Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Feb;178(4):1047–1052. doi: 10.1128/jb.178.4.1047-1052.1996

Galactofuranose biosynthesis in Escherichia coli K-12: identification and cloning of UDP-galactopyranose mutase.

P M Nassau 1, S L Martin 1, R E Brown 1, A Weston 1, D Monsey 1, M R McNeil 1, K Duncan 1
PMCID: PMC177764  PMID: 8576037

Abstract

We have cloned two open reading frames (orf6 and orf8) from the Escherichia coli K-12 rfb region. The genes were expressed in E. coli under control of the T7lac promoter, producing large quantities of recombinant protein, most of which accumulated in insoluble inclusion bodies. Sufficient soluble protein was obtained, however, for use in a radiometric assay designed to detect UDP-galactopyranose mutase activity (the conversion of UDP-galactopyranose to UDP-galactofuranose). The assay is based upon high-pressure liquid chromatography separation of sugar phosphates released from both forms of UDP-galactose by phosphodiesterase treatment. The crude orf6 gene product converted UDP-[alpha-D-U-14C]-galactopyranose to a product which upon phosphodiesterase treatment gave a compound with a retention time identical to that of synthetic alpha-galactofuranose-1-phosphate. No mutase activity was detected in extracts from cells lacking the orf6 expression plasmid or from orf8-expressing cells. The orf6 gene product was purified by anion-exchange chromatography and hydrophobic interaction chromatography. Both the crude extract and the purified protein converted 6 to 9% of the UDP-galactopyranose to the furanose form. The enzyme was also shown to catalyze the reverse reaction; in this case an approximately 86% furanose-to-pyranose conversion was observed. These observations strongly suggest that orf6 encodes UDP-galactopyranose mutase (EC 5.4.99.9), and we propose that the gene be designated glf accordingly. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified UDP-galactopyranose mutase revealed one major band, and analysis by electrospray mass spectrometry indicated a single major species with a molecular weight of 42,960 +/- 8, in accordance with that calculated for the Glf protein. N-terminal sequencing revealed that the first 15 amino acids of the recombinant protein corresponded to those expected from the published sequence. UV-visible spectra of purified recombinant enzyme indicated that the protein contains a flavin cofactor, which we have identified as flavin adenine dinucleotide.

