Characterization of the collagen-binding S-layer protein CbsA of Lactobacillus crispatus.
The cbsA gene of Lactobacillus crispatus strain JCM 5810, encoding a protein that mediates adhesiveness to collagens, was characterized and expressed in Escherichia coli. The cbsA open reading frame encoded a signal sequence of 30 amino acids and a mature polypeptide of 410 amino acids with typical features of a bacterial S-layer protein. The cbsA gene product was expressed as a His tag fusion protein, purified by affinity chromatography, and shown to bind solubilized as well as immobilized type I and IV collagens. Three other Lactobacillus S-layer proteins, SlpA, CbsB, and SlpnB, bound collagens only weakly, and sequence comparisons of CbsA with these S-layer proteins were used to select sites in cbsA where deletions and mutations were introduced. In addition, hybrid S-layer proteins that contained the N or the C terminus from CbsA, SlpA, or SlpnB as well as N- and C-terminally truncated peptides from CbsA were constructed by gene fusion. Analysis of these molecules revealed the major collagen-binding region within the N-terminal 287 residues and a weaker type I collagen-binding region in the C terminus of the CbsA molecule. The mutated or hybrid CbsA molecules and peptides that failed to polymerize into a periodic S-layer did not bind collagens, suggesting that the crystal structure with a regular array is optimal for expression of collagen binding by CbsA. Strain JCM 5810 was found to contain another S-layer gene termed cbsB that was 44% identical in sequence to cbsA. RNA analysis showed that cbsA, but not cbsB, was transcribed under laboratory conditions. S-layer-protein-expressing cells of strain JCM 5810 adhered to collagen-containing regions in the chicken colon, suggesting that CbsA-mediated collagen binding represents a true tissue adherence property of L. crispatus.
Sillanpää
J
J
Division of General Microbiology, Department of Biosciences, FIN-00014 University of Helsinki, Finland.
Martínez
B
B
Antikainen
J
J
Toba
T
T
Kalkkinen
N
N
Tankka
S
S
Lounatmaa
K
K
Keränen
J
J
Höök
M
M
Westerlund-Wikström
B
B
Pouwels
P H
PH
Korhonen
T K
TK
D000595:Amino Acid Sequence; D000818:Animals; D019677:Artificial Gene Fusion; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002645:Chickens; D003001:Cloning, Molecular; D003094:Collagen / Q000378:metabolism*; D005798:Genes, Bacterial; D007778:Lactobacillus / Q000737:chemistry / Q000235:genetics*; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D017354:Point Mutation
D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; C055764:S-layer proteins; D003094:Collagen