Extended Data Fig. 6: Three Na⁺ riboswitches in Acetivibrio cellulolyticus regulate three major processes related to sodium biology.

The bacterium A. cellulolyticus carries three Na⁺-I riboswitch representatives (red) that regulate genes associated with three major aspects of sodium biology. Sodium gradient: The pycB gene codes for oxaloacetate decarboxylase, whose activity drives Na⁺ export to yield a Na⁺ gradient [Dimroth, P., Jockel, P. & Schmid, M. Coupling mechanism of the oxaloacetate decarboxylase Na⁺ pump. Biochim. Biophys. Acta Bioenerg. 1505, 1-14 (2001)]. The gradient can then be exploited to promote solute uptake by Na⁺-dependent import proteins [Pos, K. M. & Dimroth, P. Functional properties of the purified Na⁺-dependent citrate carrier of Klebsiella pneumoniae: evidence for asymmetric orientation of the carrier protein in proteoliposomes. Biochemistry 35, 1018-1026 (1996)]. pH homeostasis: DUF1646 genes code for proteins of unknown function but some have been predicted to function as a Na⁺/H⁺ antiporters [CDD Conserved Protein Domain Family: DUF1646 (nih.gov); https://www.genome.jp/dbget-bin/www_bget?pop:7465038], which presumably can be used to increase the pH of cells growing under alkaline conditions. Osmotic stress response: Tandem Na⁺-I and c-di-AMP (brown) riboswitches are associated with dapB, which codes for dihydrodipicolinate reductase. This enzyme produces 2,3,4,5-tetrahydrodiaminopimelate, which is a precursor for both lysine and peptidoglycan biosynthesis [https://www.genome.jp/pathway/lin00300+dapB]. Peptidoglycan biosynthesis is critical for cell resistance to osmotic stress in high salt environments [Auer, G. K. & Weibel, D. B. Bacterial cell mechanics. Biochemistry 56, 3710-3724 (2017); Metris, A., George, S. M., Mulholland, F., Carter, A. T. & Baranyi, J. Metabolic shift of Escherichia coli under salt stress in the presence of glycine betaine. Appl. Environ. Microb. 80, 4745-4756 (2014)]. E. coli cells grown in 4.5% NaCl exhibit increased expression of dapB, and our findings indicate that some species directly regulate this gene using a Na⁺-responsive tandem riboswitch.