RESUMO
Pseudomonas aeruginosa PAK (serotype O6) produces a single polar, glycosylated flagellum composed of a-type flagellin. To determine whether or not flagellin glycosylation in this serotype requires O-antigen genes, flagellin was isolated from the wild type, three O-antigen-deficient mutants wbpL, wbpO, and wbpP, and a wbpO mutant complemented with a plasmid containing a wild-type copy of wbpO. Flagellin from the wbpO mutant was smaller (42 kDa) than that of the wild type (45 kDa), or other mutants strains, and exhibited an altered isoelectric point (pI 4.8) when compared with PAK flagellin (pI 4.6). These differences were because of the truncation of the glycan moiety in the wbpO-flagellin. Thus, flagellin glycosylation in P. aeruginosa PAK apparently requires a functional WbpO but not WbpP. Because WbpP was previously proposed to catalyze a metabolic step in the biosynthesis of B-band O-antigen that precedes the action of WbpO, these results prompted us to reevaluate the two-step pathway catalyzed by WbpO and WbpP. Results from WbpO-WbpP-coupled enzymatic assays showed that either WbpO or WbpP is capable of initiating the two-step pathway; however, the kinetic parameters favored the WbpO reaction to occur first, converting UDP-N-acetyl-D-glucosamine to UDP-N-acetyl-D-glucuronic acid prior to the conversion to UDP-N-acetyl-D-galacturonic acid by WbpP. This is the first report to show that a C4 epimerase could utilize UDP-N-acetylhexuronic acid as a substrate.
Assuntos
Oxirredutases do Álcool/fisiologia , Proteínas de Bactérias/metabolismo , Flagelina/química , Regulação Bacteriana da Expressão Gênica , Antígenos O/química , Pseudomonas aeruginosa/metabolismo , Oxirredutases do Álcool/genética , Catálise , Flagelina/metabolismo , Teste de Complementação Genética , Glicosilação , Espectroscopia de Ressonância Magnética , Mutação , Plasmídeos/metabolismo , Polissacarídeos/química , Racemases e Epimerases/química , Uridina Difosfato Ácido Glucurônico/química , Uridina Difosfato N-Acetilglicosamina/químicaRESUMO
The O antigen of Pseudomonas aeruginosa B-band lipopolysaccharide is synthesized by assembling O-antigen-repeat units at the cytoplasmic face of the inner membrane by nonprocessive glycosyltransferases, followed by polymerization on the periplasmic face. The completed chains are covalently attached to lipid A core by the O-antigen ligase, WaaL. In P. aeruginosa the process of ligating these O-antigen molecules to lipid A core is not clearly defined, and an O-antigen ligase has not been identified until this study. Using the sequence of waaL from Salmonella enterica as a template in a BLAST search, a putative waaL gene was identified in the P. aeruginosa genome. The candidate gene was amplified and cloned, and a chromosomal knockout of PAO1 waaL was generated. Lipopolysaccharide (LPS) from this mutant is devoid of B-band O-polysaccharides and semirough (SR-LPS, or core-plus-one O-antigen). The mutant PAO1waaL is also deficient in the production of A-band polysaccharide, a homopolymer of D-rhamnose. Complementation of the mutant with pPAJL4 containing waaL restored the production of both A-band and B-band O antigens as well as SR-LPS, indicating that the knockout was nonpolar and waaL is required for the attachment of O-antigen repeat units to the core. Mutation of waaL in PAO1 and PA14, respectively, could be complemented with waaL from either strain to restore wild-type LPS production. The waaL mutation also drastically affected the swimming and twitching motilities of the bacteria. These results demonstrate that waaL in P. aeruginosa encodes a functional O-antigen ligase that is important for cell wall integrity and motility of the bacteria.
Assuntos
Carbono-Oxigênio Ligases/fisiologia , Antígenos O/metabolismo , Pseudomonas aeruginosa/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carbono-Oxigênio Ligases/genética , Parede Celular/fisiologia , Clonagem Molecular , Deleção de Genes , Teste de Complementação Genética , Mutação , Reação em Cadeia da Polimerase , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/ultraestruturaRESUMO
The structural similarity between the pilin glycan and the O-antigen of Pseudomonas aeruginosa 1244 suggested that they have a common metabolic origin. Mutants of this organism lacking functional wbpM or wbpL genes synthesized no O-antigen and produced only non-glycosylated pilin. Complementation with plasmids containing functional wbpM or wbpL genes fully restored the ability to produce both O-antigen and glycosylated pilin. Expression of a cosmid clone containing the O-antigen biosynthetic gene cluster from P. aeruginosa PA103 (LPS serotype O11) in P. aeruginosa 1244 (LPS serotype O7) resulted in the production of strain 1244 pili that contained both O7 and O11 antigens. The presence of the O11 repeating unit was confirmed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Expression of the O-antigen biosynthesis cluster from Escherichia coli O157:H7 in strain 1244 resulted in the production of pilin that contained both the endogenous Pseudomonas as well as the Escherichia O157 O-antigens. A role for pilO in the glycosylation of pilin in P. aeruginosa is evident as the cloned pilAO operon produced glycosylated strain 1244 pilin in eight heterologous P. aeruginosa strains. Removal of the pilO gene resulted in the production of unmodified strain 1244 pilin. These results show that the pilin glycan of P. aeruginosa 1244 is a product of the O-antigen biosynthetic pathway. In addition, the structural diversity of the O-antigens used by the 1244 pilin glycosylation apparatus indicates that the glycan substrate specificity of this reaction is extremely low.