c-di-GMP targets the enhancer-binding protein SfnR1 to regulate Pseudomonas aeruginosa growth under sulfur limitation

As a ubiquitous bacterial second messenger, cyclic di-GMP (c-di-GMP) governs diverse physiological processes by directly binding to transcriptional regulators or other effector proteins. Enhancer-binding proteins (EBPs) are a distinct class of transcriptional regulators that, together with σ54-RNA polymerase, promote downstream gene expression through ATP hydrolysis. To date, no EBP other than FleQ has been reported to interact with c-di-GMP in Pseudomonas aeruginosa. By systematically screening P. aeruginosa EBPs for c-di-GMP binding, we identified SfnR1 as a novel receptor. SfnR1 is known to promote msuEDC and sfnG expression, enabling bacteria to utilize nonpreferred sulfur sources (including DMSO and DMSO₂) under sulfur limitation. However, whether SfnR1 is regulated by c-di-GMP and how this regulation affects its transcriptional activity remain unknown. Here, we demonstrate that c-di-GMP binding to SfnR1 prevents SfnR1 from binding to the msuEDC and sfnG promoters, thereby downregulating their expression. This impairs P. aeruginosa assimilation of nonpreferred sulfur sources and ultimately restricts its growth under sulfur-limiting conditions. In contrast, modulation of c-di-GMP levels in P. aeruginosa has no effect on growth under normal conditions. Collectively, our findings establish that c-di-GMP binding to SfnR1 selectively restricts P. aeruginosa growth under sulfur limitation, uncovering a novel mechanism whereby this second messenger modulates bacterial adaptation to nutrient stress.