WecA, an integral membrane protein that belongs to a family of polyisoprenyl phosphate acetylhexosamine-1-phosphate transferases, is required for the biosynthesis of O-specific LPS and enterobacterial common antigen in and other enteric bacteria. WecA functions as an UDP--acetylglucosamine (GlcNAc):undecaprenyl-phosphate GlcNAc-1-phosphate transferase. A conserved short sequence motif (His-Ile-His-His; HIHH) and a conserved arginine were identified in WecA at positions 279–282 and 265, respectively. This region is located within a predicted cytosolic segment common to all bacterial homologues of WecA. Both HIHH and the Arg are reminiscent of the HIGH motif (His-Ile-Gly-His) and a nearby upstream lysine, which contribute to the three-dimensional architecture of the nucleotide-binding site among various enzymes displaying nucleotidyltransferase activity. Thus, it was hypothesized that these residues may play a role in the interaction of WecA with UDP-GlcNAc. Replacement of the entire HIHH motif by site-directed mutagenesis produced a protein that, when expressed in the mutant MV501, did not complement the synthesis of O7 LPS. Membrane extracts containing the mutated protein failed to transfer UDP-GlcNAc into a lipid-rich fraction and to bind the UDP-GlcNAc analogue tunicamycin. Similar results were obtained by individually replacing the first histidine (H) of the HIHH motif as well as the Arg residue. The functional importance of these residues is underscored by the high level of conservation of H and Arg among bacterial WecA homologues that utilize several different UDP-acetylhexosamine substrates.


Article metrics loading...

Loading full text...

Full text loading...



