Kojima Ikuo, Kasuga Kano, Kobayashi Masayuki, Fukasawa Akira, Mizuno Satoshi, Arisawa Akira, Akagawa Hisayoshi
Department of Biotechnology, Faculty of Bioresource Sciences, Akita Prefectural University, Akita 010-0195, Japan.
J Bacteriol. 2002 Dec;184(23):6417-23. doi: 10.1128/JB.184.23.6417-6423.2002.
The occurrence of pleiotropic mutants that are defective in both antibiotic production and aerial mycelium formation is peculiar to streptomycetes. Pleiotropic mutant KSB was isolated from wild-type Streptomyces kasugaensis A1R6, which produces kasugamycin, an antifungal aminoglycoside antibiotic. A 9.3-kb DNA fragment was cloned from the chromosomal DNA of strain A1R6 by complementary restoration of kasugamycin production and aerial hypha formation to mutant KSB. Complementation experiments with deletion plasmids and subsequent DNA analysis indicated that orf5, encoding 90 amino acids, was responsible for the restoration. A protein homology search revealed that orf5 was a homolog of rpoZ, the gene that is known to encode RNA polymerase subunit omega (omega), thus leading to the conclusion that orf5 was rpoZ in S. kasugaensis. The pleiotropy of mutant KSB was attributed to a 2-bp frameshift deletion in the rpoZ region of mutant KSB, which probably resulted in a truncated, incomplete omega of 47 amino acids. Furthermore, rpoZ-disrupted mutant R6D4 obtained from strain A1R6 by insertion of Tn5 aphII into the middle of the rpoZ-coding region produced neither kasugamycin nor aerial mycelia, similar to mutant KSB. When rpoZ of S. kasugaensis and Streptomyces coelicolor, whose deduced products differed in the sixth amino acid residue, were introduced into mutant R6D4 via a plasmid, both transformants produced kasugamycin and aerial hyphae without significant differences. This study established that rpoZ is required for kasugamycin production and aerial mycelium formation in S. kasugaensis and responsible for pleiotropy.
在抗生素生产和气生菌丝体形成方面均存在缺陷的多效性突变体的出现是链霉菌所特有的。多效性突变体KSB是从野生型春日链霉菌A1R6中分离得到的,该菌株可产生抗真菌氨基糖苷类抗生素春日霉素。通过将春日霉素生产和气生菌丝形成互补恢复到突变体KSB,从菌株A1R6的染色体DNA中克隆出一个9.3 kb的DNA片段。用缺失质粒进行的互补实验及后续的DNA分析表明,编码90个氨基酸的orf5负责这种恢复。蛋白质同源性搜索显示,orf5是rpoZ的同源物,已知该基因编码RNA聚合酶亚基ω(ω),因此得出结论,orf5是春日链霉菌中的rpoZ。突变体KSB的多效性归因于突变体KSB的rpoZ区域中一个2 bp的移码缺失,这可能导致了一个截短的、不完整的47个氨基酸的ω。此外,通过将Tn5 aphII插入rpoZ编码区中间从菌株A1R6获得的rpoZ破坏突变体R6D4,与突变体KSB一样,既不产生春日霉素也不产生气生菌丝。当将推导产物在第六个氨基酸残基处不同的春日链霉菌和天蓝色链霉菌的rpoZ通过质粒导入突变体R6D4时,两个转化体均产生春日霉素和气生菌丝,且无显著差异。本研究确定rpoZ是春日链霉菌中春日霉素生产和气生菌丝体形成所必需的,并且与多效性有关。