Molecular characterization of the clumping factor (fibrinogen receptor) of Staphylococcus aureus.
Four mutants of Staphylococcus aureus strain Newman that were defective in the fibrinogen receptor (clumping factor) were isolated by transposon Tn917 mutagenesis. Southern hybridization analysis of the mutants identified transposon-host DNA junction fragments, one of which was cloned and used to generate a probe to identify and clone the wild-type clumping factor locus (clfA). The mutants failed to form clumps in soluble fibrinogen and adhered poorly to polymethylmethacrylate (PMMA) coverslips coated with fibrinogen. A single copy of the clfA gene, when introduced into the chromosome of the mutant strains, fully complemented the clumping deficiency of these strains and restored the ability of these mutants to adhere to fibrinogen-coated PMMA. In addition, the cloned clfA gene on a shuttle plasmid allowed the weakly clumping strain 8325-4 to form clumps with the same avidity as the wild-type strain Newman and also significantly enhanced the adherence of 8325-4 strains. Thus the formation of clumps in soluble fibrinogen correlated with adherence of bacteria to solid-phase fibrinogen. The clfA gene encodes a fibrinogen-binding protein with an apparent molecular mass of c. 130 kDa. The amino acid sequence of the protein was deduced from the DNA sequence; it was predicted that a 896 residue protein (molecular mass 92 kDa) would be expressed. The putative ClfA protein has features that suggest that it is associated with the cell surface. Furthermore it contains a novel 308 residue region comprising dipeptide repeats predominantly of Asp and Ser ending 28 residues upstream from the LPXTG motif common to wall-associated proteins. Significant homology was found between the ClfA protein and the fibronectin-binding proteins of S. aureus, particularly in the N- and C-termini.
McDevitt
D
D
Microbiology Department, Moyne Institute, Trinity College, Dublin, Ireland.
Francois
P
P
Vaudaux
P
P
Foster
T J
TJ
D000595:Amino Acid Sequence; D001422:Bacterial Adhesion; D015139:Blotting, Southern; D003001:Cloning, Molecular; D003030:Coagulase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D004269:DNA, Bacterial / Q000032:analysis / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000378:metabolism; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013211:Staphylococcus aureus / Q000235:genetics* / Q000378:metabolism*
C515944:ClfA protein, Staphylococcus aureus; D003030:Coagulase; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D005340:Fibrinogen