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 syst em revealed an open reading frame (modG) encoding a hypothetical 14-kD a protein. It consists of a tandem repeat of an approximately 65-amino -acid sequence that is homologous to Mop, a 7-kDa molybdopterin-bindin g 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 a nd modG in the processing of molybdate, modA, modB, and modC, which en code the component proteins of the high-affinity Mo transporter, are r equired for Mo-99 accumulation and for the nitrate reductase activity of cells growing in medium with less than 10 mu M Mo. The exchange of accumulated Mo-99 with nonradioactive Mo depends on the presence of mo dA, which encodes the periplasmic molybdate-binding protein, Mo-99 als o exchanges with tungstate but not with vanadate or sulfate, modA, mod B, and modC mutants exhibit nitrate reductase activity and Mo-99 accum ulation only when grown in more than 10 mu M Mo, indicating that A. vi nelandii also has a low-affinity Mo uptake system. The low-affinity sy stem is not expressed in a modE mutant that synthesizes the high-affin ity Mo transporter constitutively or in a spontaneous tungstate-tolera nt mutant. Like the wild type, modG mutants only show nitrate reductas e activity when grown in >10 nM Mo. However, a modE modG double mutant exhibits maximal nitrate reductase activity at a 100-fold lower Mo co ncentration. This indicates that the products of both genes affect the supply of Mo but are not essential for nitrate reductase cofactor syn thesis, However, nitrogenase-dependent growth in the presence or absen ce of Mo is severely impaired in the double mutant, indicating that th e products of modE and modG may be involved in the early steps of nitr ogenase cofactor biosynthesis in A. vinelandii.