State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, PR China.
BMC Biotechnol. 2012 Aug 20;12:52. doi: 10.1186/1472-6750-12-52.
The otrC gene of Streptomyces rimosus was previously annotated as an oxytetracycline (OTC) resistance protein. However, the amino acid sequence analysis of OtrC shows that it is a putative ATP-binding cassette (ABC) transporter with multidrug resistance function. To our knowledge, none of the ABC transporters in S. rimosus have yet been characterized. In this study, we aimed to characterize the multidrug exporter function of OtrC and evaluate its relevancy to OTC production.
In order to investigate OtrC's function, otrC is cloned and expressed in E. coli The exporter function of OtrC was identified by ATPase activity determination and ethidium bromide efflux assays. Also, the susceptibilities of OtrC-overexpressing cells to several structurally unrelated drugs were compared with those of OtrC-non-expressing cells by minimal inhibitory concentration (MIC) assays, indicating that OtrC functions as a drug exporter with a broad range of drug specificities. The OTC production was enhanced by 1.6-fold in M4018 (P = 0.000877) and 1.4-fold in SR16 (P = 0.00973) duplication mutants, while it decreased to 80% in disruption mutants (P = 0.0182 and 0.0124 in M4018 and SR16, respectively).
The results suggest that OtrC is an ABC transporter with multidrug resistance function, and plays an important role in self-protection by drug efflux mechanisms. This is the first report of such a protein in S. rimosus, and otrC could be a valuable target for genetic manipulation to improve the production of industrial antibiotics.
先前，棘孢小单孢菌的 otrC 基因被注释为一种土霉素（OTC）抗性蛋白。然而，OtrC 的氨基酸序列分析表明，它是一种具有多药耐药功能的假定三磷酸腺苷结合盒（ABC）转运蛋白。据我们所知，棘孢小单孢菌中的 ABC 转运蛋白尚未被表征。在这项研究中，我们旨在表征 OtrC 的多药外排功能，并评估其与 OTC 生产的相关性。
为了研究 OtrC 的功能，otrC 在大肠杆菌中被克隆和表达。通过 ATP 酶活性测定和溴化乙锭外排实验鉴定了 OtrC 的外排功能。此外，通过最小抑菌浓度（MIC）测定比较了 OtrC 过表达细胞与 OtrC 非表达细胞对几种结构上不相关的药物的敏感性，表明 OtrC 作为一种具有广泛药物特异性的药物外排泵发挥作用。在 M4018（P = 0.000877）和 SR16（P = 0.00973）重复突变体中，OTC 产量分别提高了 1.6 倍和 1.4 倍，而在缺失突变体中则降低到 80%（P = 0.0182 和 0.0124，分别在 M4018 和 SR16 中）。
结果表明，OtrC 是一种具有多药耐药功能的 ABC 转运蛋白，通过药物外排机制在自我保护中发挥重要作用。这是该蛋白在棘孢小单孢菌中的首次报道，otrC 可能是遗传操作的一个有价值的靶点，以提高工业抗生素的产量。
