Self-regulation of exopolysaccharide production in Bacillus subtilis by a tyrosine kinase

Abstract

We report that the Bacillus subtilis exopolysaccharide (EPS) is a signaling molecule that controls its own production. EPS synthesis depends on a tyrosine kinase that consists of a membrane component (EpsA) and a kinase component (EpsB). EPS interacts with the extracellular domain of EpsA, which is a receptor, to control kinase activity. In the absence of EPS, the kinase is inactivated by autophosphorylation. The presence of EPS inhibits autophosphorylation and instead promotes the phosphorylation of a glycosyltransferase in the biosynthetic pathway, thereby stimulating the production of EPS. Thus, EPS production is subject to a positive feedback loop that ties its synthesis to its own concentration. Tyrosine kinase-mediated self-regulation could be a widespread feature of the control of exopolysaccharide production in bacteria.

Keywords: biofilm; exopolysaccharide; tyrosine kinase.

Figure 1.

The EpsAB tyrosine kinase activity is required for biofilm formation. (A) Cartoon showing the disposition of EpsA in the membrane and the tyrosine (Y) cluster in EpsB. (B,C) Colonies of the wild type and the indicated mutants grown on biofilm-inducing medium. The strains were NCBI 3610 (WT), RL3852 (∆epsH), BAE250 (∆epsA), BAE251 (∆epsB), BAE252 (∆epsAB), BAE262 (∆epsAB amyE∷epsAB), RL3854 (∆sinR), and BAE388 (∆sinR∆epsB).

Figure 2.

Autophosphorylation inactivates EpsAB. (A) Immunoblot of His₆-tagged EpsB from the wild-type strain (BAE318) and strains producing EpsB^D81A/D83A (BAE451) and EpsB^Y225F/Y227F (BAE450) grown in biofilm-inducing medium. The blot was probed with anti-His₆ antibodies (top row) and anti-phosphotyrosine antibodies (bottom row). (B) Colony wrinkling on biofilm-inducing medium for strains NCBI 3610 (WT), BAE252 (∆epsAB), BAE320 (∆epsAB amyE∷epsAB^Y225F/Y227F), and BAE406 (∆epsAB amyE∷epsAB^Y225E/Y227E).

Figure 3.

Glycosyl transferase EpsE is phosphorylated in an EpsAB-dependent manner. (A) Immunoblot probed with anti-phosphotyrosine antibodies of affinity-purified, His₆-tagged EpsB and copurifying protein(s) from cells (BAE318) that had been grown on biofilm-inducing medium. Also shown is a control with wild-type (BAE262) cells that produced untagged EpsB. (B) Immunoblot of His₆-tagged EpsE from wild-type cells (BAE560), ∆epsAB mutant cells (BAE561), and ∆epsB mutant cells (BAE563) harboring a mutant copy of the gene producing a catalytically inactive kinase, EpsB^D81A/D83A. Cells were grown in biofilm-inducing medium, and the blot was probed with anti-His₆ antibodies (top row) and anti-phosphotyrosine antibodies (bottom row). (C) Immunoblot showing the time course of phosphorylation of EpsE and EpsB. His-tagged protein was purified from cells producing His₆-EpsE (BAE477) or cells producing His₆-EpsB (BAE318). The cells had been harvested at the indicated times during growth on biofilm-inducing medium. The blots were probed with anti-His₆ antibodies (top rows) and anti-phosphotyrosine antibodies (bottom rows).

Figure 4.

EPS stimulates phosphorylation of EpsB and EpsB. (A) Immunoblot of purified His₆-tagged EpsB from wild-type cells (BAE318) and cells mutant for epsH (BAE397) expressing the EpsAB (BAE397) or the chimeric CapA-EpsAB (BAE619) from an IPTG-inducible promoter grown in the presence or absence of polysaccharide. The wild-type cells and cells mutant for epsH were grown in biofilm-inducing medium to which IPTG was added. The epsH mutant cells were grown in LB medium to which IPTG was added at midexponential phase and that had been supplemented with no polysaccharide (−) or with purified B. subtilis EPS (20 μg/mL) or PIA (20 μg/mL) or 0.1% dextran as indicated. (B) Immunoblot showing the dose dependence of EpsB autophosphorylation on EPS. His₆-tagged EpsB from epsH mutant cells that contained an IPTG-inducible copy of epsA and his₆-epsB (BAE397) was grown in LB medium to which IPTG was added and treated with increasing concentrations of purified EPS. (C) Immunoblot of purified His₆-tagged EpsE from wild-type cells (BAE560) or epsH mutant cells that carried a xylose-inducible copy of the gene for the tagged protein (BAE562). The cells were grown in biofilm-inducing medium in the presence of xylose for 24 h and either left untreated (−) or treated with 20 µg/mL EPS. For all three panels, the top row was probed with anti-His₆ antibodies, and the bottom row was probed with anti-phosphotyrosine antibodies.

Figure 5.

The extracellular domain of EpsA binds EPS. The His-tagged extracellular domain of EpsA and CapA was immobilized on a CM5 chip surface. Shown is the binding of EPS and PIA that had been passed over the chip surface at the indicated concentrations for the EpsA receptor (A) or the CapA receptor (B).