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A LuxR Homolog Controls Production of Symbiotically Active Extracellular Polysaccharide II by Sinorhizobium meliloti. Brett J. Pellock, 2002.Production of complex extracellular polysaccharides (EPSs) by the nitrogen-fixing soil bacterium Sinorhizobium meliloti is required for efficient invasion of root nodules on the host plant alfalfa . Any one of three S . meliloti polysaccharides, succinoglycan, EPS II, or K antigen, can mediate infection thread initiation and extension (root nodule invasion) on alfalfa . Of these three polysaccharides, the only symbiotically active polysaccharide produced by S . meliloti wild-type strain Rm1021 is succinoglycan . The expR101 mutation is required to turn on production of symbiotically active forms of EPS II in strain Rm1021 . In this study, we have determined the nature of the expR101 mutation in S . meliloti . The expR101 mutation, a spontaneous dominant mutation, results from precise, reading frame-restoring excision of an insertion sequence from the coding region of expR, a gene whose predicted protein product is highly homologous to the Rhizobium leguminosarum bv . viciae RhiR protein and a number of other homologs of Vibrio fischeri LuxR that function as receptors for N-acylhomoserine lactones (AHLs) in quorum-sensing regulation of gene expression . S . meliloti ExpR activates transcription of genes involved in EPS II production in a density-dependent fashion, and it does so at much lower cell densities than many quorum-sensing systems . High-pressure liquid chromatographic fractionation of S . meliloti culture filtrate extracts revealed at least three peaks with AHL activity, one of which activated ExpR-dependent expression of the expE operon . Mechanism of cis-trans Isomerization of Unsaturated Fatty Acids in Pseudomonas putida. Angelika von Wallbrunn, 2003.We studied the pattern of the cis-trans isomerization of unsaturated fatty acids in cells of Pseudomonas putida S12 grown in a medium supplemented with oleic acid which was deuterated at both of the C atoms of its double bond . Direct evidence that isomerization does not include a transient saturation of the double bond was obtained . In addition, analysis of the amino acid sequences of the seven known Cti proteins identified them as heme-containing proteins of the cytochrome c type . Production of a Doubly Chiral Compound, (4R,6R)-4-Hydroxy-2,2,6-Trimethylcyclohexanone, by Two-Step Enzymatic Asymmetric Reduction. Masaru Wada, 2003.A practical enzymatic synthesis of a doubly chiral key compound, (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone, starting from the readily available 2,6,6-trimethyl-2-cyclohexen-1,4-dione is described . Chirality is first introduced at the C-6 position by a stereoselective enzymatic hydrogenation of the double bond using old yellow enzyme 2 of Saccharomyces cerevisiae, expressed in Escherichia coli, as a biocatalyst . Thereafter, the carbonyl group at the C-4 position is reduced selectively and stereospecifically by levodione reductase of Corynebacterium aquaticum M-13, expressed in E . coli, to the corresponding alcohol . Commercially available glucose dehydrogenase was also used for cofactor regeneration in both steps . Using this two-step enzymatic asymmetric reduction system, 9.5 mg of (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone/ml was produced almost stoichiometrically, with 94% enantiomeric excess in the presence of glucose, NAD+, and glucose dehydrogenase . To our knowledge, this is the first report of the application of S . cerevisiae old yellow enzyme for the production of a useful compound . Use of Pulsed-Field Gel Electrophoresis To Characterize the Heterogeneity and Clonality of Salmonella Isolates Obtained from the Carcasses and Feces of Swine at Slaughter. Laura Wonderling, 2003.Salmonella enterica isolates were recovered from swine at a collaborating processing plant over a 2-month period in the spring of 2000 . In the present study, molecular subtyping by pulsed-field gel electrophoresis (PFGE) was performed on the 581 confirmed Salmonella isolates from the 84 Salmonella-positive samples obtained from the previous study . A total of 32 different PFGE pulsotypes were observed visually, and a BioNumerics software analysis clustered those pulsotypes into 12 PFGE groups . The B, F, and G groups predominated throughout the sampling period and were isolated from 39, 22, and 13% of the swine, respectively . In addition, multiple isolates were obtained from 67 of the 84 Salmonella-positive samples, and subtyping revealed multiple PFGE profiles in 35 of these 67 (62%) samples . Both carcass and fecal isolates of Salmonella were recovered from 13 swine, resulting in "matched" samples . Molecular typing of the 252 isolates recovered from the matched samples revealed that 7 (54%) of the 13 carcasses were contaminated with Salmonella pulsotypes that were not isolated from the feces of the same animal . Conversely, from 6 of the 13 (46%) matched animals, Salmonella clonal types were isolated from the feces that were not isolated from the carcass of the same animal . These data establish that each lot of swine introduces new contaminants into the plant environment and that swine feces from one animal can contaminate many carcasses . In addition, these results indicate that the examination of multiple Salmonella isolates from positive samples is necessary to determine the variety of potential contaminants of swine carcasses during slaughter and processing .
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