Staphylococcus aureus cap5P encodes a UDP-N-acetylglucosamine 2-epimerase with functional redundancy

Abstract

The serotype 5 capsule gene cluster of Staphylococcus aureus comprises 16 genes (cap5A through cap5P), but little is known about how the putative gene products function in capsule biosynthesis. We propose that the N-acetylmannosaminuronic acid (ManNAcA) component of the S. aureus serotype 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 by S. aureus cap5P. Purified Cap5P converted approximately 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 in S. aureus was unaffected by insertional inactivation of cap5P. Sequence homology searches of the public S. aureus genomic databases revealed the presence of another putative UDP-GlcNAc 2-epimerase on the S. aureus chromosome that showed 61% identity to Cap5P. Redundancy of UDP-GlcNAc 2-epimerase function in S. aureus was demonstrated by cloning the cap5P homologue from strain Newman and complementing an Escherichia coli rffE mutant defective in UDP-GlcNAc 2-epimerase activity. Our results confirm the putative function of the S. aureus cap5P gene product and demonstrate the presence of a second gene on the staphylococcal chromosome with a similar function.

FIG. 1

SDS-PAGE analysis of S. aureus Cap5P expression and purification. Cell lysates were made from E. coli BL21(DE3) carrying pKBK7. Cap5P was overexpressed in the presence of 1 mM IPTG, and the soluble protein was purified over a nickel column. Lane 1, cell lysate from uninduced cells; lane 2, cell lysate from IPTG-induced cells; lane 3, column effluent reflecting unbound proteins; lane 4, purified S. aureus Cap5P. Molecular mass markers in kilodaltons are indicated on the left.

FIG. 2

GC-MS analysis of hydrolyzed and derivatized assay mixtures containing UDP-GlcNAc and S. aureus Cap5P. Elution products GlcNAc and ManNAc were identified by GC (A), in which peaks with retention times of 36 and 37 min corresponded to those of authentic GlcNAc and ManNAc standards, respectively, and MS (B), in which the peak at 37 min was shown to be an alditol acetate derivative of acetylhexosamine.

FIG. 3

Effect of incubation time on the activity of S. aureus cap5P-encoded UDP-GlcNAc 2-epimerase. Purified Cap5P was incubated with UDP-GlcNAc under standard conditions. Conversion to UDP-ManNAc was analyzed by GC-MS after alditol acetate derivatization of the samples. Results presented are pooled data from two experiments.

FIG. 4

Sequence alignment of S. aureus Cap5P and MnaA. The complete amino acid sequences of Cap5P (23) and MnaA (4a) were aligned with the BestFit program (Wisconsin Package, Genetics Computer Group). Identical and similar residues between the two sequences are connected by vertical lines and dots, respectively.