A conserved motif in S-layer proteins is involved in peptidoglycan binding in Thermus thermophilus.
There is experimental evidence to suggest that the 100-kDa S-layer protein from Thermus thermophilus HB8 binds to the peptidoglycan cell wall. This property could be related to the presence of a region (SLH) of homology with other S-layer proteins and extracellular enzymes (A. Lupas, H. Engelhardt, J. Peters, U. Santarius, S. Volker, and W. Baumeister, J. Bacteriol. 176:1224-1233, 1994). By using specific monoclonal antibodies, we show that similar regions are present in different members of the Deinococcus-Thermus phylogenetic group. To analyze the role that the SLH domain plays in vivo and in vitro in T. thermophilus, we have obtained a mutant form (slpA.X) of the S-layer gene (slpA) in which the SLH domain was deleted. The slpA.X gene was inserted into the chromosome of the thermophile by gene replacement, resulting in a mutant which expressed a major membrane protein with the size expected from the construction (90 kDa). This protein was identified as the product of slpA.X by its differential reaction with monoclonal antibodies. Mutants expressing the SlpA.X protein grow as groups of cells, surrounded by a common external envelope of trigonal symmetry that contains the SlpA.X protein as a main component, thus showing the inability of the SLH-defective protein to attach to the underlying material in vivo. In addition, averaged images of SlpA.X-rich fractions showed a regular arrangement, identical to that built up by the wild-type (SlpA) protein in the absence of peptidoglycan. Finally, we demonstrate by Western blotting (immunoblotting) the direct role of the SLH domain in the binding of the S-layer of T. thermophilus HB8 to the peptidoglycan layer.
Olabarría
G
G
Centro de Biología Molecular, Severo Ochoa, Universidad Autónoma de Madrid, Spain.
Carrascosa
J L
JL
de Pedro
M A
MA
Berenguer
J
J
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002473:Cell Wall / Q000378:metabolism / Q000648:ultrastructure; D017124:Conserved Sequence; D000939:Epitopes / Q000032:analysis; D006088:Gram-Negative Aerobic Bacteria / Q000235:genetics*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010457:Peptidoglycan / Q000378:metabolism*; D010641:Phenotype; D010802:Phylogeny; D010957:Plasmids; D011485:Protein Binding; D015183:Restriction Mapping; D013045:Species Specificity; D013824:Thermus / Q000235:genetics; D016963:Thermus thermophilus / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins; D000939:Epitopes; D010457:Peptidoglycan