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The Sinorhizobium meliloti ABC Transporter Cho Is Highly Specific for Choline and Expressed in Bacteroids from Medicago sativa Nodules. Laurence Dupont, 2004.In Sinorhizobium meliloti, choline is the direct precursor of phosphatidylcholine, a major lipid membrane component in the Rhizobiaceae family, and glycine betaine, an important osmoprotectant. Moreover, choline is an efficient energy source which supports growth . Using a PCR strategy, we identified three chromosomalgenes [choXWV] which encode components of an ABC transporter:ChoX [binding protein], ChoW [permease], and ChoV [ATPase].Whereas the best homology scores were obtained with componentsof betaine ProU-like systems, Cho is not involved in betainetransport . Site-directed mutagenesis of choX strongly reduced[60 to 75%] the choline uptake activity, and purification ofChoX, together with analysis of the ligand-binding specificity,showed that ChoX binds choline with a high affinity [KD, 2.7µM] and acetylcholine with a low affinity [KD, 145 µM]but binds none of the betaines . Uptake competition experimentsalso revealed that ectoine, various betaines, and choline derivativeswere not effective competitors for Cho-mediated choline transport.Thus, Cho is a highly specific high-affinity choline transporter.Choline transport activity and ChoX expression were inducedby choline but not by salt stress . Western blotting experimentswith antibodies raised against ChoX demonstrated the presenceof ChoX in bacteroids isolated from nitrogen-fixing nodulesobtained from Medicago sativa roots . The choX mutation did nothave an effect on growth under standard conditions, and neitherNod nor Fix phenotypes were impaired in the mutant, suggestingthat the remaining choline uptake system[s] still present inthe mutant strain can compensate for the lack of Cho transporter. On the High Value of Low Standards. Elbert Branscomb, 2002. Factors Contributing to Hydrogen Peroxide Resistance in Streptococcus pneumoniae Include Pyruvate Oxidase (SpxB) and Avoidance of the Toxic Effects of the Fenton Reaction. Christopher D. Pericone, 2003.Aerobic growth of Streptococcus pneumoniae results in production of amounts of hydrogen peroxide (H2O2) that may exceed 1 mM in the surrounding media . H2O2 production by S . pneumoniae has been shown to kill or inhibit the growth of other respiratory tract flora, as well as to have cytotoxic effects on host cells and tissue . The mechanisms allowing S . pneumoniae, a catalase-deficient species, to survive endogenously generated concentrations of H2O2 that are sufficient to kill other bacterial species is unknown . In the present study, pyruvate oxidase (SpxB), the enzyme responsible for endogenous H2O2 production, was required for survival during exposure to high levels (20 mM) of exogenously added H2O2 . Pretreatment with H2O2 did not increase H2O2 resistance in the mutant, suggesting that SpxB activity itself is required, rather than an H2O2-inducible pathway . SpxB mutants synthesized 85% less acetyl-phosphate, a potential source of ATP . During H2O2 exposure, ATP levels decreased more rapidly in spxB mutants than in wild-type cells, suggesting that the increased killing of spxB mutants was due to more rapid ATP depletion . Together, these data support the hypothesis that S . pneumoniae SpxB contributes to an H2O2-resistant energy source that maintains viability during oxidative stress . Thus, SpxB is required for resistance to the toxic by-product of its own activity . Although H2O2-dependent hydroxyl radical production and the intracellular concentration of free iron were similar to that of Escherichia coli, killing by H2O2 was unaffected by iron chelators, suggesting that S . pneumoniae has a novel mechanism to avoid the toxic effects of the Fenton reaction .
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