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Comparative Genetic Diversity of Pseudomonas stutzeri Genomovars, Clonal Structure, and Phylogeny of the Species. Aina Maria Cladera, 2004.A combined phylogenetic and multilocus DNA sequence analysis of 26 Pseudomonas stutzeri strains distributed within the 9 genomovars of the species has been performed . Type strains of the two most closely related species (P . balearica, former genomovar 6, and P . mendocina), together with P . aeruginosa, as the type species of the genus, have been included in the study . The extremely high genetic diversity and the clonal structure of the species were confirmed by the sequence analysis . Clustering of strains in the consensus phylogeny inferred from the analysis of seven nucleotide sequences (16S ribosomal DNA, internally transcribed spacer region 1, gyrB, rpoD, nosZ, catA, and nahH) confirmed the monophyletic origin of the genomovars within the Pseudomonas branch and is in good agreement with earlier DNA-DNA similarity analysis, indicating that the selected genes are representative of the whole genome in members of the species . Peritoneal Fluid Titer Test for Peritoneal Dialysis-Related Peritonitis. Christine Strijack, 2004.Standard microbiological tests (i.e., MIC) do not account for the unique factors of peritoneal dialysis (PD)-related peritonitis which can significantly influence treatment response . Our goals were to develop a peritoneal fluid titer (PFT) test and to conduct a pilot study of its association with clinical outcome . The methodology was developed by using spent dialysate collected from patients with bacterial PD-related peritonitis prior to the initiation of antibiotics . Dialysate was processed and spiked with antibiotic to simulate two standard intraperitoneal regimens: cefazolin plus tobramycin and cefazolin alone . Thirty-six clinical isolates, including Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, and Pseudomonas aeruginosa, were tested . In the pilot study, dialysate was collected from 14 patients with bacterial PD-related peritonitis . Titers were determined by using each patient's dialysate and infecting pathogen . Titers were highly reproducible, with discrepancies in only 1% of cases . Overall, PFTs were notably higher against gram-positive bacteria (P < 0.0001) . The addition of tobramycin increased titers significantly from zero to values of 1/16 to 1/64 against E . cloacae and P . aeruginosa (P < 0.0001) . In the pilot study, peritoneal fluid inhibitory titers were significantly associated with clinical outcome, with a median value of 1/96 for patients who were cured compared to 1/32 for those who failed treatment (P = 0.036) . In conclusion, this study provides preliminary support for the PFT as a pharmacodynamic index specific to the treatment of PD-related peritonitis . With further characterization and validation in patients, the PFT test may advance the study of antibiotic therapies for PD-related peritonitis . Role of Hippoboscidae Flies as Potential Vectors of Bartonella spp . Infecting Wild and Domestic Ruminants. Lénaïg Halos, 2004. Dual Role of Cysteine 172 in Redox Regulation of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Activity and Degradation. Yehouda Marcus, 2003.Alkylation and oxidation of cysteine residues significantly decrease the catalytic activity and stimulate the degradation of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) . We analyzed the role of vicinal cysteine residues in redox regulation of RuBisCO from Synechocystis sp . strain PCC 6803 . Cys172 and Cys192, which are adjacent to the catalytic site, and Cys247, which cross-links two large subunits, were replaced by alanine . Whereas all mutant cells (C172A, C192A, C172A-C192A, and C247A) and the wild type grew photoautotrophically at similar rates, the maximal photosynthesis rates of C172A mutants decreased 10 to 20% as a result of 40 to 60% declines in RuBisCO turnover number . Replacement of Cys172, but not replacement of Cys192, prominently decreased the effect of cysteine alkylation or oxidation on RuBisCO . Oxidants that react with vicinal thiols had a less inhibitory effect on the activity of either the