Plasmid-directed synthesis of enzymes required for D-mannitol transport and utilization in Escherichia coli

A transformant Escherichia coli colony bank [Clarke, L. & Carbon, J. (1976) Cell 9, 91-99] has been screened for hybrid ColE1 plasmids carrying the genes for D-mannitol utilization. Two of the plasmids, pLC11-7 and pLC15-48, were shown to contain the mannitol operon, which includes the structural genes for the mannitol-specific enzyme II of the phosphotransferase system and mannitol-1-phosphate dehydrogenase. One E. coli strain harboring plasmid pLC15-48 overproduced mannitol-1-phosphate dehydrogenase activity 4- to 5-fold. However, there was no corresponding increase in mannitol enzyme II activity. Plasmid pLC15-48 was shown to direct the synthesis of two polypeptides in E. coli minicells in the presence of cyclic AMP and mannitol. The larger (Mr = 60,000) was membrane bound and was specifically precipitated by antibody directed against purified mannitol-specific enzyme II. The smaller (Mr = 40,000) was soluble and had an electrophoretic mobility indistinguishable from that of the major component in a partially purified mannitol-1-phosphate dehydrogenase preparation. These data are consistent with previous genetic studies of the mannitol locus and confirm an independent conclusion [Jacobson, G. R., Lee, C. A. & Saier, M. H., Jr. (1979) J. Biol. Chem. 254, 249-252] that mannitol enzyme II consists of a single type of polypeptide chain that has a Mr of 60,000. The plasmid pLC15-48 DNA was characterized by mapping of restriction endonuclease cleavage sites.