Cloning and sequencing of two tandem genes involved in degradation of 2,3-dihydroxybiphenyl to benzoic acid in the polychlorinated biphenyl-degrading soil bacterium Pseudomonas sp. strain KKS102
Cloning and sequencing of two tandem genes involved in degradation of 2,3-dihydroxybiphenyl to benzoic acid in the polychlorinated biphenyl-degrading soil bacterium Pseudomonas sp. strain KKS102
复制标题
多氯联苯降解土壤细菌假单胞菌中参与将 2,3-二羟基联苯降解为苯甲酸的两个串联基因的克隆和测序。
作者：
K. Kimbara;Toshiyuki Hashimoto;Masao Fukuda;Takao Koana;Masahiro Takagi;Michio Oishi;K. Yano
利用广谱粘粒载体pKS 13，从土壤中降解多氯联苯的假单胞菌KKS 102的基因文库中分离到两个降解联苯的相关基因。当将29-kb粘粒DNA插入物的3.2-内切酶（kb）Pst I片段亚克隆到pUC 18 lacZ启动子下游的Pst I位点时，携带该重组质粒的大肠杆菌细胞表达2，3-二羟基联苯双加氧酶活性。3.2-kb Pst I片段的核苷酸测序显示有两个开放阅读框（ORFI [882碱基对]和ORFII [834碱基对]，按此基因顺序）。对Tn 5插入突变体和单向缺失突变体的分析结果表明，ORFI编码2，3-二羟基联苯双加氧酶。将ORFI基因序列与假产碱假单胞菌KF 707（K.古鲁卡河，加-地Arima和T.宫崎，细菌学杂志。169：427-429，1987），同源性为68%，两种菌株具有相同的Shine-Dalgarno序列。代谢产物的气相色谱-质谱分析结果表明，ORFII具有Meta裂解化合物水解酶活性，可产生苯甲酸。DNA测序结果表明，这两个基因包含在一个操纵子中。
Two genes involved in the degradation of biphenyl were isolated from a gene library of a polychlorinated biphenyl-degrading soil bacterium, Pseudomonas sp. strain KKS102, by using a broad-host-range cosmid vector, pKS13. When a 3.2-kilobase (kb) PstI fragment of a 29-kb cosmid DNA insert was subcloned into pUC18 at the PstI site downstream of the lacZ promoter, Escherichia coli cells carrying this recombinant plasmid expressed 2,3-dihydroxybiphenyl dioxygenase activity. Nucleotide sequencing of the 3.2-kb PstI fragment revealed that there were two open reading frames (ORFI [882 base pairs] and ORFII [834 base pairs], in this gene order). Results of analysis of Tn5 insertion mutants and unidirectional deletion mutants suggested that the ORFI coded for 2,3-dihydroxybiphenyl dioxygenase. When the sequence of ORFI was compared with that of bphC of Pseudomonas pseudoalcaligenes KF707 (K. Furukawa, N. Arima, and T. Miyazaki, J. Bacteriol. 169:427-429, 1987), the homology was 68%, with both strains having the same Shine-Dalgarno sequence. The result of gas chromatography-mass spectrometry analysis of the metabolic product suggested that the ORFII had meta cleavage compound hydrolase activity to produce benzoic acid. DNA sequencing suggested that these two genes were contained in one operon.