Fitch M W, Graham D W, Arnold R G, Agarwal S K, Phelps P, Speitel G E, Georgiou G
Department of Chemical Engineering, University of Texas at Austin 78712.
Appl Environ Microbiol. 1993 Sep;59(9):2771-6. doi: 10.1128/aem.59.9.2771-2776.1993.
Cultures of Methylosinus trichosporium OB3b grown in the presence of very low concentrations of copper synthesize a soluble methane monooxygenase (sMMO) that efficiently catalyzes the oxidation of trichloroethylene and other organic pollutants. Recently, we isolated five M. trichosporium OB3b mutants that express sMMO activity when grown in the presence of elevated copper concentrations (P.A. Phelps, S. K. Agarwal, G. E. Speitel, Jr., and G. Georgiou, Appl. Environ. Microbiol. 58:3701-3708, 1992). Here we show that, in contrast to the results for the wild-type cells, the addition of copper to mutant cultures grown on methane and nitrate as the nitrogen source has no noticeable effect on the growth rate and sMMO expression. In vitro experiments indicated that the copper-resistant phenotype does not arise from an increased stability of sMMO to copper deactivation. Furthermore, the mutant cultures exhibit altered speciation of copper in the extracellular fluid and have substantially decreased levels of cell-associated copper. On the basis of these results, we propose that the mutant phenotype arises from defects in copper uptake and metabolism rather than from changes in sMMO expression or enzyme stability.
在极低浓度铜存在的情况下培养的甲基孢囊菌OB3b能够合成一种可溶性甲烷单加氧酶（sMMO），该酶能高效催化三氯乙烯和其他有机污染物的氧化反应。最近，我们分离出了五个甲基孢囊菌OB3b突变体，它们在高浓度铜存在的情况下生长时会表达sMMO活性（P.A. 菲尔普斯、S.K. 阿加瓦尔、G.E. 斯派特尔 Jr. 和G. 乔治乌，《应用与环境微生物学》58:3701 - 3708，1992年）。在此我们表明，与野生型细胞的结果相反，向以甲烷和硝酸盐作为氮源生长的突变体培养物中添加铜，对生长速率和sMMO表达没有显著影响。体外实验表明，抗铜表型并非源于sMMO对铜失活的稳定性增加。此外，突变体培养物在细胞外液中的铜形态发生了改变，且细胞相关铜的水平大幅降低。基于这些结果，我们提出突变体表型源于铜摄取和代谢的缺陷，而非sMMO表达或酶稳定性的变化。