  1. Alexander, D. C. & Valvano, M. A. (1994). Role of rfe gene in the biosynthesis of the Escherichia coli O7-specific lipopolysaccharide and other O-specific polysaccharides containing N-acetylglucosamine. J Bacteriol 176, 7079-7084. [Google Scholar]
  2. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25, 3389-3402.[CrossRef] [Google Scholar]
  3. Amer, A. O. & Valvano, M. A. (2000). The N-terminal region of the Escherichia coli WecA (Rfe) protein, containing three predicted transmembrane helices, is required for function but not for membrane insertion. J Bacteriol 182, 498-503.[CrossRef] [Google Scholar]
  4. Amor, P. A. & Whitfield, C. (1997). Molecular and functional analysis of genes required for expression of group IB K antigens in Escherichia coli: characterization of the his region containing gene clusters for multiple cell-surface polysaccharides. Mol Microbiol 26, 145-161.[CrossRef] [Google Scholar]
  5. Anderson, M. S., Eveland, S. S. & Price, N. P. (2000). Conserved cytoplasmic motifs that distinguish sub-groups of the polyprenol phosphate:N-acetylhexosamine-1-phosphate transferase family. FEMS Microbiol Lett 191, 169-175.[CrossRef] [Google Scholar]
  6. Bordo, D. & Argos, P. (1991). Suggestions for ‘safe’ residue substitutions in site-directed mutagenesis. J Mol Biol 217, 721-729.[CrossRef] [Google Scholar]
  7. Bork, P., Holm, L., Koonin, E. V. & Sander, C. (1995). The cytidylyltransferase superfamily: identification of the nucleotide-binding site and fold prediction. Proteins 22, 259-266.[CrossRef] [Google Scholar]
  8. Bouhss, A., Mengin-Lecreulx, D., Le Beller, D. & Van Heijenoort, J. (1999). Topological analysis of the MraY protein catalysing the first membrane step of peptidoglycan synthesis. Mol Microbiol 34, 576-585.[CrossRef] [Google Scholar]
  9. Boyd, D., Manoil, C. & Beckwith, J. (1987). Determinants of membrane protein topology. Proc Natl Acad Sci USA 84, 8525-8529.[CrossRef] [Google Scholar]
  10. Boyd, D., Traxler, B. & Beckwith, J. (1993). Analysis of the topology of a membrane protein by using a minimum number of alkaline phosphatase fusions. J Bacteriol 175, 553-556. [Google Scholar]
  11. Cohen, S. N., Chang, A. C. & Hsu, L. (1972). Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci USA 69, 2110-2114.[CrossRef] [Google Scholar]
  12. Dal Nogare, A. R. & Lehrman, M. A. (1988). Conserved sequences in enzymes of the UDP-GlcNAc/MurNAc family are essential in hamster UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase. Glycobiology 8, 625-632. [Google Scholar]
  13. Dan, N., Middleton, R. B. & Lehrman, M. A. (1996). Hamster UDP-N- acetylglucosamine:dolichol-P N-acetylglucosamine-1-P transferase has multiple transmembrane spans and a critical cytosolic loop. J Biol Chem 271, 30717-30724.[CrossRef] [Google Scholar]
  14. Dower, W. J., Miller, J. F. & Ragsdale, C. W. (1988). High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res 16, 6127-6145.[CrossRef] [Google Scholar]
  15. Feldman, M. F., Marolda, C. L., Monteiro, M. A., Perry, M. B., Parodi, A. J. & Valvano, M. A. (1999). The activity of a putative polyisoprenol-linked sugar translocase (Wzx) involved in Escherichia coli O antigen assembly is independent of the chemical structure of the O repeat. J Biol Chem 274, 35129-35138.[CrossRef] [Google Scholar]
  16. Guzman, L. M., Belin, D., Carson, M. J. & Beckwith, J. (1995). Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177, 4121-4130. [Google Scholar]
  17. von Heijne, G. (1986). The distribution of positively charged residues in bacterial inner membrane proteins correlates with the trans-membrane topology. EMBO J 5, 3021-3027. [Google Scholar]
  18. von Heijne, G. (1997). Getting greasy: how transmembrane polypeptide segments integrate into the lipid bilayer. Mol Microbiol 24, 249-253.[CrossRef] [Google Scholar]
  19. Lehrman, M. A. (1994). A family of UDP-GlcNAc/MurNAc: polyisoprenol-P GlcNAc/MurNAc-1-P transferases. Glycobiology 4, 768-771.[CrossRef] [Google Scholar]
  20. L’Vov, V., Shashkov, A. S., Dmitriev, B. A., Kochetkov, N. K., Jann, B. & Jann, K. (1984). Structural studies of the O-specific side chain of the lipopolysaccharide from Escherichia coli O:7. Carbohydr Res 126, 249-259.[CrossRef] [Google Scholar]
  21. Marolda, C. L., Welsh, J., Dafoe, L. & Valvano, M. A. (1990). Genetic analysis of the O7-polysaccharide biosynthesis region from the Escherichia coli O7-K1 strain VW187. J Bacteriol 172, 3590-3599. [Google Scholar]
  22. Marolda, C. L., Feldman, M. F. & Valvano, M. A. (1999). Genetic organization of the O7-specific lipopolysaccharide biosynthesis cluster of Escherichia coli VW187 (O7:K1). Microbiology 145, 2485-2495. [Google Scholar]
  23. Osborn, M. J., Gander, J. E., Parisi, E. & Carson, J. (1972). Mechanism of assembly of the outer membrane of Salmonella typhimurium: isolation and characterization of cytoplasmic and outer membrane. J Biol Chem 247, 3962-3972. [Google Scholar]
  24. Prinz, W. A. & Beckwith, J. (1994). Gene fusion analysis of membrane protein topology: a direct comparison of alkaline phosphatase and beta-lactamase fusions. J Bacteriol 176, 6410-6413. [Google Scholar]
  25. Rick, P. D. & Silver, R. P. (1996). Enterobacterial common antigen and capsular polysaccharides. In Escherichia coli and Salmonella: Cellular and Molecular Biology, pp. 104–122. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
  26. Sekine, S., Shimada, A., Nureki, O., Cavarelli, J., Moras, D., Vassylyev, D. & Yokoyama, S. (2001). Crucial role of the HIGH-loop lysine for the catalytic activity of arginyl-tRNA synthetase. J Biol Chem 276, 3723-3726.[CrossRef] [Google Scholar]
  27. Skurnik, M. (1999). Molecular genetics of Yersinia lipopolysaccharide. In Genetics of Bacterial Polysaccharides , pp. 23-51. Edited by J. Goldberg. Boca Raton, FL:CRC Press.
  28. Sonnhammer, E. L. L., von Heijne, G. & Krogh, A. (1998). A hidden Markov model for predicting transmembrane helices in protein sequences. In Proceedings of Sixth International Conference on Intelligent Systems for Molecular Biology, pp. 175–182. Edited by J. Glasgow and others. Menlo Park, California: American Association for Artificial Intelligence.
  29. Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994).clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673-4680.[CrossRef] [Google Scholar]
  30. Venkatachalam, K. V., Fuda, H., Koonin, E. V. & Strott, C. A. (1999). Site-selected mutagenesis of a conserved nucleotide binding HXGH motif located in the ATP sulfurylase domain of human bifunctional 3′-phosphoadinosine 5′-phosphosulfate synthase. J Biol Chem 274, 2601-2604.[CrossRef] [Google Scholar]
  31. Zhang, L., Radziejewska-Lebrecht, J., Krajewska-Pietrasik, D., Toivanen, P. & Skurnik, M. (1997). Molecular and chemical characterization of the lipopolysaccharide O-antigen and its role in virulence of Yersinia enterocolitica serotype O:8. Mol Microbiol 23, 63-76.[CrossRef] [Google Scholar]

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error