Ganguly Tridib, Kajfasz Jessica K, Abranches Jacqueline, Lemos José A
Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, FL, USA.
Mol Microbiol. 2020 Jul;114(1):109-126. doi: 10.1111/mmi.14499. Epub 2020 Apr 8.
Spx is a major regulator of stress responses in Firmicutes. In Streptococcus mutans, two Spx homologues, SpxA1 and SpxA2, were identified as mediators of oxidative stress responses but the regulatory circuits controlling their levels and activity are presently unknown. Comparison of SpxA1 and SpxA2 protein sequences revealed differences at the C-terminal end, with SpxA1 containing an unusual number of acidic residues. Here, we showed that a green fluorescence protein (GFP) reporter becomes unstable when fused to the last 10 amino acids of SpxA2 but remained stable when fused to the C-terminal acidic tail of SpxA1. Inactivation of clpP or simultaneous inactivation of clpC and clpE stabilized the GFP::SpxA2 fusion protein. Addition of acidic amino acids to the GFP::SpxA2 chimera stabilized GFP, while deletion of the acidic residues destabilized GFP::SpxA1 . Promoter reporter fusions revealed that spxA1 transcription is co-repressed by the metalloregulators PerR and SloR while spxA2 transcription is largely dependent on the envelope stress regulator LiaFSR. In agreement with spxA2 being part of the LiaR regulon, SpxA2 was found to be critical for the growth of S. mutans under envelope stress conditions. Finally, we showed that redox sensing is essential for SpxA1-dependent activation of oxidative stress responses but dispensable for SpxA2-mediated envelope stress responses.
Spx是厚壁菌门应激反应的主要调节因子。在变形链球菌中，两个Spx同源物SpxA1和SpxA2被鉴定为氧化应激反应的介质，但目前控制它们水平和活性的调节回路尚不清楚。SpxA1和SpxA2蛋白质序列的比较揭示了C末端的差异，SpxA1含有异常数量的酸性残基。在这里，我们表明，绿色荧光蛋白（GFP）报告基因与SpxA2的最后10个氨基酸融合时变得不稳定，但与SpxA1的C末端酸性尾巴融合时保持稳定。clpP失活或clpC和clpE同时失活可使GFP::SpxA2融合蛋白稳定。向GFP::SpxA2嵌合体中添加酸性氨基酸可使GFP稳定，而删除酸性残基则会使GFP::SpxA1不稳定。启动子报告基因融合显示，spxA1转录受到金属调节因子PerR和SloR的共同抑制，而spxA2转录在很大程度上依赖于包膜应激调节因子LiaFSR。与spxA2是LiaR调节子的一部分一致，发现SpxA2对于变形链球菌在包膜应激条件下的生长至关重要。最后，我们表明氧化还原感应对于SpxA1依赖的氧化应激反应激活至关重要，但对于SpxA2介导的包膜应激反应则是可有可无的。
