Coordinated cyclic-di-GMP repression of Salmonella motility through YcgR and cellulose

Cyclic di-GMP (c-di-GMP) is a secondary messenger that controls a variety of cellular processes, including the switch between a biofilm and a planktonic bacterial lifestyle. This nucleotide binds to cellular effectors in order to exert its regulatory functions. In Salmonella, two proteins, BcsA and YcgR, both of them containing a c-di-GMP binding PilZ domain, are the only known c-di-GMP receptors. BcsA, upon c-di-GMP binding, synthesizes cellulose, the main exopolysaccharide of the biofilmmatrix. YcgR is dedicated to c-di-GMP-dependent inhibition of motility through its interaction with flagellar motor proteins. However, previ-ous evidences indicate that in the absence of YcgR, there is still an additional element that mediates motility impairment under high c-di-GMP levels. Here we have uncovered that cellulose per se is the factor that further promotes inhibition of bacterial mo-tility once high c-di-GMP contents drive the activation of a sessile lifestyle. Inactivation of different genes of the bcsABZC operon, mutation of the conserved residues in the RxxxRmotif of the BcsA PilZ domain, or degradation of the cellulose pro-duced by BcsA rescued the motility defect ofycgR strains in which high c-di-GMP levels were reached through the overexpres-sion of diguanylate cyclases. High c-di-GMP levels provoked cellulose accumulation around cells that impeded flagellar rotation, probably by means of steric hindrance, without affecting flagellum gene expression, exportation, or assembly. Our results high-light the relevance of cellulose in Salmonella lifestyle switching as an architectural element that is both essential for biofilm de-velopment and required, in collaboration with YcgR, for complete motility inhibition. Bis-(3=–5=)-cyclic dimeric GMP (c-di-GMP), discovered byBenziman and colleagues in 1987 as an allosteric activator o