Biomedical Sciences Department, School of Public Health, State University of New York at Albany, Albany, USA.
Wadsworth Center, New York Department of Health, 120 New Scotland Avenue, Albany, NY, 12208, USA.
Sci Rep. 2024 Sep 5;14(1):20664. doi: 10.1038/s41598-024-71248-y.
The recA gene, encoding Recombinase A (RecA) is one of three Mycobacterium tuberculosis (Mtb) genes encoding an in-frame intervening protein sequence (intein) that must splice out of precursor host protein to produce functional protein. Ongoing debate about whether inteins function solely as selfish genetic elements or benefit their host cells requires understanding of interplay between inteins and their hosts. We measured environmental effects on native RecA intein splicing within Mtb using a combination of western blots and promoter reporter assays. RecA splicing was stimulated in bacteria exposed to DNA damaging agents or by treatment with copper in hypoxic, but not normoxic, conditions. Spliced RecA was processed by the Mtb proteasome, while free intein was degraded efficiently by other unknown mechanisms. Unspliced precursor protein was not observed within Mtb despite its accumulation during ectopic expression of Mtb recA within E. coli. Surprisingly, Mtb produced free N-extein in some conditions, and ectopic expression of Mtb N-extein activated LexA in E. coli. These results demonstrate that the bacterial environment greatly impacts RecA splicing in Mtb, underscoring the importance of studying intein splicing in native host environments and raising the exciting possibility of intein splicing as a novel regulatory mechanism in Mtb.
RecA 基因，编码重组酶 A（RecA），是编码三个分枝杆菌（Mtb）基因之一，编码一个框架内的介入蛋白序列（intein），该序列必须从前体宿主蛋白中剪接出来，以产生有功能的蛋白。关于整合子是否仅作为自私的遗传元件发挥作用，还是对其宿主细胞有益，这一持续存在的争论需要了解整合子与其宿主之间的相互作用。我们使用 Western blot 和启动子报告基因分析相结合的方法，测量了环境对结核分枝杆菌天然 RecA 整合子剪接的影响。在缺氧条件下，暴露于 DNA 损伤剂或铜处理的细菌中，RecA 剪接受到刺激，但在正常氧条件下则不受刺激。在结核分枝杆菌中，拼接的 RecA 被蛋白酶体加工，而游离的整合子则通过其他未知机制被有效降解。尽管在大肠埃希菌中异位表达 Mtb recA 会导致其积累，但在结核分枝杆菌中并未观察到未剪接的前体蛋白。令人惊讶的是，在某些条件下，结核分枝杆菌会产生游离的 N-外显子，并且异位表达 Mtb N-外显子会激活大肠埃希菌中的 LexA。这些结果表明，细菌环境极大地影响了结核分枝杆菌中 RecA 的剪接，强调了在天然宿主环境中研究整合子剪接的重要性，并提出了整合子剪接作为结核分枝杆菌中一种新的调控机制的令人兴奋的可能性。
