Biochemical and genetic analysis of the nifUSVWZM cluster from Azotobacter vinelandii

Abstract

Azotobacter vinelandii genes contained within the major nif-cluster and designated orf6, nifU, nifS, nifV, orf7, orf8, nifW, nifZ, nifM, and orf9 are organized into at least two overlapping transcriptional units. Nitrogenase derepressed crude extracts of Azotobacter vinelandii mutant strains having individual deletions located within nifU, nifS, nifV, nifW, nifZ, or nifM were examined for nitrogenase component protein activities. The results of these experiments indicated that, in A. vinelandii, the nifU, nifS and nifM gene products are required for the full activation or the catalytic stability of the nitrogenase Fe protein. Deletion of the nifV gene resulted in lower MoFe protein activity, probably resulting from the accumulation of an altered FeMo-cofactor. The nifW and nifZ gene products were required for the full activation or catalytic stability of the MoFe protein. Deletion of nifZ alone or nifM alone did not appear to affect FeMo-cofactor biosynthesis. However, deletion of both nifZ and nifM eleminated either FeMo-cofactor biosynthesis or the insertion of FeMo-cofactor into the apo-MoFe protein. Other genes contained within the nifUSVWZM gene cluster (orf6, orf7, orf8, and orf9) were not required for Mo-dependent diazotrophic growth.

Iron-Sulfur Cluster Assembly

“…One possibility we have considered is that the product of a gene located immediately upstream from nifU, designated Nif IscA, which has also been shown to be capable of serving as an [Fe-S] cluster assembly scaffold in vitro (34), could serve this function. To test this possibility a strain was constructed where a 93-base pair deletion within Nif iscA (12) was placed in combination with substitutions that inactivate both the N-terminal and C-terminal domains of NifU. This strain is not further impaired in diazotrophic growth, so it seems unlikely that Nif IscA significantly contributes to nitrogenase maturation under the conditions used in the present work.…”

“…A version of a nifUS expression plasmid for which eight histidine codons were placed between nifS codons 396 and 397 was constructed by inserting a synthetic DNA fragment into the unique StuI restriction site of pDB1289. For the construction of mutant A. vinelandii strains, the appropriate plasmids were used in DNA transformation experiments, resulting in double-reciprocal recombination between the genome and the plasmid vector as previously described in detail (12,21). Plasmids used in this work are not capable of autonomous replication in A. vinelandii, and they are listed in Table I.…”

“…NifU-In previous work it was shown that deletion of the A. vinelandii nifU gene nearly eliminates diazotrophic growth capacity (12). In contrast, substitution of alanine for any of the three N-terminal cysteines proposed to be involved in providing ligands for [Fe-S] cluster assembly lowers diazotrophic capacity but not as severely as for the nifU deletion strain (3).…”

“…Thus, under nitrogen fixing conditions NifU is apparently unable to effectively substitute for IscU function. Similarly, NifU deletion strains are severely impaired in their capacity for diazotrophic growth, indicating that IscU cannot effectively replace the function of NifU (12). In contrast, the iscSUAhscBAfdx genes are not essential in E. coli because there is some redundant function provided by another [Fe-S] cluster assembly apparatus encoded by sufABCDSE.…”

Du fer et du soufre dans les protéines

“…Les protéines HscB et HscA forment un duo cochaperon/chaperon du type Hsp40/Hsp70, dont l'action est spécifiquement dédiée à la biogenèse des centres Fe-S [20]. Les protéines HscBA interviennent dans la libération et le transfert du centre Fe-S de la protéine laboratoire de D. Dean (Virginia Tech) découvrit, par des approches génétiques, que l'activité de la nitrogénase d'Azotobacter vinelandii nécessitait in vivo l'assistance de plusieurs protéines, toutes apparemment requises pour l'insertion des centres Fe-S [4]. Depuis, les études menées sur des organismes tels que Escherichia coli et A. vinelandii pour les procaryotes, et Saccharomyces cerevisiae, Arabidopsis thaliana et l'homme pour les eucaryotes, ont permis de distinguer trois machineries nécessaires à la formation des centres Fe-S et à leur insertion dans des protéines cellulaires « clientes » : la machinerie ISC (iron-sulfur cluster), présente chez les bactéries et dans les mitochondries, la machinerie SUF, présente chez les bactéries et dans les chloroplastes, et la machinerie NIF, celle-là même initialement identifiée par D. Dean et ses collègues, qui est spécifique de la maturation de la nitrogénase.…”