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

• Published in: Microbiology (Society for General Microbiology) 2001-01, Vol.147 (1), p.75-86
• Main Authors: Behrens, Susanne, Mitchell, Wilfrid J, Bahl, Hubert
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacteriology; Biological and medical sciences; Clostridium - genetics; Clostridium - metabolism; Escherichia coli Proteins; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial; Genetics; Mannitol - metabolism; Microbiology; Molecular Sequence Data; Operon; Phosphoenolpyruvate Sugar Phosphotransferase System - genetics; Phosphoenolpyruvate Sugar Phosphotransferase System - metabolism; Phosphorylation; Repressor Proteins - genetics; Repressor Proteins - metabolism; RNA, Messenger - metabolism; Sequence Analysis, DNA
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/00221287-147-1-75

Institut für Mikrobiologie, Georg-August-Universität Göttingen, Grisebachstraße 8, D-37077 Göttingen, Germany 1 Department of Biological Sciences, Heriot-Watt University, Riccarton, Edinburgh EH14 4AS, UK 2 Author for correspondence: Hubert Bahl. Tel: [phone]. Fax: [phone]. e-mail: hubert.bahl{at}biologie.uni-rostock.de Clostridium acetobutylicum DSM 792 accumulates and phosphorylates mannitol via a phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS). PEP-dependent mannitol phosphorylation by extracts of cells grown on mannitol required both soluble and membrane fractions. Neither the soluble nor the membrane fraction could be complemented by the opposite fraction prepared from glucose-grown cells, indicating that the mannitol-specific PTS consists of both a soluble (IIA) and a membrane-bound (IICB) component. The mannitol ( mtl ) operon of C. acetobutylicum DSM 792 comprises four genes in the order mtlARFD . Sequence analysis of deduced protein products indicated that the mtlA and mtlF genes respectively encode the IICB and IIA components of the mannitol PTS, which is a member of the fructose-mannitol (Fru) family. The mtlD gene product is a mannitol-1-phosphate dehydrogenase, while mtlR encodes a putative transcriptional regulator. MtlR contains two PTS regulatory domains (PRDs), which have been found in a number of DNA-binding transcriptional regulators and in transcriptional antiterminators of the Escherichia coli BglG family. Also, near the C-terminus is a well-conserved signature motif characteristic of members of the IIA Fru /IIA Mtl /IIA Ntr PTS protein family. These regions are probably the sites of PTS-dependent phosphorylation to regulate the activity of the protein. A helix–turn–helix DNA-binding motif was not found in MtlR. Transcriptional analysis of the mtl genes by Northern blotting indicated that the genes were transcribed as a polycistronic operon, expression of which was induced by mannitol and repressed by glucose. Primer extension experiments identified a transcriptional start point 42 bp upstream of the mtlA start codon. Two catabolite-responsive elements (CREs), one of which overlapped the putative -35 region of the promoter, were located within the 100 bp upstream of the start codon. These sequences may be involved in regulation of expression of the operon. Keywords: mtl genes, catabolite-responsive elements, PTS regulatory domain (PRD), carbohydrate accumulation Abbreviations: CRE, catabolite-respo