Gas chromatographic assay for in vitro complementation of Pseudomonas aeruginosa mutants deficient in nitrate reduction
Gas chromatographic assay for in vitro complementation of Pseudomonas aeruginosa mutants deficient in nitrate reduction
复制标题
气相色谱法测定硝酸盐还原缺陷的铜绿假单胞菌突变体的体外补充
DOI：
10.1128/aem.49.1.24-27.1985
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发表时间：
1985
影响因子：
4.4
通讯作者：
J. Rowe
中科院分区：
文献类型：
--
作者：
D. Hernandez;J. Rowe
开发了一种电子捕获气相色谱技术，以连续测量硝酸盐（NO3-）还原在体外互补试验中与提取物铜绿假单胞菌突变体缺乏同化和异化硝酸盐还原作为一个单一的基因突变的结果。该程序涉及耦合硝酸盐还原一氧化二氮（N2 O）的演变通过一系列特定的反硝化途径的反应。该测定依赖于硝酸盐浓度，并且在0.2%硝酸钾的终浓度下获得最佳活性。还原表现出2：1的化学计量比（NO3-/N2 O），和琥珀酸是最好的电子源的反应，其次是葡萄糖，丙酮酸，和苹果酸。该技术还可用于连续监测硝酸盐还原。亚硝酸还原酶测定的最佳亚硝酸盐浓度为0.025%。突变体提取物的初步互补研究表明，铜绿假单胞菌中的两个硝酸盐还原途径之间至少有两个基因是共享的。
An electron capture gas-chromatographic technique was developed to continuously measure nitrate (NO3-) reduction during in vitro complementation tests with extracts from Pseudomonas aeruginosa mutants deficient in both assimilatory and dissimilatory nitrate reduction as a result of a single genetic mutation. The procedure involves coupling nitrate reduction to nitrous oxide (N2O) evolution via a series of reactions specific to the denitrification pathway. The assay was dependent on nitrate concentration, and optimal activity was obtained with a final concentration of 0.2% potassium nitrate. The reduction exhibited a stoichiometry of 2:1 (NO3-/N2O), and succinate was the best electron source for the reaction, followed by glucose, pyruvate, and malate. The technique can also be used for continuously monitoring nitrate reduction. The optimal nitrite concentration in the nitrite reductase assay was 0.025%. The initial complementation studies of mutant extracts demonstrated that at least two genes are shared between the two nitrate reduction pathways in P. aeruginosa.