Activation of Inactive Nitrogenase by Acid-Treated Component I

When Azotobacter vinelandii was derepressed for nitrogenase synthesis in a N-free medium containing tungstate instead of molybdate, an inactive component I was synthesized. Although this inactive component I could be activated in vivo upon addition of molybdate to the medium, it could not be activated in vitro when molybdate was added to the extracts. Activation occurred, however, when an acid-treated component I was added to extracts of cells derepressed in medium containing tungstate. Acid treatment completely abolished component I activity. Mutant strains UW45 and UW10 were unable to fix N(2). Both strains synthesized normal levels of component II but produced inactive component I. Acid-treated component I activated inactive component I in extracts of mutant strain UW45 but not mutant strain UW10. This activating factor could be obtained from N(2)-fixing Klebsiella pneumoniae, Clostridium pasteurianum, and Rhodospirillum rubrum.     

Molybdenum cofactor amounts in Chlamydomonas reinhardtii depend on the Nit5 gene function related to molybdate transport

Strain 21gr from Chlamydomonas reinhardtii is a cryptic mutant defective in the Nit5 gene related to the biosynthesis of molybdenum cofactor (MoCo). In spite of this mutation, this strain has active MoCo and can grow on nitrate media. In genetic crosses, the Nit5 mutation cosegregated with a phenotype of resistance to high concentrations of molybdate and tungstate. Molybdate/tungstate toxicity was much higher in nitrate than in ammonium media. Strain 21gr showed lower amounts of MoCo activity than the wild type both when grown in nitrate and after growth in ammonium and nitrate induction. However, nitrate reductase (NR) specific activity was similar in wild type and 21gr cells. Tungstate, either at nanomolar concentrations in nitrate media or at micromolar concentrations during growth in ammonium and nitrate induction, strongly decreased MoCo and NR amounts in wild‐type cells but had a slight effect in 21gr cells. Molybdate uptake activity of ammonium‐grown cells from both the wild‐type and 21gr strains was small and blocked by sulphate 0·3 mM . However, cells from nitrate medium showed a molybdate uptake activity insensitive to sulphate. This uptake activity was much higher and more sensitive to inhibition by tungstate in the wild type than in strain 21gr. These results suggest that strain 21gr has a high affinity and low capacity molybdate transport system able to discriminate efficiently tungstate, and lacks a high capacity molybdate/tungstate transport system, which operates in wild‐type cells upon nitrate induction. This high capacity molybdate transport system would account for both the stimulating effect of molybdate on MoCo amounts and the toxic effects of tungstate and molybdate when present at high concentrations.     

Transport of molybdate in the cyanobacterium Anabaena variabilis ATCC 29413

Heterocyst-forming filamentous cyanobacteria, such as Anabaena variabilis ATCC 29413, require molybdenum as a component of two essential cofactors for the enzymes nitrate reductase and nitrogenase. A. variabilis efficiently transported (99)Mo (molybdate) at concentrations less than 10(-9) M. Competition experiments with other oxyanions suggested that the molybdate-transport system of A. variabilis also transported tungstate but not vanadate or sulfate. Although tungstate was probably transported, tungsten did not function in place of molybdenum in the Mo-nitrogenase. Transport of (99)Mo required prior starvation of the cells for molybdate, suggesting that the Mo-transport system was repressed by molybdate. Starvation, which required several generations of growth for depletion of molybdate, was enhanced by growth under conditions that required synthesis of nitrate reductase or nitrogenase. These data provide evidence for a molybdate storage system in A. variabilis. NtcA, a regulatory protein that is essential for synthesis of nitrate reductase and nitrogenase, was not required for transport of molybdate. The closely related strain Anabaena sp. PCC 7120 transported (99)Mo in a very similar way to A. variabilis.     

