in a murine model that mimics melioidosis and glanders. All vaccine-immunized mice developed a specific antibody response to B. mallei and B. pseudomallei O antigen and to B. thailandensis and were significantly protected against challenge with a lethal dose of B. thailandensis. These results suggest that live-attenuated SL3261 expressing B. mallei O antigen is a promising platform for developing a safe and effective vaccine.

Nuclear magnetic resonance structure and dynamics of the response regulator Sma0114 from Sinorhizobium meliloti.

Receiver domains control intracellular responses triggered by signal transduction in bacterial two-component systems. Here, we report the solution nuclear magnetic resonance structure and dynamics of Sma0114 from the bacterium Sinorhizobium meliloti, the first such characterization of a receiver domain from the HWE-kinase family of two-component systems. The structure of Sma0114 adopts a prototypical α(5)/ß(5) Rossman fold but has features that set it apart from other receiver domains. The fourth ß-strand of Sma0114 houses a PFxFATGY sequence motif, common to many HWE-kinase-associated receiver domains. This sequence motif in Sma0114 may substitute for the conserved Y-T coupling mechanism, which propagates conformational transitions in the 455 (α4-ß5-α5) faces of receiver domains, to prime them for binding downstream effectors once they become activated by phosphorylation. In addition, the fourth α-helix of the consensus 455 face in Sma0114 is replaced with a segment that shows high flexibility on the pico- to nanosecond time scale by (15)N relaxation data. Secondary structure prediction analysis suggests that the absence of helix α4 may be a conserved property of the HWE-kinase-associated family of receiver domains to which Sma0114 belongs. In spite of these differences, Sma0114 has a conserved active site, binds divalent metal ions such as Mg(2+) and Ca(2+) that are required for phosphorylation, and exhibits micro- to millisecond active-site dynamics similar to those of other receiver domains. Taken together, our results suggest that Sma0114 has a conserved active site but differs from typical receiver domains in the structure of the 455 face that is used to effect signal transduction following activation.

NMR assignments for the Sinorhizobium meliloti response regulator Sma0114.

Response regulators are terminal ends of bacterial two-component systems that undergo extensive structural reorganization in response to phosphoryl transfer from their cognate histidine kinases. The response regulator encoded by the gene sma0114 of Sinorhizobium meliloti is a part of a unique class of two-component systems that employ HWE histidine kinases. The distinct features of Sma0114 include a PFxFATGY motif that houses the conserved threonine in the "Y-T coupling" conformational switch which mediates output response through downstream protein-protein interactions, and the replacement of the conserved phenylalanine/tyrosine in Y-T coupling by a leucine. Here we present (1)H, (15)N, and (13)C NMR assignments for Sma0114. We identify the secondary structure of the protein based on TALOS chemical shift analysis, (3)J(HNHα) coupling constants and hydrogen-deuterium exchange. The secondary structure determined by NMR is in good agreement with that predicted from the sequence. Both methods suggest that Sma0114 differs from standard CheY-like folds by missing the fourth α-helix. Our initial NMR characterization of Sma0114 paves the way to a full investigation of the structure and dynamics of this response regulator.

Characterization of a two-component regulatory system that regulates succinate-mediated catabolite repression in Sinorhizobium meliloti.

When they are available, Sinorhizobium meliloti utilizes C(4)-dicarboxylic acids as preferred carbon sources for growth while suppressing the utilization of some secondary carbon sources such as α- and ß-galactosides. The phenomenon of using succinate as the sole carbon source in the presence of secondary carbon sources is termed succinate-mediated catabolite repression (SMCR). Genetic screening identified the gene sma0113 as needed for strong SMCR when S. meliloti was grown in succinate plus lactose, maltose, or raffinose. sma0113 and the gene immediately downstream, sma0114, encode the proteins Sma0113, an HWE histidine kinase with five PAS domains, and Sma0114, a CheY-like response regulator lacking a DNA-binding domain. sma0113 in-frame deletion mutants show a relief of catabolite repression compared to the wild type. sma0114 in-frame deletion mutants overproduce polyhydroxybutyrate (PHB), and this overproduction requires sma0113. Sma0113 may use its five PAS domains for redox level or energy state monitoring and use that information to regulate catabolite repression and related responses.

Compartment-specific phosphorylation of rat thyroid hormone receptor alpha1 regulates nuclear localization and retention.

The thyroid hormone receptor alpha1 (TRalpha1) is a transcription factor, which can activate or repress gene expression in response to thyroid hormone. In addition, some of its actions, including DNA binding and transcriptional activation, are thought to be regulated by phosphorylation. Results presented here, using Xenopus oocyte microinjection assays, demonstrate that a phosphorylated form of rat TRalpha1 is present in the nucleus, whereas unphosphorylated TRalpha1 remains cytoplasmic. Changes in the phosphorylation state of TRalpha1 occur rapidly and point to the possibility that phosphorylation occurs in the nucleus. Furthermore, increasing the overall phosphorylation state of the cell leads to enhanced nuclear retention of TRalpha1, suggesting that compartment-specific phosphorylation regulates nuclear localization of TRalpha1. Enhanced nuclear retention of TRalpha1 is not dependent on phosphorylation of serine 12, a well-characterized casein kinase II site, nor is phosphorylation of this site necessary for import of TRalpha1 into the Xenopus oocyte nucleus. Similarly, mutational analysis in mammalian cells shows that nuclear localization and partitioning of TRalpha1 to the nuclear matrix are independent of serine 12 phosphorylation. Taken together, these studies suggest that phosphorylation of one or more sites in TRalpha1, excluding serine 12, enhances nuclear retention and/or inhibits nuclear export but is not directly involved in nuclear import.

Use of antibiotic resistance analysis for representativeness testing of multiwatershed libraries.

The use of antibiotic resistance analysis (ARA) for microbial source tracking requires the generation of a library of isolates collected from known sources in the watershed. The size and composition of the library are critical in determining if it represents the diversity of patterns found in the watershed. This study was performed to determine the size that an ARA library needs to be to be representative of the watersheds for which it will be used and to determine if libraries from different watersheds can be merged to create multiwatershed libraries. Fecal samples from known human, domesticated, and wild animal sources were collected from six Virginia watersheds. From these samples, enterococci were isolated and tested by ARA. Based on cross-validation discriminant analysis, only the largest of the libraries (2,931 isolates) were found to be able to classify nonlibrary isolates as well as library isolates (i.e., were representative). Small libraries tended to have higher average rates of correct classification, but were much less able to correctly classify nonlibrary isolates. A merged multiwatershed library (6,587 isolates) was created and was found to be large enough to be representative of the isolates from the contributing watersheds. When isolates that were collected from the contributing watersheds approximately 1 year later were analyzed with the multiwatershed library, they were classified as well as the isolates in the library, suggesting that the resistance patterns are temporally stable for at least 1 year. The ability to obtain a representative, temporally stable library demonstrates that ARA can be used to identify sources of fecal pollution in natural waters.

Genome-wide transcriptional profiling in a histidine kinase mutant of Helicobacter pylori identifies members of a regulon.

To identify putative members of a regulon controlled by the H. pylori sensory histidine kinase HP0164 (HK0164), we constructed HK0164 null mutant H. pylori strains and analyzed bacterial gene transcription using DNA arrays. Seven genes were differentially expressed in multiple HK0164 mutant strains compared to their expression in control strains. Strain-dependent variations in differential expression were also detected. These results indicate that the signal transduction circuit utilizing HK0164 controls the transcription of at least seven genes in H. pylori.