Bistability and biofilm formation in Bacillus subtilis

Abstract

Biofilms of Bacillus subtilis consist of long chains of cells that are held together in bundles by an extracellular matrix of exopolysaccharide and the protein TasA. The exopolysaccharide is produced by enzymes encoded by the epsA-O operon and the gene encoding TasA is located in the yqxM-sipW-tasA operon. Both operons are under the control of the repressor SinR. Derepression is mediated by the antirepressor SinI, which binds to SinR with a 1:1 stoichiometry. Paradoxically, in medium promoting derepression of the matrix operons, the overall concentration of SinR in the culture greatly exceeded that of SinI. We show that under biofilm-promoting conditions sinI, which is under the control of the response regulator Spo0A, was expressed only in a small subpopulation of cells, whereas sinR was expressed in almost all cells. Activation of Spo0A is known to be subject to a bistable switch, and we infer that SinI reaches levels sufficient to trigger matrix production only in the subpopulation of cells in which Spo0A is active. Additionally, evidence suggests that sinI is expressed at intermediate, but not low or high, levels of Spo0A activity, which may explain why certain nutritional conditions are more effective in promoting biofilm formation than others.

Figure 1. SinI levels greatly exceed that of SinR

(A) Assays of β-galactosidase specific activity of cells carrying either the P_sinR-lacZ (filled squares; strain YC108) or the P_sinI-lacZ (filled diamonds; strain YC127) fusion at the amyE locus on the chromosome. Assays were performed for cells grown in MSgg medium and harvested at the indicated times. Time zero refers to the end of exponential phase growth. (B), (C) Quantitative immunoblots of SinR and SinI. Left-hand panels show affinity-purified, recombinant SinR and SinI proteins that were loaded at the indicated amounts. In the right-hand panels, cleared protein lysates prepared from early stationary phase cultures (one hour into stationary phase) were loaded on the same gel in a series of dilutions.

Figure 2. Fluorescence microscopy of cells expressing P_sinR-gfp and P_sinI-gfp

In all panels, cells were treated with the red membrane stain FM4-64. Green is fluorescence from GFP. (A) Shows cells containing P_sinR-gfp (YC173) or P_sinI-gfp (YC162) integrated into the chromosome at the amyE locus of strain 3610 at one hour after the end of exponential phase in MSgg medium. (B) Shows cells containing P_sinI-gfp and a construct (YC170) in which the spo0A gene was replaced with the mutant allele sad67 under the control of an IPTG-inducible promoter. (C) Shows cells containing P_sinI-gfp (YC162) at the indicated times before and after the end of exponential phase growth in MSgg medium.

Figure 3. Effect of medium on expression of sinI, eps, and spoIIG

(A) Morphology of colonies of strain 3610 formed on agar plates containing MSgg (left-hand panel) or DS (middle panel) medium. The colony shown in the right-hand panel was formed on solid DS medium by a 3610 derivative overexpressing a functional fusion of SinI with GFP (YC227). (B) Assays of β-galactosidase specific activity for P_eps-lacZ (left-hand panel; YC130) and P_sinI-lacZ (right-hand panel; YC127) in either DS or MSgg medium. Activities of P_eps-lacZ and P_sinI-lacZ were measured one hour after the end of exponential phase growth. (C) and (D) Distribution of cells expressing P_spoIIG-cfp (columns in 3C; FC476) or P_sinI-gfp (columns in 3D; YC162) at various intensities in the indicated media and at the indicated times. Intensities for individual cells were measured using Metamorph. Each bar represents the percentage (Y axis) of cells whose fluorescence intensity was within ±5 in panel C and ±10 in panel D to the unit shown in the X axis, versus total cells in the population. The horizontal bracket in panel D identifies a subpopulation of cells that expressed P_sinI-gfp highly.

Figure 4. Fluorescence microscopy of cells expressing P_eps-gfp or P_yqxM-cfp

The P_eps-gfp or P_yqxM-cfp was in a wild background (YC164 or YC189) or in a sinR (YC167 or YC221) or sinI (YC168 or YC190) mutant background. The cells were treated with the red membrane stain FM4-64. Fluorescence from P_yqxM-cfp was false-colored green.

Figure 5. Fluorescence microscopy of cells harboring both P_yqxM-yfp and either P_sinI-cfp (YC243) or P_sinR-cfp (YC244)

Fluorescence from P_yqxM-yfp was false-colored red. Fluorescence from P_sinI-cfp and P_sinR-cfp was false-colored green. Images in panel D are overlays from the corresponding images in B and C.