The S-layer Protein of Lactobacillus acidophilus ATCC 4356: Identification and Characterisation of Domains Responsible for S-protein Assembly and Cell Wall Binding

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Lactobacillus acidophilus, like many other bacteria, harbors a surface layer consisting of a protein (S A-protein) of 43 kDa. S A-protein could be readily extracted and crystallized in vitro into large crystalline patches on lipid monolayers with a net negative charge but not on lipids with a net neutral charge. Reconstruction of the S-layer from crystals grown on dioleoylphosphatidylserine indicated an oblique lattice with unit cell dimensions ( a=118 Å; b=53 Å, and γ=102 °) resembling those determined for the S-layer of Lactobacillus helveticus ATCC 12046. Sequence comparison of S A-protein with S-proteins from L. helveticus , Lactobacillus 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-terminal one-third (SAC) of S A-protein. The sequence of the N-terminal domains is variable, while that of the C-terminal domain is highly conserved in the S-proteins of these organisms and contains a tandem repeat. Proteolytic digestion of S A-protein showed that SAN was protease-resistant, suggesting a compact structure. SAC was rapidly degraded by proteases and therefore probably has a more accessible structure. DNA sequences encoding SAN or Green Fluorescent Protein fused to SAC (GFP-SAC) were efficiently expressed in Escherichia coli. Purified SAN could crystallize into mono and multi-layered crystals with the same lattice parameters as those found for authentic S A-protein. A calculated S A-protein minus SAN density-difference map revealed the probable location, in projection, of the SAC domain, which is missing 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 cells from which the S-layer had been removed, but not to non-stripped cells or to Lactobacillus casei.