Fang Zhuo, Newton-Foot Mae, Sampson Samantha Leigh, Gey van Pittius Nicolaas Claudius
DST/NRF Centre of Excellence in Biomedical Tuberculosis Research, US/MRC Centre for Molecular and Cellular Biology, Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, University of Stellenbosch, Francie van Zijl Drive, Tygerberg, 7505, South Africa.
Division of Medical Microbiology, Department of Pathology, Faculty of Medicine and Health Sciences, University of Stellenbosch, Tygerberg, South Africa.
BMC Res Notes. 2017 Aug 25;10(1):426. doi: 10.1186/s13104-017-2752-0.
The ESX secretion system, also known as the Type VII secretion system, is mostly found in mycobacteria and plays important roles in nutrient acquisition and host pathogenicity. One of the five ESXs, ESX-3, is associated with mycobactin-mediated iron acquisition. Although the functions of some of the membrane-associated components of the ESX systems have been described, the role of by mycosin-3 remains elusive. The esx-3 gene cluster encoding ESX-3 in both Mycobacterium tuberculosis and Mycobacterium smegmatis has two promoters, suggesting the presence of two transcriptional units. Previous studies indicated that the two promoters only showed a difference in response under acid stress (pH 4.2). This study aimed to study the effect of a mycosin-3 deletion on the physiology of M. smegmatis and to assess the promoter activities in wildtype, mycosin-3 mutant and complementation strains.
The gene mycP was deleted from wildtype M. smegmatis via homologous recombination. The mycP gene was complemented in the deletion mutant using each of the two intrinsic promoters from the M. smegmatis esx-3 gene cluster. The four strains were compared in term of bacterial growth and intracellular iron content. The two promoter activities were assessed under iron-rich, iron-deprived and iron-rescued conditions by assessing the mycP expression level. Although the mycP gene deletion did not significantly impact bacterial growth or intracellular iron levels in comparison to the wild-type and complemented strains, the two esx-3 promoters were shown to respond inversely to iron deprivation and iron rescue.
This finding correlates with the previously published data that the first promoter upstream of msmeg0615, is upregulated under low iron levels but downregulated under high iron levels. In addition, the second promoter, upstream of msmeg0620, behaves in an inverse fashion to the first promoter implying that the genes downstream may have additional roles when the iron levels are high.
ESX分泌系统,也称为VII型分泌系统,主要存在于分枝杆菌中,在营养获取和宿主致病性方面发挥重要作用。五个ESX系统之一的ESX-3与分枝杆菌素介导的铁获取有关。尽管已经描述了ESX系统中一些膜相关成分的功能,但肌球蛋白-3的作用仍然难以捉摸。在结核分枝杆菌和耻垢分枝杆菌中编码ESX-3的esx-3基因簇有两个启动子,表明存在两个转录单元。先前的研究表明,这两个启动子仅在酸胁迫(pH 4.2)下表现出响应差异。本研究旨在研究肌球蛋白-3缺失对耻垢分枝杆菌生理学的影响,并评估野生型、肌球蛋白-3突变体和互补菌株中的启动子活性。
通过同源重组从野生型耻垢分枝杆菌中删除了mycP基因。使用耻垢分枝杆菌esx-3基因簇的两个内在启动子之一在缺失突变体中对mycP基因进行互补。比较了这四种菌株的细菌生长和细胞内铁含量。通过评估mycP表达水平,在富铁、缺铁和铁挽救条件下评估了两个启动子的活性。尽管与野生型和互补菌株相比,mycP基因缺失对细菌生长或细胞内铁水平没有显著影响,但两个esx-3启动子对铁剥夺和铁挽救的反应相反。
这一发现与先前发表的数据相关,即msmeg0615上游的第一个启动子在低铁水平下上调,但在高铁水平下下调。此外,msmeg0620上游的第二个启动子与第一个启动子的行为相反,这意味着当铁水平较高时,下游基因可能具有额外的作用。