Regulatory Pathway of ECF-Sig25 in Streptomyces avermitilis

doi:10.13304/j.nykjdb.2023.0003

Abstract

Abstract:

The linkage regulation of extracytoplasmic function （ECF） sigma factors and two-component system （TCS） play essential roles for bacteria adaptation to various environment and stress condition. In order to study the ECF-sig25 gene in Streptomycesavermitilis and explore the regulatory pathway of Sig25， the response of Sig25 to stress was detected by spore spot test， its influence on morphological differentiation was analyzed by plate culture， and the target gene of Sig25 was verified by bioinformatics and RT-qPCR. The results showed that TCS gene smrAB was located upstream of ECF-sig25， and the phosphorylation of SmrA depended solely on SmrB. Stress tests showed that Sig25 responded to high pH， osmotic stress， SDS and heat stress， but not to oxidative stress， acid stress and EDTA stress. Bioinformatic prediction， RT-qPCR and electrophoretic mobility shift assay （EMSA） results revealed that Sig25 directly regulated transcription of whiG， SAV3434， SAV4621， SAV5010， SAV6537， gdhA1， glnA2 and amtB1， which were involved in morphological development， methyl group supply， DNA replication， signal transduction， secondary metabolism and nitrogen metabolism. The plate culture showed that deletion of sig25 had no effect on morphological developmentin of S. avermitilis.

Key words: Streptomyces avermitilis, ECF-sigma factor, Sig25, two-component systems, SmrAB

摘要：

ECF-Sigma因子和双组份调控系统连锁调控在细菌适应不同的生存环境和压力条件中起着重要作用。为研究阿维链霉菌中的ECF-sig25基因，通过孢子点接试验检测Sig25对胁迫的响应，通过平板划线分析其对形态分化的影响，通过生物信息学、RT-qPCR等验证Sig25的靶基因，以探索Sig25的调控通路。结果显示，ECF-sig25基因上游存在1个双组份调控系统基因smrAB；且上游调控元件SmrA的磷酸化严格依赖于SmrB。压力胁迫试验显示，Sig25响应碱、渗透压、去污剂和热激胁迫，不响应氧化、酸和EDTA胁迫。生物信息学预测、RT-qPCR和EMSA（electrophoretic mobility shift assay）揭示，Sig25直接调控whiG、SAV3434、SAV4621、SAV5010、SAV6537、SAV7165、gdhA1、glnA2和amtB1基因的转录，这些基因分别参与形态分化、甲基供给、DNA复制、信号传导、次级代谢和氮代谢等生理过程。平板划线结果显示，缺失sig25不影响阿维链霉菌的形态分化。

关键词: 阿维链霉菌, ECF-Sigma因子, Sig25, 双组份调控系统, SmrAB

CLC Number:

Shuai LUO, Xiaojie WANG, Hucheng ZHANG, Linying LIU, Weijie JIAN, Hua ZHU, Jianya ZHU. Regulatory Pathway of ECF-Sig25 in Streptomyces avermitilis[J]. Journal of Agricultural Science and Technology, 2024, 26(6): 122-132.

罗帅, 王晓杰, 张虎成, 刘霖颖, 简伟杰, 朱华, 朱建亚. 阿维链霉菌中ECF-Sig25的调控通路[J]. 中国农业科技导报, 2024, 26(6): 122-132.

Figures/Tables 14

Fig. 1 Organization of sig25 and smrAB on S. avermitilis chromosome［9］

Fig. 2 Conserved binding sequence of SmrA［9］

Fig. 3 Growth of Dsig25 and WT in different stress mediums

Fig. 4 Relative expression levels of putative target genes in Dsig25 and DsmrAB mutant strainsNote： The expression level of wild type is as 1； N indicates no significant difference with wild type.

Fig. 5 Confirmation of SmrA targetsNote： Each lane contains 0.15 nmol·L-1 labeled probe. 1—Free probe； 2—100 nmol·L-1 GST-SmrA； 3—300 nmol·L-1 GST-SmrA； 4—300 nmol·L-1 GST-SmrA and 100-fold specific unlabeled probe； 5—GST.

Fig. 6 Confirmation of Sig25 targetsNote： Each lane contains 0.15 nmol·L-1 labeled probe. 1—Free probe； 2—3.5 μmol·L-1 His6-Sig25； 3—7.0 μmol·L-1 His6-Sig25； 4—14.0 μmol·L-1 His6-Sig25； 5—14.0 μmol·L-1 His6-Sig25 and 100-fold specific unlabeled competitive probe.

Table 2 Transcription levels of putative target genes in various strains and binding of their promoter regions with SmrA and Sig25

基因Gene	SmrA	Sig25	DsmrAB	Dsig25	基因Gene	SmrA	Sig25	DsmrAB	Dsig25
SAV681	+	-	→	↓	SAV4621	+	+	↑	→
SAV1003	+	-	→	↓	SAV5010	+	+	↓	↑
SAV1689	+	-	↓	↓	SAV6537	+	+	→	↑
SAV2630	+	+	↓	↓	SAV6710	+	-	↑	↑
SAV3434	+	+	↓	↓	SAV7165	-	+	↓	↓

Fig. 7 Detection of interaction between SmrA and promoter regions of nitrogen metabolism genesNote：Each lane contains 0.15 nmol·L-1 labeled probe. 1—Free probe； 2—0.1 μmol·L-1 GST-SmrA； 3—0.3 μmol·L-1 GST-SmrA； 4—0.3 μmol·L-1 GST-SmrA （+） with ~100-fold excess of unlabeled probe （S）； 5—2.0 μmol·L-1GST protein as negative control.

Fig. 8 Detection of interaction between His6-Sig25 and promoter regions of nitrogen metabolism genesNote： 1—Free probe without protein； 2—7 μmol·L-1 His6-Sig25； 3—14 μmol·L-1 His6-Sig25； 4—14 μmol·L-1 His6-Sig25+100-fold excess of unlabeled probe.

Fig. 9 Relative expression levels of SmrA and Sig25 common nitrogen metabolism target genes in different strainsNote： The expression level of wild type is as 1.0.

Fig. 10 Avermectin yield in DsmrAB， Dsig25 and various related complementation strainsNote： Different lowercase letters indicate significant differences between different strains at P<0.05 level.

Fig. 11 Phenotypes of sig25 and smrAB strains grown on YMS and MM mediumsA~B： YMS medium； C~D： MM medium；WT—wild-type；Dsig25—sig25 deletion mutant；Csig25—sig25 complementation mutant；DsmrAB—smrAB deletion mutant；CsmrAB—smrAB complementation mutant

Fig. 12 Phenotypes of mutant strains with smrAB and sig25 in MM medium with 100 mmol·L-1 glutamic acidNote： WT—Wild-type；Dsig25—sig25 deletion mutant；Csig25—sig25 complementation mutant；DsmrAB—smrAB deletion mutant；CsmrAB—smrAB complementation mutant

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