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Overexpression of gnsA, a Multicopy Suppressor of the secG Null Mutation, Increases Acidic Phospholipid Contents by Inhibiting Phosphatidylethanolamine Synthesis at Low Temperatures. Rie Sugai, 2004.GnsA overproduction was previously found to suppress both the secG null mutation and the fabA6 mutation in Escherichia coli by increasing the unsaturated fatty acid contents . We report here that it also increased the acidic phospholipid contents at 20°C but not at 37°C . GnsA overproduction at 20°C specifically inhibited phosphatidylethanolamine synthesis and therefore caused the increase in the proportion of acidic phospholipids . Mutational Analysis of Transmembrane Regions 3 and 4 of SecY, a Central Component of Protein Translocase. Hiroyuki Mori, 2004.The SecYEG heterotrimeric membrane protein complex functions as a channel for protein translocation across the Escherichia coli cytoplasmic membrane . SecY is the central subunit of the SecYEG complex and contains 10 transmembrane segments (TM1 to TM10) . Previous mutation studies suggested that TM3 and TM4 are particularly important for SecY function . To further characterize TM3 and TM4, we introduced a series of cysteine-scanning mutations into these segments . With one exception (an unstable product), all the mutant proteins complemented the cold-sensitive growth defect of the secY39 mutant . A combination of this secY mutation and the secG deletion resulted in synthetic lethality, and the TM3 and TM4 SecY cysteine substitution mutations were examined for their ability to complement this lethality . Although they were all positive for complementation, some of the complemented cells exhibited significant retardation of protein export . The substitution-sensitive residues in TM3 can be aligned to one side of the alpha-helix, and those in TM4 revealed a tendency for residues closer to the cytosolic side of the membrane to be more severely affected . Disulfide cross-linking experiments identified a specific contact point for TM3 and SecG TM2 as well as for TM4 and SecG TM1 . Thus, although TM3 and TM4 do not contain any single residue that is absolutely required, they include functionally important helix surfaces and specific contact points with SecG . These results are discussed in light of the structural information available for the SecY complex . Biodegradation of Chlorpyrifos by Enterobacter Strain B-14 and Its Use in Bioremediation of Contaminated Soils. Brajesh K. Singh, 2004.Six chlorpyrifos-degrading bacteria were isolated from an Australian soil and compared by biochemical and molecular methods . The isolates were indistinguishable, and one (strain B-14) was selected for further analysis . This strain showed greatest similarity to members of the order Enterobacteriales and was closest to members of the Enterobacter asburiae group . The ability of the strain to mineralize chlorpyrifos was investigated under different culture conditions, and the strain utilized chlorpyrifos as the sole source of carbon and phosphorus . Studies with ring or uniformly labeled [14C]chlorpyrifos in liquid culture demonstrated that the isolate hydrolyzed chlorpyrifos to diethylthiophospshate (DETP) and 3, 5, 6-trichloro-2-pyridinol, and utilized DETP for growth and energy . The isolate was found to possess mono- and diphosphatase activities along with a phosphotriesterase activity . Addition of other sources of carbon (glucose and succinate) resulted in slowing down of the initial rate of degradation of chlorpyrifos . The isolate degraded the DETP-containing organophosphates parathion, diazinon, coumaphos, and isazofos when provided as the sole source of carbon and phosphorus, but not fenamiphos, fonofos, ethoprop, and cadusafos, which have different side chains . Studies of the molecular basis of degradation suggested that the degrading ability could be polygenic and chromosome based . Further studies revealed that the strain possessed a novel phosphotriesterase enzyme system, as the gene coding for this enzyme had a different sequence from the widely studied organophosphate-degrading gene (opd) . The addition of strain B-14 (106 cells g–1) to soil with a low indigenous population of chlorpyrifos-degrading bacteria treated with 35 mg of chlorpyrifos kg–1 resulted in a higher degradation rate than was observed in noninoculated soils . These results highlight the potential of this bacterium to be used in the cleanup of contaminated pesticide waste in the environment . Performance of Standard Phenotypic Assays for TonB Activity, as Evaluated by Varying the Level of Functional, Wild-Type TonB. Ray A. Larsen, 2003.The ability of gram-negative bacterial cells to transport cobalamin and iron-siderophore complexes and their susceptibility to killing by some bacteriophages and colicins are characteristics routinely used to assay mutations of proteins in the TonB-dependent energy transduction system . These assays vary greatly in sensitivity and are subject to perturbation by overexpression of TonB and, perhaps, other proteins that contribute to the process . Thus, the choice of assay and the means by which a potential mutant is expressed can greatly influence the interpretation and recognition of a given mutant . In the present study, we expressed TonB at several different quantified levels in cells that were then subjected to a panel of assays . Our results suggest that it is reasonable to regard the assays as having windows of sensitivity . Thus, while no single assay satisfactorily spans the potential range of TonB activity, it is evident that certain assays are better suited for resolving small deviations from wild-type levels of activity, with others most useful when activity levels are very low . It is apparent from the results that the application of all possible assays to the characterization of new mutants will yield the most meaningful results .
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