Transcriptional regulation of nitrogen fixation by molybdenum in Azotobacter vinelandii

Jacobson, Marty R.; Premakumar, R.; Bishop, Paul E.

doi:10.1128/jb.167.2.480-486.1986

Cited by 101 publications

(63 citation statements)

References 33 publications

(25 reference statements)

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“…Since both of these fractions had dinitrogenase 3 activity, the fraction eluting first was designated dinitrogenase 3F (F denotes faster migrating) and the fraction eluting second was designated dinitrogenase 3s (S denotes slower migrating). The third major brown band had migrated only 20 mm through the separation gel after 16 h and was not collected.…”

Section: Methodsmentioning

confidence: 99%

Purification of a second alternative nitrogenase from a nifHDK deletion strain of Azotobacter vinelandii

1988

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A second alternative nitrogenase complex (nitrogenase 3) was purified from a nifHDK deletion strain of Azotobacter vinelandii. The active complex is made up of two components, dinitrogenase 3 and dinitrogenase reductase 3. Dinitrogenase 3 contains two protein subunits (alpha, Mr 58,000, and beta, Mr 50,000) which assemble into at least two active configurations: alpha 2 beta 2 (dinitrogenase 3s) and alpha 1 beta 2 (dinitrogenase 3F). Dinitrogenase 3s contains 24 Fe and 18 acid-labile S2-ions per Mr 216,000, and dinitrogenase 3F contains 11 Fe and 9 acid-labile S2-ions per Mr 158,000. Dinitrogenase reductase 3 is composed of two protein subunits of identical Mr (32,500) and contains four Fe and four acid-labile S2- ions per Mr 65,000. On two-dimensional gels, the protein subunits of the nitrogenase 3 complex comigrated with the four Mo-, V-, and NH4+-repressible proteins originally designated as N2ase B: the nitrogenase hypothesized to exist in the alternative N2 fixation system first described in 1980 (P.E. Bishop, D. M. L. Jarlenski, and D. R. Hetherington, Proc. Natl. Acad. Sci. USA 77:7342-7346, 1980). Neutron activation analysis indicated that the nitrogenase 3 complex lacked significant amounts of Mo, V, Cr, Re, and W. Some Zn, however, was found in the dinitrogenase 3S and dinitrogenase 3F preparations. The pattern of substrate reduction efficiency was H+ greater than N2 greater than C2H2. The maximum specific activity found for N2 reduction was 38 nmol of NH3 per min per mg of protein (dinitrogenase 3S). Nitrogenase 3 was found to be extremely sensitive to O2, and activities could not be reproducibly maintained during freezing and thawing.

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Section: Methodsmentioning

confidence: 99%

Purification of a second alternative nitrogenase from a nifHDK deletion strain of Azotobacter vinelandii

1988

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“…In this paper, we present the nucleotide sequence of the (28). In addition, we provide genetic evidence indicating that this genomic region contains the genes encoding the subunits of the components of nitrogenase 3.…”

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“…The genetic basis of the V nitrogenase from A. vinelandii is not as well understood as it is for the V nitrogenase from A. chroococcum, but extensive similarities appear to exist within the two organisms (49, 55; our unpublished results). The vnfH genes in both organisms are organized in an operon also containing a ferredoxin-like gene downstream from vnfH (28,54). The vnJfl-ferredoxin operon is separated from an operon containing the structural genes (vnfD vnfK) for dinitrogenase 2 by approximately 2 kilobase pairs (kbp) in A. chroococcum and by 1 kbp in A. vinelandii (55 and our unpublished results).…”

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Nucleotide sequence and mutational analysis of the structural genes (anfHDGK) for the second alternative nitrogenase from Azotobacter vinelandii

et al. 1989

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The nucleotide sequence of a region of the Azotobacter vinelandii genome exhibiting sequence similarity to nifH has been determined. The order of open reading frames within this 6.1-kilobase-pair region was found to be anfH (alternative nitrogen fixation, nifH-like gene), anfD (nifD-like gene), anfG (potentially encoding a protein similar to the product of vnfG from Azotobacter chroococcum), anfK (nifK-like gene), followed by two additional open reading frames. The 5'-flanking region of anfH contains a nif promoter similar to that found in the A. vinelandii nifHDK gene cluster. The presumed products of anfH, anfD, and anfK are similar in predicted Mr and pI to the previously described subunits of nitrogenase 3. Deletion plus insertion mutations introduced into the anfHDGK region of wild-type strain A. vinelandii CA resulted in mutant strains that were unable to grow in Mo-deficient, N-free medium but grew in the presence of 1 microM Na2MoO4 or V2O5. Introduction of the same mutations into the nifHDK deletion strain CA11 resulted in strains that grew under diazotrophic conditions only in the presence of vanadium. The lack of nitrogenase 3 subunits in these mutant strains was demonstrated through two-dimensional gel analysis of protein extracts from cells derepressed for nitrogenase under Mo and V deficiency. These results indicate that anfH, anfD, and anfK encode structural proteins for nitrogenase 3.

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“…The absence of molybdoenzyme activity at low Mo concentrations is of interest in the context of the physiology of A. vinelandii, which has an alternative nitrogenase (nitrogenase-3) that does not appear to contain either molybdenum or vanadium (6,43). Transcription of Mo nitrogenase structural genes in A. vinelandii ceases and that of the alternative nitrogenase genes is induced when the Mo concentration decreases from 50 to 25 nM Mo (27). Within this range of concentrations, both the Mo and alternative nitrogenases are expressed.…”

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Mutational analysis of genes of the mod locus involved in molybdenum transport, homeostasis, and processing in Azotobacter vinelandii

1995

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99Mo also exchanges with tungstate but not with vanadate or sulfate. modA, modB, and modC mutants exhibit nitrate reductase activity and 99 Mo accumulation only when grown in more than 10 M 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.

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Transcriptional regulation of nitrogen fixation by molybdenum in Azotobacter vinelandii

Cited by 101 publications

References 33 publications

Purification of a second alternative nitrogenase from a nifHDK deletion strain of Azotobacter vinelandii

Purification of a second alternative nitrogenase from a nifHDK deletion strain of Azotobacter vinelandii

Nucleotide sequence and mutational analysis of the structural genes (anfHDGK) for the second alternative nitrogenase from Azotobacter vinelandii

Mutational analysis of genes of the mod locus involved in molybdenum transport, homeostasis, and processing in Azotobacter vinelandii

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