The mob locus of Escherichia coli K12 required for molybdenum cofactor biosynthesis is expressed at very low levels

• Published in: Microbiology (Society for General Microbiology) Vol. 141; no. 7; pp. 1663 - 1671
• Main Authors: lobbi-Nivol, Chantal, Palmer, Tracy, Whitty, Patrick W, McNairn, Elizabeth, Boxer, David H
• Format: Journal Article
• Language: English
• Published: Reading Soc General Microbiol 01.07.1995; Society for General Microbiology
• Subjects: Amino Acid Sequence; Anaerobiosis; Bacterial Proteins - biosynthesis; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacteriology; Base Sequence; Biological and medical sciences; Chlorates - pharmacology; Cloning, Molecular; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial; Genes, Bacterial; Genetics; Guanine Nucleotides - biosynthesis; Guanine Nucleotides - genetics; Guanine Nucleotides - metabolism; Microbiology; Molecular Sequence Data; Mutagenesis, Insertional; Nitrate Reductases - genetics; Operon; Peptide Elongation Factor Tu - genetics; Promoter Regions, Genetic; Pterins - metabolism; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - metabolism; Restriction Mapping; Sequence Homology, Amino Acid; Transduction, Genetic; Gene; Nucleotide sequence; Transcription; Escherichia coli; Bacteria; Biosynthesis; Gene expression; Cofactor; Molybdenum; Aminoacid sequence; Enterobacteriaceae
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/13500872-141-7-1663

1 Department of Biochemistry, Medical Sciences Institute, Dundee University, Dundee DD1 4HN, UK ABSTRACT Summary: The mob locus of Escherichia coli encodes functions which catalyse the synthesis of active molybdenum cofactor, molybdopterin guanine dinucleotide, from molybdopterin and GTP. Reporter translational lac fusion mutations in the mobA gene have been constructed using plac Mu9 mutagenesis. The mob locus is expressed at very low levels under both aerobic and anaerobic growth conditions. Neither additions to the growth media (nitrate, tungstate or molybdate) nor secondary mutations at the moa, mob, mod, moe or mog loci affected the level of expression. Two transcription initiation sites and their associated promoter regions have been identified upstream of mobA . Both of the promoter regions show a poor match to the –35 and –10 consensus sequences for –70 promoters. A 2-2 kb chromosomal DNA fragment which complemented all available mob mutants has been sequenced. Two ORFs were identified, arranged as a single transcription unit. The encoded polypeptides have predicted molecular masses of 21642 Da and 19362 Da, respectively. The DNA has been subcloned into a T7 overexpression system and the predicted products identified. The mobA gene encodes protein FA, which has been purified to homogeneity and brings about the activation of inactive molybdoenzymes in cell extracts of mob mutants. The mobB gene encodes a polypeptide with a putative nucleotide binding site. All available mob mutations which have been selected for by their ability to grow anaerobically in the presence of chlorate are located in the mobA gene. 2 Author for correspondence: David H. Boxer. Tel: +44 1382 344880. Fax: +44 1382 322558 e-mail: dhboxer@ bad.dundee.ac.uk Keywords: Escherichia coli K12, mob locus, molybdenum cofactor biosynthesis, molybdopterin guanine dinucleotide Present address: Laboratoire de Chimie Bacterienne, CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille, Cedex 9, France.