Distinct functional domains of the Salmonella enterica WbaP transferase that is involved in the initiation reaction for synthesis of the O antigen subunit.
WbaP is a membrane enzyme that initiates O antigen synthesis in Salmonella enterica by catalysing the transfer of galactose 1-phosphate (Gal-1-P) onto undecaprenyl phosphate (Und-P). WbaP possesses at least three predicted structural domains: an N-terminal region containing four transmembrane helices, a large central periplasmic loop, and a C-terminal domain containing the last transmembrane helix and a large cytoplasmic tail. In this work, we investigated the contribution of each region to WbaP function by constructing a series of mutant WbaP proteins and using them to complement O antigen synthesis in DeltawbaP mutants of S. enterica serovars Typhi and Typhimurium. Truncated forms of WbaP lacking the periplasmic loop exhibited altered chain-length distributions in O antigen polymerization, suggesting that this central domain is involved in modulating the chain-length distribution of the O polysaccharide. The N-terminal and periplasmic domains were dispensable for complementation of O antigen synthesis in vivo, suggesting that the C-terminal domain carries the sugar-phosphate transferase activity. However, despite the fact that they complemented the synthesis of O antigen in the DeltawbaP mutant in vivo, membrane extracts containing WbaP derivatives without the N-terminal domain failed to transfer radioactive Gal from UDP-Gal into a lipid-rich fraction. These results suggest that the N-terminal region of WbaP, which contains four transmembrane domains, is essential for the insertion or stability of the protein in the bacterial membrane. We propose that the domain structure of WbaP enables this protein not only to function in the transfer of Gal-1-P to Und-P but also to establish critical interactions with additional proteins required for the correct assembly of O antigen in S. enterica.
Saldías
M Soledad
MS
Infectious Diseases Research Group, Siebens-Drake Research Institute, Department of Microbiology and Immunology, University of Western Ontario, London, ON N6A 5C1, Canada.|Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, PO Box 174 Correo 22, Santiago, Chile.
Patel
Kinnari
K
Infectious Diseases Research Group, Siebens-Drake Research Institute, Department of Microbiology and Immunology, University of Western Ontario, London, ON N6A 5C1, Canada.
Marolda
Cristina L
CL
Infectious Diseases Research Group, Siebens-Drake Research Institute, Department of Microbiology and Immunology, University of Western Ontario, London, ON N6A 5C1, Canada.
Bittner
Mauricio
M
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, PO Box 174 Correo 22, Santiago, Chile.
Contreras
Inés
I
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, PO Box 174 Correo 22, Santiago, Chile.
Valvano
Miguel A
MA
Infectious Diseases Research Group, Siebens-Drake Research Institute, Department of Medicine, University of Western Ontario, London, ON N6A 5C1, Canada.|Infectious Diseases Research Group, Siebens-Drake Research Institute, Department of Microbiology and Immunology, University of Western Ontario, London, ON N6A 5C1, Canada.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004926:Escherichia coli / Q000254:growth & development; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019081:O Antigens / Q000378:metabolism*; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics; D033903:Periplasmic Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D019779:Salmonella enterica / Q000201:enzymology* / Q000378:metabolism; D017421:Sequence Analysis; D014166:Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D019081:O Antigens; D009842:Oligopeptides; D010455:Peptides; D033903:Periplasmic Proteins; C085706:FLAG peptide; D014166:Transferases