Yakovleva G M, Kim S K, Wanner B L
Department of Biological Science, Purdue University, West Lafayette, IN 47907, USA.
Appl Microbiol Biotechnol. 1998 May;49(5):573-8. doi: 10.1007/s002530051215.
On the basis of mutational analysis, the genes for phosphonate uptake and degradation in Escherichia coli were shown to be organized in a 10.9-kb operon of 14 genes (named phnC to phnP) and induced by phosphate (P(i)) starvation [Metcalf and Wanner (1993) J Bacteriol 175: 3430-3442]. The repression of phosphonate utilization by P(i) has hindered both the biochemical characterization of the carbon-phosphorus (C-P) lyase activity and the development of improved methods for phosphonate biodegradation in biotechnology. We have cloned the genes phnG to phnP (associated with C-P lyase activity) with the lac promoter to provide expression of C-P lyase in the presence of P(i). A number of strains lacking portions of the phn operon have been constructed. In vivo complementation of the strains, in which phnC to phnP (including both Pn transport and catalysis genes) or phnH to phnP (including only catalysis genes) was deleted, with plasmids carrying various fragments of the phn operon revealed that the expression of phnC-phnP gene products is essential to restore growth on minimal medium with phosphonate as the sole phosphorus source, while phnG-phnM gene products are required for C-P lyase activity as assessed by in vivo methane production from methylphosphonic acid. The minimum size of the DNA required for the whole-cell C-P lyase activity has been determined to be a 5.8-kb fragment, encompassing the phnG to phnM genes. Therefore, there is no requirement for the phn CDE-encoded phosphate transport system, suggesting that cleavage of the C-P bond may occur on the outer surface of the inner membrane of E. coli cells, releasing the carbon moiety into the periplasm. These data are in agreement with the observation that phosphonates cannot serve as the carbon source for E. coli growth.
基于突变分析，已表明大肠杆菌中负责膦酸盐摄取和降解的基因被组织在一个由14个基因组成的10.9 kb操纵子中（命名为phnC至phnP），并受磷酸盐（P(i)）饥饿诱导[梅特卡夫和万纳（1993年）《细菌学杂志》175: 3430 - 3442]。P(i)对膦酸盐利用的抑制作用既阻碍了碳 - 磷（C - P）裂解酶活性的生化特性研究，也阻碍了生物技术中膦酸盐生物降解改进方法的开发。我们已克隆了phnG至phnP基因（与C - P裂解酶活性相关）并带有lac启动子，以便在有P(i)存在的情况下表达C - P裂解酶。已构建了许多缺失phn操纵子部分片段的菌株。用携带phn操纵子各种片段的质粒对缺失phnC至phnP（包括Pn转运和催化基因）或phnH至phnP（仅包括催化基因）的菌株进行体内互补实验表明，phnC - phnP基因产物的表达对于在以膦酸盐作为唯一磷源的基本培养基上恢复生长至关重要，而phnG - phnM基因产物是C - P裂解酶活性所必需的，这可通过体内甲基膦酸产生甲烷来评估。已确定全细胞C - P裂解酶活性所需的最小DNA大小为一个5.8 kb的片段，包含phnG至phnM基因。因此，不需要phn CDE编码的磷酸盐转运系统，这表明C - P键的裂解可能发生在大肠杆菌细胞内膜的外表面，将碳部分释放到周质中。这些数据与膦酸盐不能作为大肠杆菌生长的碳源这一观察结果一致。
