The activity of a putative polyisoprenol-linked sugar translocase (Wzx) involved in Escherichia coli O antigen assembly is independent of the chemical structure of the O repeat.
During O antigen lipopolysaccharide (LPS) synthesis in bacteria, transmembrane migration of undecaprenylpyrophosphate (Und-P-P)-bound O antigen subunits occurs before their polymerization and ligation to the rest of the LPS molecule. Despite the general nature of the translocation process, putative O-antigen translocases display a low level of amino acid sequence similarity. In this work, we investigated whether complete O antigen subunits are required for translocation. We demonstrate that a single sugar, GlcNAc, can be incorporated to LPS of Escherichia coli K-12. This incorporation required the functions of two O antigen synthesis genes, wecA (UDP-GlcNAc:Und-P GlcNAc-1-P transferase) and wzx (O-antigen translocase). Complementation experiments with putative O-antigen translocases from E. coli O7 and Salmonella enterica indicated that translocation of O antigen subunits is independent of the chemical structure of the saccharide moiety. Furthermore, complementation with putative translocases involved in synthesis of exopolysaccharides demonstrated that these proteins could not participate in O antigen assembly. Our data indicate that recognition of a complete Und-P-P-bound O antigen subunit is not required for translocation and suggest a model for O antigen synthesis involving recognition of Und-P-P-linked sugars by a putative complex made of Wzx translocase and other proteins involved in the processing of O antigen.
Feldman
M F
MF
Instituto de Investigaciones Bioquímicas Fundación Campomar, Buenos Aires, Argentina.
Marolda
C L
CL
Monteiro
M A
MA
Perry
M B
MB
Parodi
A J
AJ
Valvano
M A
MA
D000371:Agglutination; D001426:Bacterial Proteins; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D006602:Hexosyltransferases / Q000378:metabolism; D008050:Lipid A / Q000378:metabolism; D008565:Membrane Proteins; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D016296:Mutagenesis; D019081:O Antigens / Q000096:biosynthesis* / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D016339:Spectrometry, Mass, Fast Atom Bombardment
D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008050:Lipid A; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D019081:O Antigens; D009844:Oligosaccharides; D011994:Recombinant Proteins; C117254:RfbX protein, Pseudomonas; C543926:Wzx protein, E coli; D006602:Hexosyltransferases; C085652:O-antigen polymerase