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

Aguilar C, Vlamakis H, Guzman A, Losick R, Kolter R - MBio (2010)

Bottom Line: We have found that Spo0A~P levels are maintained at low levels in the matrix-deficient mutant, thereby delaying expression of sporulation-specific genes.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts, USA.

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.

No MeSH data available.


Related in: MedlinePlus

A kinD deletion restores sporulation to the eps tasA matrix mutant under biofilm-inducing conditions. (A) Flow cytometry of cells harboring the PsspB-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.
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Figure 5: A kinD deletion restores sporulation to the eps tasA matrix mutant under biofilm-inducing conditions. (A) Flow cytometry of cells harboring the PsspB-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.

Mentions: To determine if KinD’s ability to decrease the levels of Spo0A~P is necessary for the sporulation delay in the mutant lacking matrix, we introduced the kinD deletion into the eps tasA matrix-deficient strain and analyzed the expression of the PsspB-yfp sporulation reporter in the kinD eps tasA triple mutant. Our results indicate that while expression of the sporulation reporter was severely diminished in the double eps tasA mutant at 72 h of biofilm development, the level of PsspB-yfp reporter expression in the kinD eps tasA triple mutant was closer to wild-type levels (Fig. 5A). This increase in sporulation gene expression is independent of biofilm architecture, as the triple mutant colonies phenocopy the eps tasA mutant (see Fig. S1 in the supplemental material).


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

Aguilar C, Vlamakis H, Guzman A, Losick R, Kolter R - MBio (2010)

A kinD deletion restores sporulation to the eps tasA matrix mutant under biofilm-inducing conditions. (A) Flow cytometry of cells harboring the PsspB-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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2912670&req=5

Figure 5: A kinD deletion restores sporulation to the eps tasA matrix mutant under biofilm-inducing conditions. (A) Flow cytometry of cells harboring the PsspB-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.
Mentions: To determine if KinD’s ability to decrease the levels of Spo0A~P is necessary for the sporulation delay in the mutant lacking matrix, we introduced the kinD deletion into the eps tasA matrix-deficient strain and analyzed the expression of the PsspB-yfp sporulation reporter in the kinD eps tasA triple mutant. Our results indicate that while expression of the sporulation reporter was severely diminished in the double eps tasA mutant at 72 h of biofilm development, the level of PsspB-yfp reporter expression in the kinD eps tasA triple mutant was closer to wild-type levels (Fig. 5A). This increase in sporulation gene expression is independent of biofilm architecture, as the triple mutant colonies phenocopy the eps tasA mutant (see Fig. S1 in the supplemental material).

Bottom Line: We have found that Spo0A~P levels are maintained at low levels in the matrix-deficient mutant, thereby delaying expression of sporulation-specific genes.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts, USA.

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.

No MeSH data available.


Related in: MedlinePlus