Coordinated Cyclic-Di-GMP Repression of Salmonella Motility through YcgR and Cellulose

Abstract

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 biofilm matrix. YcgR is dedicated to c-di-GMP-dependent inhibition of motility through its interaction with flagellar motor proteins. However, previous 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 motility 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 RxxxR motif of the BcsA PilZ domain, or degradation of the cellulose produced by BcsA rescued the motility defect of ⌬ycgR strains in which high c-di-GMP levels were reached through the overexpression 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 highlight the relevance of cellulose in Salmonella lifestyle switching as an architectural element that is both essential for biofilm development and required, in collaboration with YcgR, for complete motility inhibition.

Bistable Expression of CsgD in Salmonella enterica Serovar Typhimurium Connects Virulence to Persistence

“…The importance of SPI-1 in this process was confirmed, since planktonic cells and multicellular aggregates from a ⌬SPI-1 mutant strain had nearly equal colonization efficiencies. Furthermore, the reduction in virulence that we observed for the ⌬csgD strain, together with recent connections between c-di-GMP, virulence (28), and motility (52), suggest that the master biofilm regulator, CsgD, can modulate the virulence capacity of S. Typhimurium.…”

“…In contrast, high expression of ycgR and ydiV in planktonic cells at TP2 did not match the predicted phenotypes. YcgR contains a PilZ domain for binding c-di-GMP and represses motility by acting as a type of flagellar brake (51,52). Based on the published function, we would have expected ycgR to be more highly expressed in multicellular aggregates and to function at higher c-di-GMP levels inside the cell.…”

Cellulose as an Architectural Element in Spatially Structured Escherichia coli Biofilms

“…Cellulose can also counteract the proinflammatory effect of curli fibers when expressed in the mammalian host (55,56). Finally, cellulose can inhibit the rotation of flagella, which was observed in a mutant deficient for the c-di-GMP-binding protein YcgR, a protein that shuts down rotation by direct interaction with the flagellar basal body (57). In fact, we could visually demonstrate a tight entanglement of cellulose with flagella in the transition area between the growth and stationary phase layers of the macrocolony (Fig.…”

Type III secretion systems: the bacterial flagellum and the injectisome

“…Additionally, c-di-GMP plays an important role in the functional control of flagella, in addition to its effects on the transcriptional and post-transcriptional regulation of motility: the c-di-GMP-binding protein YcgR represses motility by binding to the MotAB stator [6,9], as well as to the C-ring [7,8]. In addition, high c-di-GMP levels lead to accumulation of extracellular cellulose in Salmonella, which sterically reduces motility [244]. Heterogeneity in the cellular levels of c-di-GMP, caused by the activity of a phosphodiesterase, which in turn is controlled and asymmetrically partitioned by binding to the chemotaxis histidine kinase CheA in P. aeruginosa, accordingly leads to different motility and chemotactic response between the two daughter cells after division [245,246].…”