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

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Abstract
A transformant E. coli colony bank was screened for hybrid ColE1 plasmids carrying the genes for D-mannitol utilization. Two of the plasmids, pLC11-7 and pLC15-48, 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. There was no corresponding increase in mannitol enzyme II activity. Plasmid pLC15-48 directed the synthesis of 2 polypeptides in E. coli minicells in the presence of cAMP and mannitol. The larger peptide (MW 60,000) was membrane bound and was specifically precipitated by antibody directed against purified mannitol-specific enzyme II. The smaller peptide (MW 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. et al., 1979) that mannitol enzyme II consists of a single type of polypeptide chain that has a MW of 60,000. The plasmid pLC15-48 DNA was characterized by mapping of restriction endonuclease cleavage sites.