Abstract
We previously showed that the product of the wbaP gene of Salmonella enterica serovar Typhimurium has two functions: it is involved in the first step of O-antigen synthesis (the galactosyltransferase [GT] function) and in a later step (the T function), first thought to be the flipping of the O-antigen subunit on undecaprenyl pyrophosphate from the cytoplasmic face to the periplasmic face of the cytoplasmic membrane. We now locate two wbaP(T) mutations within the first half of the wbaP gene by sequencing. Both mutants retain GT activity, although one was a frameshift mutation resulting in a stop codon 10 codons after the frameshift to give an open reading frame containing only 138 of the 476 codons in WbaP. We also show that there is a secondary translation starting within the wbaP gene resulting in the synthesis of a polypeptide with GT activity. These results indicate that the N- and C-terminal halves of WbaP are the T and GT functional domains, respectively. We now propose that the T block operates prior to the flippase function, probably at the release of undecaprenyl pyrophosphate-linked galactose from WbaP.
Full Text
The Full Text of this article is available as a PDF (310.8 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Arakawa Y., Wacharotayankun R., Nagatsuka T., Ito H., Kato N., Ohta M. Genomic organization of the Klebsiella pneumoniae cps region responsible for serotype K2 capsular polysaccharide synthesis in the virulent strain Chedid. J Bacteriol. 1995 Apr;177(7):1788–1796. doi: 10.1128/jb.177.7.1788-1796.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Borthakur D., Barker R. F., Latchford J. W., Rossen L., Johnston A. W. Analysis of pss genes of Rhizobium leguminosarum required for exopolysaccharide synthesis and nodulation of peas: their primary structure and their interaction with psi and other nodulation genes. Mol Gen Genet. 1988 Jul;213(1):155–162. doi: 10.1007/BF00333413. [DOI] [PubMed] [Google Scholar]
- Bugert P., Geider K. Molecular analysis of the ams operon required for exopolysaccharide synthesis of Erwinia amylovora. Mol Microbiol. 1995 Mar;15(5):917–933. doi: 10.1111/j.1365-2958.1995.tb02361.x. [DOI] [PubMed] [Google Scholar]
- Bugg T. D., Brandish P. E. From peptidoglycan to glycoproteins: common features of lipid-linked oligosaccharide biosynthesis. FEMS Microbiol Lett. 1994 Jun 15;119(3):255–262. doi: 10.1111/j.1574-6968.1994.tb06898.x. [DOI] [PubMed] [Google Scholar]
- Coplin D. L., Cook D. Molecular genetics of extracellular polysaccharide biosynthesis in vascular phytopathogenic bacteria. Mol Plant Microbe Interact. 1990 Sep-Oct;3(5):271–279. doi: 10.1094/mpmi-3-271. [DOI] [PubMed] [Google Scholar]
- Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Eisenberg D., Schwarz E., Komaromy M., Wall R. Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J Mol Biol. 1984 Oct 15;179(1):125–142. doi: 10.1016/0022-2836(84)90309-7. [DOI] [PubMed] [Google Scholar]
- Glucksmann M. A., Reuber T. L., Walker G. C. Family of glycosyl transferases needed for the synthesis of succinoglycan by Rhizobium meliloti. J Bacteriol. 1993 Nov;175(21):7033–7044. doi: 10.1128/jb.175.21.7033-7044.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Guidolin A., Morona J. K., Morona R., Hansman D., Paton J. C. Nucleotide sequence analysis of genes essential for capsular polysaccharide biosynthesis in Streptococcus pneumoniae type 19F. Infect Immun. 1994 Dec;62(12):5384–5396. doi: 10.1128/iai.62.12.5384-5396.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ielpi L., Couso R. O., Dankert M. A. Sequential assembly and polymerization of the polyprenol-linked pentasaccharide repeating unit of the xanthan polysaccharide in Xanthomonas campestris. J Bacteriol. 1993 May;175(9):2490–2500. doi: 10.1128/jb.175.9.2490-2500.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ivashina T. V., Khmelnitsky M. I., Shlyapnikov M. G., Kanapin A. A., Ksenzenko V. N. The pss4 gene from Rhizobium leguminosarum by viciae VF39: cloning, sequence and the possible role in polysaccharide production and nodule formation. Gene. 1994 Dec 2;150(1):111–116. doi: 10.1016/0378-1119(94)90868-0. [DOI] [PubMed] [Google Scholar]
- Jiang X. M., Neal B., Santiago F., Lee S. J., Romana L. K., Reeves P. R. Structure and sequence of the rfb (O antigen) gene cluster of Salmonella serovar typhimurium (strain LT2). Mol Microbiol. 1991 Mar;5(3):695–713. doi: 10.1111/j.1365-2958.1991.tb00741.x. [DOI] [PubMed] [Google Scholar]
- Kent J. L., Osborn M. J. Properties of the O-specific hapten formed in vivo by mutant strains of Salmonella typhimurium. Biochemistry. 1968 Dec;7(12):4396–4408. doi: 10.1021/bi00852a036. [DOI] [PubMed] [Google Scholar]
