CHARACTERIZATION OF THE N-ACETYLGLUCOSAMINYLTRANSFERASE ACTIVITY INVOLVED IN THE BIOSYNTHESIS OF THE STAPHYLOCOCCUS-EPIDERMIDIS POLYSACCHARIDE INTERCELLULAR ADHESIN

The polysaccharide intercellular adhesin (PIA) is an important factor in the colonization of medical devices by Staphylococcus epidermidis. The genes encoding PIA production are organized in the icaADBC (interc ellular adhesion) operon. To study the function of the individual gene s, we have established an in vitro assay with UDP-N-acetylglucosamine, the substrate for PIA biosynthesis, and analyzed the products by thin -layer chromatography and mass spectrometry. IcaA alone exhibited a lo w N-acetylglucosaminyltransferase activity and represents the catalyti c enzyme. Coexpression of icaA with icaD led to a significant increase in activity. The newly identified icaD gene is located between icaA a nd icaB and overlaps both genes, N-Acetylglucosamine oligomers produce d by IcaAD reached a maximal length of 20 residues. Only when icaA and icaD were expressed together with icaC were oligomer chains that reac t with PIA-specific antiserum synthesized. IcaA and IcaD are located i n the cytoplasmic membrane, and IcaC also has all the structural featu res of an integral membrane protein. These results indicate a close in teraction between IcaA, IcaD, and IcaC. Tunicamycin and bacitracin did not affect the in vitro synthesis of PIA intermediates or the complet e PIA biosynthesis in vivo, suggesting that a undecaprenyl phosphate c arrier is not involved. IcaAD represents a novel protein combination a mong P-glycosyltransferases.