MSMEG 5850, a stress-induced TetR protein, involved in global transcription regulation in Mycobacterium smegmatis - Supplementry tables

Aim: To decipher the role of MSMEG 5850 in the physiology of mycobacteria. Methods: MSMEG 5850 was knocked out and RNA sequencing was performed. MSMEG 5850 protein was purified from the Escherichia coli pET28a system. Electrophoretic mobility shift assay and size exclusion chromatography were used to determine the binding of MSMEG 5850 to its motif and binding stoichiometry. The effect of nutritional stress was monitored. Results: Transcriptome analysis revealed the differential expression of 148 genes in an MSMEG 5850 knockout strain. MSMEG 5850 had control over 50 genes because those genes had a binding motif upstream of their sequence. The electrophoretic mobility shift assay showed MSMEG 5850 bound to its motif as a monomer. MSMEG 5850 was upregulated under nutritional stress and promoted the survival of mycobacteria. Conclusion: The study confirms the role of MSMEG 5850 in global transcriptional regulation.

目的：本研究旨在阐明MSMEG 5850在分枝杆菌生理过程中的作用。方法：敲除MSMEG 5850并开展RNA测序（RNA Sequencing）；从大肠杆菌（Escherichia coli）pET28a表达系统中纯化MSMEG 5850蛋白；采用电泳迁移率变动分析（Electrophoretic Mobility Shift Assay）与尺寸排阻色谱法（Size Exclusion Chromatography），明确MSMEG 5850与其结合基序的结合特性及结合化学计量比；同时监测营养胁迫对菌体的影响。结果：转录组分析显示，MSMEG 5850敲除菌株中共存在148个差异表达基因；其中50个基因的序列上游带有MSMEG 5850的结合基序，提示MSMEG 5850可对这些基因实施调控。电泳迁移率变动分析结果证实，MSMEG 5850以单体形式与其结合基序相结合。MSMEG 5850在营养胁迫条件下表达上调，并可促进分枝杆菌的存活。结论：本研究明确证实了MSMEG 5850在全局转录调控中的作用。