J. Bacteriol., 10 1996, 5586-5591, Vol 178, No. 19
Copyright © 1996, American Society for Microbiology

Cloning, expression, and isolation of the mannitol transport protein from the thermophilic bacterium Bacillus stearothermophilus

SA Henstra, B Tolner, RH ten Hoeve Duurkens, WN Konings and GT Robillard
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.

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(Mt1) 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 mtl4 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 degrees C of B. stearothermophilus HCB(Mt1) 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 degrees 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.

This article has been cited by other articles:

• Jorge, C. D., Fonseca, L. L., Boos, W., Santos, H. (2008). Role of Periplasmic Trehalase in Uptake of Trehalose by the Thermophilic Bacterium Rhodothermus marinus. J. Bacteriol. 190: 1871-1878 [Abstract] [Full Text]  
• Deutscher, J., Francke, C., Postma, P. W. (2006). How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria. Microbiol. Mol. Biol. Rev. 70: 939-1031 [Abstract] [Full Text]  
• Watanabe, S., Hamano, M., Kakeshita, H., Bunai, K., Tojo, S., Yamaguchi, H., Fujita, Y., Wong, S.-L., Yamane, K. (2003). Mannitol-1-Phosphate Dehydrogenase (MtlD) Is Required for Mannitol and Glucitol Assimilation in Bacillus subtilis: Possible Cooperation of mtl and gut Operons. J. Bacteriol. 185: 4816-4824 [Abstract] [Full Text]  
• Tran, H. L., Kathariou, S. (2002). Restriction Fragment Length Polymorphisms Detected with Novel DNA Probes Differentiate among Diverse Lineages of Serogroup 4 Listeria monocytogenes and Identify Four Distinct Lineages in Serotype 4b. Appl. Environ. Microbiol. 68: 59-64 [Abstract] [Full Text]  
• Behrens, S., Mitchell, W. J., Bahl, H. (2001). Molecular analysis of the mannitol operon of Clostridium acetobutylicum encoding a phosphotransferase system and a putative PTS-modulated regulator. Microbiology 147: 75-86 [Abstract] [Full Text]  
• Honeyman, A. L., Curtiss, R. III (2000). The mannitol-specific enzyme II (mtlA) gene and the mtlR gene of the PTS of Streptococcus mutans. Microbiology 146: 1565-1572 [Abstract] [Full Text]  
• Henstra, S. A., Duurkens, R. H., Robillard, G. T. (2000). Multiple Phosphorylation Events Regulate the Activity of the Mannitol Transcriptional Regulator MtlR of the Bacillus stearothermophilus Phosphoenolpyruvate-dependent Mannitol Phosphotransferase System. J. Biol. Chem. 275: 7037-7044 [Abstract] [Full Text]  
• Boyd, D. A., Thevenot, T., Gumbmann, M., Honeyman, A. L., Hamilton, I. R. (2000). Identification of the Operon for the Sorbitol (Glucitol) Phosphoenolpyruvate:Sugar Phosphotransferase System in Streptococcus mutans. Infect. Immun. 68: 925-930 [Abstract] [Full Text]  
• Henstra, S. A., Tuinhof, M., Duurkens, R. H., Robillard, G. T. (1999). The Bacillus stearothermophilus Mannitol Regulator, MtlR, of the Phosphotransferase System. A DNA-BINDING PROTEIN, REGULATED BY HPr AND IICBmtl-DEPENDENT PHOSPHORYLATION. J. Biol. Chem. 274: 4754-4763 [Abstract] [Full Text]  
• Stoop, J. M. H., Mooibroek, H. (1998). Cloning and Characterization of NADP-Mannitol Dehydrogenase cDNA from the Button Mushroom, Agaricus bisporus, and Its Expression in Response to NaCl Stress. Appl. Environ. Microbiol. 64: 4689-4696 [Abstract] [Full Text]  
• van Montfort, B. A., Schuurman-Wolters, G. K., Duurkens, R. H., Mensen, R., Poolman, B., Robillard, G. T. (2001). Cysteine Cross-linking Defines Part of the Dimer and B/C Domain Interface of the Escherichia coli Mannitol Permease. J. Biol. Chem. 276: 12756-12763 [Abstract] [Full Text]