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Journal of Bacteriology, May 2001, p. 2795-2802, Vol. 183, No. 9
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.9.2795-2802.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Dissection of the Functional and Structural Domains of Phosphorelay Histidine Kinase A of Bacillus subtilis†

Ling Wang, Céline Fabret, Kyoko Kanamaru, Keith Stephenson, Veronique Dartois, Marta Perego, and James A. Hoch*

Division of Cellular Biology, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037

Received 10 October 2000/Accepted 9 January 2001

The initiation of sporulation in Bacillus subtilis results primarily from phosphoryl group input into the phosphorelay by histidine kinases, the major kinase being kinase A. Kinase A is active as a homodimer, the protomer of which consists of an approximately 400-amino-acid N-terminal putative signal-sensing region and a 200-amino-acid C-terminal autokinase. On the basis of sequence similarity, the N-terminal region may be subdivided into three PAS domains: A, B, and C, located from the N- to the C-terminal end. Proteolysis experiments and two-hybrid analyses indicated that dimerization of the N-terminal region is accomplished through the PAS-B/PAS-C region of the molecule, whereas the most amino-proximal PAS-A domain is not dimerized. N-terminal deletions generated with maltose binding fusion proteins showed that an intact PAS-A domain is very important for enzymatic activity. Amino acid substitution mutations in PAS-A as well as PAS-C affected the in vivo activity of kinase A, suggesting that both PAS domains are required for signal sensing. The C-terminal autokinase, when produced without the N-terminal region, was a dimer, probably because of the dimerization required for formation of the four-helix-bundle phosphotransferase domain. The truncated autokinase was virtually inactive in autophosphorylation with ATP, whereas phosphorylation of the histidine of the phosphotransfer domain by back reactions from Spo0F~P appeared normal. The phosphorylated autokinase lost the ability to transfer its phosphoryl group to ADP, however. The N-terminal region appears to be essential both for signal sensing and for maintaining the correct conformation of the autokinase component domains.

* Corresponding author. Mailing address: Department of Molecular and Experimental Medicine, MEM-116, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037. Phone: (858) 784-7905. Fax: (858) 784-7966. E-mail: hoch{at}scripps.edu.

dagger Publication 13630-MEM from the Department of Molecular and Experimental Medicine at The Scripps Research Institute.

Journal of Bacteriology, May 2001, p. 2795-2802, Vol. 183, No. 9
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.9.2795-2802.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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