Selective removal of molybdenum traces from growth media of N2-fixing bacteria

A new method for the selective removal of traces of molybdenum from growth media of N2-fixing bacteria (Rhodobacter capsulatus and Klebsiella pneumoniae) was developed. This method is based on the filtration of nutrient solutions through a layer of activated carbon (pulverized charcoal). The adsorption of Mo (molybdate) to activated carbon was optimal if a charcoal suspension (50 g/liter) was degassed by boiling before use and if the pH of the solutions, which had to be purified, was adjusted to values between 1.5 and 4. In this pH region no or only negligible amounts of other metal ions were adsorbed. The activated carbon method was compared with other Mo-eliminating procedures, including 8-hydroxyquinoline/dichloromethane extraction, Chelex 100 chromatography, and treatment with Mo-starved Azotobacter vinelandii cells. The activated carbon filtration appeared to be the most effective, specific, and rapid method. Whereas the untreated Rhodobacter growth medium was contaminated with 1.2 ppb Mo, as analyzed by inductively coupled plasma mass spectrometry (ICP-MS), the activated carbon-treated medium was below the ICP-MS detection limit (less than 0.05 ppb). A similarly effective removal of Mo impurities was obtained by the Azotobacter treatment. Even at low optical densities (2-5 at 436 nm) Mo traces were removed very rapidly within 10-15 min. However, because the Mo uptake/Mo adsorption capacity of A. vinelandii depended on freshly cultivated cells and on the growth phase at which the cells were harvested, this microbiological method was generally more time-consuming and less reproducible than the activated carbon method.(ABSTRACT TRUNCATED AT 250 WORDS)     

4-hydroxybenzoyl coenzyme A reductase (dehydroxylating) is required for anaerobic degradation of 4-hydroxybenzoate by Rhodopseudomonas palustris and shares features with molybdenum-containing hydroxylases.

The anaerobic degradation of 4-hydroxybenzoate is initiated by the formation of 4-hydroxybenzoyl coenzyme A, with the next step proposed to be a dehydroxylation to benzoyl coenzyme A, the starting compound for a central pathway of aromatic compound ring reduction and cleavage. Three open reading frames, divergently transcribed from the 4-hydroxybenzoate coenzyme A ligase gene, hbaA, were identified and sequenced from the phototrophic bacterium Rhodopseudomonas palustris. These genes, named hbaBCD, specify polypeptides of 17.5, 82.6, and 34.5 kDa, respectively. The deduced amino acid sequences show considerable similarities to a group of hydroxylating enzymes involved in CO, xanthine, and nicotine metabolism that have conserved binding sites for [2Fe-2S] clusters and a molybdenum cofactor. Cassette disruption of the hbaB gene yielded a mutant that was unable to grow anaerobically on 4-hydroxybenzoate but grew normally on benzoate. The hbaB mutant cells did not accumulate [14C]benzoyl coenzyme A during short-term uptake of [14C]4-hydroxybenzoate, but benzoyl coenzyme A was the major radioactive metabolite formed by the wild type. In addition, crude extracts of the mutant failed to convert 4-hydroxybenzoyl coenzyme A to benzoyl coenzyme A. This evidence indicates that the hbaBCD genes encode the subunits of a 4-hydroxybenzoyl coenzyme A reductase (dehydroxylating). The sizes of the specified polypeptides are similar to those reported for 4-hydroxybenzoyl coenzyme A reductase isolated from the denitrifying bacterium Thauera aromatica. The amino acid consensus sequence for a molybdenum cofactor binding site is in HbaC. This cofactor appears to be an essential component because anaerobic growth of R. palustris on 4-hydroxybenzoate, but not on benzoate, was retarded unless 0.1 microM molybdate was added to the medium. Neither tungstate nor vanadate replaced molybdate, and tungstate competitively inhibited growth stimulation by molybdate.     

Role of molybdate and other transition metals in the accumulation of protochelin by Azotobacter vinelandii