C172A or C192A enzyme variants, suggesting that a disulfide bond was formed upon oxidation . Thiol oxidation induced RuBisCO dissociation into subunits . This effect was either reduced in the C172A and C192A mutant enzymes or eliminated by carboxypentitol bisphosphate (CPBP) binding to the activated enzyme form . The CPBP effect presumably resulted from a conformational change in the carbamylated CPBP-bound enzyme, as implied from an alteration in the electrophoretic mobility . Stress conditions, provoked by nitrate deprivation, decreased the RuBisCO contents and activities in the wild type and in the C192A and C247A mutants but not in the C172A and C172A-C192A mutants . These results suggest that although Cys172 does not participate in catalysis, it plays a role in redox regulation of RuBisCO activity and degradation . Molecular Analysis of a Saccharomyces cerevisiae Mutant with Improved Ability To Utilize Xylose Shows Enhanced Expression of Proteins Involved in Transport, Initial Xylose Metabolism, and the Pentose Phosphate Pathway. C. Fredrik Wahlbom, 2003.Differences between the recombinant xylose-utilizing Saccharomyces cerevisiae strain TMB 3399 and the mutant strain TMB 3400, derived from TMB 3399 and displaying improved ability to utilize xylose, were investigated by using genome-wide expression analysis, physiological characterization, and biochemical assays . Samples for analysis were withdrawn from chemostat cultures . The characteristics of S . cerevisiae TMB 3399 and TMB 3400 grown on glucose and on a mixture of glucose and xylose, as well as of S . cerevisiae TMB 3400 grown on only xylose, were investigated . The strains were cultivated under chemostat conditions at a dilution rate of 0.1 h-1, with feeds consisting of a defined mineral medium supplemented with 10 g of glucose liter-1, 10 g of glucose plus 10 g of xylose liter-1 or, for S . cerevisiae TMB 3400, 20 g of xylose liter-1 . S . cerevisiae TMB 3400 consumed 31% more xylose of a feed containing both glucose and xylose than S . cerevisiae TMB 3399 . The biomass yields for S . cerevisiae TMB 3400 were 0.46 g of biomass g of consumed carbohydrate-1 on glucose and 0.43 g of biomass g of consumed carbohydrate-1 on xylose . A Ks value of 33 mM for xylose was obtained for S . cerevisiae TMB 3400 . In general, the percentage error was <20% between duplicate microarray experiments originating from independent fermentation experiments . Microarray analysis showed higher expression in S . cerevisiae TMB 3400 than in S . cerevisiae TMB 3399 for (i) HXT5, encoding a hexose transporter; (ii) XKS1, encoding xylulokinase, an enzyme involved in one of the initial steps of xylose utilization; and (iii) SOL3, GND1, TAL1, and TKL1, encoding enzymes in the pentose phosphate pathway . In addition, the transcriptional regulators encoded by YCR020C, YBR083W, and YPR199C were expressed differently in the two strains . Xylose utilization was, however, not affected in strains in which YCR020C was overexpressed or deleted . The higher expression of XKS1 in S . cerevisiae TMB 3400 than in TMB 3399 correlated with higher specific xylulokinase activity in the cell extracts . The specific activity of xylose reductase and xylitol dehydrogenase was also higher for S . cerevisiae TMB 3400 than for TMB 3399, both on glucose and on the mixture of glucose and xylose . Identification of a Novel Cryptosporidium Genotype in Pigs. U. M. Ryan, 2003.Over a 3-year period, a total of 646 fecal samples from pigs in 22 indoor and outdoor herds from Western Australia were screened for Cryptosporidium spp . by microscopy . Results revealed that 39 of 646 samples (6.03%) were positive for Cryptosporidium . Cryptosporidium was much more common in outdoor herds (17.2%) than in indoor herds (0.5%) and was more common in animals between the ages of 5 and 8 weeks (69.2%) than in younger animals (P < 0.0001) . Molecular characterization of the positive samples at the 18S ribosomal DNA locus identified two distinct genotypes of Cryptosporidium: the previously identified pig genotype I and a novel pig genotype (pig genotype II), both of which warrant species status .
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