Abstract
otrA resembles elongation factor G (EF-G) and is considered to be an oxytetracycline (OTC)-resistance determinant in Streptomyces rimosus. In order to determine whether otrA also conferred resistance to OTC and other aminoglycosides to Streptomyces coelicolor, the otrA gene from S. rimosus M527 was cloned under the control of the strong ermE* promoter. The resulting plasmid, pIB139-otrA, was introduced into S. coelicolor M145 by intergeneric conjugation, yielding the recombinant strain S. coelicolor M145-OA. As expected S. coelicolor M145-OA exhibited higher resistance levels specifically to OTC and aminoglycosides gentamycin, hygromycin, streptomycin, and spectinomycin. However, unexpectedly, S. coelicolor M145-OA on solid medium showed an accelerated aerial mycelia formation, a precocious sporulation, and an enhanced actinorhodin (Act) production. Upon growth in 5-L fermentor, the amount of intra- and extracellular Act production was 6-fold and 2-fold higher, respectively, than that of the original strain. Consistently, reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that the transcriptional level of pathway-specific regulatory gene actII-orf4 was significantly enhanced in S. coelicolor M145-OA compared with in S. coelicolor M145.
摘要
目的
将来源于龟裂链霉菌(Streptomyces rimosus)M527 的 otrA 基因在天蓝色链霉菌M145 中异源表达, 通过考察宿主菌对土霉素以及氨基糖苷类抗生 素的抗性、放线紫红素的合成、菌株形态等方面 的变化鉴定 otrA 基因的功能。
创新点
S. rimosus 中的 otrA 基因与转录延伸因子EF-G 具 有很高的同源性,被认为是土霉素的抗性基因之 一,但其具体的生物学功能目前尚未有研究报 道。本文首次实现 otrA 基因在天蓝色链霉菌 M145 中的异源表达,不仅提高了宿主对土霉素 以及氨基糖苷类抗生素的抗性,还促进了菌株产 孢和放线紫红素的合成,从而初步证实了OTRA 生物学功能。
方法
克隆来源于S. rimosus M527 的 otrA 基因,将其 置于链霉菌整合型载体 pIB139 强启动子 PermE* 的下游,构建重组质粒pIB139-otrA;通过接合转 移将其转入天蓝色链霉菌M145 获得重组菌 M145-OA,实现otrA 基因在天蓝色链霉菌M145 中的异源表达;通过扫描电镜观察菌株的形态变 化;通过含有不同浓度的不同抗生素的抗性平板 筛选测试菌株的抗性水平变化;通过5-L 发酵罐 发酵实验考察次级代谢产物放线紫红素的合成 能力变化;通过荧光定量PCR 考察放线紫红素合 成途径中的调控基因actII-orf4 的转录水平变化。
结论
来源于S. rimosus M527 的 otrA 基因在天蓝色链 霉菌M145 中实现异源表达。一方面对宿主天蓝 色链霉菌形态分化、放线紫红素产量的影响表明 otrA 可能作为一种类似延伸因子的发挥重要作 用;另一方面宿主对土霉素及氨基糖苷类抗生素 抗性的提高可能归因于OTRA对核糖体的保护作 用。
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Acknowledgements
Thanks to Prof. ZX DENG (Shanghai Jiao Tong University, Shanghai, China) for giving the plasmid pIB139 for this study.
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Project supported by the National Natural Science Foundation of China (No. 31772213) and the Excellent Youth Fund of Zhejiang Province, China (No. LR17C140002)
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Impact of otrA expression on morphological differentiation, actinorhodin production, and resistance to aminoglycosides in Streptomyces coelicolor M145
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Zhao, Yf., Lu, Dd., Bechthold, A. et al. Impact of otrA expression on morphological differentiation, actinorhodin production, and resistance to aminoglycosides in Streptomyces coelicolor M145. J. Zhejiang Univ. Sci. B 19, 708–717 (2018). https://doi.org/10.1631/jzus.B1800046
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DOI: https://doi.org/10.1631/jzus.B1800046