Both molybdate and iron are metals that are required by the obligately aerobic organism Azotobacter vinelandii to survive in the nutrient-limited conditions of its natural soil environment. Previous studies have shown that a high concentration of molybdate (1 mM) affects the formation of A. vinelandii siderophores such that the tricatecholate protochelin is formed to the exclusion of the other catecholate siderophores, azotochelin and aminochelin. It has been shown previously that molybdate combines readily with catecholates and interferes with siderophore function. In this study, we found that the manner in which each catecholate siderophore interacted with molybdate was consistent with the structure and binding potential of the siderophore. The affinity that each siderophore had for molybdate was high enough that stable molybdo-siderophore complexes were formed but low enough that the complexes were readily destabilized by Fe(3+). Thus, competition between Fe(3+) and molybdate did not appear to be the primary cause of protochelin accumulation; in addition, we determined that protochelin accumulated in the presence of vanadate, tungstate, Zn(2+), and Mn(2+). We found that all five of these metal ions partially inhibited uptake of (55)Fe-protochelin and (55)Fe-azotochelin complexes. Also, each of these metal ions partially inhibited the activity of ferric reductase, an enzyme important in the deferration of ferric siderophores. Our results suggest that protochelin accumulates in the presence of molybdate because protochelin uptake and conversion into its component parts, azotochelin and aminochelin, are inhibited by interference with ferric reductase.     

Inhibition of cultured cell growth by tungstate and molybdate

The growth of suspension cultured cells of Nicotiana tabacum (tobacco) was inhibited completely by 100 M tungstate. Even though molybdate reversed the tungstate inactivation of nitrate reductase activity, the growth inhibition was not reversed. The growth inhibition of N. tabacum, Daucus carota, Glycine max and Solanum tuberosum suspension cultured cells by tungstate was similar in media with or without amino acids as a source of reduced nitrogen. Only in the case of G. max was a slight reversal caused by the amino acids. Tungstate was slightly less inhibitory to the growth of a nitrate reductase-lacking mutant N. tabacum line (nia-63) than to the line with nitrate reductase. These results indicate that tungstate must inhibit the cell growth of the four species used, predominantly, in some way other than by inhibiting nitrate reductase activity. Similar studies with molybdate, a sulfate analog which apparently competes with sulfate at the ATP sulfury-lase enzyme, showed that 1 mM concentrations were completely inhibitory to cell growth. The addition of sulfate or cysteine, as a source of reduced sulfur, and amino acids, as a source of reduced nitrogen, in most cases did not reverse the molybdate inhibition appreciably. Some reversal was seen only by sulfate with D. carota cells and by cysteine plus amino acids with D. carota and G. max. These results indicate that selection for tungstate or molybdate resistance will in general not select for higher levels or other alterations in the activity of nitrate reductase or ATP sulfurylase, respectively, since these ions do not inhibit growth by primarily affecting these enzymatic steps in cultured cells of the four species studied.     

Tungsten transport protein A (WtpA) in Pyrococcus furiosus: the first member of a new class of tungstate and molybdate transporters

A novel tungstate and molybdate binding protein has been discovered from the hyperthermophilic archaeon Pyrococcus furiosus. This tungstate transport protein A (WtpA) is part of a new ABC transporter system selective for tungstate and molybdate. WtpA has very low sequence similarity with the earlier-characterized transport proteins ModA for molybdate and TupA for tungstate. Its structural gene is present in the genome of numerous archaea and some bacteria. The identification of this new tungstate and molybdate binding protein clarifies the mechanism of tungstate and molybdate transport in organisms that lack the known uptake systems associated with the ModA and TupA proteins, like many archaea. The periplasmic protein of this ABC transporter, WtpA (PF0080), was cloned and expressed in Escherichia coli. Using isothermal titration calorimetry, WtpA was observed to bind tungstate (dissociation constant [K(D)] of 17 +/- 7 pM) and molybdate (K(D) of 11 +/- 5 nM) with a stoichiometry of 1.0 mol oxoanion per mole of protein. These low K(D) values indicate that WtpA has a higher affinity for tungstate than do ModA and TupA and an affinity for molybdate similar to that of ModA. A displacement titration of molybdate-saturated WtpA with tungstate showed that the tungstate effectively replaced the molybdate in the binding site of the protein.     

Association of molybdopterin guanine dinucleotide with Escherichia coli dimethyl sulfoxide reductase: effect of tungstate and a mob mutation.

