MOLECULAR-CLONING OF A 32-KILODALTON LIPOPROTEIN COMPONENT OF A NOVELIRON-REGULATED STAPHYLOCOCCUS-EPIDERMIDIS ABC TRANSPORTER

Our previous studies identified two iron-regulated cytoplasmic membran e proteins of 32 and 36 kDa expressed by both Staphylococcus epidermid is and Staphylococcus aureus, In this study we show by Triton X-114 ph ase partitioning and tritiated palmitic acid labelling that these prot eins are lipoproteins which are anchored into the cytoplasmic membrane by their lipid-modified N termini, In common with those of some other grampositive bacteria, these highly immunogenic lipoproteins were rel eased from the bacterial cell into the culture supernatants, with rele ase being promoted by growth of the bacteria under iron-restricted con ditions. Immunoelectron microscopy with a monospecific rabbit antiseru m to the 32-kDa S. epidermidis lipoprotein showed that the majority of the antigen was distributed throughout the staphylococcal cell wall. Only minor quantities were detected in the cytoplasmic membrane, and e xposure of the lipoprotein on the bacterial surface was minimal. A mon oclonal antibody raised to the 32-kDa lipoprotein of S, aureus was use d in immunoblotting studies to investigate the conservation of this an tigen among a variety of staphylococci, The monoclonal antibody reacte d with polypeptides of 32 kDa in S. epidermidis and S, aureus and of 4 0 kDa in Staphylococcus hominis, No reactivity was detected with Staph ylococcus lugdunensis, Staphylococcus cohni, or Staphylococcus haemoly ticus. The gene encoding the 32-kDa lipoprotein from S. epidermidis ha s been isolated from a Lambda Zap II genomic DNA library and found to be a component of an iron-regulated operon encoding a novel ABC-type t ransporter. The operon contains three genes, designated sitA, -B, and -C, encoding an ATPase, a cytoplasmic membrane protein, and the 32-kDa lipoprotein, respectively, SitC shows significant homology both with a number of bacterial adhesins, including FimA of Streptococcus parasa nguis and ScaA of Streptococcus gordonii, and with lipoproteins of a r ecently described family of ABC transporters with proven or putative m etal ion transport functions. Although the solute specificity of this novel transporter has not yet been determined, we speculate that it ma y be involved in either siderophore- or transferrin-mediated iron upta ke in S. epidermidis.