Characterization of an Escherichia coli rff mutant defective in transfer of N-acetylmannosaminuronic acid (ManNAcA) from UDP-ManNAcA to a lipid-linked intermediate involved in enterobacterial common antigen synthesis

J Bacteriol. 1988 Jan;170(1):228-33. doi: 10.1128/jb.170.1.228-233.1988.

Abstract

The rff genes of Salmonella typhimurium include structural genes for enzymes involved in the conversion of UDP N-acetyl-D-glucosamine (UDP-GlcNAc) to UDP N-acetyl-D-mannosaminuronic acid (UDP-ManNAcA), the donor of ManNAcA residues in enterobacterial common antigen (ECA) synthesis. An rff mutation (rff-726) of Escherichia coli has been described (U. Meier and H. Mayer, J. Bacteriol. 163:756-762, 1985) that abolished ECA synthesis but which did not affect the synthesis of UDP-ManNAcA or any other components of ECA. The nature of the enzymatic defect resulting from the rff-726 lesion was investigated in the present study. The in vitro synthesis of GlcNAc-pyrophosphorylundecaprenol (lipid I), an early intermediate in ECA synthesis, was demonstrated by using membranes prepared from a mutant of E. coli possessing the rff-726 lesion. However, in vitro synthesis of the next lipid-linked intermediate in the biosynthetic sequence, ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid II), was severely impaired. Transduction of wild-type rff genes into the mutant restored the ability to synthesize both lipid II and ECA as determined by in vitro assay and Western blot (immunoblot) analyses done with anti-ECA monoclonal antibody, respectively. Our results are consistent with the conclusion that the rff-726 mutation is located in the structural gene for the transferase that catalyzes the transfer of ManNAcA from UDP-ManNAcA to lipid I.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Antigens, Bacterial / biosynthesis*
  • Antigens, Bacterial / genetics
  • Chemical Phenomena
  • Chemistry
  • Escherichia coli / enzymology
  • Escherichia coli / genetics*
  • Escherichia coli / immunology
  • Escherichia coli / metabolism
  • Genes*
  • Genes, Bacterial
  • Hexosyltransferases / genetics*
  • Hexosyltransferases / metabolism
  • Lipid Metabolism
  • Mutation
  • N-Acetylhexosaminyltransferases*
  • Transduction, Genetic
  • Transferases / genetics*
  • Transferases / metabolism
  • Uronic Acids / metabolism*

Substances

  • Antigens, Bacterial
  • Uronic Acids
  • enterobacterial common antigen
  • N-acetylmannosaminuronic acid
  • Transferases
  • Hexosyltransferases
  • N-Acetylhexosaminyltransferases
  • N-acetylmannosaminuronic acid transferase