Carbon substrate re-orders relative growth of a bacterium using Mo-, V-, or Fe-nitrogenase for nitrogen fixation.

Biological nitrogen fixation is catalyzed by the molybdenum (Mo), vanadium (V) and iron (Fe)-only nitrogenase metalloenzymes. Studies with purified enzymes have found that the 'alternative' V- and Fe-nitrogenases generally reduce N2 more slowly and produce more byproduct H2 than the Mo-nitrogenase, leading to an assumption that their usage results in slower growth. Here we show that, in the metabolically versatile photoheterotroph Rhodopseudomonas palustris, the type of carbon substrate influences the relative rates of diazotrophic growth based on different nitrogenase isoforms. The V-nitrogenase supports growth as fast as the Mo-nitrogenase on acetate but not on the more oxidized substrate succinate. Our data suggest that this is due to insufficient electron flux to the V-nitrogenase isoform on succinate compared with acetate. Despite slightly faster growth based on the V-nitrogenase on acetate, the wild-type strain uses exclusively the Mo-nitrogenase on both carbon substrates. Notably, the differences in H2 :N2 stoichiometry by alternative nitrogenases (~1.5 for V-nitrogenase, ~4-7 for Fe-nitrogenase) and Mo-nitrogenase (~1) measured here are lower than prior in vitro estimates. These results indicate that the metabolic costs of V-based nitrogen fixation could be less significant for growth than previously assumed, helping explain why alternative nitrogenase genes persist in diverse diazotroph lineages and are broadly distributed in the environment.

Siderophore production in Azotobacter vinelandii in response to Fe-, Mo- and V-limitation.

Azotobacter vinelandii is a terrestrial diazotroph well studied for its siderophore production capacity and its role as a model nitrogen fixer. In addition to Fe, A. vinelandii siderophores are used for the acquisition of the nitrogenase co-factors Mo and V. However, regulation of siderophore production by Mo- and V-limitation has been difficult to confirm and knowledge of the full suite of siderophores synthesized by this organism has only recently become available. Using this new information, we conducted an extensive study of siderophore production in N2 -fixing A. vinelandii under a variety of trace metal conditions. Our results show that under Fe-limitation the production of all siderophores increases, while under Mo-limitation only catechol siderophore production is increased, with the strongest response seen in protochelin. We also find that the newly discovered A. vinelandii siderophore vibrioferrin is almost completely repressed under Mo- and V-limitation. An examination of the potential nitrogen 'cost' of siderophore production reveals that investments in siderophore N can represent as much as 35% of fixed N, with substantial differences between cultures using the Mo- as opposed to the less efficient V-nitrogenase.

Diversity and Activity of Alternative Nitrogenases in Sequenced Genomes and Coastal Environments.

The nitrogenase enzyme, which catalyzes the reduction of N2 gas to NH4+, occurs as three separate isozyme that use Mo, Fe-only, or V. The majority of global nitrogen fixation is attributed to the more efficient 'canonical' Mo-nitrogenase, whereas Fe-only and V-('alternative') nitrogenases are often considered 'backup' enzymes, used when Mo is limiting. Yet, the environmental distribution and diversity of alternative nitrogenases remains largely unknown. We searched for alternative nitrogenase genes in sequenced genomes and used PacBio sequencing to explore the diversity of canonical (nifD) and alternative (anfD and vnfD) nitrogenase amplicons in two coastal environments: the Florida Everglades and Sippewissett Marsh (MA). Genome-based searches identified an additional 25 species and 10 genera not previously known to encode alternative nitrogenases. Alternative nitrogenase amplicons were found in both Sippewissett Marsh and the Florida Everglades and their activity was further confirmed using newly developed isotopic techniques. Conserved amino acid sequences corresponding to cofactor ligands were also analyzed in anfD and vnfD amplicons, offering insight into environmental variants of these motifs. This study increases the number of available anfD and vnfD sequences ∼20-fold and allows for the first comparisons of environmental Mo-, Fe-only, and V-nitrogenase diversity. Our results suggest that alternative nitrogenases are maintained across a range of organisms and environments and that they can make important contributions to nitrogenase diversity and nitrogen fixation.

Biological nitrogen fixation by alternative nitrogenases in boreal cyanolichens: importance of molybdenum availability and implications for current biological nitrogen fixation estimates.

