Biosynthesis of enterobacterial common antigen

Abstract

Cultures of Salmonella typhimurium pulse-labeled with N-acetyl-D-[3H]glucosamine ([3H]GlcNAc) incorporated isotope into a GIcNAc-linkced lipid that was tentatively identified as GlcNAc-pyrophosphorylundecaprenol. The incorporation of [3H]GlcNAc into this compound was abolished when cells were pulse-labeled in the presence of the antibiotic tunicamycin. Tunicamycin also abolished the in vivo synthesis of the haptenic form of enterobacterial common antigen (ECA) in S. typhimurium as determined by the passive hemagglutination test. These data indicated that the synthesis of the GlcNAc-linked lipid is related to ECA synthesis. Support for this conclusion was provided by the following observations. Cultures of Escherichia coli and S. typhimurium incorporated [3H]GlcNAc into cell envelope components that migrated as a homologous series of polymers when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The [3H]GIcNAc-labeled polymers were not detected in mutants of E. coli and S. typhimurium defective in ECA synthesis due to lesions in either the rfe or rff gene clusters. These polymers were identified as ECA based on Western blot analyses employing anti-ECA monoclonal antibody. The incorporation of [3H]GIcNAc into ECA polymers was abolished by tunicamycin when the drug was added to cultures to give a minimum concentration of 3 ,ug/ml. In addition, pulse-chase experiments provided evidence for a prvcursor-product relationship between the GlcNAc-linked lipid and ECA. These results strongly suggest that the GlcNAc-linked lipid is involved in the biosynthesis of ECA in a manner analogous to the role of carrier lipid in the biosynthesis of 0-antigen and peptidoglycan.

ECA, the enterobacterial common antigen

“…ECApc to phospholipase D allowed the removal of the lipid moiety and the identification of GIcNAc as the reducing end of the sugar chain [11]. This result is in good agreement with the recent discovery of diphosphorylundecaprenolbound GIcNAc as the first intermediate in ECA biosynthesis [24]. Fig.…”

“…1), indicating a size heterogeneity of chain lengths as demonstrated for smooth lipopolysaccharides [22]. Such patterns were also observed with monoclonal ECA antibodies for crude bacterial extracts [23,24]. From the number of bands ( Fig.…”

“…Recently, the apparent M r of ECA in crude extracts of Salmonella typhimurium was described to range over 12000-35 000 [24].…”

Genes affecting progression of bacteriophage P22 infection in Salmonella identified by transposon and single gene deletion screens

“…Enterobacterial common antigen is a surface glycolipid that is conserved among enteric bacteria and contributes to pathogenicity and virulence in S. enterica (Gilbreath et al, 2012). In Salmonella, it is composed of a linear polysaccharide with a tri-saccharide repeat composed of 4-acetamide-4,6-dideoxy-D-galactose-N-acetyl-D-mannosaminuronic acid-N-acetyl-D-glucosamine and can be anchored to the outer membrane by attachment to either the O-antigen core (above) or a phosphoglyceride (Rick et al, 1985). Seven genes in the ECA synthesis pathway, including five in the wec cluster as well as glmU and wzxE, were identified in the Tn5 assay as genes whose products inhibit progression of P22 infection (Supporting Information Table S2 and Fig.…”

The biology of colicin M

“…The synthesis of a number of cell envelope constituents in both Gram-negative and Grampositive bacteria is known or thought to require the bactoprenol carrier lipid. Aside from peptidoglycan and O-antigen, these include enterobacterial common antigen [96], K1 capsular polysaccharide [97,98], lipomannan [99], teichoic acid [1~30,101], teichuronic acid [102,103] and colanic add [104]. The synthesis of membrane derived oligosaccharides (MDO) has also been suggested to use the bactoprenol carrier [105,106].…”