Staying in the loop to make ends meet: roles and regulation of GlmR in Bacillus subtilis

Abstract

The metabolic networks of most life forms integrate cost-benefit analysis to properly budget carbon and other essential nutrients through continuous assessment of nutrient availability and environmental threats. Bacillus subtilis is a Gram-positive model bacterium found in diverse ecological niches such as soil, marine environments, and the human gut. As such, B. subtilis cells finetune metabolic pathways by monitoring signals indicating the presence of nutrients and stressors. A highly conserved protein, GlmR, is a key player in rationing carbon for the production of cell envelope precursors. This function of GlmR can be attributed to its role in cell shape regulation and antibiotic resistance. Given its central position in carbon utilization, GlmR is under post-translational regulation by phosphorylation and UDP-N-acetylglucosamine (UDP-GlcNAc) binding. GlmR is also linked to cyclic-di-AMP (c-di-AMP), a nucleotide second messenger involved in stress response. In this study, we probed the importance of GlmR in cell morphogenesis, c-di-AMP signaling, and investigated the physiological significance of post-translational regulation. Our results reveal that cells lacking glmR exhibit: (i) increased susceptibility to tunicamycin, a cell envelope targeting antibiotic; (ii) impaired division site positioning; and (iii) elevated intracellular c-di-AMP concentration. Furthermore, we show that the function of GlmR is finetuned by UDP-GlcNAc binding, phosphorylation, and acetylation. Additionally, we provide evidence showing that the recently discovered enzymatic activity of GlmR is integral for its function. We show that GlmR is a cell width determinant and propose a model suggesting close cooperation with an actin-like protein, MreB. Overall, our studies highlight that GlmR is at the crux of carbon flux with an important role in maintaining cell envelope integrity. IMPORTANCEBacteria must integrate feedback from multiple metabolic processes to efficiently allocate carbon to produce essential building blocks such as nucleotides, amino acids, and cell wall precursors to support life. GlmR is a critical metabolic factor involved in the making of cell envelope precursors in diverse bacterial phyla. In Bacillus subtilis, cells lacking GlmR are deformed and hypersensitive to cell wall targeting antibiotics. As siphoning off too much carbon from other essential processes is detrimental to cell viability, GlmR activity is tightly regulated. Here we report that absence of GlmR leads to aberrant placement of cytokinetic machinery and an increase in the levels of cyclic-di-AMP, a nucleotide second messenger that assists in the cell wall stress response. We also show that GlmR function is post-translationally finetuned by phosphorylation and acetylation. Furthermore, our data reveals that the catalytic activity of GlmR is required for its function. Thus, the activity of GlmR is tightly calibrated through multiple means for efficient carbon utilization.