title: Biosynthesis of enterobacterial common antigen in Escherichia coli. Biochemical characterization of Tn10 insertion mutants defective in enterobacterial common antigen synthesis.
abstract: Twelve independent Tn10 insertion mutants of Escherichia coli K12 were isolated that were defective in the synthesis of enterobacterial common antigen (ECA). The mutants were identified by screening a random pool of Tn10 insertion mutants for their ECA phenotype using a colony-immunoblot assay. All 12 of the Tn10 insertion mutants were found to be located in the chromosomal region of the rff-rfe genes. Four of the Tn10 insertions were in rfe genes while the remaining eight Tn10 insertions were in rff genes. All of the rfe::Tn10 insertion mutants were defective in the synthesis of GlcNAc-pyrophosphorylundecaprenol (C55-PP-GlcNAc, lipid I), the first lipid-linked intermediate involved in ECA synthesis. Biochemical characterization of the rff::Tn10 insertion mutants revealed that they were defective in various steps of ECA synthesis subsequent to the synthesis of lipid I. These defects included: (i) the inability to synthesize UDP-ManNAcA due to Tn10 insertions in the structural genes for UDP-GlcNAc-2-epimerase (rffE) and UDP-ManNAcA (N-acetyl-D-mannosaminuronic acid) dehydrogenase (rffD), (ii) defects in the synthesis of C55-GlcNAc-ManNAcA (lipid II) due to insertion of transposon Tn10 in the structural gene for the UDP-ManNAcA transferase (rffM), (iii) the inability to synthesize TDP-Fuc4NAc (4-acetamido-4,6-dideoxy-D-galactose) due to Tn10 insertions in the structural gene for the transaminase that catalyzes the conversion of TDP-4-keto-6-deoxy-D-glucose to TDP-4-amino-4,6-dideoxy-D-galactose (rffA), and (iv) defects in steps subsequent to the synthesis of C55-GlcNAc-ManNAcA-Fuc4NAc (lipid III). In addition, a re-examination of a mutant possessing the rff-726 lesion revealed that it was defective in the synthesis of lipid III due to a defect in the structural gene for the Fuc4NAc transferase (rffT).
authors.affiliation: Department of Microbiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
authors.forename: U
authors.initials: U
authors.lastname: Meier-Dieter
authors.forename: R
authors.initials: R
authors.lastname: Starman
authors.forename: K
authors.initials: K
authors.lastname: Barr
authors.forename: H
authors.initials: H
authors.lastname: Mayer
authors.forename: P D
authors.initials: PD
authors.lastname: Rick
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism; D000942:Antigens, Bacterial / Q000235:genetics*; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology*; D005838:Genotype; D009154:Mutation; D010641:Phenotype; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000942:Antigens, Bacterial; D004251:DNA Transposable Elements; D011135:Polysaccharides, Bacterial; C017286:enterobacterial common antigen; D000117:Acetylglucosamine