Evidence that an N-terminal S-layer protein fragment triggers the release of a cell-associated high-molecular-weight amylase in Bacillus stearothermophilus ATCC 12980.
During growth on starch medium, the S-layer-carrying Bacillus stearothermophilus ATCC 12980 and an S-layer-deficient variant each secreted three amylases, with identical molecular weights of 58,000, 122,000, and 184,000, into the culture fluid. Only the high-molecular-weight amylase (hmwA) was also identified as cell associated. Extraction and reassociation experiments showed that the hmwA had a high-level affinity to the peptidoglycan-containing layer and to the S-layer surface, but the interactions with the peptidoglycan-containing layer were stronger than those with the S-layer surface. For the S-layer-deficient variant, no changes in the amount of cell-associated and free hmwA could be observed during growth on starch medium, while for the S-layer-carrying strain, cell association of the hmwA strongly depended on the growth phase of the cells. The maximum amount of cell-associated hmwA was observed 3 h after inoculation, which corresponded to early exponential growth. The steady decrease in cell-associated hmwA during continued growth correlated with the appearance and the increasing intensity of a protein with an apparent molecular weight of 60,000 on sodium dodecyl sulfate gels. This protein had a high-level affinity to the peptidoglycan-containing layer and was identified as an N-terminal S-layer protein fragment which did not result from proteolytic cleavage of the whole S-layer protein but seems to be a truncated copy of the S-layer protein which is coexpressed with the hmwA under certain culture conditions. During growth on starch medium, the N-terminal S-layer protein fragment was integrated into the S-layer lattice, which led to the loss of its regular structure over a wide range and to the loss of amylase binding sites. Results obtained in the present study provide evidence that the N-terminal part of the S-layer protein is responsible for the anchoring of the subunits to the peptidoglycan-containing layer, while the surface-located C-terminal half could function as a binding site for the hmwA.
Egelseer
E M
EM
Zentrum für Ultrastrukturforschung and Ludwig Bolzmann-Institut für Molekulare Nanotechnologie, Universität für Bodenkultur, Vienna, Austria.
Schocher
I
I
Sleytr
U B
UB
Sára
M
M
D000681:Amylases / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001426:Bacterial Proteins; D002462:Cell Membrane / Q000648:ultrastructure; D002473:Cell Wall / Q000378:metabolism; D014644:Genetic Variation; D001411:Geobacillus stearothermophilus / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D008562:Membrane Glycoproteins; D016253:Microscopy, Immunoelectron; D008954:Models, Biological; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D006146:Guanidines; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; C055764:S-layer proteins; D000681:Amylases; D019791:Guanidine