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Journal of Bacteriology, June 2001, p. 3506-3514, Vol. 183, No. 11
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.11.3506-3514.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Biosynthesis of the Glycolipid Anchor in Lipoteichoic Acid of Staphylococcus aureus RN4220: Role of YpfP, the Diglucosyldiacylglycerol Synthase

Michael Y. Kiriukhin,1,dagger Dmitri V. Debabov,1,Dagger Dean L. Shinabarger,2 and Francis C. Neuhaus1,*

Department of Biochemistry, Molecular and Cell Biology, Northwestern University, Evanston, Illinois 60208,1 and Pharmacia Corporation, Kalamazoo, Michigan 490072

Received 1 December 2000/Accepted 15 March 2001

In Staphylococcus aureus RN4220, lipoteichoic acid (LTA) is anchored in the membrane by a diglucosyldiacylglycerol moiety. The gene (ypfP) which encodes diglucosyldiacylglycerol synthase was recently cloned from Bacillus subtilis and expressed in Escherichia coli (P. Jorasch, F. P. Wolter, U. Zahringer, and E. Heinz, Mol. Microbiol. 29:419-430, 1998). To define the role of ypfP in this strain of S. aureus, a fragment of ypfP truncated from both ends was cloned into the thermosensitive replicon pVE6007 and used to inactivate ypfP. Chloramphenicol-resistant (ypfP::cat) clones did not synthesize the glycolipids monoglucosyldiacylglycerol and diglucosyldiacylglycerol. Thus, YpfP would appear to be the only diglucosyldiacylglycerol synthase in S. aureus providing glycolipid for LTA assembly. In LTA from the mutant, the glycolipid anchor is replaced by diacylglycerol. Although the doubling time of the mutant was identical to that of the wild type in Luria-Bertani (LB) medium, growth of the mutant in LB medium containing 1% glycine was not observed. This inhibition was antagonized by either L- or D-alanine. Moreover, viability of the mutant at 37°C in 0.05 M phosphate (pH 7.2)-saline for 12 h was reduced to <0.1%. Addition of 0.1% D-glucose to the phosphate-saline ensured viability under these conditions. The autolysis of the ypfP::cat mutant in the presence of 0.05% Triton X-100 was 1.8-fold faster than that of the parental strain. Electron microscopy of the mutant revealed not only a small increase in cell size but also the presence of pleomorphic cells. Each of these phenotypes may be correlated with either (or both) a deficiency of free glycolipid in the membrane or the replacement of the usual glycolipid anchor of LTA with diacylglycerol.

* Corresponding author. Mailing address: Department of Biochemistry, Molecular and Cell Biology, Northwestern University, 2153 Sheridan Road, Evanston, IL 60208. Phone: (847) 491-5656. Fax: (847) 467-1380. E-mail: f-neuhaus{at}northwestern.edu.

dagger Present address: Department of Biochemistry and Molecular Biology, University of Maryland, Baltimore MD 21201-1503.

Dagger Present address: Advanced Medicine, Inc., South San Francisco, CA 94080.

Journal of Bacteriology, June 2001, p. 3506-3514, Vol. 183, No. 11
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.11.3506-3514.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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