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.A rapidly emerging research field involves bacterial surface expression of metal-binding peptides (16,17,36,37,40) for potential generation of novel biosorbents for removal of toxic metals from wastewater. Bacterial sequestration of toxic metals has previously been investigated using nonengineered bacteria (23), but recombinant DNA technology offers the possibility of improving the metal binding capacity of the bacteria. Such engineered bacteria have in fact been evaluated for removal of Cd 2ϩ from actual factory wastewater (2). Periplasmic expression of a Neurospora crassa metallothionein in Escherichia coli generated cells that were superior to bacteria with cytoplasmic metallothionein localization in terms of metal ion adsorption (29). Surface expression on E. coli cells of yeast or mammalian metallothioneins resulted in recombinant bacteria with increased ability to bind Cd 2ϩ ions (37). Surface expression of hexahistidyl peptides by genetic insertion into the outer membrane protein LamB generated recombinant E. coli cells with improved metalloadsorption capacity (36). Histidine clusters have also been expressed in fimbrial proteins (33), generating bacteria with improved metal binding. Other metal-binding peptides have been expressed in the periplasm (30) or at the cell surface (16,17,33) of E. coli, yielding bacteria with enhanced capacity to bind to divalent metal ions.

Generation of Metal-Binding Staphylococci through Surface Display of Combinatorially Engineered Cellulose-Binding Domains

“…4, bar 2) have gained improved metal-binding capacity. Similar observations have, however, been demonstrated previously (43).…”

“…Using this strategy, both Escherichia coli (46) and Staphylococcus carnosus (43) strains with increased ability to bind Ni 2ϩ and Cd 2ϩ ions have been generated. The introduction of combinatorial protein engineering has also made it possible to select peptides, from large peptide libraries with increased selectivity for certain metals (4,30,37,44), and such peptides might become interesting for surface display applications (44).…”

Bioadsorption Strategies with Yeast Molecular Display Technology

“…3 has been investigated as an alternative strategy to bioaccumulation. To achieve the bioremediation of metalpolluted water, various metal-binding proteins or peptides have been displayed on the microbial cell surfaces of E. coli, Ralstonia eutropha, S. cerevisiae, Staphylococcus xylosus, and Staphylococcus carnosus, such as metallothioneins Kuroda and Ueda, 2006;Valls et al, 2000 , mercury-responsive metalloregulatory proteins Bae et al, 2003, hexa-histidine Kuroda et al, 2001Samuelson et al, 2000 , histidine-rich peptides GHHPHG; HP , and cysteine-rich peptides GCGCPCGCG; CP Kotrba et al, 1999 . The genetic strategy for metal ion bioadsorption in the case of a yeast cell is shown in Fig.…”

Engineering Bacteria for Bioremediation

“…Nevertheless, a Gram-positive surface display system also possesses its own merits, compared to Gram-negative bacteria (Malik et. al., 1998;Samuelson et. al., 2000): (a) translocation through only one membrane is required; and (b) Grampositive bacteria have been shown to be more rigid and, therefore, less sensitive to shear forces (Kelemen & Sharpe, 1979) due to the thick cell wall surrounding the cells, which potentially make them more suitable for field applications, such as bioadsorption.…”