The cyclic-di-GMP phosphodiesterase BinA negatively regulates cellulose-containing biofilms in Vibrio fischeri

Abstract

Bacteria produce different types of biofilms under distinct environmental conditions. Vibrio fischeri has the capacity to produce at least two distinct types of biofilms, one that relies on the symbiosis polysaccharide Syp and another that depends upon cellulose. A key regulator of biofilm formation in bacteria is the intracellular signaling molecule cyclic diguanylate (c-di-GMP). In this study, we focused on a predicted c-di-GMP phosphodiesterase encoded by the gene binA, located directly downstream of syp, a cluster of 18 genes critical for biofilm formation and the initiation of symbiotic colonization of the squid Euprymna scolopes. Disruption or deletion of binA increased biofilm formation in culture and led to increased binding of Congo red and calcofluor, which are indicators of cellulose production. Using random transposon mutagenesis, we determined that the phenotypes of the DeltabinA mutant strain could be disrupted by insertions in genes in the bacterial cellulose biosynthesis cluster (bcs), suggesting that cellulose production is negatively regulated by BinA. Replacement of critical amino acids within the conserved EAL residues of the EAL domain disrupted BinA activity, and deletion of binA increased c-di-GMP levels in the cell. Together, these data support the hypotheses that BinA functions as a phosphodiesterase and that c-di-GMP activates cellulose biosynthesis. Finally, overexpression of the syp regulator sypG induced binA expression. Thus, this work reveals a mechanism by which V. fischeri inhibits cellulose-dependent biofilm formation and suggests that the production of two different polysaccharides may be coordinated through the action of the cellulose inhibitor BinA.

FIG. 1.

The binA gene and its predicted protein. (A) The binA gene (VFA1038) is located downstream from and oriented in the same direction as the syp gene locus. Individual genes are indicated by block arrows, and the four known or putative promoters within the syp locus are indicated by line arrows. (B) The BinA protein (630 amino acids [aa]) is predicted to have three domains, GAF (∼Q20 to L151), GGDEF (∼H205 to A338), and EAL (∼L374 to D611), as indicated. Only the EAL domain is well conserved.

FIG. 2.

Biofilm formation by the binA mutant. V. fischeri strains ES114 (wild type), KV4131 (ΔbinA), and KV4196 (ΔbinA/attTn7::binA⁺) were grown statically for 72 h in HMM at room temperature and then analyzed for biofilm formation by crystal violet staining (A). Crystal violet staining was subsequently quantified from a triplicate set of tubes (B). Error bars represent ±1 standard deviation.

FIG. 3.

Biofilm formation by binA syp double mutants. Mutants defective for both binA and various syp genes (KV4205, KV4197, KV4209, and KV4601) were analyzed for biofilm formation using the crystal violet assay following 72 h of static growth in HMM at room temperature. The assay was performed multiple times in triplicate using the double mutants indicated in the text; shown are a representative data set.

FIG. 4.

BinA inhibits Congo red binding, wrinkled-colony formation, and Calcofluor binding. Congo red binding (A) and colony morphology (B) were assessed after 48 h of growth at room temperature on Congo red plates. (C) Calcofluor staining was assayed after 72 h of static growth in HMM at room temperature. The strains are the same as those indicated in Fig. 2.

FIG. 5.

Role of the cellulose operon in biofilm formation. Congo red binding (A) and colony morphology (B) were assessed after 48 h of growth at room temperature on Congo red plates. Crystal violet staining was visualized (C) and quantified (D) after 72 h of static growth in HMM at room temperature. Error bars represent ±1 standard deviation from a triplicate set of tubes. (E) Calcofluor staining was assessed after 72 h of static growth in HMM at room temperature. (F) The bcs locus (VFA0887 to VFA0881) with the locations of the Tn insertions indicated by inverted triangles.

FIG. 6.

Motility of binA mutant and overexpression strains. The motility of V. fischeri strains was monitored by measuring the diameter of the outer chemotaxis ring formed in TBS-Mg²⁺ soft agar over time. (A) Motility of the ΔbinA mutant (KV4131) and its wild-type parent ES114. (B) Motility of wild-type strain ES114 carrying the control vector pKV69 or the binA overexpression plasmid pCMA9 (pBinA). Error bars represent ±1 standard deviation.

FIG. 7.

Role of the EAL motif in BinA activity. The effect on BinA activity of mutating the EAL motif to AAL or EAA was monitored using Congo red binding (A) and crystal violet staining (B) of cultures grown in HMM-Tc under shaking conditions for 48 h at 28°C. Crystal violet staining was subsequently quantified from a triplicate set of tubes (C); error bars represent ±1 standard deviation. (D) Western immunoblot analysis of tagged versions of the BinA protein. BinA-expressing plasmids pMSM17, -18, and -19 were introduced into binA mutant KV4131. Whole-cell extracts were processed as described in Materials and Methods.

FIG. 8.

Intracellular c-di-GMP concentrations. Each bar represents the average intracellular c-di-GMP concentration of three samples. The sample concentrations ranged from 0 nM to 53.4 nM for wild-type V. fischeri and from 86.7 nM to 413 nM for the ΔbinA mutant (KV4131). The error bars represent ±1 standard error of the mean.

FIG. 9.

Impact of the syp regulator SypG on binA expression and activity. (A) Diagram of the chromosomal region of the reporter strain KV4612, which contains pEAH50 inserted by homologous recombination with the sypR-binA intergenic region; this region is labeled P_binA for simplicity, although it is not known whether a promoter exists there. The 3′ end of sypR, the sypR-binA intergenic region, and the 5′ end of the binA gene are duplicated in the strain; one wild-type copy of binA is present. The drawing is not to scale. (B) β-Galactosidase activities of the binA mutant reporter strain KV4612 carrying the control plasmid pKV69 (vector), SypG overexpression plasmid pEAH73 (SypG), and SypG-D53E increased-activity allele plasmid pKV276 (SypG*). (C) Congo red binding by sypN mutant strain KV1838 carrying the indicated plasmids after 48 h of growth on a Congo red plate at room temperature.