In vivo domain-based functional analysis of the major sporulation sensor kinase, KinA, in Bacillus subtilis.
Sensor histidine kinases are widely used by bacteria to detect and respond to environmental signals. In Bacillus subtilis, KinA is a major kinase providing phosphate input to the phosphorelay that activates the sporulation pathway upon starvation via the phosphorylated Spo0A transcription factor. KinA contains three PAS domains in its amino-terminal sensor domain, which appear to be involved in the sensing of an unidentified sporulation signal(s) produced upon starvation. Prior biochemical studies have suggested that KinA forms a homodimer as a functional enzyme and that the most amino-terminal PAS domain (PAS-A) plays an important role in sensing the signal(s) to activate an ATP-dependent autophosphorylation reaction to a histidine residue. To analyze the structure and function of the kinase in vivo, we have used a strain in which the synthesis of KinA is under the control of an isopropyl-beta-d-thiogalactopyranoside (IPTG)-inducible promoter. In vivo functional studies in combination with domain-based deletion analysis show that the cytosolic KinA forms a homo-oligomer as an active form under both nutrient-rich and nutrient-depleted conditions via its amino- and carboxyl-terminal domains independently. Furthermore, we found that a mutant in which the PAS-A domain was deleted was still able to induce sporulation at a wild-type level irrespective of nutrient availability, suggesting that PAS-BC domains are sufficient to maintain the kinase activity. Based on these results, we propose that the primary role of the amino-terminal sensor domain is to form a stable complex as a functional kinase, but possibly not for the binding of an unidentified sporulation signal(s).
Eswaramoorthy
Prahathees
P
Department of Biology and Biochemistry, University of Houston, Houston, TX 77204-5001, USA.
Guo
Tao
T
Fujita
Masaya
M
D001412:Bacillus subtilis / Q000502:physiology*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D010766:Phosphorylation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism*; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D015398:Signal Transduction
D001426:Bacterial Proteins; C075521:kinA protein, Bacillus subtilis; D011494:Protein Kinases