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.
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.
Barr
K
K
Department of Microbiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814.
Ward
S
S
Meier-Dieter
U
U
Mayer
H
H
Rick
P D
PD
D000942:Antigens, Bacterial / Q000096:biosynthesis* / Q000235:genetics; D055598:Chemical Phenomena; D002621:Chemistry; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D005796:Genes; D005798:Genes, Bacterial; D006602:Hexosyltransferases / Q000235:genetics* / Q000378:metabolism; D050356:Lipid Metabolism; D009154:Mutation; D017349:N-Acetylhexosaminyltransferases; D014161:Transduction, Genetic; D014166:Transferases / Q000235:genetics* / Q000378:metabolism; D014574:Uronic Acids / Q000378:metabolism*
D000942:Antigens, Bacterial; D014574:Uronic Acids; C017286:enterobacterial common antigen; C043028:N-acetylmannosaminuronic acid; D014166:Transferases; D006602:Hexosyltransferases; D017349:N-Acetylhexosaminyltransferases; C054493:N-acetylmannosaminuronic acid transferase