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1.
J Bacteriol ; 193(22): 6113-22, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21926229

ABSTRACT

Upon starvation, Bacillus subtilis cells switch from growth to sporulation. It is believed that the N-terminal sensor domain of the cytoplasmic histidine kinase KinA is responsible for detection of the sporulation-specific signal(s) that appears to be produced only under starvation conditions. Following the sensing of the signal, KinA triggers autophosphorylation of the catalytic histidine residue in the C-terminal domain to transmit the phosphate moiety, via phosphorelay, to the master regulator for sporulation, Spo0A. However, there is no direct evidence to support the function of the sensor domain, because the specific signal(s) has never been found. To investigate the role of the N-terminal sensor domain, we replaced the endogenous three-PAS repeat in the N-terminal domain of KinA with a two-PAS repeat derived from Escherichia coli and examined the function of the resulting chimeric protein. Despite the introduction of a foreign domain, we found that the resulting chimeric protein, in a concentration-dependent manner, triggered sporulation by activating Spo0A through phosphorelay, irrespective of nutrient availability. Further, by using chemical cross-linking, we showed that the chimeric protein exists predominantly as a tetramer, mediated by the N-terminal domain, as was found for KinA. These results suggest that tetramer formation mediated by the N-terminal domain, regardless of the origin of the protein, is important and sufficient for the kinase activity catalyzed by the C-terminal domain. Taken together with our previous observations, we propose that the primary role of the N-terminal domain of KinA is to form a functional tetramer, but not for sensing an unknown signal.


Subject(s)
Bacillus subtilis/enzymology , Bacterial Proteins/metabolism , Protein Kinases/metabolism , Spores, Bacterial/growth & development , Bacillus subtilis/chemistry , Bacillus subtilis/genetics , Bacillus subtilis/growth & development , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial , Phosphorus-Oxygen Lyases/chemistry , Phosphorus-Oxygen Lyases/genetics , Phosphorus-Oxygen Lyases/metabolism , Protein Kinases/chemistry , Protein Kinases/genetics , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction , Spores, Bacterial/enzymology , Spores, Bacterial/genetics
2.
J Bacteriol ; 192(15): 3870-82, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20511506

ABSTRACT

Sporulation in Bacillus subtilis is controlled by a complex gene regulatory circuit that is activated upon nutrient deprivation. The initial process is directed by the phosphorelay, involving the major sporulation histidine kinase (KinA) and two additional phosphotransferases (Spo0F and Spo0B), that activates the master transcription factor Spo0A. Little is known about the initial event and mechanisms that trigger sporulation. Using a strain in which the synthesis of KinA is under the control of an IPTG (isopropyl-beta-d-thiogalactopyranoside)-inducible promoter, here we demonstrate that inducing the synthesis of the KinA beyond a certain level leads to the entry of the irreversible process of sporulation irrespective of nutrient availability. Moreover, the engineered cells expressing KinA under a sigma(H)-dependent promoter that is similar to but stronger than the endogenous kinA promoter induce sporulation during growth. These cells, which we designated COS (constitutive sporulation) cells, exhibit the morphology and properties of sporulating cells and express sporulation marker genes under nutrient-rich conditions. Thus, we created an engineered strain displaying two cell cycles (growth and sporulation) integrated into one cycle irrespective of culture conditions, while in the wild type, the appropriate cell fate decision is made depending on nutrient availability. These results suggest that the threshold level of the major sporulation kinase acts as a molecular switch to determine cell fate and may rule out the possibility that the activity of KinA is regulated in response to the unknown signal(s).


Subject(s)
Bacillus subtilis/physiology , Bacterial Proteins/metabolism , Protein Kinases/metabolism , Bacterial Proteins/genetics , Cell Cycle , Gene Expression Regulation, Bacterial/physiology , Gene Expression Regulation, Enzymologic/physiology , Phosphotransferases/metabolism , Protein Kinases/genetics , Sigma Factor/genetics , Sigma Factor/metabolism , Spores, Bacterial/physiology
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