KinD is a checkpoint protein linking spore formation to extracellular-matrix production in Bacillus subtilis biofilms

Abstract

Bacillus subtilis cells form multicellular biofilm communities in which spatiotemporal regulation of gene expression occurs, leading to differentiation of multiple coexisting cell types. These cell types include matrix-producing and sporulating cells. Extracellular matrix production and sporulation are linked in that a mutant unable to produce matrix is delayed for sporulation. Here, we show that the delay in sporulation is not due to a growth advantage of the matrix-deficient mutant under these conditions. Instead, we show that the link between matrix production and sporulation is through the Spo0A signaling pathway. Both processes are regulated by the phosphorylated form of the master transcriptional regulator Spo0A. When cells have low levels of phosphorylated Spo0A (Spo0A~P), matrix genes are expressed; however, at higher levels of Spo0A~P, sporulation commences. We have found that Spo0A~P levels are maintained at low levels in the matrix-deficient mutant, thereby delaying expression of sporulation-specific genes. This is due to the activity of one of the components of the Spo0A phosphotransfer network, KinD. A deletion of kinD suppresses the sporulation defect of matrix mutants, while its overproduction delays sporulation. Our data indicate that KinD displays a dual role as a phosphatase or a kinase and that its activity is linked to the presence of extracellular matrix in the biofilms. We propose a novel role for KinD in biofilms as a checkpoint protein that regulates the onset of sporulation by inhibiting the activity of Spo0A until matrix, or a component therein, is sensed.

FIG 1

Matrix mutants are delayed in sporulation. (A) Sporulation of wild-type cells (WT) compared to the level for tasA, eps, or eps tasA matrix mutants. Cells were grown on MSgg solid medium at 30°C for the indicated time prior to harvesting, and viable spores were quantified after heat treatment for 20 min at 80°C. Error bars indicate standard errors of the means. (B) Total cells in biofilms grown on MSgg solid medium at 30°C for the indicated times. The entire colonies were harvested at the indicated times and disrupted using mild sonication conditions. Cells were resuspended in water, and absorbance at 600 nm was recorded immediately. Each strain was assayed in duplicate in at least three independent experiments. Error bars indicate standard errors of the means.

FIG 2

The matrix mutant sporulation defect is medium dependent. Images are top views of cells grown under each condition. Spore counts of cells grown on solid 100% MSgg (black bars) or 10% MSgg (open bars) at 30°C for 72 h. Error bars indicate standard errors of the means.

FIG 3

Matrix mutant cells retain low Spo0A~P levels. Flow cytometry of wild-type (black lines) and eps tasA (red lines) mutant cells. The grey line represents a control with cells not expressing yellow fluorescent protein (YFP). Cells were grown on MSgg solid medium at 30°C for 72 h prior to harvesting and disruption of the biofilm for flow cytometry. Cells harbored the indicated reporter fusions: P_yqxM-yfp, P_skfA-yfp, or P_sdpA-yfp. AU, arbitrary units.

FIG 4

A kinD mutant shows early sporulation. Flow cytometry of wild-type, kinD, or kinC mutant cells harboring the P_sspB-yfp promoter fusion. Cells were grown on MSgg solid medium at 30°C for 24 h prior to harvesting and disruption of the biofilm for flow cytometry. The control is wild-type cells with no fluorescent protein. AU, arbitrary units.

FIG 5

A kinD deletion restores sporulation to the eps tasA matrix mutant under biofilm-inducing conditions. (A) Flow cytometry of cells harboring the P_sspB-yfp promoter fusion. Cells were grown on MSgg solid medium at 30°C for 72 h prior to harvesting and disruption of the biofilm for flow cytometry. The control is wild-type cells with no fluorescent protein. AU, arbitrary units. (B) Sporulation of wild type, eps tasA, kinD, and kinD eps tasA cells. Cells were grown on MSgg solid medium at 30°C for the indicated time prior to harvesting and counting of viable spores after heat treatment. Error bars represent standard errors of the means.

FIG 6

Sporulation is delayed in a strain overexpressing kinD. Flow cytometry of wild-type and P_hyperspank-kinD cells harboring the P_sspB-yfp promoter fusion. Cells were grown on solid medium with IPTG at 30°C for 48 h prior to harvesting and disrupting of the biofilm for flow cytometry. The control is cells with no fluorescent protein. (A) One hundred percent MSgg medium. (B) Ten percent MSgg medium. AU, arbitrary units.