We have identified the organic component of the molybdenum cofactor in Escherichia coli dimethyl sulfoxide reductase (DmsABC) to be molybdopterin (MPT) guanine dinucleotide (MGD) and have studied the effects of tungstate and a mob mutation on cofactor (Mo-MGD) insertion. Tungstate severely inhibits anaerobic growth of E. coli on a glycerol-dimethyl sulfoxide minimal medium, and this inhibition is partially overcome by overexpression of DmsABC. Isolation and characterization of an oxidized derivative of MGD (form A) from DmsABC overexpressed in cells grown in the presence of molybdate or tungstate indicate that tungstate inhibits insertion of Mo-MGD. No electron paramagnetic resonance evidence for the assembly of tungsten into DmsABC was found between Eh = -450 mV and Eh = +200 mV. The E. coli mob locus is responsible for the addition of a guanine nucleotide to molybdo-MPT (Mo-MPT) to form Mo-MGD. DmsABC does not bind Mo-MPT or Mo-MGD in a mob mutant, indicating that nucleotide addition must precede cofactor insertion. No electron paramagnetic resonance evidence for the assembly of molybdenum into DmsABC in a mob mutant was found between Eh = -450 mV and Eh = +200 mV. These data support a model for Mo-MGD biosynthesis and assembly into DmsABC in which both metal chelation and nucleotide addition to MPT precede cofactor insertion.     

Endogenous metabolism of Azotobacter agilis

Sobek, J. M. (University of Southwestern Louisiana, Lafayette), J. F. Charba, and W. N. Foust. Endogenous metabolism of Azotobacter agilis. J. Bacteriol. 92:687-695. 1966-Ribonucleic acid, deoxyribonucleic acid, cellular carbohydrate, and the cold trichloroacetic acid and acidic alcohol fractions of the cell do not appear to function as endogenous reserves for Azotobacter agilis. The immediate endogenous reserve of cells grown on glucose, acetate, or succinate was poly-beta-hydroxybutyric acid (PHB). Viability of the cells during starvation was dependent upon the initial levels of PHB and the growth substrate. Cells with high initial PHB levels survived longer than cells with lower levels. Cells from succinate-grown cultures had lower PHB levels than cells from glucose-grown cultures, but were capable of maintaining their viability longer. Cellular protein may also serve as a secondary endogenous reserve substrate for this organism.     

Detection of alternative nitrogenases in aerobic gram-negative nitrogen-fixing bacteria.

Strains of aerobic, microaerobic, nonsymbiotic, and symbiotic dinitrogen-fixing bacteria were screened for the presence of alternative nitrogenase (N2ase) genes by DNA hybridization between genomic DNA and DNA encoding structural genes for components 1 of three different enzymes. A nifDK gene probe was used as a control to test for the presence of the commonly occurring Mo-Fe N2ase, a vnfDGK gene probe was used to show the presence of V-Fe N2ase, and an anfDGK probe was used to detect Fe N2ase. Hitherto, all three enzymes have been identified in Azotobacter vinelandii OP, and all but the Fe N2ase are present in Azotobacter chroococcum ATCC 4412 (MCD1). Mo-Fe N2ase and V-Fe N2ase structural genes only were confirmed in this strain and in two other strains of A. chroococcum (ATCC 480 and ATCC 9043). A similar pattern was observed with Azotobacter beijerinckii ATCC 19360 and Azotobacter nigricans ATCC 35009. Genes for all three systems are apparently present in two strains of Azotobacter paspali (ATCC 23367 and ATCC 23833) and also in Azomonas agilis ATCC 7494. There was no good evidence for the existence of any genes other than Mo-Fe N2ase structural genes in several Rhizobium meliloti strains, cowpea Rhizobium strain 32H1, or Bradyrhizobium japonicum. Nitrogenase and nitrogenase genes in Azorhizobium caulinodans behaved in an intermediate fashion, showing (i) the formation of ethane from acetylene under Mo starvation, a characteristic of alternative nitrogenases, and (ii) a surprising degree of cross-hybridization to the vnfDGK, but not the anfDGK, probe. vnfDGK- and anfDGK-like sequences were not detected in two saccharolytic Pseudomonas species or Azospirillum brasilense Sp7. The occurrence of alternative N2ases seems restricted to members of the family Azotobacteraceae among the aerobic and microaerobic diazotrophs tested, suggesting that an ability to cope with O2 when fixing N2 may be an important factor influencing the distribution of alternative nitrogenases.     