Cryptogamic species and their associated cyanobacteria have attracted the attention of biogeochemists because of their critical roles in the nitrogen cycle through symbiotic and asymbiotic biological fixation of nitrogen (BNF). BNF is mediated by the nitrogenase enzyme, which, in its most common form, requires molybdenum at its active site. Molybdenum has been reported as a limiting nutrient for BNF in many ecosystems, including tropical and temperate forests. Recent studies have suggested that alternative nitrogenases, which use vanadium or iron in place of molybdenum at their active site, might play a more prominent role in natural ecosystems than previously recognized. Here, we studied the occurrence of vanadium, the role of molybdenum availability on vanadium acquisition and the contribution of alternative nitrogenases to BNF in the ubiquitous cyanolichen Peltigera aphthosa s.l. We confirmed the use of the alternative vanadium-based nitrogenase in the Nostoc cyanobiont of these lichens and its substantial contribution to BNF in this organism. We also showed that the acquisition of vanadium is strongly regulated by the abundance of molybdenum. These findings show that alternative nitrogenase can no longer be neglected in natural ecosystems, particularly in molybdenum-limited habitats.

Nitrogen isotope fractionation by alternative nitrogenases and past ocean anoxia.

Biological nitrogen fixation constitutes the main input of fixed nitrogen to Earth's ecosystems, and its isotope effect is a key parameter in isotope-based interpretations of the N cycle. The nitrogen isotopic composition (δ(15)N) of newly fixed N is currently believed to be ∼-1‰, based on measurements of organic matter from diazotrophs using molybdenum (Mo)-nitrogenases. We show that the vanadium (V)- and iron (Fe)-only "alternative" nitrogenases produce fixed N with significantly lower δ(15)N (-6 to -7‰). An important contribution of alternative nitrogenases to N2 fixation provides a simple explanation for the anomalously low δ(15)N (<-2‰) in sediments from the Cretaceous Oceanic Anoxic Events and the Archean Eon. A significant role for the alternative nitrogenases over Mo-nitrogenase is also consistent with evidence of Mo scarcity during these geologic periods, suggesting an additional dimension to the coupling between the global cycles of trace elements and nitrogen.

Molybdenum and phosphorus interact to constrain asymbiotic nitrogen fixation in tropical forests.

Biological di-nitrogen fixation (N(2)) is the dominant natural source of new nitrogen to land ecosystems. Phosphorus (P) is thought to limit N(2) fixation in many tropical soils, yet both molybdenum (Mo) and P are crucial for the nitrogenase reaction (which catalyzes N(2) conversion to ammonia) and cell growth. We have limited understanding of how and when fixation is constrained by these nutrients in nature. Here we show in tropical forests of lowland Panama that the limiting element on asymbiotic N(2) fixation shifts along a broad landscape gradient in soil P, where Mo limits fixation in P-rich soils while Mo and P co-limit in P-poor soils. In no circumstance did P alone limit fixation. We provide and experimentally test a mechanism that explains how Mo and P can interact to constrain asymbiotic N(2) fixation. Fixation is uniformly favored in surface organic soil horizons--a niche characterized by exceedingly low levels of available Mo relative to P. We show that soil organic matter acts to reduce molybdate over phosphate bioavailability, which, in turn, promotes Mo limitation in sites where P is sufficient. Our findings show that asymbiotic N(2) fixation is constrained by the relative availability and dynamics of Mo and P in soils. This conceptual framework can explain shifts in limitation status across broad landscape gradients in soil fertility and implies that fixation depends on Mo and P in ways that are more complex than previously thought.

Role of the siderophore azotobactin in the bacterial acquisition of nitrogenase metal cofactors.

Fixation of dinitrogen by soil bacteria is catalyzed by the enzyme nitrogenase which requires iron, molybdenum, and/or vanadium as metal cofactors. Under conditions of iron deficiency, the ubiquitous N2-fixing bacterium Azotobacter vinelandii produces azotobactin, a fluorescent pyoverdine-like compound which serves as a siderophore. Azotobatin's hydroxamate, catechol, and alpha-hydroxy-acid moieties endow it with a very high affinity for Fe(III), and the Fe complex is taken up by the bacterium. Here we show that azotobactin also serves for the uptake of Mo and V. Azotobactin forms strong complexes with molybdate and vanadate and the complexes are taken up by regulated transport systems. The kinetics of complexation of molybdate and vanadate by azotobactin are faster than the complexation of Fe(III), which is either precipitated or bound to strong complexing agents. As a result of this kinetic advantage, the Mo and V complexes of azotobactin form despite the higher affinity of the compound for Fe, which is present in large excess in the environment. The results obtained here for azotobactin and previous data for the bis- and tris-catechols produced by A. vinelandii show that those "siderophores" are really "metallophores" that promote the bacterial acquisition of Mo and V in addition to Fe.

