Staphylococcus aureus cap5P encodes a UDP-N-acetylglucosamine 2-epimerase with functional redundancy.
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.
Kiser
K B
KB
Channing Laboratory, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Bhasin
N
N
Deng
L
L
Lee
J C
JC
D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002238:Carbohydrate Epimerases / Q000235:genetics* / Q000378:metabolism; D002352:Carrier Proteins; D002876:Chromosomes, Bacterial; D017931:DNA Primers; D004789:Enzyme Activation / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010641:Phenotype; D012689:Sequence Homology, Nucleic Acid; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics*
D001426:Bacterial Proteins; C107803:Cap5 protein, Staphylococcus; D002352:Carrier Proteins; D017931:DNA Primers; D002238:Carbohydrate Epimerases; C024519:N-acyl-D-glucosamine 2-epimerase