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Journal of Bacteriology, November 2008, p. 6940-6947, Vol. 190, No. 21
0021-9193/08/$08.00+0     doi:10.1128/JB.00851-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Use of CDP-Glycerol as an Alternate Acceptor for the Teichoic Acid Polymerase Reveals that Membrane Association Regulates Polymer Length{triangledown}

Jeffrey W. Schertzer{dagger} and Eric D. Brown*

Antimicrobial Research Centre and Department of Biochemistry and Biomedical Sciences, McMaster University, 1200 Main St. W., Hamilton, Ontario, Canada L8N 3Z5

Received 21 June 2008/ Accepted 14 August 2008

The study of bacterial extracellular polysaccharide biosynthesis is hampered by the fact that these molecules are synthesized on membrane-resident carrier lipids. To get around this problem, a practical solution has been to synthesize soluble lipid analogs and study the biosynthetic enzymes using a soluble system. This has been done for the Bacillus subtilis teichoic acid polymerase, TagF, although several aspects of catalysis were inconsistent with the results obtained with reconstituted membrane systems or physiological observations. In this work we explored the acceptor substrate promiscuity and polymer length disregulation that appear to be characteristic of TagF activity away from biological membranes. Using isotope labeling, steady-state kinetics, and chemical lability studies, we demonstrated that the enzyme can synthesize poly(glycerol phosphate) teichoic acid using the elongation substrate CDP-glycerol as an acceptor. This suggests that substrate specificity is relaxed in the region distal to the glycerol phosphate moiety in the acceptor molecule under these conditions. Polymer synthesis proceeded at a rate (27 min–1) comparable to that in the reconstituted membrane system after a distinct lag period which likely represented slower initiation on the unnatural CDP-glycerol acceptor. We confirmed that polymer length became disregulated in the soluble system as the polymers synthesized on CDP-glycerol acceptors were much larger than the polymers synthesized on the membrane or previously found attached to bacterial cell walls. Finally, polymer synthesis on protease-treated membranes suggested that proper length regulation is retained in the absence of accessory proteins and provided evidence that such regulation is conferred through proper association of the polymerase with the membrane.

* Corresponding author. Mailing address: Department of Biochemistry and Biomedical Sciences, McMaster University, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada. Phone: (905) 525-9140, ext. 22392. Fax: (905) 522-9033. E-mail: ebrown{at}mcmaster.ca

{triangledown} Published ahead of print on 19 August 2008.

{dagger} Present address: Department of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, TX 78712.

Journal of Bacteriology, November 2008, p. 6940-6947, Vol. 190, No. 21
0021-9193/08/$08.00+0     doi:10.1128/JB.00851-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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