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Papers In Press, published online ahead of print October 29, 2001
Microbiology Department, Trinity College, Dublin, Dublin 2
Corresponding Author: tfoster{at}tcd.ie
The elastin binding proteins EbpS of Staphylococcus aureus strains Cowan and 8325-4 were predicted from sequence analysis to comprise 486 residues. Specific antibodies were raised against an N-terminal domain (residues 1-267) and a C-terminal domain (residues 343-486) expressed as recombinant proteins in Escherichia coli. Western blotting of lysates of wild-type 8325-4 and Newman and the corresponding ebpS mutants showed that EbpS migrated with an apparent molecular weight of 83 kDa. The protein was found exclusively in cytoplasmic membrane fractions purified from protoplasts or lysed cells, in contrast to the clumping factor ClfA, which was cell-wall-associated. EbpS was predicted to have three hydrophobic domains H1 (205-224), H2 (265-280) and H3 (315-342). A series of hybrid proteins was formed between EbpS at the N-terminus and either alkaline phosphatase or b-galactosidase at the C-terminus (EbpS-PhoA, EbpS-LacZ). PhoA and LacZ were fused to EbpS between hydrophobic domains H1-H2, H2-H3 and distal to H3. Expression of enzymatic activity in E.coli showed that EbpS is an integral membrane protein with two membrane spanning domains H1 and H3. N-terminal residues 1-205 and C-terminal residues 343-486 were predicted to be exposed on the outer face of the cytoplasmic membrane. The ligand-binding domain of EbpS is known from previous studies to be present in the N-terminus between residues 14-34 and probing whole cells with anti-EbpS1-267 antibodies indicated that this region is exposed on the surface of intact cells. This was also confirmed by the observation that wild-type S.aureus Newman cells bound labelled tropoelastin whereas the ebpS mutant bound 72% less. In contrast, the C-terminus, which carries a putative LysM peptidoglycan binding domain, is not exposed on the surface of intact cells and presumably remains buried within the peptidoglycan.
J. Biol. Chem, 10.1074/jbc.M107621200
Submitted on August 9, 2001
Revised on October 25, 2001
Accepted on October 29, 2001
The elastin binding protein of staphylococcus aureus (EbpS) is expressed at the cell surface as an integral membrane protein and not as a cell wall-associated protein
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