Catechol siderophores control tungsten uptake and toxicity in the nitrogen-fixing bacterium Azotobacter vinelandii.

Molybdenum (Mo) and tungsten (W), which have similar chemistry, are present at roughly the same concentration in the earth's continental crust, and both are present in oxic systems as oxoanions, molybdate and tungstate. Molybdenum is a cofactor in the molybdenum-nitrogenase enzyme and is thus an important micronutrient for N2-fixing bacteria such as Azotobacter vinelandii (A. vinelandii). Tungsten is known to be toxic to N2-fixing bacteria, partly by substituting for Mo in nitrogenase. We showthatthe catechol siderophores produced by A. vinelandii, in addition to being essential for iron acquisition, modulate the relative uptake of Mo and W. These catechol siderophores (particularly protochelin), whose concentrations in the growth medium increase sharply at high W, complex all the tungstate along with molybdate and some of the iron. The molybdenum-catechol complex is taken up much more rapidly than the W complex, allowing A. vinelandii to satisfy its Mo requirement and avoid W toxicity. Mutants deficient in the production of catechol siderophores are more sensitive to tungstate and have higher cellular W quotas than the wild type. The binding of metals by excreted catechol siderophores allows A. vinelandii to discriminate in its uptake of essential metals, such as Fe and Mo, over that of toxic metals, such as W, and to sustain high growth rates under adverse environmental conditions.

Complexation of oxoanions and cationic metals by the biscatecholate siderophore azotochelin.

Azotochelin is a biscatecholate siderophore produced by the nitrogen-fixing soil bacterium Azotobacter vinelandii. The complexation properties of azotochelin with a series of oxoanions [Mo(VI), W(VI) and V(V)] and divalent cations [Cu(II), Zn(II), Co(II) and Mn(II)] were investigated by potentiometry, UV-vis and X-ray spectroscopy. Azotochelin forms a strong 1:1 complex with molybdate (log K=7.6+/-0.4) and with tungstate and vanadate; the stability of the complexes increases in the order Mo

Sources and variations of mercury in tuna.

While the bulk of human exposure to mercury is through the consumption of marine fish, most of what we know about mercury methylation and bioaccumulation is from studies of freshwaters. We know little of where and how mercury is methylated in the open oceans, and there is currently a debate whether methylmercury concentrations in marine fish have increased along with global anthropogenic mercury emissions. Measurements of mercury concentrations in Yellowfin tuna caught off Hawaii in 1998 show no increase compared to measurements of the same species caught in the same area in 1971. On the basis of the known increase in the global emissions of mercury over the past century and of a simple model of mercury biogeochemistry in the Equatorial and Subtropical Pacific ocean, we calculate that the methylmercury concentration in these surface waters should have increased between 9 and 26% over this 27 years span if methylation occurred in the mixed layer or in the thermocline. Such an increase is statistically inconsistent with the constant mercury concentrations measured in tuna. We conclude tentatively that mercury methylation in the oceans occurs in deep waters or in sediments.

Acquisition of inorganic carbon by the marine diatom Thalassiosira weissflogii.

Recent data on the physiology of inorganic carbon acquisition by the model marine diatom Thalassiosira weissflogii (Grunow) demonstrate the importance of the catalytic equilibration of HCO3-and CO2by carbonic anhydrases located in the periplasm and in the cytoplasm. These enzymes can use Zn, Co or Cd as their metal centre, and their activity increases at low ambient CO2. The silica frustule provides buffering for extracellular CA activity, The transmembrane transport of CO2 may occur by passive diffusion. Under CO2 limitation, the cytoplasmic HCO3-is used to form malate and oxaloacetic acid via phosphoenolpyruvate carboxylase. It appears that subsequent decarboxylation of these compounds in the chloroplast regenerates CO2 near the site of Rubisco, and thus provides the organism with an effective unicellular C4 photosynthetic pathway. These results, together with other published data, bring up two major questions regarding inorganic carbon acquisition in diatoms: What is the major species of inorganic carbon (CO2 or HCO3-) transported across the membrane under natural conditions? And what is the form of carbon (inorganic or organic) accumulated by the cells?