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Cysteine-Scanning Analysis of the Dimerization Domain of EnvZ, an Osmosensing Histidine Kinase. Ling Qin, 2003.EnvZ and OmpR are a transmembrane sensor and its cognate response regulator, respectively, regulating the transcription of porin genes in response to medium osmolarity in Escherichia coli . The cytoplasmic domain of EnvZ (EnvZc) possesses both kinase and phosphatase activities and can be dissected into two functional domains, A and B . Here, we performed a cysteine-scanning analysis of domain A, a 67-residue central dimerization and phosphatase domain containing His-243 as the phosphorylation site, and we examined the effects of the cysteine substitution mutations on the enzymatic activities of domain A . The substitution mutations were made at 31 residues, from which 24 mutant domain A proteins were biochemically characterized . From the analysis of the phosphatase activity of purified mutant proteins, it was found that there are two regions in domain A which are important for this activity . Cysteine mutations in these regions dramatically reduce or completely abolish the phosphatase activity of domain A . The mutations that have the most-severe effects on domain A phosphatase activity also significantly reduce the phosphatase activity of EnvZc containing the same mutation . Using an in vitro complementation system with EnvZc(H243V), these cysteine mutants were further characterized for their autophosphorylation activities as well as their phosphotransfer activities . The results indicate that some mutations are specific either for the phosphatase activity or for the kinase activity . Activation of Antibiotic Biosynthesis by Specified Mutations in the rpoB Gene (Encoding the RNA Polymerase ß Subunit) of Streptomyces lividans. Haifeng Hu, 2002.We found that the biosynthesis of actinorhodin (Act), undecylprodigiosin (Red), and calcium-dependent antibiotic (CDA) are dramatically activated by introducing certain mutations into the rpoB gene that confer resistance to rifampin to Streptomyces lividans 66, which produces less or no antibiotics under normal growth conditions . Activation of Act and/or Red biosynthesis by inducing mutations in the rpoB gene was shown to be dependent on the mutation's position and the amino acid species substituted in the ß-subunit of the RNA polymerase . Mutation analysis identified 15 different kinds of point mutations, which are located in region I, II, or III of the rpoB gene and, in addition, two novel mutations (deletion of nucleotides 1287 to 1289 and a double substitution at nucleotides 1309 and 1310) were also found . Western blot analyses and S1 mapping analyses demonstrated that the expression of actII-ORF4 and redD, which are pathway-specific regulatory genes for Act and Red, respectively, was activated in the mutants able to produce Act and Red . The ActIV-ORF1 protein (an enzyme for Act biosynthesis) and the RedD protein were produced just after the upregulation of ActII-ORF4 and RedZ, respectively . These results indicate that the mutation in the rpoB gene of S . lividans, resulting in the activation of Act and/or Red biosynthesis, functions at the transcription level by activating directly or indirectly the key regulatory genes, actII-ORF4 and redD . We propose that the mutated RNA polymerase may function by mimicking the ppGpp-bound form in activating the onset of secondary metabolism in Streptomyces . Potential Rates of Fermentation in Digesta from the Gastrointestinal Tract of Pigs: Effect of Feeding Fermented Liquid Feed. Ole Højberg, 2003.Microbial catabolic capacity in digesta from the gastrointestinal tract of pigs fed either dry feed or fermented liquid feed (FLF) was determined with the PhenePlate multisubstrate system . The in vitro technique was modified to analyze the kinetics of substrate catabolism mediated by the standing stock of enzymes (potential rates of fermentation), allowing a quantitative evaluation of the dietary effect on the catabolic capacity of the microbiota . In total, the potential rates of fermentation were significantly reduced in digesta from the large intestine (cecum, P < 0.1; colon, P < 0.01; and rectum, P < 0.0001) of pigs fed FLF compared to pigs fed dry feed . No effect of diet was observed in the stomach (P = 0.71) or the distal part of the small intestine (P = 0.97) . The highest rates of fermentation and the most significant effect of diet were observed for readily fermentable carbohydrates like maltose, sucrose, and lactose . Feeding FLF to pigs also led to a reduction in the large intestine of the total counts of anaerobic bacteria in general and lactic acid bacteria specifically, as well as of microbial activity, as determined by the concentration of ATP and short-chain fatty acids . The low-molecular-weight carbohydrates were fermented mainly to lactic acid in the FLF before being fed to the animals . This may have limited microbial nutrient availability in the digesta reaching the large intestine of pigs fed FLF and may have caused the observed reduction in activity and density of the cecal and colonic microbial population . On the other hand, feeding FLF to pigs reduced the viable counts of coliform bacteria (indicator of Escherichia coli and Salmonella spp.) most profoundly in the stomach and the distal part of the small intestine, probably due to the bactericidal effect of lactic acid and low pH . The results presented clearly demonstrate that feeding FLF to pigs had a great impact on the indigenous microbiota, as reflected in bacterial numbers, short-chain fatty acid concentration, and substrate utilization . However, completely different mechanisms may be involved in the proximal and the distal parts of the gastrointestinal tract . The present study illustrates the utility of the PhenePlate system for quantifying the catabolic capacity of the indigenous gastrointestinal tract microbiota . Population Genetics of Vibrio vulnificus: Identification of Two Divisions and a Distinct Eel-Pathogenic Clone. Michaela Gutacker, 2003.Genetic relationships among 62 Vibrio vulnificus strains of different geographical and host origins were analyzed by multilocus enzyme electrophoresis (MLEE), random amplification of polymorphic DNA (RAPD), and sequence analyses of the recA and glnA genes . Out of 15 genetic loci analyzed by MLEE, 11 were polymorphic . Cluster analysis identified 43 distinct electrophoretic types (ETs) separating the V . vulnificus population into two divisions (divisions I and II) . One ET (ET 35) included all indole-negative isolates from diseased eels worldwide (biotype 2) . A second ET (ET 2) marked all of the strains from Israel isolated from patients who handled St . Peter's fish (biotype 3) . RAPD analysis of the 62 V . vulnificus isolates identified 26 different profiles separated into two divisions as well . In general, this subdivision was comparable (but not identical) to that observed by MLEE . Phylogenetic analysis of 543 bp of the recA gene and of 402 bp of the glnA gene also separated the V . vulnificus population into two major divisions in a manner similar to that by MLEE and RAPD . Sequence data again indicated the overall subdivision of the V . vulnificus population into different biotypes . In particular, indole-negative eel-pathogenic isolates (biotype 2) on one hand and the Israeli isolates (biotype 3) on the other tended to cluster together in both gene trees . None of the methods showed an association between distinct clones and human clinical manifestations . Furthermore, except for the Israeli strains, only minor clusters comprising geographically related isolates were observed . In conclusion, all three approaches (MLEE, RAPD, and DNA sequencing) generated comparable but not always equivalent results . The significance of the two divisions (divisions I and II) still remains to be clarified, and a reevaluation of the definition of the biotypes is also needed .
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