Surface display of heterologous proteins on bacterial cells is an important objective for many applications in microbiology and molecular biology. In gram-negative bacteria, different types of surface proteins have been exploited for this purpose. These include the following: (i) outer membrane proteins LamB (4), PhoE (1), and OmpA (35); (ii) lipoproteins TraT (19) and the peptidoglycan-associated lipoprotein (10); (iii) the fimbria protein fimbrillin (20, 21); and (iv) the flagellar protein flagellin (32). The different surface display systems have been used extensively to express heterologous antigenic determinants on the bacterial cells for the purpose of developing live bacterial vaccine vehicles. Enteric bacteria, such as Escherichia coli and Salmonella typhimurium, have been studied in this context, and the cell surface presentation has been considered advantageous to induce an antibody response to the exposed antigens with live cells for immunization (11,28,41).The expression of functional single-chain antibodies on the surface of E. coli cells (7, 10) has opened the discussion of whether this strategy would be an alternative to the rapidly developing phage technology for the selection of peptides or recombinant antibody fragments from large libraries (31). An interesting application for bacterial cells exposing heterologous proteins might be the development of whole-cell adsorbents by the surface expression of suitable protein ligands. Immobilized recombinant bacteria cannot be considered for separation processes in pharmaceutical industries but might because of their low cost constitute competitive adsorbents for certain separations (3, 11). The use of enzyme-coated bacteria as novel biocatalysts has also been envisioned, because enzymes with retained activity have been surface displayed on E. coli cells (8, 9).Investigations with gram-positive bacteria for cell surface display of heterologous proteins have recently been initiated. Expression systems for the mouth commensal bacterium Streptococcus gordinii (37) and the nonpathogenic bacterium Staphylococcus xylosus (18) have been developed on the basis of the fibrillar M6 protein from Streptococcus pyogenes and protein A from Staphylococcus aureus, respectively. These cell surface display systems have been used for the surface expression of several antigenic determinants, and immunization with live recombinant bacteria induces both local and systemic antibody responses to the hybrid receptors (33, 37), suggesting that gram-positive bacteria might constitute potential live bacterial vaccine delivery systems. The surface receptors of gram-positive bacteria seem to be more permissive for the insertion of extended sequences of foreign proteins (6) than the different gram-negative systems, in which both translocation through the cytoplasmic membrane and correct integration into the outer membrane are required for proper surface exposure of the heterologous polypeptide. Considering the development of whole-cell adsorbents or bacterial biocatalysts by surface ...

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The twin arginine translocation (Tat) pathway of bacteria and plant chloroplasts mediates translocation of essentially folded proteins across the cytoplasmic membrane. The detailed understanding of the mechanism of protein targeting to the Tat pathway has been hampered by the lack of screening or selection systems suitable for genetic analysis. We report here the development of a highly quantitative protein reporter for genetic analysis of Tat-specific export. Specifically, export via the Tat pathway rescues green fluorescent protein (GFP) fused to an SsrA peptide from degradation by the cytoplasmic proteolytic ClpXP machinery. As a result, cellular fluorescence is determined by the amount of GFP in the periplasmic space. We used the GFP-SsrA reporter to isolate gain-of-function mutants of a Tatspecific leader peptide and for the genetic analysis of the "invariant" signature RR dipeptide motif. Flow cytometric screening of trimethylamine N-oxide reductase (TorA) leader peptide libraries resulted in isolation of six gain-of function mutants that conferred significantly higher steady-state levels of export relative to the wildtype TorA leader. All the gain-of-function mutations occurred within or near the (S/T)RRXFLK consensus motif, highlighting the significance of this region in interactions with the Tat export machinery. Randomization of the consensus RR dipeptide in the TorA leader revealed that a basic side chain (R/K) is required at the first position whereas the second position can also accept Gln and Asn in addition to basic amino acids. This result indicates that twin arginine translocation does not require the presence of an arginine dipeptide within the conserved sequence motif.

Two different host-vector expression systems designed for cell surface display of chimeric receptors on Staphylococcus xylosus and Staphylococcus carnosus have been evaluated for surface display of a mouse immunoglobulin G1() [IgG1()] anti-human IgE single-chain Fv (scFv) antibody fragment. To achieve surface anchoring of the chimeric receptors containing the scFv, the cell surface attachment regions from Staphylococcus aureus protein A were used in both expression systems. The different chimeric receptors could be recovered from cell wall extracts of both S. xylosus and S. carnosus, and surface localization was demonstrated by taking advantage of a serum albumin-binding reporter region present within the two types of receptors. In addition, the two different recombinant staphylococci carrying hybrid receptors containing the scFv were demonstrated to react with the antigen, which was human IgE, in whole-cell enzyme-linked immunosorbent assays. This is the first report of an antibody fragment expressed in a functional form anchored to the surface of gram-positive bacteria. The potential use of recombinant gram-positive bacteria as whole-cell diagnostic devices or alternatives to filamentous phages for surface display of scFv libraries is discussed.

Two different host-vector expression systems, designed for cell surface display of heterologous receptors on Staphylococcus xylosus and Staphylococcus carnosus, respectively, were compared for the surface display of four variants of a 101 amino acid region derived from the G glycoprotein of human respiratory syncytial virus (RSV). Surface localization of the different chimeric receptors was evaluated by a colorimetric assay and by fluorescence-activated cell sorting. It was concluded that the S. carnosus system was better both in the ability to translocate inefficiently secreted peptides and in the number of exposed hybrid receptors. The potential use of the described staphylococci as live bacterial vaccine vehicles or alternatives to filamentous phages for surface display of protein libraries is discussed.

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.

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The heterologous surface expression of the cholera toxin B subunit (CTB) from Vibrio cholerae in two staphylococcal species, Staphylococcus xylosus and Staphylococcus carnosus, has been investigated. The gene encoding native CTB (103 amino acids) was introduced into gene constructs encoding chimeric receptors designed to be translocated and anchored on the outer cell surface of the staphylococci. Since functionality of CTB is correlated with its ability to form pentamers and the capacity of the pentameric CTB to bind the GM1 ganglioside, both the surface accessibility and the functionality of the surface-displayed CTB receptors were evaluated. It could be concluded that the chimeric receptors were targeted to the cell wall of the staphylococci, since they could be released by lysostaphin treatment and, after subsequent affinity purification, identified as full-length products by immunoblotting. Surface accessibility of the chimeric receptors was demonstrated by a colorimetric assay and by immunofluorescence staining with a CTB-reactive rabbit antiserum. Pentamerization was investigated by using a monoclonal antibody described to be specific for pentameric CTB, and the functionality of the receptors was tested in a binding assay with digoxigenin-labelled GM1. It was concluded that functional CTB was present on both types of staphylococci, and for S. carnosus, the reactivity to the pentamer-specific monoclonal antibody and in the GM1 binding assay was indeed significant. The implications of the results for the design of live bacterial vaccine delivery systems intended for administration by the mucosal route are discussed.