Ledger T, Pieper D H, González B
Laboratorio de Microbiología, Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Casilla 114-D, Santiago, Chile.
Appl Environ Microbiol. 2006 Apr;72(4):2783-92. doi: 10.1128/AEM.72.4.2783-2792.2006.
Phenoxyalkanoic compounds are used worldwide as herbicides. Cupriavidus necator JMP134(pJP4) catabolizes 2,4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA), using tfd functions carried on plasmid pJP4. TfdA cleaves the ether bonds of these herbicides to produce 2,4-dichlorophenol (2,4-DCP) and 4-chloro-2-methylphenol (MCP), respectively. These intermediates can be degraded by two chlorophenol hydroxylases encoded by the tfdB(I) and tfdB(II) genes to produce the respective chlorocatechols. We studied the specific contribution of each of the TfdB enzymes to the 2,4-D/MCPA degradation pathway. To accomplish this, the tfdB(I) and tfdB(II) genes were independently inactivated, and growth on each chlorophenoxyacetate and total chlorophenol hydroxylase activity were measured for the mutant strains. The phenotype of these mutants shows that both TfdB enzymes are used for growth on 2,4-D or MCPA but that TfdB(I) contributes to a significantly higher extent than TfdB(II). Both enzymes showed similar specificity profiles, with 2,4-DCP, MCP, and 4-chlorophenol being the best substrates. An accumulation of chlorophenol was found to inhibit chlorophenoxyacetate degradation, and inactivation of the tfdB genes enhanced the toxic effect of 2,4-DCP on C. necator cells. Furthermore, increased chlorophenol production by overexpression of TfdA also had a negative effect on 2,4-D degradation by C. necator JMP134 and by a different host, Burkholderia xenovorans LB400, harboring plasmid pJP4. The results of this work indicate that codification and expression of the two tfdB genes in pJP4 are important to avoid toxic accumulations of chlorophenols during phenoxyacetic acid degradation and that a balance between chlorophenol-producing and chlorophenol-consuming reactions is necessary for growth on these compounds.
苯氧基链烷酸化合物在全球范围内被用作除草剂。食酸铜绿假单胞菌JMP134(pJP4)利用质粒pJP4上携带的tfd功能分解代谢2,4 - 二氯苯氧基乙酸(2,4 - D)和4 - 氯 - 2 - 甲基苯氧基乙酸(MCPA)。TfdA分别裂解这些除草剂的醚键，生成2,4 - 二氯苯酚(2,4 - DCP)和4 - 氯 - 2 - 甲基苯酚(MCP)。这些中间体可被tfdB(I)和tfdB(II)基因编码的两种氯酚羟化酶降解，生成相应的氯儿茶酚。我们研究了每种TfdB酶在2,4 - D/MCPA降解途径中的具体作用。为此，分别使tfdB(I)和tfdB(II)基因失活，并测定突变菌株在每种氯苯氧基乙酸上的生长情况以及总氯酚羟化酶活性。这些突变体的表型表明，两种TfdB酶都可用于在2,4 - D或MCPA上生长，但TfdB(I)的贡献程度明显高于TfdB(II)。两种酶表现出相似的底物特异性谱，2,4 - DCP、MCP和4 - 氯苯酚是最佳底物。发现氯酚的积累会抑制氯苯氧基乙酸的降解，tfdB基因的失活增强了2,4 - DCP对食酸铜绿假单胞菌细胞的毒性作用。此外，通过TfdA的过表达增加氯酚的产生，也对食酸铜绿假单胞菌JMP134以及携带质粒pJP4的另一个宿主——嗜麦芽窄食单胞菌LB400的2,4 - D降解产生负面影响。这项工作的结果表明，pJP4中两个tfdB基因的编码和表达对于避免苯氧基乙酸降解过程中氯酚的毒性积累很重要，并且氯酚生成反应和氯酚消耗反应之间的平衡对于在这些化合物上生长是必要的。
