Abstract
N-Acetyl-l-fucosamine is a constituent of surface polysaccharide structures of Pseudomonas aeruginosa and Staphylococcus aureus. The three P. aeruginosa enzymes WbjB, WbjC, and WbjD, as well as the S. aureus homologs Cap5E, Cap5F, and Cap5G, involved in the biosynthesis of N-acetyl-l-fucosamine have been overexpressed and purified to near homogeneity. Capillary electrophoresis (CE), mass spectroscopy (MS), and nuclear magnetic resonance spectroscopy have been used to elucidate the biosynthesis pathway, which proceeds in five reaction steps. WbjB/Cap5E catalyzed 4,6-dehydration of UDP-N-acetyl-d-glucosamine and 3- and 5-epimerization to yield a mixture of three keto-deoxy-sugars. The third intermediate compound was subsequently reduced at C-4 to UDP-2-acetamido-2,6-dideoxy-l-talose by WbjC/Cap5F. Incubation of UDP-2-acetamido-2,6-dideoxy-l-talose (UDP-TalNAc) with WbjD/Cap5G resulted in a new peak separable by CE that demonstrated identical mass and fragmentation patterns by CE-MS/MS to UDP-TalNAc. These results are consistent with WbjD/Cap5G-mediated 2-epimerization of UDP-TalNAc to UDP-FucNAc. A nonpolar gene knockout of wbjB, the first of the genes associated with this pathway, was constructed in P. aeruginosa serotype O11 strain PA103. The corresponding mutant produced rough lipopolysaccharide devoid of B-band O antigen. This lipopolysaccharide deficiency could be complemented with P. aeruginosa wbjB or with the S. aureus homolog cap5E. Insertional inactivation of either the cap5G or cap5F genes abolished capsule polysaccharide production in the S. aureus strain Newman. Providing the appropriate gene in trans, thereby complementing these mutants, fully restored the capsular polysaccharide phenotype.
Highlights
N-Acetyl-L-fucosamine is a constituent of surface polysaccharide structures of Pseudomonas aeruginosa and Staphylococcus aureus
WbjB/Cap5E is moderately homologous to WbpM, which is putatively involved in UDP-N-acetyl-D-fucosamine/quinovosamine biosynthesis and shows reactivity on UDP-D-GlcNAc [42]
This report describes for the first time the biosynthesis steps from the precursor UDP-D-GlcNAc to a nucleotide precursor sugar that is consistent with UDP-L-FucNAc; five distinct enzymatic steps, catalyzed by three enzymes, are sufficient
Summary
Materials—UDP-N-acetyl-D-glucosamine, UDP-N-acetyl-D-galactosamine, UDP-D-glucose, NADϩ, NADH, NADPϩ, NADPH, and the antibiotics used in this study were obtained from Sigma-Aldrich. DNA Manipulations, PCR, DNA Sequencing, and Protein Sequencing—All of the standard DNA recombinant procedures were performed according to the methods described by Sambrook et al [32] or as recommended by the corresponding manufacturer. The corresponding plasmids pFuc (wbjB), pFuc (wbjC), pFuc (wbjD), and pFuc (cap5E) were confirmed by DNA sequencing and used for amplification of PCR products for complementation studies and/or overexpression of N-terminal histidine-tagged fusion proteins. S. aureus cap5E, capF, and capG genes were PCR-amplified from strain Newman chromosomal DNA with High Fidelity PCR Supermix (Invitrogen). To construct the plasmids containing a nonpolar gene disruption of P. aeruginosa wbjB, the PCR product described above was ligated into SmaI-cut pEX100T, taking advantage of the blunt-end PCR product made by Pwo polymerase. SM10 BL21(DE3) P. aeruginosa IATS O11 PA103 BK103B S. aureus VI-114 Newman G01 F4 RN4220 8325–4 Plasmids pET23der pET28a pET24aϩ pET24cϩ pEX100T pUCGm pUCP27 pFuc pFuc pFuc pFuc pKOB1 pKOB2 pPAB27 pSAE27 pKBK50d pET5F1.1 pKBK6a pJCL69 pJCL69–2 pERMB pJCL69–2ermB pCL10 pKOR1 pAP1.2EX pKOR3 pKOR4
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