Horinouchi S, Kumada Y, Beppu T
J Bacteriol. 1984 May;158(2):481-7. doi: 10.1128/jb.158.2.481-487.1984.
We cloned a DNA fragment directing synthesis of A-factor from the total cellular DNA of streptomycin-producing Streptomyces bikiniensis on the plasmid vector pIJ385 . Introduction of the recombinant plasmid ( pAFB1 ) into A-factor-deficient S. bikiniensis and Streptomyces griseus mutants led to A-factor production in the host cells, as a result of which streptomycin production, streptomycin resistance, and spore formation of these mutants were simultaneously restored. The plasmid pAFB1 also complemented both afsA and afsB mutations of Streptomyces coelicolor A3(2). These results indicated that the cloned DNA fragment contained the genetic determinant of A-factor biosynthesis. The cloned fragment, when carried on a multicopy vector plasmid, induced production of a large amount of A-factor in several Streptomyces hosts. In Southern blot DNA/DNA hybridization analyses with a trimmed 5-kilobase fragment containing the intact A-factor determinant as probe, total cellular DNA from A-factor-deficient mutants gave no positive hybridization. The DNA blot experiment also showed a wide distribution of sequences homologous to the S. bikiniensis A-factor determinant among most, but not all, A-factor-producing actinomycetes with a varying extent of homology and the absence of these sequences from most A-factor nonproducers .
我们从产生链霉素的比基尼链霉菌的总细胞 DNA 中克隆了一个指导 A 因子合成的 DNA 片段，并将其连接到质粒载体 pIJ385 上。将重组质粒（pAFB1）导入缺乏 A 因子的比基尼链霉菌和灰色链霉菌突变体中，导致宿主细胞产生 A 因子，结果这些突变体的链霉素产量、链霉素抗性和孢子形成同时得以恢复。质粒 pAFB1 还互补了天蓝色链霉菌 A3(2)的 afsA 和 afsB 突变。这些结果表明，克隆的 DNA 片段包含 A 因子生物合成的遗传决定因素。当克隆片段携带在多拷贝载体质粒上时，可在几种链霉菌宿主中诱导产生大量 A 因子。在用包含完整 A 因子决定因素的 5 千碱基切割片段作为探针进行的 Southern 印迹 DNA/DNA 杂交分析中，缺乏 A 因子的突变体的总细胞 DNA 未产生阳性杂交信号。DNA 印迹实验还表明，在大多数（但不是全部）产生 A 因子的放线菌中，存在与比基尼链霉菌 A 因子决定因素同源的序列，同源程度各不相同，而大多数不产生 A 因子的菌株中则没有这些序列。
