Molecular analysis of the mannitol operon of Clostridium acetobutylicum encoding a phosphotransferase system and a putative PTS-modulated regulator

Clostridium acetobutylicum DSM 792 accumulates and phosphorylates mannitol via a phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PIS). PEP-dependent mannitol phosphorylation by extracts of cells grown on mannit ol required both soluble and membrane fractions. Neither soluble nor the me mbrane fraction could be complemented by the opposite soluble nor the membr ane fraction could De complemented ay me opposite fraction prepared from gl ucose-grown cells, indicating that the mannitol-specific PTS consists of ba th a soluble (IIA) and a membrane-bound (IICB) component. The mannitol (mtl ) operon of C. acetobutylicum DSM 792 comprises four genes in the order mtI ARFD. Sequence analysis of deduced protein products indicated that the mtIA and mtIF genes respectively encode the IICB and IIA components of the mann itol PTS, which is a member of the fructose-mannitol (Fru) family. The mtID gene product is a mannitol-1-phosphate dehydrogenase, while mtIR encodes a putative transcriptional regulator. MtIR contains two PTS regulatory domai ns (PRDs), which have been found in a number of DNA-binding transcriptional regulators and in transcriptional antiterminators of the Escherichia coli BgIG family. Also, near the C-terminus is a well-conserved signature motif characteristic of members of the IIA(Fru)/IIIA(Mtl)/IIAN(Ntr) PTS protein f amily. These regions are probably the sites of PTS-dependent phosphorylatio n to regulate the activity of the protein. A helix-turn-helix DNA-binding m otif was not found in MtIR. Transcriptional analysis of the mtl genes by No rthern blotting indicated that the genes were transcribed as a polycistroni c operon, expression of which was induced by mannitol and repressed by gluc ose, Primer extension experiments identified a transcriptional start point 42 bp upstream of the mtIA start codon. Two catabolite-responsive elements (CREs), one of which overlapped the putative -35 region of the promoter, we re located within the 100 bp upstream of the start codon. These sequences m ay be involved in regulation of expression of the operon.