Molecular cloning of a 32-kilodalton lipoprotein component of a novel iron-regulated Staphylococcus epidermidis ABC transporter

Abstract

Our previous studies identified two iron-regulated cytoplasmic membrane proteins of 32 and 36 kDa expressed by both Staphylococcus epidermidis and Staphylococcus aureus. In this study we show by Triton X-114 phase partitioning and tritiated palmitic acid labelling that these proteins are lipoproteins which are anchored into the cytoplasmic membrane by their lipid-modified N termini. In common with those of some other gram-positive bacteria, these highly immunogenic lipoproteins were released from the bacterial cell into the culture supernatants, with release being promoted by growth of the bacteria under iron-restricted conditions. Immunoelectron microscopy with a monospecific rabbit antiserum 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 exposure of the lipoprotein on the bacterial surface was minimal. A monoclonal antibody raised to the 32-kDa lipoprotein of S. aureus was used in immunoblotting studies to investigate the conservation of this antigen among a variety of staphylococci. The monoclonal antibody reacted with polypeptides of 32 kDa in S. epidermidis and S. aureus and of 40 kDa in Staphylococcus hominis. No reactivity was detected with Staphylococcus lugdunensis, Staphylococcus cohni, or Staphylococcus haemolyticus. The gene encoding the 32-kDa lipoprotein from S. epidermidis has 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 transporter. 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 parasanguis and ScaA of Streptococcus gordonii, and with lipoproteins of a recently described family of ABC transporters with proven or putative metal ion transport functions. Although the solute specificity of this novel transporter has not yet been determined, we speculate that it may be involved in either siderophore- or transferrin-mediated iron uptake in S. epidermidis.

FIG. 1

SDS-PAGE (A) and immunoblots (B) of Triton X-114 extracts prepared from S. aureus BB and S. epidermidis 901 grown under iron-restricted growth conditions. Lanes: 1, molecular mass markers; 2 and 3, whole-cell polypeptide profiles; 4 and 5, Triton X-114 extracts of intact cells; 6 and 7, Triton X-114 extracts of lysostaphin-digested cells; lanes 8 and 9, Triton X-114 extracts of filtered culture supernatants; 2, 4, 6, and 8, S. aureus BB; 3, 5, 7, and 9, S. epidermidis 901. The gel in panel A was silver stained, and immunoblots were reacted with a pool of monospecific rabbit antisera to the 32- and 36-kDa iron-regulated cytoplasmic membrane proteins of S. epidermidis (36).

FIG. 2

Autoradiograph showing [³H]palmitate labelling of Triton X-114-extractable lipoproteins of S. aureus BB and S. epidermidis 901. Lanes: 1 and 2, whole-cell [³H]palmitate-labelled profiles; 3 and 4, Triton X-114 extracts of [³H]palmitate-labelled lysostaphin-digested cells; 5 and 6, Triton X-114 extracts of [³H]palmitate-labelled filtered culture supernatants; 1, 3, and 5, S. aureus BB; 2, 4, and 6, S. epidermidis 901.

FIG. 3

Electron micrographs of thin sections of S. epidermidis 901 cells reacted with monospecific antiserum to the 32-kDa S. epidermidis lipoprotein. (A) Sections reacted with monospecific rabbit anti-32-kDa lipoprotein antibody and protein A-gold conjugate; (B) sections reacted with nonimmune rabbit serum and protein A-gold conjugate. Bars, 1 μm.

FIG. 4

Immunoblots of recombinant E. coli SOLR pW32 reacted with anti-S. aureus BB wall antiserum (A) or monospecific anti-32-kDa S. epidermidis lipoprotein antiserum (B). Lanes: 1, BB wall extract; 2 and 4, E. coli SOLRpW32 whole-cell extract; 3 and 5, E. coli SOLR whole-cell extract. The position of the 32-kDa lipoprotein antigen is indicated by the arrowhead.

FIG. 5

Organization of the S. epidermidis ABC transporter operon.

FIG. 6

Northern blot showing transcript analysis of the S. epidermidis ABC transporter operon and differential transcription under iron-rich and iron-restricted growth conditions. Lanes: 1, molecular size markers; 2, RNA extracted from iron-restricted culture; 3, RNA extracted from iron-rich culture. The arrowhead indicates the position of the single 2.7-kb RNA transcript detected in S. epidermidis 901.