Evidence for an alternative nitrogen fixation system in Azotobacter vinelandii.

Bishop, Paul E.; Jarlenski, D M; Hetherington, D R

doi:10.1073/pnas.77.12.7342

Cited by 242 publications

(154 citation statements)

References 22 publications

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“…Most importantly, V is found at the active center of an alternative form of the enzyme nitrogenase, which fixes atmospheric N 2 gas into bioavailable ammonia and is responsible for the natural input of new nitrogen into the earth's ecosystems. The molybdenum (Mo)-nitrogenase, which is the most common and efficient form of the enzyme, has a Mo cofactor at its active site, but when Mo is not available, some bacteria can express an alternative V-nitrogenase, which uses a V cofactor in place of Mo (3,4). Some organisms also have an Fe-only nitrogenase, which requires only Fe at its active center and is used when neither Mo nor V is available (14,20).…”

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confidence: 99%

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Vanadium Requirements and Uptake Kinetics in the Dinitrogen-Fixing Bacterium Azotobacter vinelandii

Bellenger

Wichard

Kraepiel

2008

Appl Environ Microbiol

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Vanadium is a cofactor in the alternative V-nitrogenase that is expressed by some N 2 -fixing bacteria when Mo is not available. We investigated the V requirements, the kinetics of V uptake, and the production of catechol compounds across a range of concentrations of vanadium in diazotrophic cultures of the soil bacterium Azotobacter vinelandii. In strain CA11.70, a mutant that expresses only the V-nitrogenase, V concentrations in the medium between 10 ؊8 and 10 ؊6 M sustain maximum growth rates; they are limiting below this range and toxic above. A. vinelandii excretes in its growth medium micromolar concentrations of the catechol siderophores azotochelin and protochelin, which bind the vanadate oxoanion. The production of catechols increases when V concentrations become toxic. Short-term uptake experiments with the radioactive isotope 49 V show that bacteria take up the V-catechol complexes through a regulated transport system(s), which shuts down at high V concentrations. The modulation of the excretion of catechols and of the uptake of the V-catechol complexes allows A. vinelandii to precisely manage its V homeostasis over a range of V concentrations, from limiting to toxic.

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“…Unlike most transition metals (but like Mo), the most stable form of V in oxic environments is a negatively charged ion, the oxoanion vanadate (H 2 VO 4 Ϫ /H 2 VO 4 2Ϫ ). Vanadate is a structural and electronic analogue of phosphate, and it competes against phosphate for uptake in freshwater algae (24).…”

mentioning

confidence: 99%

Vanadium Requirements and Uptake Kinetics in the Dinitrogen-Fixing Bacterium Azotobacter vinelandii

Bellenger

Wichard

Kraepiel

2008

Appl Environ Microbiol

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“…This allows the independent expression of dinitrogenase reductase 2 (vnfH gene product) and dinitrogenase 2 (vnfDGK products). Unlike dinitrogenase 2, dinitrogenase reductase 2 is present not only under diazotrophic conditions in the presence of V but also in the absence of Mo and V, conditions where nitrogenase 3 is present (3,8,25). Dinitrogenase reductase 2 is unlikely to function in a catalytic role under Mo-and V-deficient conditions because purified dinitrogenase reductase 2 does not effectively complement dinitrogenase 3 in in vitro assays (8).…”

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confidence: 99%

“…The A. vinelandii strains were derepressed for nitrogenases 1 and 2 for 3 to 5 h, and, for nitrogenase 3, the derepression time was 12 h. When possible, cells were grown to a cell density of 70 to 100 Klett units in N-free medium. Cell-free protein extracts were prepared as previously described (2). Isoelectric focusing and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of proteins in cell-free extracts were conducted by the method of O'Farrell (22) I8-Galactosidase assays.…”

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confidence: 99%

The gene encoding dinitrogenase reductase 2 is required for expression of the second alternative nitrogenase from Azotobacter vinelandii

1991

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Under diazotrophic conditions in the absence of molybdenum (Mo) and vanadium (V), Azotobacter vinelandii reduces N2 to NH4+ by using nitrogenase 3 (encoded by anfHDGK). However, dinitrogenase reductase 2 (encoded by vnfH) is also expressed under these conditions even though this protein is a component of the Vcontaining alternative nitrogenase. Mutant strains that lack dinitrogenase reductase 2 (VnfH-) grow slower than the wild-type strain in N-free, Mo-, and V-deficient medium. In this medium, these strains synthesize dinitrogenase reductase 1 (a component of the Mo-containing nitrogenase encoded by nifH), even though this component is not normally synthesized in the absence of Mo. Strains that lack both dinitrogenase reductases 1 and 2 (NifH-VnfH-) are unable to grow diazotrophically in Mo-and V-deficient medium. In this medium, NifH-VnfH-strains containing an anfH--lacZ transcriptional fusion exhibited less than 3% of the ,B-galactosidase activity observed in the wild type with the same fusion. P-Galactosidase activity expressed by VnfHmutants containing the anJH-lacZ fusion ranged between 57 and 78% of that expressed by the wild type containing the samne fusion. Thus, expression of dinitrogenase reductase 2 seems to be required for transcription of the anfHDGK operon, although, in Vnft-mutants, dinitrogenase reductase 1 appears to serve this function. Active dinitrogenase reductase 1 or 2 is probably required for this function since a nijM deletion mutant containing the anffl-lacZ fusion was unable to synthesize ,-galactosidase above background levels. An anfA deletion strain containing the anpfH-lacZ fusion exhibited ,B-galactosidase activity at 16% of that of the wild type containing the same fusion. However, in the presence of NH4', the ,B-galactosidase activity expressed by this strain more than doubled. This indicates that AnfA is required not only for normal levels of anfHDGK transcription but also for NH4'-and, to a lesser extent, Mo-mediated repression of this transcription.The regulation of the expression of the three nitrogenases in Azotobacter vinelandii is responsive to the presence or absence of ammonium (NH4'), molybdenum (Mo), and vanadium (V) in the culture medium. The synthesis of all three nitrogenases is repressed by NH4'. Nitrogenase 1 is found in cells grown in the presence of Mo and nitrogenase 2 is expressed in the presence of V but in the absence of Mo, whereas nitrogenase 3 is synthesized only in the absence of both Mo and V (4, 9, 13, and references therein). Our knowledge of the molecular basis for nitrogen and metal regulation in A. vinelandii is still rudimentary. The regulatory genes nifA, vnfA, and anfA have been identified, and some of their functions have been described on the basis of the phenotypes of NifA-, VnfA-, and AnfA-mutants (1, 14). The nifA gene product is required for transcription of the structural genes for nitrogenase 1 (1). NifA binds to an upstream activator sequence (6) or indirectly represses the synthesis of nitrogenase 1 in cells grown in Mo-deficient me...

