Purified, recombinant TagF protein from Bacillus subtilis 168 catalyzes the polymerization of glycerol phosphate onto a membrane acceptor in vitro.
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-[(14)C]glycerol into a membrane fraction in vitro. This activity showed a saturable dependence on the concentration of CDP-glycerol (K(m) of 340 microm) and the membrane acceptor (half-maximal activity at 650 microg of protein/ml of purified B. subtilis membranes). High pressure liquid chromatography analysis confirmed the polymeric nature of the reaction product, approximately 35 glycerol phosphate units in length.
Schertzer
Jeffrey W
JW
Antimicrobial Research Centre, the Department of Biochemistry, McMaster University, Hamilton, Ontario L8N 3Z5, Canada.
Brown
Eric D
ED
D001412:Bacillus subtilis / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005816:Genetic Complementation Test; D005990:Glycerol / Q000378:metabolism; D005994:Glycerophosphates / Q000737:chemistry*; D007700:Kinetics; D008956:Models, Chemical; D010457:Peptidoglycan / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011108:Polymers / Q000737:chemistry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D013682:Teichoic Acids / Q000737:chemistry; D013696:Temperature; D013997:Time Factors; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D005994:Glycerophosphates; D010457:Peptidoglycan; D011108:Polymers; D011994:Recombinant Proteins; D013682:Teichoic Acids; D017855:Transferases (Other Substituted Phosphate Groups); C072175:CDP glycerol glycerophosphotransferase; D005990:Glycerol