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Biochemical Characterization of the Naturally Occurring Oxacillinase OXA-50 of Pseudomonas aeruginosa. Delphine Girlich, 2004.The blaOXA-50 gene (formerly known as the PA5514 gene) is an oxacillinase gene identified in silico in the genome of Pseudomonas aeruginosa PAO1 . By using a mutant strain of P . aeruginosa PAO1 that had an inactivated blaAmpC cephalosporinase gene, the blaOXA-50 gene was shown to be expressed constitutively in P . aeruginosa . This ß-lactamase gene was cloned onto a multicopy plasmid and expressed in P . aeruginosa and Escherichia coli . It conferred decreased susceptibility to ampicillin and ticarcillin and, interestingly, to moxalactam and meropenem in P . aeruginosa but not in E . coli . Overexpression and purification enabled us to determine the molecular mass (25 kDa), the pI value (8.6), and the hydrolysis spectrum of the OXA-50 ß-lactamase . It is a narrow-spectrum oxacillinase that uncommonly hydrolyzes imipenem, although at a low level . Very similar oxacillinase genes were identified in all P . aeruginosa isolates from various geographical origins tested . The weak variability of the nucleotide sequence of this gene (0 to 2%) corresponded to that found for the naturally occurring blaAmpC cephalosporinase gene of P . aeruginosa . The study indicated that P . aeruginosa harbors two naturally encoded ß-lactamase genes, one of which encodes an inducible cephalosporinase and the other of which encodes a constitutively expressed oxacillinase . Lack of Protective Osmolytes Limits Final Cell Density and Volumetric Productivity of Ethanologenic Escherichia coli KO11 during Xylose Fermentation. S. A. Underwood, 2004.Limited cell growth and the resulting low volumetric productivity of ethanologenic Escherichia coli KO11 in mineral salts medium containing xylose have been attributed to inadequate partitioning of carbon skeletons into the synthesis of glutamate and other products derived from the citrate arm of the anaerobic tricarboxylic acid pathway . The results of nuclear magnetic resonance investigations of intracellular osmolytes under different growth conditions coupled with those of studies using genetically modified strains have confirmed and extended this hypothesis . During anaerobic growth in mineral salts medium containing 9% xylose (600 mM) and 1% corn steep liquor, proline was the only abundant osmolyte (71.9 nmol ml–1 optical density at 550 nm [OD550] unit–1), and growth was limited . Under aerobic conditions in the same medium, twice the cell mass was produced, and cells contained a mixture of osmolytes: glutamate (17.0 nmol ml–1 OD550 unit–1), trehalose (9.9 nmol ml–1 OD550 unit–1), and betaine (19.8 nmol ml–1 OD550 unit–1) . Two independent genetic modifications of E . coli KO11 (functional expression of Bacillus subtilis citZ encoding NADH-insensitive citrate synthase; deletion of ackA encoding acetate kinase) and the addition of a metabolite, such as glutamate (11 mM) or acetate (24 mM), as a supplement each increased the intracellular glutamate pool during fermentation, doubled cell growth, and increased volumetric productivity . This apparent requirement for a larger glutamate pool for increased growth and volumetric productivity was completely eliminated by the addition of a protective osmolyte (2 mM betaine or 0.25 mM dimethylsulfoniopropionate), consistent with adaptation to osmotic stress rather than relief of a specific biosynthetic requirement . Mutational Analysis of the Residue at Position 48 in the Salmonella enterica Serovar Typhimurium PhoQ Sensor Kinase. Sarah Sanowar, 2003.The PhoP/PhoQ two-component regulatory system of Salmonella enterica serovar Typhimurium plays an essential role in controlling virulence by mediating the adaptation to Mg2+ depletion . The pho-24 allele of phoQ harbors a single amino acid substitution (T48I) in the periplasmic domain of the PhoQ histidine kinase sensor . This mutation has been shown to increase net phosphorylation of the PhoP response regulator . We analyzed the effect on signaling by PhoP/PhoQ of various amino acid substitutions at this position (PhoQ-T48X [X = A, S, V, I, or L]) . Mutations T48V, T48I, and T48L were found to affect signaling by PhoP/PhoQ both in vivo and in vitro . Mutations PhoQ-T48V and PhoQ-T48I increased both the expression of the mgtA::lacZ transcriptional fusion and the net phosphorylation of PhoP, conferring to cells a PhoP constitutively active phenotype . In contrast, mutation PhoQ-T48L barely responded to changes in the concentration of external Mg2+, in vivo and in vitro, conferring to cells a PhoP constitutively inactive phenotype . By analyzing in vitro the individual catalytic activities of the PhoQ-T48X sensors, we found that the PhoP constitutively active phenotype observed for the PhoQ-T48V and PhoQ-T48I proteins is solely due to decreased phosphatase activity . In contrast, the PhoP constitutively inactive phenotype observed for the PhoQ-T48L mutant resulted from both decreased autokinase activity and increased phosphatase activity . Our data are consistent with a model in which the residue at position 48 of PhoQ contributes to a conformational switch between kinase- and phosphatase-dominant states . A Synechococcus PglnA::luxAB Fusion for Estimation of Nitrogen Bioavailability to Freshwater Cyanobacteria. Osnat Gillor, 2003.In contrast to extensive studies of phosphorus, widely considered the main nutrient limiting phytoplankton biomass in freshwater ecosystems, there have been few studies on the role of nitrogen in controlling phytoplankton populations . This situation may be due partly to the complexity in estimating its utilization and bioavailability . In an attempt to provide a novel tool for this purpose, we fused the promoter of the glutamine synthetase-encoding gene, P glnA, from Synechococcus sp . strain PCC7942 to the luxAB luciferase-encoding genes of the bioluminescent bacterium Vibrio harveyi . The resulting construct was introduced into a neutral site on the Synechococcus chromosome to yield the reporter strain GSL . Light emission by this strain was dependent upon ambient nitrogen concentrations . The linear response range of the emitted luminescence was 1 mM to 1 µM for the inorganic nitrogen species tested (ammonium, nitrate, and nitrite) and 10- to 50-fold lower for glutamine and urea . When water samples collected from along a depth profile in Lake Kinneret (Israel) were exposed to the reporter strain, the bioluminescence of the reporter strain mirrored the total dissolved nitrogen concentrations determined for the same samples and was shown to be a sensitive indicator of the concentration of bioavailable nitrogen .
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