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“…The formation of CH 4 , H 2 and NH 3 as a function of NaCN concentration is presented in Figure 1 (top). Methylamine was not determined from these assays and no adjustment for this omission has been made.…”

Section: Product Formation As a Function Of Nacn Concentrationmentioning

confidence: 99%

Azotobacter vinelandiiVanadium Nitrogenase: Formaldehyde Is a Product of Catalyzed HCN Reduction, and Excess Ammonia Arises Directly from Catalyzed Azide Reduction

2006

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The nitrogenase-catalyzed reduction of both cyanide and azide results in the production of "excess NH 3 ", which is an amount of NH 3 over and above that expected to be formed from the wellrecognized reactions. Several suggestions have been made as to the possible sources of "excess NH 3 ", but previous attempts to characterize these reactions have met with either limited (or no) success or controversy. Because V-nitrogenase has a propensity to release partially reduced intermediates, e.g., N 2 H 4 during N 2 reduction, it was selected to probe the reduction of cyanide and azide. Sensitive assay procedures were developed and employed to monitor the production of either HCHO or CH 3 OH (its further two-electron-reduced product) from HCN. Like Monitrogenase, V-nitrogenase suffered electron-flux inhibition by CN − (but was much less sensitive than Mo-nitrogenase) but, unlike Mo-nitrogenase, MgATP hydrolysis was also inhibited by CN − . V-nitrogenase also released more of the four-electron-reduced intermediate, CH 3 NH 2 , than did Mo-nitrogenase. At high NaCN concentrations, V-nitrogenase directed a significant percentage of electron flux into "excess NH 3 " and, under these conditions, substantial amounts of HCHO, but no CH 3 OH, were detected for the first time. With azide, in contrast to Mo-nitrogenase, both total electron flux and MgATP hydrolysis with V-nitrogenase were inhibited. V-nitrogenase, unlike Mo-nitrogenase, showed no preference between the two-electron reduction to N 2 -plus-NH 3 and the six-electron reduction to N 2 H 4 -plus-NH 3 . V-nitrogenase formed more "excess NH 3 ", but reduction of the N 2 produced by the two-electron reduction of N 3 − was not its source. Rather, it was formed directly by the eight-electron reduction of N 3 − . Unlike Mo-nitrogenase, CO could not completely eliminate either cyanide or azide reduction by V-nitrogenase. CO did, however, eliminate the inhibition of both electron flux and MgATP hydrolysis by CN − , but not that caused by azide. These different responses to CO suggest different sites or modes of interaction for these two substrates with V-nitrogenase. † Support from the National Institutes of Health (Grant #DK 37255 to W.E.N.) is gratefully acknowledged. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2015 January 16. Azotobacter vinelandii is a strictly aerobic N 2 -fixing organism that harbors three related, but genetically distinct, nitrogenases. Which one of these enzymes is operational at any particular time depends on which metal ions are present (1). For more than 50 years, the requirement of molybdenum for nitrogenase was thought to be mandatory (2), even though vanadium was demonstrated early on to be almost as stimulatory as Mo to the growth of some bacteria on N 2 as the sole nitrogen source (3). Despite these and other early experiments, the essentiality of Mo became entrenched. However, it is now clear that Mo is not essential for biological nitrogen fixation. Progress through the 1980's clearly identi...

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Evidence for an alternative nitrogen fixation system in Azotobacter vinelandii.

Cited by 242 publications

References 22 publications

Vanadium Requirements and Uptake Kinetics in the Dinitrogen-Fixing Bacterium Azotobacter vinelandii

Vanadium Requirements and Uptake Kinetics in the Dinitrogen-Fixing Bacterium Azotobacter vinelandii

The gene encoding dinitrogenase reductase 2 is required for expression of the second alternative nitrogenase from Azotobacter vinelandii

Azotobacter vinelandiiVanadium Nitrogenase: Formaldehyde Is a Product of Catalyzed HCN Reduction, and Excess Ammonia Arises Directly from Catalyzed Azide Reduction

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