Identification of an alternative 2,3-dihydroxybiphenyl 1,2-dioxygenase in Rhodococcus sp strain RHA1 and cloning of the gene
Identification of an alternative 2,3-dihydroxybiphenyl 1,2-dioxygenase in Rhodococcus sp strain RHA1 and cloning of the gene
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DOI：
10.1128/aem.62.8.2940-2946.1996
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发表时间：
1996-08-01
影响因子：
4.4
通讯作者：
Yano, K
中科院分区：
文献类型：
--
作者：
Hauschild, JE;Masai, E;Yano, K
革兰氏阳性红球菌菌株RHA 1具有很强的多氯联苯降解能力。Masai，A.山田，J.M.希利，T. Hatta，K. Kimbara，M. Fukuda和K. Yano，Appl. Microbiol. 61：2079-2085，1995）。另一种2，3-二羟基联苯1，2-双加氧酶（2，3-DHBD），命名为EtbC，在生长在大肠杆菌上的RDC 1细胞中鉴定。EtbC含有迄今为止在红球菌属菌株中鉴定的任何Meta裂解双加氧酶的最广泛的底物特异性，包括RHA 1 BphC。从生长在大肠杆菌上的RDC 1细胞中纯化出几乎均一的EtbC，并测定了58个氨基酸的NH 2-末端序列。NH 2-末端氨基酸序列用于从RDC 1染色体DNA 2，3-DHBD表达文库中鉴定etbC基因。成功克隆了etbC基因，并测定了其核苷酸序列。细胞提取物的底物特异性模式和天然非变性聚丙烯酰胺凝胶电泳分析确定了两个2，3-DHBD（BphC和EtbC）在RHA 1细胞中的共表达，无论是在联苯或联苯上生长。共表达的BphC extradiol双加氧酶在RHA 1 nas的强多氯联苯降解活性中的可能含义表明。
Gram-positive Rhodococcus sp. strain RHA1 possesses strong polychlorinated biphenyl-degrading capabilities, An RHA1 bphC gene mutant, strain RDC1, had been previously constructed (E. Masai, A. Yamada, J. M. Healy, T. Hatta, K. Kimbara, M. Fukuda, and K. Yano, Appl. Environ. Microbiol. 61:2079-2085, 1995). An alternative 2,3-dihydroxybiphenyl 1,2-dioxygenase (2,3-DHBD), designated EtbC, was identified in RDC1 cells grown on ethylbenzene. EtbC contained the broadest substrate specificity of any meta cleavage dioxygenase identified in a Rhodococcus strain to date, including RHA1 BphC. EtbC was purified to near homogeneity from RDC1 cells grown on ethylbenzene, and a 58-amino-acid NH2-terminal sequence was determined, The NH2-terminal amino acid sequence was used for the identification of the etbC gene from an RDC1 chromosomal DNA 2,3-DHBD expression library. The etbC gene was successfully cloned, and we report here the determination of its nucleotide sequence. The substrate specificity patterns of cell extract and native nondenaturing polyacrylamide gel electrophoresis analysis identified the coexpression of two 2,3-DHBDs (BphC and EtbC) in RHA1 cells grown on either biphenyl or ethylbenzene. The possible implication of coexpressed BphC extradiol dioxygenases in the strong polychlorinated-biphenyl degradation activity of RHA1 nas suggested.