Table_1_RskA Is a Dual Function Activator-Inhibitor That Controls SigK Activity Across Distinct Bacterial Genera.XLSX

It has been previously shown that RskA, the anti-Sigma factor K of Mycobacterium tuberculosis, inhibits SigK and that mutations in RskA promote high expression of the SigK regulon. The latter observation led us to hypothesize that RskA mutations lead to loss of the anti-Sigma factor function. In this report, we used natural and artificial mutations in RskA to determine the basis of the SigK-RskA partnership. Consistent with predictions, the N-terminal cytoplasmic portion of RskA was sufficient on its own to inhibit SigK. Unexpectedly, RskA also served as an activator of SigK. This activation depended on the same N-terminal region and was enhanced by the membrane-extracellular portion of RskA. Based on this, we engineered similar truncations in a Gram-negative bacterium, namely Yersinia enterocolitica. Again, we observed that, with specific alterations of RskA, we were able to enhance SigK activity. Together these results support an alternative mechanism of anti-Sigma factor function, that we could term modulator (activator-inhibitor) in both Actinobacteria and Gram-negative bacteria, suggesting that Sigma factor activation by anti-Sigma factors could be under-recognized.

既往研究表明，结核分枝杆菌（Mycobacterium tuberculosis）的抗σ因子K（anti-Sigma factor K）RskA能够抑制SigK的活性，且RskA的突变可上调SigK调控子（SigK regulon）的表达水平。上述观察结果促使我们提出如下假说：RskA突变会导致其抗σ因子功能丧失。本研究利用RskA的天然突变体与人工突变体，解析SigK与RskA互作的分子基础。与理论预测相符的是，RskA的N端胞质结构域单独即可抑制SigK的活性。令人意外的是，RskA同时可作为SigK的激活因子，该激活作用依赖于同一N端区域，且可被RskA的膜-胞外结构域进一步增强。基于上述发现，我们在革兰氏阴性菌（Gram-negative bacterium）小肠结肠炎耶尔森菌（Yersinia enterocolitica）中构建了类似的截短突变体。实验结果再次显示，通过对RskA进行特异性改造，可有效增强SigK的活性。综上，本研究结果证实抗σ因子存在一种替代作用机制——我们可将其命名为‘双功能调节因子（兼具激活与抑制功能）’，该机制同时存在于放线菌门（Actinobacteria）与革兰氏阴性菌中，这提示抗σ因子介导的σ因子激活现象可能长期被学界低估。