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 (intercellular adhesion) operon. To study the function of the individual genes, 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 low N-acetylglucosaminyltransferase activity and represents the catalytic enzyme. Coexpression of icaA with icaD led to a significant increase in activity. The newly identified icaD gene is located between icaA and icaB and overlaps both genes. N-Acetylglucosamine oligomers produced by IcaAD reached a maximal length of 20 residues. Only when icaA and icaD were expressed together with icaC were oligomer chains that react with PIA-specific antiserum synthesized. IcaA and IcaD are located in the cytoplasmic membrane, and IcaC also has all the structural features of an integral membrane protein. These results indicate a close interaction between IcaA, IcaD, and IcaC. Tunicamycin and bacitracin did not affect the in vitro synthesis of PIA intermediates or the complete PIA biosynthesis in vivo, suggesting that a undecaprenyl phosphate carrier is not involved. IcaAD represents a novel protein combination among beta-glycosyltransferases.
Gerke
C
C
Mikrobielle Genetik, Universität Tübingen, Waldhäuser Strasse 70/8, 72076 Tübingen, Germany.
Kraft
A
A
Süssmuth
R
R
Schweitzer
O
O
Götz
F
F
D000595:Amino Acid Sequence; D001422:Bacterial Adhesion; D001483:Base Sequence; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis*; D016415:Sequence Alignment; D013212:Staphylococcus epidermidis / Q000201:enzymology* / Q000378:metabolism; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism
D011135:Polysaccharides, Bacterial; C113579:polysaccharide intercellular adhesin; D014537:Uridine Diphosphate N-Acetylglucosamine; D017351:N-Acetylglucosaminyltransferases; C037649:N-acetyllactosaminide beta-1,6-N-acetylglucosaminyltransferase