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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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Author: Smit, E. · Oling, F. · Demel, R. · Martinez, B. · Pouwels, P.H.
Institution: Centraal Instituut voor Voedingsonderzoek TNO
Source:Journal of Molecular Biology, 2, 305, 245-257
Identifier: 41810
doi: doi:10.1006/jmbi.2000.4258
Keywords: Biology · Amino Acid Sequence · Bacterial Proteins · Cell Wall · Crystallization · Electrophoresis, Polyacrylamide Gel · Escherichia coli · Lactobacillus acidophilus · Membrane Glycoproteins · Membrane Proteins · Microscopy, Electron · Models, Molecular · Molecular Sequence Data · Peptide Fragments · Phosphatidylserines · Protein Binding · Protein Structure, Quaternary · Protein Structure, Secondary · Protein Structure, Tertiary · Recombinant Fusion Proteins · Sequence Alignment · Sequence Analysis, Protein · Solutions · Trypsin · Escherichia coli · Lactobacillus acidophilus · Lactobacillus casei · Lactobacillus crispatus · Lactobacillus helveticus


Lactobacillus acidophilus, like many other bacteria, harbors a surface layer consisting of a protein (SA-protein) of 43 kDa. SA-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 SA-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 SA-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 SA-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 SA-protein. A calculated SA-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. © 2001 Academic Press. Chemicals/CAS: 1,2-dioleoylphosphatidylserine, 70614-14-1; Bacterial Proteins; Membrane Glycoproteins; Membrane Proteins; Peptide Fragments; Phosphatidylserines; Recombinant Fusion Proteins; Solutions; surface array protein, bacteria; Trypsin, EC