CLONING, NUCLEOTIDE-SEQUENCE AND CHARACTERIZATION OF THE MANNITOL DEHYDROGENASE GENE FROM RHODOBACTER-SPHAEROIDES

Transposon mutagenesis and antibiotic enrichment were employed to isol ate a mutant of Rhodobacter sphaeroides Si4 designated strain M22, tha t had lost the ability to grow on D-mannitol and to produce the enzyme mannitol dehydrogenase (MDH). DNA flanking the transposon in the muta nt strain was used as a probe for the identification and cloning of th e MDH gene (mtlK). A 5.5 kb EcoRI/Bg/II fragment from R. sphaeroides S i4 was isolated and shown to complement the mutation in R. sphaeroides M22. Successful complementation required that a promoter of the vecto r-plasmid pRK415 be present, suggesting that the mtlK gene is part of a larger operon. Using oligonucleotides derived from the N-terminal se quence of MDH as probes mtlK was located on the complementing fragment and the gene was sequenced. The mtlK open reading frame encodes a pro tein of 51 404 Da with an N-terminal sequence identical to that obtain ed from amino acid analysis of the purified MDH. The MDH of R. sphaero ides Si4 exhibits distant similarity to the mannitol-1-phosphate dehyd rogenases from Escherichia coli and Enterococcus faecalis, with 28.1 % and 26.3 % identity, respectively. Mutant strains deficient in MtlK d isplayed substantial levels of sorbitol dehydrogenase activity, origin ally thought to be only a minor activity associated with the MDH enzym e. It is likely that we have uncovered an additional polyol dehydrogen ase with activity for sorbitol. The mtlK gene can be used for overexpr ession of MDH in E. coli in order to obtain sufficient amounts of enzy me for further investigations and applications.