Resistance Mechanisms to Bleomycin in a Producer Organism.

生产生物体对博莱霉素的耐药机制。

基本信息

批准号：
01550765
负责人：
SUGIYAMA Masanori
金额：
$ 1.28万
依托单位：
Hiroshima University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

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.

必须保护产生抗生素的生物体免受其自身抗生素的致命作用。产生蛋白质合成抗生素抑制剂的链霉菌的抗性机制已在酶失活、靶位点抗性和膜渗透性方面进行了研究。博莱霉素是一种糖肽抗生素，用作抗肿瘤剂；有趣的是，链霉菌产生DNA合成抗生素抑制剂的耐药机制是否不同于蛋白质合成抗生素抑制剂。在本研究中，我们试图研究博莱霉素生产微生物的自身抗性机制，并克隆编码抗性决定簇的基因。轮枝链霉菌ATCC15003被用作博莱霉素生产微生物。初步实验表明，博莱霉素的抗菌活性在与无细胞提取物和乙酰辅酶A一起孵育后消失。当提取物在65℃下孵育5分钟时，即使存在乙酰辅酶A，其灭活活性也消失。接下来，我尝试克隆一个基因，该基因决定博莱霉素生产者中的博莱霉素乙酰化酶。用作宿主的生物体是 S. lividans 66。我从 S. verticillus ATCC15003 中获得了编码该酶的基因，作为 7 kb DNA 片段，并将其命名为 blm B。我发现了另一个基因 (blmA)，该基因赋予 S. verticillus ATCC15003 博莱霉素抗性变青丹66的7kb片段含有blmB基因。对作为 700 bp 片段获得的 blmA 基因进行 DNA 序列分析。结果，blmA基因编码的蛋白是由122个氨基酸组成的多肽，分子量为13197。我在本研究中发现blumA和blmB基因在大肠杆菌中表达。初步实验表明blmA基因编码的蛋白是博来霉素的结合蛋白。我正在详细研究 blm A 蛋白的一些特性。