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Journal of Zhejiang University SCIENCE B 2018 Vol.19 No.9 P.708-717

http://doi.org/10.1631/jzus.B1800046


Impact of otrA expression on morphological differentiation, actinorhodin production, and resistance to aminoglycosides in Streptomyces coelicolor M145


Author(s):  Yan-Fang Zhao, dan-Dan Lu, Andreas Bechthold, Zheng Ma, Xiao-Ping Yu

Affiliation(s):  Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University, Hangzhou 310018, China; more

Corresponding email(s):   mazheng520@163.com, yuxiaoping19630306@163.com

Key Words:  otrA gene, Streptomyces coelicolor, Actinorhodin, Morphological differentiation, actII-orf4


Yan-Fang Zhao, dan-Dan Lu, Andreas Bechthold, Zheng Ma, Xiao-Ping Yu. Impact of otrA expression on morphological differentiation, actinorhodin production, and resistance to aminoglycosides in Streptomyces coelicolor M145[J]. Journal of Zhejiang University Science B, 2018, 19(9): 708-717.

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author="Yan-Fang Zhao, dan-Dan Lu, Andreas Bechthold, Zheng Ma, Xiao-Ping Yu",
journal="Journal of Zhejiang University Science B",
volume="19",
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pages="708-717",
year="2018",
publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.B1800046"
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%T Impact of otrA expression on morphological differentiation, actinorhodin production, and resistance to aminoglycosides in Streptomyces coelicolor M145
%A Yan-Fang Zhao
%A dan-Dan Lu
%A Andreas Bechthold
%A Zheng Ma
%A Xiao-Ping Yu
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T1 - Impact of otrA expression on morphological differentiation, actinorhodin production, and resistance to aminoglycosides in Streptomyces coelicolor M145
A1 - Yan-Fang Zhao
A1 - dan-Dan Lu
A1 - Andreas Bechthold
A1 - Zheng Ma
A1 - Xiao-Ping Yu
J0 - Journal of Zhejiang University Science B
VL - 19
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SP - 708
EP - 717
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PB - Zhejiang University Press & Springer
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DOI - 10.1631/jzus.B1800046


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.

天蓝色链霉菌M145中otrA基因的表达对菌株形态分化、放线紫红素合成及氨基糖苷类抗生素抗性的影响

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.

关键词: otrA基因;天蓝色链霉菌;放线紫红素;形态分化;actII-orf4

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article

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[37]List of electronic supplementary materials

[38]Fig. S1 Phenotypic verification of seven randomly recombinant strains of S. coelicolor M145-OA

[39]Fig. S2 PCR analysis of otrA gene from S. coelicolor M145-OA

[40]Fig. S3 Morphological analyses of S. rimosus M527 and S. rimosus M527-OA on MS medium

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