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 stearoth ermophilus ATCC 12980 and an S-layer-deficient variant each secreted t hree amylases, with identical molecular weights of 58,000, 122,000, an d 184,000, into the culture fluid, Only the high-molecular-weight amyl ase (hmwA) was also identified as cell associated, Extraction and reas sociation experiments showed that the hmwA had a high-level affinity t o the peptidoglycan-containing layer and to the S-layer surface, but t he 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 o bserved 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 obs erved 3 h after inoculation, which corresponded to early exponential g rowth, The steady decrease in cell-associated hmwA during continued gr owth 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 peptidogly can-containing layer and was identified as an N-terminal S-layer prote in fragment which did not result from proteolytic cleavage of the whol e S-layer protein but seems to be a truncated copy of the S-layer prot ein which is coexpressed with the hmwA under certain culture condition s. During growth on starch medium, the N-terminal S-layer protein frag ment 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 bi nding sites, Results obtained in the present study provide evidence th at the N-terminal part of the S-layer protein is responsible for the a nchoring of the subunits to the peptidoglycan containing layer, while the surface-located C-terminal half could function as a binding site f or the hmwA.