- Klena J. D., Schnaitman C. A. Function of the rfb gene cluster and the rfe gene in the synthesis of O antigen by Shigella dysenteriae 1. Mol Microbiol. 1993 Jul;9(2):393–402. doi: 10.1111/j.1365-2958.1993.tb01700.x. [DOI] [PubMed] [Google Scholar]
- Liu D., Cole R. A., Reeves P. R. An O-antigen processing function for Wzx (RfbX): a promising candidate for O-unit flippase. J Bacteriol. 1996 Apr;178(7):2102–2107. doi: 10.1128/jb.178.7.2102-2107.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Liu D., Haase A. M., Lindqvist L., Lindberg A. A., Reeves P. R. Glycosyl transferases of O-antigen biosynthesis in Salmonella enterica: identification and characterization of transferase genes of groups B, C2, and E1. J Bacteriol. 1993 Jun;175(11):3408–3413. doi: 10.1128/jb.175.11.3408-3413.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Liu D., Lindqvist L., Reeves P. R. Transferases of O-antigen biosynthesis in Salmonella enterica: dideoxyhexosyltransferases of groups B and C2 and acetyltransferase of group C2. J Bacteriol. 1995 Jul;177(14):4084–4088. doi: 10.1128/jb.177.14.4084-4088.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lugtenberg B., Meijers J., Peters R., van der Hoek P., van Alphen L. Electrophoretic resolution of the "major outer membrane protein" of Escherichia coli K12 into four bands. FEBS Lett. 1975 Oct 15;58(1):254–258. doi: 10.1016/0014-5793(75)80272-9. [DOI] [PubMed] [Google Scholar]
- Marino P. A., McGrath B. C., Osborn M. J. Energy dependence of O-antigen synthesis in Salmonella typhimurium. J Bacteriol. 1991 May;173(10):3128–3133. doi: 10.1128/jb.173.10.3128-3133.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McGrath B. C., Osborn M. J. Localization of the terminal steps of O-antigen synthesis in Salmonella typhimurium. J Bacteriol. 1991 Jan;173(2):649–654. doi: 10.1128/jb.173.2.649-654.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Meier-Dieter U., Barr K., Starman R., Hatch L., Rick P. D. Nucleotide sequence of the Escherichia coli rfe gene involved in the synthesis of enterobacterial common antigen. Molecular cloning of the rfe-rff gene cluster. J Biol Chem. 1992 Jan 15;267(2):746–753. [PubMed] [Google Scholar]
- Müller P., Keller M., Weng W. M., Quandt J., Arnold W., Pühler A. Genetic analysis of the Rhizobium meliloti exoYFQ operon: ExoY is homologous to sugar transferases and ExoQ represents a transmembrane protein. Mol Plant Microbe Interact. 1993 Jan-Feb;6(1):55–65. doi: 10.1094/mpmi-6-055. [DOI] [PubMed] [Google Scholar]
- Osborn M. J., Tze-Yuen R. Y. Biosynthesis of bacterial lipopolysaccharide. VII. Enzymatic formation of the first intermediate in biosynthesis of the O-antigen of Salmonella typhimurium. J Biol Chem. 1968 Oct 10;243(19):5145–5152. [PubMed] [Google Scholar]
- Reed J. W., Capage M., Walker G. C. Rhizobium meliloti exoG and exoJ mutations affect the exoX-exoY system for modulation of exopolysaccharide production. J Bacteriol. 1991 Jun;173(12):3776–3788. doi: 10.1128/jb.173.12.3776-3788.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Reuber T. L., Walker G. C. Biosynthesis of succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti. Cell. 1993 Jul 30;74(2):269–280. doi: 10.1016/0092-8674(93)90418-p. [DOI] [PubMed] [Google Scholar]
- Rubens C. E., Heggen L. M., Haft R. F., Wessels M. R. Identification of cpsD, a gene essential for type III capsule expression in group B streptococci. Mol Microbiol. 1993 May;8(5):843–855. doi: 10.1111/j.1365-2958.1993.tb01631.x. [DOI] [PubMed] [Google Scholar]
- Smith T. F., Waterman M. S. Identification of common molecular subsequences. J Mol Biol. 1981 Mar 25;147(1):195–197. doi: 10.1016/0022-2836(81)90087-5. [DOI] [PubMed] [Google Scholar]
- Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wang L., Reeves P. R. Involvement of the galactosyl-1-phosphate transferase encoded by the Salmonella enterica rfbP gene in O-antigen subunit processing. J Bacteriol. 1994 Jul;176(14):4348–4356. doi: 10.1128/jb.176.14.4348-4356.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wilbur W. J., Lipman D. J. Rapid similarity searches of nucleic acid and protein data banks. Proc Natl Acad Sci U S A. 1983 Feb;80(3):726–730. doi: 10.1073/pnas.80.3.726. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wilkinson R. G., Gemski P., Jr, Stocker B. A. Non-smooth mutants of Salmonella typhimurium: differentiation by phage sensitivity and genetic mapping. J Gen Microbiol. 1972 May;70(3):527–554. doi: 10.1099/00221287-70-3-527. [DOI] [PubMed] [Google Scholar]
