Zhang Hao, Ouyang Zhilin, Zhao Nannan, Han Shuangyan, Zheng Suiping
Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, China.
Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, China.
Front Bioeng Biotechnol. 2022 Feb 9;10:816628. doi: 10.3389/fbioe.2022.816628. eCollection 2022.
In the genus , AmtR is a key component of the nitrogen regulatory system, and it belongs to the TetR family of transcription regulators. There has been much research on AmtR structure, functions, and regulons in the type strain ATCC 13032, but little research in other strains. In this study, chromatin immunoprecipitation and massively parallel DNA sequencing (ChIP-seq) was performed to identify the AmtR regulon in ATCC 14067. Ten peaks were obtained in the ATCC 14067 genome including two new peaks related to three operons (RS_01910-RS_01915, RS_15995, and RS_16000). The interactions between AmtR and the promoter regions of the three operons were confirmed by electrophoretic mobility shift assays (EMSAs). The RS_01910, RS_01915, RS_15995, and RS_16000 are not present in the type strain ATCC 13032. Sequence analysis indicates that RS_01910, RS_01915, RS_15995, and RS_16000, are related to the degradation of creatine and creatinine; RS_01910 may encode a protein related to creatine transport. The genes RS_01910, RS_01915, RS_15995, and RS_16000 were given the names A, T, A, and B, respectively. Real-time quantitative PCR (RT-qPCR) analysis and sfGFP (superfolder green fluorescent protein) analysis reveal that AmtR directly and negatively regulates the transcription and expression of A, T, A, and B. A growth test shows that ATCC 14067 can use creatine or creatinine as a sole nitrogen source. In comparison, a T deletion mutant strain is able to grow on creatinine but loses the ability to grow on creatine. This study provides the first genome-wide captures of the dynamics of AmtR binding events and the regulatory network they define. These elements provide more options for synthetic biology by extending the scope of the AmtR regulon.
在该属中，AmtR是氮调节系统的关键组成部分，属于转录调节因子的TetR家族。关于模式菌株ATCC 13032中的AmtR结构、功能和调控子已有很多研究，但在其他菌株中的研究较少。在本研究中，进行了染色质免疫沉淀和大规模平行DNA测序（ChIP-seq）以鉴定ATCC 14067中的AmtR调控子。在ATCC 14067基因组中获得了10个峰，包括与三个操纵子（RS_01910 - RS_01915、RS_15995和RS_16000）相关的两个新峰。通过电泳迁移率变动分析（EMSA）证实了AmtR与这三个操纵子启动子区域之间的相互作用。RS_01910、RS_01915、RS_15995和RS_16000在模式菌株ATCC 13032中不存在。序列分析表明，RS_01910、RS_01915、RS_15995和RS_16000与肌酸和肌酐的降解有关；RS_01910可能编码一种与肌酸转运相关的蛋白质。基因RS_01910、RS_01915、RS_15995和RS_16000分别被命名为A、T、A和B。实时定量PCR（RT-qPCR）分析和超折叠绿色荧光蛋白（sfGFP）分析表明，AmtR直接且负向调节A、T、A和B的转录和表达。生长试验表明，ATCC 14067可以使用肌酸或肌酐作为唯一氮源。相比之下，T缺失突变株能够在肌酐上生长，但失去了在肌酸上生长的能力。本研究首次在全基因组范围内捕获了AmtR结合事件的动态变化及其所定义的调控网络。这些元件通过扩展AmtR调控子的范围为合成生物学提供了更多选择。
