Staphylococcal surface display of metal-binding polyhistidyl peptides

Abstract

Recombinant Staphylococcus xylosus and Staphylococcus carnosus strains were generated with surface-exposed chimeric proteins containing polyhistidyl peptides designed for binding to divalent metal ions. Surface accessibility of the chimeric surface proteins was demonstrated and the chimeric surface proteins were found to be functional in terms of metal binding, since the recombinant staphylococcal cells were shown to have gained Ni(2+)- and Cd(2+)-binding capacity, suggesting that such bacteria could find use in bioremediation of heavy metals. This is, to our knowledge, the first time that recombinant, surface-exposed metal-binding peptides have been expressed on gram-positive bacteria. Potential environmental or biosensor applications for such recombinant staphylococci as biosorbents are discussed.

FIG. 1

Expression cassettes of the different expression vectors designed for surface displays on S. xylosus and S. carnosus shown with their encoded gene products anchored to the cell surface. The names of the constructed expression vectors are given below the expression cassettes, the molecular masses of the encoded surface proteins are indicated, and the abbreviated names of the recombinant staphylococci are shown to the right. Note that the propeptide (PP) from the S. hyicus lipase gene construct is not processed in S. carnosus but is processed in its homologous host, S. hyicus (32). This propeptide has been suggested to be essential for secretion of heterologous gene fusion products from S. carnosus (4) when using the lipase signal peptide for secretion. M′, the processed and covalently anchored form of the M sequence of SpA; p_SPA, promoter region from the SpA gene; p_Lip, S. hyicus promoter region designed for production in S. carnosus (6).

FIG. 2

Characterization of gene products by SDS-PAGE (10 to 20% polyacrylamide) analysis under reducing conditions. Coomassie brilliant blue was used for staining. The chimeric surface proteins were extracted from the cell wall of the recombinant staphylococci and subsequently subjected to single-step ABP-mediated HSA affinity chromatography. Lane 1, Sx:ABP, lane 2, Sx:H1ABP; lane 3, Sx:H2ABP; lane 4, Sc:ABP; lane 5, Sc:H1ABP; lane 6, Sc:H2ABP; lane M, marker proteins with molecular masses (in kilodaltons).

FIG. 3

Histogram representation of results from the whole-cell Ni²⁺-binding assay. Wild-type and recombinant S. xylosus or S. carnosus cells were incubated with nickel-chelated alkaline phosphatase conjugate. Upon addition of substrate, the color response was monitored in five separate samples from each construct. Hatched bars indicate the A₄₀₅ response for S. xylosus cells: wild type (bar 1), Sx:ABP (bar 2), Sx:H1ABP (bar 3), and Sx:H2ABP (bar 4). Open bars indicate the A₄₀₅ response for S. carnosus cells: wild type (bar 5), Sc:ABP (bar 6), Sc:H1ABP (bar 7), and Sc:H2ABP (bar 8). Error bars represent standard deviations.

FIG. 4

Histogram representation of results from the Cd²⁺ bioadsorption assay. Wild-type and recombinant S. xylosus or S. carnosus cells were incubated with radioactive ¹⁰⁹Cd, and residual radioactivity in the cell supernatants, monitored by liquid scintillation, corresponds to the amount of nonadsorbed Cd²⁺. Each supernatant was analyzed as five separate samples. Hatched bars indicate Cd²⁺ concentrations for S. xylosus cells: wild type (bar 1), Sx:ABP (bar 2), and Sx:H2ABP (bar 3). Open bars indicate Cd²⁺ concentrations for S. carnosus cells: wild type (bar 4), Sc:ABP (bar 5), and Sc:H2ABP (bar 6). Error bars represent standard deviations.