Staphylococcus aureus cap5P Encodes a UDP- N -Acetylglucosamine 2-Epimerase with Functional Redundancy

Abstract

ABSTRACTThe serotype 5 capsule gene cluster ofStaphylococcus aureuscomprises 16 genes (cap5Athroughcap5P), but little is known about how the putative gene products function in capsule biosynthesis. We propose that theN-acetylmannosaminuronic acid (ManNAcA) component of theS. aureusserotype 5 capsular polysaccharide (CP5) is synthesized from a UDP-N-acetylglucosamine (UDP-GlcNAc) precursor that is epimerized to UDP-N-acetylmannosamine (UDP-ManNAc) and then oxidized to UDP-ManNAcA. We report the purification and biochemical characterization of a recombinant UDP-GlcNAc 2-epimerase encoded byS. aureus cap5P. Purified Cap5P converted ∼10% of UDP-GlcNAc to UDP-ManNAc as detected by gas chromatography-mass spectrometry. The epimerization of UDP-GlcNAc to UDP-ManNAc occurred over a wide pH range and was unaffected by divalent cations. Surprisingly, CP5 expression inS. aureuswas unaffected by insertional inactivation ofcap5P. Sequence homology searches of the publicS. aureusgenomic databases revealed the presence of another putative UDP-GlcNAc 2-epimerase on theS. aureuschromosome that showed 61% identity to Cap5P. Redundancy of UDP-GlcNAc 2-epimerase function inS. aureuswas demonstrated by cloning thecap5Phomologue from strain Newman and complementing anEscherichia coli rffEmutant defective in UDP-GlcNAc 2-epimerase activity. Our results confirm the putative function of theS. aureus cap5Pgene product and demonstrate the presence of a second gene on the staphylococcal chromosome with a similar function.