Mutational analysis of genes of the mod locus involved in molybdenum transport, homeostasis, and processing in Azotobacter vinelandii.
DNA sequencing of the region upstream from the Azotobacter vinelandii operon (modEABC) that contains genes for the molybdenum transport system revealed an open reading frame (modG) encoding a hypothetical 14-kDa protein. It consists of a tandem repeat of an approximately 65-amino-acid sequence that is homologous to Mop, a 7-kDa molybdopterin-binding protein of Clostridium pasteurianum. The tandem repeat is similar to the C-terminal half of the product of modE. The effects of mutations in the mod genes provide evidence for distinct high- and low-affinity Mo transport systems and for the involvement of the products of modE and modG in the processing of molybdate. modA, modB, and modC, which encode the component proteins of the high-affinity Mo transporter, are required for 99Mo accumulation and for the nitrate reductase activity of cells growing in medium with less than 10 microM Mo. The exchange of accumulated 99Mo with nonradioactive Mo depends on the presence of modA, which encodes the periplasmic molybdate-binding protein. 99Mo also exchanges with tungstate but not with vanadate or sulfate. modA, modB, and modC mutants exhibit nitrate reductase activity and 99Mo accumulation only when grown in more than 10 microM Mo, indicating that A. vinelandii also has a low-affinity Mo uptake system. The low-affinity system is not expressed in a modE mutant that synthesizes the high-affinity Mo transporter constitutively or in a spontaneous tungstate-tolerant mutant. Like the wild type, modG mutants only show nitrate reductase activity when grown in > 10 nM Mo. However, a modE modG double mutant exhibits maximal nitrate reductase activity at a 100-fold lower Mo concentration. This indicates that the products of both genes affect the supply of Mo but are not essential for nitrate reductase cofactor synthesis. However, nitrogenase-dependent growth in the presence or absence of Mo is severely impaired in the double mutant, indicating that the products of modE and modG may be involved in the early steps of nitrogenase cofactor biosynthesis in A. vinelandii.
Mouncey
N J
NJ
Nitrogen Fixation Laboratory, University of Sussex, Brighton, United Kingdom.
Mitchenall
L A
LA
Pau
R N
RN
D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000235:genetics* / Q000254:growth & development; D001692:Biological Transport; D004252:DNA Mutational Analysis; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics*; D006706:Homeostasis; D008291:Malate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D008982:Molybdenum / Q000378:metabolism* / Q000494:pharmacology; D009154:Mutation; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000378:metabolism; D016366:Open Reading Frames / Q000235:genetics*; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D017973:Tungsten Compounds / Q000494:pharmacology
D017973:Tungsten Compounds; C025399:sodium tungstate(VI); D008982:Molybdenum; D008291:Malate Dehydrogenase; C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+); D009565:Nitrate Reductases; D050901:Nitrate Reductase