PHYSICAL AND FUNCTIONAL S-LAYER RECONSTITUTION IN AEROMONAS-SALMONICIDA

The various functions attributed to the S-layer of Aeromonas salmonici da have been previously identified by their conspicuous absence in S-l ayer-defective mutants. As a different approach to establish the multi functional nature of this S-layer, we established methods for reconsti tution of the S-layer of A. salmonicida. Then we investigated the func tional competence of the reconstituted S-layer. S-layers were reconsti tuted in different systems: on inert membranes or immobilized lipopoly saccharide (LPS) from purified S-layer protein (A-protein) or on viabl e cells from either A-protein or preassembled S-layer sheets, In the a bsence of divalent cations and LPS, purified A-protein in solution spo ntaneously assembled into tetrameric oligomers and, upon concentration by ultrafiltration, into macroscopic, semicrystalline sheets formed b y oligomers loosely organized in a tetragonal arrangement, In the pres ence of Ca2+, purified A-protein assembled into normal tetragonal arra ys of interlocked subunits, A-protein bound with high affinity (K-d, 1 .55 x 10(-7) M) and specificity to high-molecular-weight LPS from A, s almonicida but not to the LPSs of several other bacterial species. In vivo, A-protein could be reconstituted only on A, salmonicida cells wh ich contained LPS, and Ca2+ affected both a regular tetragonal organiz ation 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-layer-secreting mutant) to an S-layer-negati ve mutant occurred consistently and efficiently when the two mutant st rains were cocultured on calcium-replete solid media. Reattached A-pro tein (exposed on the surface of S-layer-negative mutants) was able to bind porphyrins and an S-layer specific phage hut largely lacked regul ar organization, as judged by its inability to bind immunoglobulins. R eattached S-layer sheets were regularly organized and imparted the pro perties of porphyrin binding, hydrophobicity, autoaggregation, adheren ce tb and invasion of fish macrophages and epithelial cells, and resis tance to macrophage cytotoxicity. However, cells with reconstituted S- layers were still sensitive to complement and insensitive to the antib iotics streptonigrin and chloramphenicol, indicating incomplete functi onal reconstitution.