The gene encoding the IgG‐binding protein G from Streptococcus G148 was isolated by molecular cloning. A subclone containing a 1.5‐kb insert gave a functional product in Escherichia coli. Protein analysis of affinity‐purified polypeptides revealed two gene products, both smaller than protein G spontaneously released from streptococci, but with identical IgG‐binding properties. The complete nucleotide sequence of the insert revealed a repeated structure probably evolved through duplications of fragments of different sizes. The deduced amino acid sequence revealed an open reading frame extending throughout the insert, terminating in a TAA stop codon. Analysis of the two gene products by N‐terminal amino acid determination suggests that two different TTG codons are recognized in E. coli for initiation of translation to yield the two products. Based on these results several truncated gene constructions were expressed and analysed. The results suggest that the C‐terminal part of streptococcal protein G consists of three IgG‐binding domains followed by a region which anchors the protein to the cell surface. Structural and functional comparisons with streptococcal M protein and staphylococcal protein A have been made.

The gene encoding the fibronectin‐binding protein (FNBP) from Staphylococcus aureus strain 8325‐4 was isolated from a gene bank in pBR322. The original clone, containing a 6.5‐kb insert, gave a functional product present in the periplasm of Escherichia coli. Analysis of polypeptides isolated after affinity chromatography on fibronectin‐Sepharose followed by ion‐exchange chromatography revealed two gene products, 87 and 165 kd in mol. wt. The amino acid compositions of these two polypeptides and a native FNBP from S. aureus strain Newman were very similar. Antibodies raised against the native FNBP from strain Newman precipitated the 125I‐labelled 165‐kd polypeptide, and unlabeled 165‐ and 87‐kd polypeptides as well as native FNBP inhibited the immunoprecipitation reactions. The region of the fnbp‐gene encoding the fibronectin‐binding activity has been identified and subcloned in an expression vector based on the staphylococcal protein A gene. The resulting product in E. coli is an extracellular fusion protein consisting of two IgG‐binding domains of protein A followed by a fibronectin‐binding region. The fusion protein binds to fibronectin and completely inhibits the binding of fibronectin to intact cells of S. aureus.

The present study reports on fibrinogen (Fg) binding ofStaphylococcus epidermidis. Adhesion of differentS. epidermidis strains to immobilized Fg was found to vary significantly between different strains, and the component responsible was found to be proteinaceous in nature. To further characterize the Fg-binding activity, a shotgun phage display library covering the S. epidermidis chromosome was constructed. By affinity selection (panning) against immobilized Fg, a phagemid clone, pSEFG1, was isolated, which harbors an insert with an open reading frame of ∼1.7 kilobases. Results from binding and inhibition experiments demonstrated that the insert of pSEFG1 encodes a specific Fg-binding protein. Furthermore, affinity-purified protein encoded by pSEFG1 completely inhibited adhesion of S. epidermidis to immobilized Fg. By additional cloning and DNA sequence analyses, the complete gene, termed fbe, was found to consist of an open reading frame of 3,276 nucleotides encoding a protein, called Fbe, with a deduced molecular mass of ∼119 kDa. With a second phage display library made from another clinical isolate ofS. epidermidis, it was possible to localize the Fg-binding region to a 331-amino-acid-long fragment. PCR analysis showed that the fbe gene was found in 40 of 43 clinical isolates of S. epidermidis. The overall organization of Fbe resembles those of other extracellular surface proteins of staphylococci and streptococci. Sequence comparisons with earlier known proteins revealed that this protein is related to an Fg-binding protein of Staphylococcus aureus called clumping factor.

The sequence of region X of staphylococcal protein A has been determined. The hypothesis has been put forward that this region spans the Staphylococcus aureus cell wall and is responsible for the binding to the peptidoglycan. The primary amino acid sequence of region X was determined for two strains exhibiting cell-wall-bound protein A, Cowan I and 8325 -4. The sequence determination of the Cowan I material is partial and was performed by Edman degradation, in contrast to the sequence of the 8325 -4 material which was completely analyzed by nucleotide sequencing of the corresponding gene. The region consists of two structurally different domains, a highly repetitive region (X,), with an octapeptide structure repeated approximately 12 times, and a C-terminal domain (X,) with an unique sequence. A comparison between the two strains reveals a high mutual homology as well as a high internal homology between the octapeptide structures. Six out of eight amino acids are identical in the repetition of this structure throughout region X, in both proteins and the other two are changed in a rather regular pattern.

No abstract