Resistance Mechanisms to Bleomycin in a Producer Organism.

产生抗生素的生物体必须受到保护，使其免受自身抗生素的致命影响。从酶失活、靶位抗性和膜通透性等方面研究了产生蛋白质合成抗生素抑制剂的链霉菌的耐药机制。博莱霉素是一种糖肽抗生素，被用作抗肿瘤药物；人们有兴趣了解产生DNA合成抗生素抑制剂的链霉菌的耐药机制是否与蛋白质合成的机制不同。在本研究中，我们试图研究一株博莱霉素产生菌的自身抗性机制，并克隆其抗性决定基因(S)的编码基因(S)。初步实验表明，博莱霉素与无细胞提取物和乙酰辅酶A孵育后，其抗菌活性消失。当提取物在65℃下孵育5min时，即使在乙酰辅酶A存在的情况下，也失去了灭活活性。下一步，我试图克隆一个基因(S)，它决定了博莱霉素产生菌中的一种博莱霉素乙酰化酶。用作宿主的微生物是变铅青链霉菌66。在含有blmB基因的7kb片段中，我发现了另一个基因blmA，该基因与变铅青链霉菌66对博莱霉素的抗性有关。对扩增出的700bp的blmA基因进行DNA序列分析。结果表明，该基因编码的蛋白质是一个由122个氨基酸组成的多肽，其相对分子质量为13197。我在目前的研究中发现，bluma和blmB基因在大肠杆菌中表达。初步实验表明，blmA基因编码的蛋白是与博莱霉素结合的蛋白。我正在详细研究BLMA蛋白的一些性质。

Organisms which produce antibiotics must be protected from the lethal effect of their own antibiotics. The resistance mechanisms in streptomycetes which produce antibiotic inhibitors of protein synthesis have been studied with respect to enzymatic inactivation, resistance of target site and membrane permeability. Bleomycin is a glycopeptide antibiotic and is used as an antitumor agent ; it is of interest to know whether the resistance mechanisms in streptomycetes which produce antibiotic inhibitors of DNA synthesis are different from those of protein synthesis. In the present study, we attempted to investigate self-resistance mechanisms in a bleomycin-producing microorganism and to clone the gene (s) encoding the resistant determinant (s).Streptomyces verticillus ATCC15003 was used as a bleomycin-producing microorganism. Initial experiments indicated that the antibacterial activity of bleomycin disappeared after incubation with the cell-free extract and acetyl coenzyme A. When the extract was incubated at 65 C for 5 min, the inactivating activity was lost, even in the presence of acetyl CoA. Next, I attempted to clone a gene (s) which determined a bleomycin-acetylating enzyme in the bleomycin producer. The organism used as a host was S. lividans 66. I obtained a gene coding for the enzyme as a 7 kb DNA fragment from S. verticillus ATCC15003 and named blm B. I found another gene (blmA) which conferred the bleomycin resistance to S. lividans 66 in the 7 kb fragment containing blmB gene. The blmA gene obtained as a 700 bp fragment was analyzed for DNA sequences. As a result, the protein encoded by blmA gene was a polypeptide consisting of 122 amino acids and the molecular weight was 13197. I found in the present study that blumA and blmB genes were expressed in Escherichia coli. The preliminary experiments suggested that the protein encoded by blmA gene was a binding protein to bleomycin. I am investigating some properties of the blm A protein in detail.