CLONING, EXPRESSION, AND ISOLATION OF THE MANNITOL TRANSPORT PROTEIN FROM THE THERMOPHILIC BACTERIUM BACILLUS-STEAROTHERMOPHILUS

A mannitol phosphotransferase system (PTS) was identified in Bacillus stearothermophilus by in vitro complementation with Escherichia coli E I, HPr, and IIA(Mtl). Degenerate primers based on regions of high amin o acid similarity in the E. coli and Staphylococcus carnosus EII(Mtl) were used to develop a digoxigenin-labeled probe by PCR, Using this pr obe, ale isolated three overlapping DNA fragments totaling 7.2 kb whic h contain the genes mtlA, mtlR, mtlF, and mtlD, encoding the mannitol IICB, a regulator, IIA, and a mannitol-1-phosphate dehydrogenase, resp ectively, The mtlA gene consists of 1,413 bp coding for a 471-amino-ac id protein with a calculated mass of 50.1 kDa, The amino acid sequence shows high similarity with the sequence of IICBMtl of S. carnosus and the IICB part of the IICBA(Mtl)s of E. coli and B. subtilis. The enzy me could be functionally expressed in E. coil by placing it behind the strong tac promoter, The rate of thermal inactivation at 60 degrees C of B. stearothermophilus IICBMtl expressed in E. coli was two times l ower than that of E. coli IICBMtl, IICBMtl in B. stearothermophilus is maximally active at 85 degrees C and thus very thermostable. The enzy me was purified on Ni-nitrilotriacetic acid resin to greater than 95% purity after six histidines were fused to the C-terminal part of the t ransporter.