title: Functional characterization of UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferases of Escherichia coli and Caulobacter crescentus.
abstract: Escherichia coli K-12 WcaJ and the Caulobacter crescentus HfsE, PssY, and PssZ enzymes are predicted to initiate the synthesis of colanic acid (CA) capsule and holdfast polysaccharide, respectively. These proteins belong to a prokaryotic family of membrane enzymes that catalyze the formation of a phosphoanhydride bond joining a hexose-1-phosphate with undecaprenyl phosphate (Und-P). In this study, in vivo complementation assays of an E. coli K-12 wcaJ mutant demonstrated that WcaJ and PssY can complement CA synthesis. Furthermore, WcaJ can restore holdfast production in C. crescentus. In vitro transferase assays demonstrated that both WcaJ and PssY utilize UDP-glucose but not UDP-galactose. However, in a strain of Salmonella enterica serovar Typhimurium deficient in the WbaP O antigen initiating galactosyltransferase, complementation with WcaJ or PssY resulted in O-antigen production. Gas chromatography-mass spectrometry (GC-MS) analysis of the lipopolysaccharide (LPS) revealed the attachment of both CA and O-antigen molecules to lipid A-core oligosaccharide (OS). Therefore, while UDP-glucose is the preferred substrate of WcaJ and PssY, these enzymes can also utilize UDP-galactose. This unexpected feature of WcaJ and PssY may help to map specific residues responsible for the nucleotide diphosphate specificity of these or similar enzymes. Also, the reconstitution of O-antigen synthesis in Salmonella, CA capsule synthesis in E. coli, and holdfast synthesis provide biological assays of high sensitivity to examine the sugar-1-phosphate transferase specificity of heterologous proteins.
authors.affiliation: Departments of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada.
authors.forename: Kinnari B
authors.initials: KB
authors.lastname: Patel
authors.forename: Evelyn
authors.initials: E
authors.lastname: Toh
authors.forename: Ximena B
authors.initials: XB
authors.lastname: Fernandez
authors.forename: Anna
authors.initials: A
authors.lastname: Hanuszkiewicz
authors.forename: Gail G
authors.initials: GG
authors.lastname: Hardy
authors.forename: Yves V
authors.initials: YV
authors.lastname: Brun
authors.forename: Mark A
authors.initials: MA
authors.lastname: Bernards
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Valvano
mesh_terms: D001483:Base Sequence; D016935:Caulobacter crescentus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D009154:Mutation; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D011134:Polysaccharides / Q000096:biosynthesis; D013045:Species Specificity
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011134:Polysaccharides; C004275:colanic acid; D017856:Phosphotransferases (Phosphate Group Acceptor); C573281:WcaJ protein, E coli