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 ΔycgR 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.

Fig 1

High c-di-GMP levels inhibit Salmonella motility in the absence of YcgR. Representative swimming motility plates after incubation at 23°C for 16 h are shown. Quantitative measurement of motility is also shown. The total area of growth was measured, and the percent motility relative to that of the parental strain was calculated. Means and standard deviations of results from three repeats on three separate days are shown. Overexpression of the DGC-encoding gene stm1987 in a wild-type S. Enteritidis strain (WT pBR328::stm1987) completely inhibited motility even in the absence of YcgR (ΔycgR pBR328::stm1987). Overexpression of a heterologous DGC-encoding gene, hmsT, in a multiple S. Enteritidis mutant in all genes encoding GGDEF domain proteins (ΔXII P_sen4316::hmsT) also resulted in motility blockage that was only slightly restored when the ycgR gene was mutated (ΔXII ΔycgR P_sen4316::hmsT). Inhibition of motility depended on the capacity of STM1987 and HmsT to synthesize c-di-GMP (see WT pBR328::stm1987-GS and ΔXII P_sen4316::hmsT-GS strains, respectively).

Fig 2

Expression of hmsT gene under the sen4316 promoter results in a high accumulation of the HmsT protein. Western blot analysis of HmsT and SEN4316 expression in ΔXII derivative strains in which the hmsT or sen4316 genes with a 3×Flag epitope coding sequence were chromosomally restored under the sen4316 promoter is shown. Strains were grown under tethering assay conditions, that is, at 28°C for 6 h, or under swimming conditions, that is, at 23°C for 16 h. HmsT was highly produced under both conditions compared to the expression of the original Salmonella DGC-encoding gene, sen4316. A Western blot using anti-GroEL antibodies was used as a loading control.

Fig 3

High c-di-GMP levels do not regulate flagellum gene expression, flagellar exportation, and assembly. (A) Analysis of fliC promoter activity. Samples of the wild-type strain, ΔXII, and ΔXII P_sen4316::hmsT harboring gfp[LVA] transcriptional fusions to the fliC promoter were analyzed by Western blotting using anti-GFP antibodies. A Western blot using anti GroEL antibodies was used as a loading control. (B) Western blot analysis of exported flagellin from the wild type, ΔXII, and derivative strains overexpressing stm1987 and hmsT, respectively. (C) Flagellin immunofluorescence of the wild type, ΔXII, and derivative strains overexpressing a DGC. Blue fluorescence corresponds to the Hoechst 33342 dsDNA stain. In all cases, bacteria were grown under tethering assay conditions.

Fig 4

C-di-GMP binding to BcsA is responsible for motility inhibition in the absence of YcgR. Representative swimming motility plates and quantitative measurement of motility after incubation at 23°C for 16 h are shown. (A) Swimming motility of a strain that expresses two unique sources of c-di-GMP and presents a mutation in ycgR was completely rescued by means of an additional mutation of the cellulose synthase-encoding gene, bcsA. The same result was obtained when the bcsA mutation was transduced to a ycgR single mutant overexpressing stm1987 and to ΔXII strain containing a ycgR mutation and overexpressing hmsT. (B) Deletion of both ycgR and bcsA in strains that present high levels of c-di-GMP is needed to recuperate swimming behavior. Restoration of motility is also achieved by mutating the c-di-GMP binding motif of the PilZ domain of BcsA.

Fig 5

A premature cellulose production that has taken place as a consequence of high intracellular c-di-GMP levels in the cell is responsible for motility inhibition. (A) Endoglucanase activity of BcsZ was confirmed by assessing carboxymethylcellulose (CMC)-degrading activity of the wild-type S. Enteritidis strain harboring the bcsZ-overexpressing plasmid pUA1108::bcsZ. As a control, the endoglucanase phenotype of the wild-type strain harboring an empty pUA1108 overexpression vector is also presented. (B) Degradation of the cellulose produced by a strain that overexpresses a unique and very active source of c-di-GMP and lacks YcgR is enough to recuperate swimming motility. Strains assayed were transformed with an empty pUA1108 plasmid or with the overexpressing plasmid pUA1108::bcsZ. Representative swimming motility plates and quantitative measurement of motility after incubation at 23°C for 16 h are shown. (C) Correlation between swimming motility, rotation behavior in the tethering assay, and cellulose production. Early cellulose synthesis was detected in strains that overexpressed a DGC and were grown under tethering assay conditions, that is, at 28°C for 6 h. Detection of cellulose production by calcofluor staining (CF) is shown in the right panel. Membrane staining with FM4-64 is shown in the intermediate panel.

Fig 6

The motility defect of double yhjH and ycgR mutants is rescued by inactivation of cellulose production. (A) Representative swimming motility plates after incubation at 23°C for 16 h. (B) Quantitative measurement of motility. The total area of growth was measured, and the percent motility relative to that of the parental strain was calculated. Means and standard deviations of results from three repeats on three separate days are shown. Inactivation of ycgR is able to substantially but not totally restore the bacterial motility defect of S. Enteritidis or S. Typhimurium ΔyhjH mutants. An additional mutation of the cellulose synthase-encoding gene, bcsA, leads to a total recuperation of wild-type motility in both Salmonella species.