The various functions attributed to the S-layer of Aeromonas salmonicida have been previously identified by their conspicuous absence in S-layer-defective mutants. As a different approach to establish the multifunctional nature of this S-layer, we established methods for reconstitution of the S-layer of A. salmonicida. Then we investigated the functional competence of the reconstituted S-layer. S-layers were reconstituted in different systems: on inert membranes or immobilized lipopolysaccharide (LPS) from purified S-layer protein (Aprotein) or on viable cells from either A-protein or preassembled S-layer sheets. In the absence of divalent cations and LPS, purified A-protein in solution spontaneously assembled into tetrameric oligomers and, upon concentration by ultrafiltration, into macroscopic, semicrystalline sheets formed by oligomers loosely organized in a tetragonal arrangement. In the presence of Ca
2؉, purified A-protein assembled into normal tetragonal arrays of interlocked subunits. A-protein bound with high affinity (K d , 1.55 ؋ 10 ؊7 M) and specificity to high-molecular-weight LPS from A. salmonicida but not to the LPSs of several other bacterial species. In vivo, A-protein could be reconstituted only on A. salmonicida cells which contained LPS, and Ca
2؉affected both a regular tetragonal organization of the reattached A-protein and an enhanced reattachment of the A-protein to the cell surface. The reconstitution of preformed S-layer sheets (produced by an S-layersecreting mutant) to an S-layer-negative mutant occurred consistently and efficiently when the two mutant strains were cocultured on calcium-replete solid media. Reattached A-protein (exposed on the surface of S-layer-negative mutants) was able to bind porphyrins and an S-layer-specific phage but largely lacked regular organization, as judged by its inability to bind immunoglobulins. Reattached S-layer sheets were regularly organized and imparted the properties of porphyrin binding, hydrophobicity, autoaggregation, adherence to and invasion of fish macrophages and epithelial cells, and resistance to macrophage cytotoxicity. However, cells with reconstituted S-layers were still sensitive to complement and insensitive to the antibiotics streptonigrin and chloramphenicol, indicating incomplete functional reconstitution.S-layers cover the cell surfaces of a wide range of bacteria and have been hypothesized to be involved in protection, molecular sieving, ion trapping, cell adhesion, surface recognition, and morphogenesis (35). However, only in a few instances have any functions of eubacterial S-layers been rigorously demonstrated, e.g., the protective role of S-layers against Bdellovibrio predation (34) or against nonspecific host defenses (5, 36) and the cell adhesion properties of the S-layer of the fish pathogenic bacterium Aeromonas salmonicida (16,17,43).The main cell surface antigenic determinants of A. salmonicida are a smooth layer of lipopolysaccharide (LPS), possessing O polysaccharides of homogeneous lengths, and an S-layer, composed...