Full Text

The Full Text of this article is available as a PDF (292.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Araki Y., Ito E. Linkage units in cell walls of gram-positive bacteria. Crit Rev Microbiol. 1989;17(2):121–135. doi: 10.3109/10408418909105745. [DOI] [PubMed] [Google Scholar]
  2. Berst M., Hellerqvist C. G., Lindberg B., Lüderitz O., Svensson S., Westphal O. Structural investigations on T1 lipopolysaccharides. Eur J Biochem. 1969 Dec;11(2):353–359. doi: 10.1111/j.1432-1033.1969.tb00779.x. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Daffe M., Brennan P. J., McNeil M. Predominant structural features of the cell wall arabinogalactan of Mycobacterium tuberculosis as revealed through characterization of oligoglycosyl alditol fragments by gas chromatography/mass spectrometry and by 1H and 13C NMR analyses. J Biol Chem. 1990 Apr 25;265(12):6734–6743. [PubMed] [Google Scholar]
  5. GANDER J. E. Biogenesis of galactocarolose by Penicillium charlesii. I. Incorporation of C14 from glucose-C14 and DL-tartrate-1,4-C14 into polyglactose. Arch Biochem Biophys. 1960 Dec;91:307–309. doi: 10.1016/0003-9861(60)90505-1. [DOI] [PubMed] [Google Scholar]
  6. Gruber P. R., Gray G. R. Isolation and analysis by the reductive-cleavage method of linkage positions and ring forms in the Mycobacterium smegmatis cell-wall arabinogalactan. Carbohydr Res. 1990 Aug 1;203(1):79–90. doi: 10.1016/0008-6215(90)80047-7. [DOI] [PubMed] [Google Scholar]
  7. Haworth W. N., Raistrick H., Stacey M. Polysaccharides synthesised by micro-organisms: The molecular structure of galactocarolose produced from glucose by Penicillium Charlesii G. Smith. Biochem J. 1937 Apr;31(4):640–644. doi: 10.1042/bj0310640. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Latgé J. P., Kobayashi H., Debeaupuis J. P., Diaquin M., Sarfati J., Wieruszeski J. M., Parra E., Bouchara J. P., Fournet B. Chemical and immunological characterization of the extracellular galactomannan of Aspergillus fumigatus. Infect Immun. 1994 Dec;62(12):5424–5433. doi: 10.1128/iai.62.12.5424-5433.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Liu D., Reeves P. R. Escherichia coli K12 regains its O antigen. Microbiology. 1994 Jan;140(Pt 1):49–57. doi: 10.1099/13500872-140-1-49. [DOI] [PubMed] [Google Scholar]
  10. Mendelzon D. H., Parodi A. J. N-linked high mannose-type oligosaccharides in the protozoa Crithidia fasciculata and Crithidia harmosa contain galactofuranose residues. J Biol Chem. 1986 Feb 15;261(5):2129–2133. [PubMed] [Google Scholar]
  11. Moraes C. T., Bosch M., Parodi A. J. Structural characterization of several galactofuranose-containing, high-mannose-type oligosaccharides present in glycoproteins of the trypanosomatid Leptomonas samueli. Biochemistry. 1988 Mar 8;27(5):1543–1549. doi: 10.1021/bi00405a022. [DOI] [PubMed] [Google Scholar]
  12. Nikaido H., Sarvas M. Biosynthesis of T1 antigen in Salmonella: biosynthesis in a cell-free system. J Bacteriol. 1971 Mar;105(3):1073–1082. doi: 10.1128/jb.105.3.1073-1082.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Sarvas M., Nikaido H. Biosynthesis of T1 antigen in Salmonella: origin of D-galactofuranose and D-ribofuranose residues. J Bacteriol. 1971 Mar;105(3):1063–1072. doi: 10.1128/jb.105.3.1063-1072.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Stevenson G., Neal B., Liu D., Hobbs M., Packer N. H., Batley M., Redmond J. W., Lindquist L., Reeves P. Structure of the O antigen of Escherichia coli K-12 and the sequence of its rfb gene cluster. J Bacteriol. 1994 Jul;176(13):4144–4156. doi: 10.1128/jb.176.13.4144-4156.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  16. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  17. Trejo A. G., Chittenden G. J., Buchanan J. G., Baddiley J. Uridine diphosphate alpha-D-galactofuranose, an intermediate in the biosynthesis of galactofuranosyl residues. Biochem J. 1970 Apr;117(3):637–639. doi: 10.1042/bj1170637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Unkefer C. J., Gander J. E. The 5-O-beta-D-galactofuranosyl-containing glycopeptide from Penicillium charlesii. Carbon 13 nuclear magnetic resonance studies. J Biol Chem. 1979 Dec 10;254(23):12131–12135. [PubMed] [Google Scholar]
  19. Whitfield C., Richards J. C., Perry M. B., Clarke B. R., MacLean L. L. Expression of two structurally distinct D-galactan O antigens in the lipopolysaccharide of Klebsiella pneumoniae serotype O1. J Bacteriol. 1991 Feb;173(4):1420–1431. doi: 10.1128/jb.173.4.1420-1431.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Wierenga R. K., Terpstra P., Hol W. G. Prediction of the occurrence of the ADP-binding beta alpha beta-fold in proteins, using an amino acid sequence fingerprint. J Mol Biol. 1986 Jan 5;187(1):101–107. doi: 10.1016/0022-2836(86)90409-2. [DOI] [PubMed] [Google Scholar]
  21. Yao Z., Valvano M. A. Genetic analysis of the O-specific lipopolysaccharide biosynthesis region (rfb) of Escherichia coli K-12 W3110: identification of genes that confer group 6 specificity to Shigella flexneri serotypes Y and 4a. J Bacteriol. 1994 Jul;176(13):4133–4143. doi: 10.1128/jb.176.13.4133-4143.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES