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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

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.
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Figure 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.

Mentions: We previously reported that cells lacking either the TasA protein or the exopolysaccharide (EPS) component of the matrix were defective in sporulation when grown under biofilm-inducing conditions but not when grown in dispersed culture (5). To further understand this sporulation defect, we analyzed the kinetics of spore formation in biofilms of single mutants lacking either tasA or the eps operon as well as in a double mutant lacking both major matrix components (eps tasA) (Fig. 1A). As expected, the kinetics of spore formation in biofilms formed by the wild type showed an increasing number of spores over time, with the highest increase in the number of spores observed between 24 h and 48 h of development. In contrast, cells from biofilms of each single mutant or of the double eps tasA mutant showed a minimal increase in the number of spores up to 72 h of growth. It was only after 72 h of growth that a steady increase in the number of spores was observed. By 120 h of development, the number of spores was about 10- to 100-fold less than the wild-type level for all three mutant strains (Fig. 1A). Since the rate of spore formation of the double eps tasA mutant was similar to that of the individual tasA and eps mutants, we focused our analyses on the double mutant for the remaining experiments.


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)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 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.
Mentions: We previously reported that cells lacking either the TasA protein or the exopolysaccharide (EPS) component of the matrix were defective in sporulation when grown under biofilm-inducing conditions but not when grown in dispersed culture (5). To further understand this sporulation defect, we analyzed the kinetics of spore formation in biofilms of single mutants lacking either tasA or the eps operon as well as in a double mutant lacking both major matrix components (eps tasA) (Fig. 1A). As expected, the kinetics of spore formation in biofilms formed by the wild type showed an increasing number of spores over time, with the highest increase in the number of spores observed between 24 h and 48 h of development. In contrast, cells from biofilms of each single mutant or of the double eps tasA mutant showed a minimal increase in the number of spores up to 72 h of growth. It was only after 72 h of growth that a steady increase in the number of spores was observed. By 120 h of development, the number of spores was about 10- to 100-fold less than the wild-type level for all three mutant strains (Fig. 1A). Since the rate of spore formation of the double eps tasA mutant was similar to that of the individual tasA and eps mutants, we focused our analyses on the double mutant for the remaining experiments.

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