Chodur Daniel M, Guo Linda, Pu Meng, Bruger Eric, Fernandez Nico, Waters Chris, Rowe-Magnus Dean A
Department of Biology, Indiana University, Bloomington, Indiana, USA.
Alcon Canada Inc., Mississauga, ON, Canada.
J Bacteriol. 2017 Sep 5;199(19). doi: 10.1128/JB.00344-17. Print 2017 Oct 1.
is an estuarine bacterium and potent opportunistic human pathogen. It enters the food chain by asymptomatically colonizing a variety of marine organisms, most notably oysters. Expression of the -encoded extracellular polysaccharide, which enhances cell-surface adherence, is regulated by cyclic di-GMP (c-di-GMP) and the activator BrpT. The and homologs VpsT and CpsQ, directly bind c-di-GMP via a novel W[F/L/M][T/S]R motif, and c-di-GMP binding is absolutely required for activity. Notably, BrpT belongs to a distinct subclass of VpsT-like regulators that harbor a proline in the third position of the c-di-GMP binding motif (WLPR), and the impact of this change on activity is unknown. We show that the locus is organized as two linked operons with BrpT specifically binding to promoters upstream of and Expression data and structural modeling suggested that BrpT might be less dependent on c-di-GMP binding for activity than VpsT or CpsQ. We show that the affinity of BrpT for c-di-GMP is low and that signal binding is not a requisite for BrpT function. Furthermore, a BrpT mutant engineered to carry a canonical WLTR motif (BrpT) bound c-di-GMP with high affinity and its activity was now c-di-GMP dependent. Conversely, introduction of the WLPR motif into VpsT suppressed its dependence on c-di-GMP for activity. This is the first demonstration of reduced dependence on signal association for regulator function within this motif family. Thus, BrpT defines a new class of VpsT-like transcriptional regulators, and the WLPR motif variant may similarly liberate the activity of other subclass members. A genome may encode nearly 100 proteins that make, break, and bind c-di-GMP, underscoring its central role in the physiology of these bacteria. The activity of the biofilm regulators VpsT of and CpsQ of is regulated by the direct binding of c-di-GMP via a novel W[F/L/M][T/S]R motif. The homolog, BrpT, bears an unusual WLPR variant and remains active at low intracellular c-di-GMP levels. This suggests that the WLPR motif may also liberate the activity of other members of this subclass. A single point mutation at the 3rd position of the motif was sufficient to moderate dependence on c-di-GMP binding for activator function, highlighting the simplicity with which complex bacterial signaling networks can be rewired.
是一种河口细菌，也是一种强大的机会性人类病原体。它通过无症状地定殖于多种海洋生物（最显著的是牡蛎）进入食物链。由其编码的细胞外多糖可增强细胞表面黏附性，其表达受环二鸟苷酸（c-di-GMP）和激活剂BrpT调控。其同源物VpsT和CpsQ通过一个新的W[F/L/M][T/S]R基序直接结合c-di-GMP，且c-di-GMP结合对于活性是绝对必需的。值得注意的是，BrpT属于VpsT样调节因子的一个独特亚类，在c-di-GMP结合基序（WLPR）的第三位含有一个脯氨酸，而这种变化对活性的影响尚不清楚。我们发现该基因座由两个连锁的操纵子组成，BrpT特异性结合和上游的启动子。表达数据和结构建模表明，与VpsT或CpsQ相比，BrpT的活性可能对c-di-GMP结合的依赖性较小。我们发现BrpT对c-di-GMP的亲和力较低，且信号结合不是BrpT功能的必要条件。此外，一个经过改造携带典型WLTR基序的BrpT突变体（BrpT）与c-di-GMP具有高亲和力结合，其活性现在依赖于c-di-GMP。相反，将WLPR基序引入VpsT可抑制其对c-di-GMP活性的依赖性。这是首次证明该基序家族内调节因子功能对信号关联的依赖性降低。因此，BrpT定义了一类新的VpsT样转录调节因子，且WLPR基序变体可能同样释放其他亚类成员的活性。一个基因组可能编码近100种制造、破坏和结合c-di-GMP的蛋白质，强调了其在这些细菌生理学中的核心作用。生物膜调节因子霍乱弧菌的VpsT和副溶血弧菌的CpsQ的活性通过一个新的W[F/L/M][T/S]R基序直接结合c-di-GMP来调控。其同源物BrpT具有一个不寻常的WLPR变体，在低细胞内c-di-GMP水平下仍保持活性。这表明WLPR基序也可能释放该亚类其他成员的活性。基序第三位的单个点突变足以调节激活剂功能对c-di-GMP结合的依赖性，突出了复杂细菌信号网络重新布线的简单性。
