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

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

Smit, E.
Oling, F.
Demel, R.
Martinez, B.
Pouwels, P.H.
Centraal Instituut voor Voedingsonderzoek TNO

2001

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 3.4.21.4

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

http://resolver.tudelft.nl/uuid:84d8c382-7e26-4867-a041-361d09f3a803

https://doi.org/10.1006/jmbi.2000.4258

41810

0022-2836

Journal of Molecular Biology, 305 (2), 245-257

article