Abstract
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.
Highlights
P. aeruginosa PAK produces a single polar flagellum containing a-type flagellin, which has been shown to be post-translationally modified at two sites with O-linked heterogeneous glycans [14]
We investigated the potential role that O-antigen biosynthetic genes might play in the synthesis of these complex glycans by comparing flagellin proteins isolated from wild-type PAK and three mutant strains defective in O-antigen biosynthesis
The mass of the glycosylated wild-type flagellin in our study determined by MALDITOF mass spectrometry (MS) was found to be 42,577 (Ϯ10) m/z, and this mass is consistent with that reported by Schirm et al [14], whose results suggested that PAK flagellin was modified at two sites with a broad distribution of glycoforms, including one corresponding to 42,573 Da
Summary
In Pseudomonas aeruginosa strains, dTDP-L-rhamnose is channeled into synthesis of numerous L-rhamnose-containing cell-surface structures, including rhamnolipids [4], core oligosaccharide, and O-antigen polysaccharide [5] This sugar-nucleotide is synthesized by proteins encoded in the rmlBDAC gene cluster, which is unlinked to the gene clusters that are required for synthesis of these glycoconjugates. We isolated and compared flagellin from wild-type PAK and three mutants, wbpL, wbpO, and wbpP Null mutations in these genes, contained within the O6 O-antigen cluster, have been generated, and analysis of the LPS phenotypes from these mutants showed the absolute requirement of these genes for O-antigen production [17]. WbpP has been characterized as a UDP-N-acetyl-D-glucosamine (UDP-D-GlcNAc) 4-epimerase [19], and WbpO has been characterized as a 6-dehydrogenase that accepts UDP-N-acetyl-Dgalactosamine (UDP-D-GalNAc), the product of WbpP/UDPD-GlcNAc reactions, and produces UDP-D-GalNAcA [20]
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