Furukawa K, Hayashida S, Taira K
Department of Agricultural Chemistry, Kyushu University, Fukuoka, Japan.
Gene. 1991 Feb 1;98(1):21-8. doi: 10.1016/0378-1119(91)90099-w.
A transposon, Tn5-B21, was gene-specifically inserted into the chromosomal biphenyl/polychlorinated biphenyl-catabolic operon (bph operon) of soil bacteria. The cloned bphA, bphB and bphC genes of Pseudomonas pseudoalcaligenes KF707, coding for conversion of biphenyl into a ring meta-cleavage product (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid), carried random insertions of Tn5-B21. The mutagenized bphABC DNA, carried by a suicide plasmid, was introduced back into the parent strain KF707, resulting in the appearance of gene-specific transposon mutants by double crossover homologous recombination: the bphA::Tn5-B21 mutant did not attack 4-chlorobiphenyl, the bphB::Tn5-B21 mutant accumulated dihydrodiol, and the bphC::Tn5-B21 mutant produced dihydroxy compound. Gene-specific transposon mutants of the bph operon were also obtained for some other biphenyl-utilizing strains which possess bph operons nearly identical to that of KF707.
转座子Tn5-B21被基因特异性地插入到土壤细菌的染色体联苯/多氯联苯分解代谢操纵子（bph操纵子）中。假产碱假单胞菌KF707的克隆bphA、bphB和bphC基因编码联苯转化为间位环裂解产物（2-羟基-6-氧代-6-苯基己-2,4-二烯酸），这些基因带有Tn5-B21的随机插入。由自杀质粒携带的诱变bphABC DNA被重新导入亲本菌株KF707，通过双交换同源重组产生基因特异性转座子突变体：bphA::Tn5-B21突变体不攻击4-氯联苯，bphB::Tn5-B21突变体积聚二氢二醇，bphC::Tn5-B21突变体产生二羟基化合物。对于其他一些具有与KF707几乎相同的bph操纵子的联苯利用菌株，也获得了bph操纵子的基因特异性转座子突变体。
