A mannitol phosphotransferase system (PTS) was identified in Bacillus stearothermophilus by in vitro complementation with Escherichia coli EI, HPr, and IIA Mtl . Degenerate primers based on regions of high amino acid similarity in the E. coli and Staphylococcus carnosus EII Mtl were used to develop a digoxigenin-labeled probe by PCR. Using this probe, we isolated three overlapping DNA fragments totaling 7.2 kb which contain the genes mtlA, mtlR, mtlF, and mtlD, encoding the mannitol IICB, a regulator, IIA, and a mannitol-1-phosphate dehydrogenase, respectively. The mtlA gene consists of 1,413 bp coding for a 471-amino-acid protein with a calculated mass of 50.1 kDa. The amino acid sequence shows high similarity with the sequence of IICB Mtl of S. carnosus and the IICB part of the IICBA Mtl s of E. coli and B. subtilis. The enzyme could be functionally expressed in E. coli by placing it behind the strong tac promoter. The rate of thermal inactivation at 60؇C of B. stearothermophilus IICB Mtl expressed in E. coli was two times lower than that of E. coli IICB Mtl . IICB Mtl in B. stearothermophilus is maximally active at 85؇C and thus very thermostable. The enzyme was purified on Ni-nitrilotriacetic acid resin to greater than 95% purity after six histidines were fused to the C-terminal part of the transporter.Many bacteria transport D-mannitol and other carbohydrates via a phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS). Two general cytoplasmic proteins, EI and HPr, are responsible for the transfer of the phosphoryl group from PEP to different sugar-specific PTS proteins (15,18,22 In general, the genes of proteins involved in the uptake and phosphorylation of a specific sugar are located in a single operon. To date, the mtlA genes of E. coli (13), S. carnosus (7), and Bacillus subtilis (1) and the cryptic mtlA gene of E. coli (29), all encoding homologous mannitol transporters, have been cloned. Our attempts to crystallize the E. coli protein have been unsuccessful, possibly because of limited stability. Since membrane proteins in thermophilic bacteria were found to be more stable than their mesophilic counterparts, a search for a mannitol PTS was done in these organisms. A mannitol PTS had been found in the obligate anaerobic thermophile Clostridium thermocellum (21). This paper reports the identification of a mannitol PTS in the aerobic thermophile Bacillus stearothermophilus and presents the cloning of the mannitol operon, sequencing of the mtlA gene, functional expression of B. stearothermophilus IICB Mtl in E. coli, and purification and partial characterization of the mannitol transporter.
MATERIALS AND METHODSStrains and plasmids used in this study are listed in Table 1. Restriction enzymes, Taq DNA polymerase, Klenow enzyme, T4 DNA ligase, digoxigenin (DIG) labeling mix, and DIG nucleic acid detection kit were purchased from Boehringer Mannheim. The DNA sequencing kit was obtained from Pharmacia. Sodium deoxycholate (DOC), n-decyl--D-maltopyranoside (d-maltopyranoside), PEP, and mannitol...