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.

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.

Receiver domains control intracellular responses triggered by signal transduction in bacterial two-component systems. Here, we report the solution NMR 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 α5/β5 Rossman-fold but has features that set it apart from other receiver domains. The fourth β-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-β5-α5) faces of receiver domains, to prime them for binding downstream effectors once they become activated by phosphorylation. In addition, Sma0114 lacks the fourth α-helix of the consensus 455 face and 15N relaxation data show that it is replaced by a segment that is flexible on the ps-ns timescale. Secondary structure prediction of Sma0114 and other HWE-kinase-associated receiver domains suggests that the absence of helix α4 may be a conserved property of this family. In spite of these differences, Sma0114 has a conserved active site, binds divalent metal ions such as Mg2+ and Ca2+ that are required for phosphorylation, and exhibits μs-ms active site dynamics similar to 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.

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 ␣-and ␤-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.Sinorhizobium, Rhizobium, Bradyrhizobium, and Azorhizobium (rhizobia) are important nitrogen-fixing prokaryotes. These grow in the soil as free-living organisms but can also live as nitrogen-fixing symbionts inside roots of plants belonging to the family Leguminosae (8,11,21,33,41,54). Rhizobia are able to utilize a wide range of compounds as carbon sources, such as sugars, amino acids, and tricarboxylic acid (TCA) cycle intermediates. Studies have shown that the C 4 -dicarboxylic TCA cycle intermediates succinate, fumarate, and malate support high rates of growth in laboratory medium and are used by rhizobia in preference to carbon sources including glucose, fructose, galactose, lactose, and myo-inositol (23,26,44,63).The phenomenon of Sinorhizobium meliloti utilizing succinate and similar C 4 -dicarboxylic acids in preference to other compounds is called succinate-mediated catabolite repression (SMCR) (9). One of the first reports of catabolite repression in S. meliloti (then Rhizobium meliloti) showed that S. meliloti exhibited diauxic growth in a medium containing 0.2% succinate and 0.2% lactose as carbon sources (63). This study also showed that the production of ␤-galactosidase was repressed when succinate and lactose were present together and that it increased to higher levels after succinate had been exhausted from the medium.Succinate and other C 4 -dicarboxylic acids are sensed and transported by the Dct (dicarboxylate transport) system which is encoded by dctA, dctB, and dctD (48,66,(69)(70)(71). DctB is activated by the presence of C 4 -dicarboxylic acids and autophosphorylates. Activated DctB phosphorylates DctD, which then binds upstream of dctA along with 54 -RNA polymerase to initiate transcription (70). dctA encodes the permease required for transport of succinate and other C 4 -dicarboxylic acids. When succinate is in abundance, S. meliloti will preferentially import this carbon source for metabolism and in...