The S-layer protein of Lactobacillus acidophilus ATCC 4356: Identificationand characterisation of domains responsible for S-protein assembly and cell wall binding

Lactobacillus acidophilus, like many other bacteria, harbors a surface laye r consisting of a protein (S-A-protein) of 43 kDa. S-A-protein could be rea dily extracted and crystallized in vitro into large crystalline patches on lipid monolayers with a net negative charge but not on lipids with a net ne utral charge. Reconstruction of the S-layer from crystals grown on dioleoyl phosphatidylserine indicated an oblique lattice with unit cell dimensions ( a = 118 Angstrom; b = 53 Angstrom, and gamma = 102 degrees) resembling thos e determined for the S-layer of Lactobacillus helveticus ATCC 12046. Sequen ce comparison of S-A-protein with S-proteins from L. helveticus, Lactobacil lus crispatus and the S-proteins encoded by the silent S-protein genes from L. acidophilus and L, crispatus suggested the presence of two domains, one comprising the N-terminal two-thirds (SAN), and another made up of the C-t erminal one-third (SAC) of S-A-protein. The sequence of the N-terminal doma ins is variable, while that of the C-terminal domain is highly conserved in the S-proteins of these organisms and contains a tandem repeat. Proteolyti c digestion of S-A-protein showed that SAN was protease-resistant, suggesti ng a compact structure. SAC was rapidly degraded by proteases and therefore probably has a more accessible structure. DNA sequences encoding SAN or Gr een Fluorescent Protein fused to SAC (GFP-SAC) were efficiently expressed i n Escherichia coli. Purified SAN could crystallize into mono and multi-laye red crystals with the same lattice parameters as those found for authentic S-A-protein. A calculated S-A-protein minus SAN density-difference map reve aled the probable location, in projection, of the SAC domain, which is miss ing from the truncated SAN peptide. The GFP-SAC fusion product was shown to bind to the surface of L. acidophilus, L. helveticus and L. crispatus cell s from which the S-layer had been removed, but not to non-stripped cells or to Lactobacillus casei. (C) 2001 Academic Press.