MOLECULAR CHARACTERIZATION OF THE CLUMPING FACTOR (FIBRINOGEN RECEPTOR) OF STAPHYLOCOCCUS-AUREUS

Four mutants of Staphylococcus aureus strain Newman that were defectiv e in the fibrinogen receptor (clumping factor) were isolated by transp oson Tn917 mutagenesis. Southern hybridization analysis of the mutants identified transposon-host DNA junction fragments, one of which was c loned and used to generate a probe to identify and clone the wild-type clumping factor locus (clfA). The mutants failed to form clumps in so luble fibrinogen and adhered poorly to polymethylmethacrylate (PMMA) c overslips coated with fibrinogen. A single copy of the clfA gene, when introduced into the chromosome of the mutant strains, fully complemen ted 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 str ain 8325-4 to form clumps with the same avidity as the wild-type strai n Newman and also significantly enhanced the adherence of 8325-4 strai ns. Thus the formation of clumps in soluble fibrinogen correlated with adherence of bacteria to solid-phase fibrinogen. The clfA gene encode s a fibrinogen-binding protein with an apparent molecular mass of c. 1 30 kDa. The amino acid sequence of the protein was deduced from the DN A sequence; it was predicted that a 896 residue protein (molecular mas s 92 kDa) would be expressed. The putative ClfA protein has features t hat suggest that it is associated with the cell surface. Furthermore i t contains a novel 308 residue region comprising dipeptide repeats pre dominantly of Asp and Ser ending 28 residues upstream from the LPXTG m otif common to wall-associated proteins. Significant homology was foun d between the ClfA protein and the fibronectin-binding proteins of S. aureus, particularly in the N- and C-termini.