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J. Biol. Chem., Vol. 278, Issue 20, 18002-18007, May 16, 2003
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From the Antimicrobial Research Centre, the Department of
Biochemistry, McMaster University, Hamilton,
Ontario L8N 3Z5, Canada
We report the first characterization of a
recombinant protein involved in the polymerization of wall teichoic
acid. Previously, a study of the teichoic acid polymerase activity
associated with membranes from Bacillus subtilis 168 strains bearing thermosensitive mutations in tagB, tagD,
and tagF implicated TagF as the poly(glycerol phosphate)
polymerase (Pooley, H. M., Abellan, F. X., and Karamata, D. (1992) J. Bacteriol. 174, 646-649). In the work reported
here, we have demonstrated an unequivocal role for tagF in
the thermosensitivity of one such mutant (tagF1) by
conditional complementation at the restrictive temperature with
tagF under control of the xylose promoter at the
amyE locus. We have overexpressed and purified recombinant
B. subtilis TagF protein, and we provide direct biochemical evidence that this enzyme is responsible for polymerization of poly(glycerol phosphate) teichoic acid in B. subtilis
168. Recombinant hexahistidine-tagged TagF protein was purified from
Escherichia coli and was used to develop a novel membrane
pelleting assay to monitor poly(glycerol phosphate) polymerase
activity. Purified TagF was shown to incorporate radioactivity from its
substrate CDP-[14C]glycerol into a membrane fraction
in vitro. This activity showed a saturable dependence on
the concentration of CDP-glycerol (Km of 340 µM) and the membrane acceptor (half-maximal activity at 650 µg of protein/ml of purified B. subtilis membranes).
High pressure liquid chromatography analysis confirmed the polymeric nature of the reaction product, ~35 glycerol phosphate units in length.
Purified, Recombinant TagF Protein from Bacillus
subtilis 168 Catalyzes the Polymerization of Glycerol Phosphate
onto a Membrane Acceptor in Vitro*
*
This work was supported by the Canadian Institutes of Health
Research Grant MOP-15496 and by a Canada Research Chair in Microbial Biochemistry (to E. D. B.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Antimicrobial Research
Centre, Dept. of Biochemistry, McMaster University, 1200 Main St. W.,
Hamilton, Ontario L8N 3Z5, Canada. Tel.: 905-525-9140 (ext. 22392);
Fax: 905-522-9033; E-mail: ebrown@mcmaster.ca.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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