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Journal of Bacteriology, September 2009, p. 5358-5368, Vol. 191, No. 17
0021-9193/09/$08.00+0     doi:10.1128/JB.00503-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

In Vivo Domain-Based Functional Analysis of the Major Sporulation Sensor Kinase, KinA, in Bacillus subtilis{triangledown} ,{dagger}

Prahathees Eswaramoorthy, Tao Guo,{ddagger} and Masaya Fujita*

Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204-5001

Received 10 April 2009/ Accepted 16 June 2009

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-β-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).

* Corresponding author. Mailing address: Department of Biology and Biochemistry, University of Houston, Houston, TX 77204-5001. Phone: (713) 743-9479. Fax: (713) 743-8351. E-mail: mfujita{at}uh.edu

{triangledown} Published ahead of print on 26 June 2009.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

{ddagger} Present address: College of Pharmacy, University of Houston, Texas Medical Center, 1441 Moursund Street, Room 522, Houston, TX 77030.

Journal of Bacteriology, September 2009, p. 5358-5368, Vol. 191, No. 17
0021-9193/09/$08.00+0     doi:10.1128/JB.00503-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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