The mechanism of action of molybdenum and tungsten upon collagen structures in vivo

The effect of in vivo administration of molybdenum (as sodium molybdate) and tungsten (as sodium tungstate) was investigated in the skin of laboratory rats. It was proved that the amount of both bound molybdenum and tungsten in collagen is relatively small being 0.05 and 0.06 moles per mole respectively. Besides the fraction of firmly bound molybdenum and tungsten a much higher extractable pool of both these metals was found. It was also demonstrated that in vivo shadowing of collagen is caused by the fraction of loosely bound metals. On the other hand pronounced changes were shown in the mechanical properties of connective tissue after molybdate and tungstate administration. Surprisingly, the change in mechanical properties indicated a lower level of cross-linking after the administration of the investigated metals. It is therefore concluded that bitopical binding of molybdenum and tungsten in the collagen structure is unlikely. It also appears that the biological effect of these metals is due to the competition with copper and the interference with the physiological cross-linking reactions based on the partial blockade of lysyloxidase.     

Simultaneous Involvement of a Tungsten-Containing Aldehyde:Ferredoxin Oxidoreductase and a Phenylacetaldehyde Dehydrogenase in Anaerobic Phenylalanine Metabolism

Anaerobic phenylalanine metabolism in the denitrifying betaproteobacterium Aromatoleum aromaticum is initiated by conversion of phenylalanine to phenylacetate, which is further metabolized via benzoyl-coenzyme A (CoA). The formation of phenylacetate is catalyzed by phenylalanine transaminase, phenylpyruvate decarboxylase, and a phenylacetaldehyde-oxidizing enzyme. The presence of these enzymes was detected in extracts of cells grown with phenylalanine and nitrate. We found that two distinct enzymes are involved in the oxidation of phenylacetaldehyde to phenylacetate, an aldehyde:ferredoxin oxidoreductase (AOR) and a phenylacetaldehyde dehydrogenase (PDH). Based on sequence comparison, growth studies with various tungstate concentrations, and metal analysis of the enriched enzyme, AOR was shown to be a tungsten-containing enzyme, necessitating specific cofactor biosynthetic pathways for molybdenum- and tungsten-dependent enzymes simultaneously. We predict from the genome sequence that most enzymes of molybdopterin biosynthesis are shared, while the molybdate/tungstate uptake systems are duplicated and specialized paralogs of the sulfur-inserting MoaD and the metal-inserting MoeA proteins seem to be involved in dedicating biosynthesis toward molybdenum or tungsten cofactors. We also characterized PDH biochemically and identified both NAD⁺ and NADP⁺ as electron acceptors. We identified the gene coding for the enzyme and purified a recombinant Strep-tagged PDH variant. The homotetrameric enzyme is highly specific for phenylacetaldehyde, has cooperative kinetics toward the substrate, and shows considerable substrate inhibition. Our data suggest that A. aromaticum utilizes PDH as the primary enzyme during anaerobic phenylalanine degradation, whereas AOR is not essential for the metabolic pathway. We hypothesize a function as a detoxifying enzyme if high aldehyde concentrations accumulate in the cytoplasm, which would lead to substrate inhibition of PDH.     

Reduced nicotinamide–adenine dinucleotide–nitrite reductase from Azotobacter chroococcum

1. The assimilatory nitrite reductase of the N(2)-fixing bacterium Azotobacter chroococcum was prepared in a soluble form from cells grown aerobically with nitrate as the nitrogen source, and some of its properties have been studied. 2. The enzyme is a FAD-dependent metalloprotein (mol.wt. about 67000), which stoicheiometrically catalyses the direct reduction of nitrite to NH(3) with NADH as the electron donor. 3. NADH-nitrite reductase can exist in two either active or inactive interconvertible forms. Inactivation in vitro can be achieved by preincubation with NADH. Nitrite can specifically protect the enzyme against this inactivation and reverse the process once it has occurred. 4. A. chroococcum nitrite reductase is an adaptive enzyme whose formation depends on the presence of either nitrate or nitrite in the nutrient solution. 5. Tungstate inhibits growth of the microorganism very efficiently, by competition with molybdate, when nitrate is the nitrogen source, but does not interfere when nitrite or NH(3) is substituted for nitrate. The addition of tungstate to the culture media results in the loss of nitrate reductase activity but does not affect nitrite reductase.     

Aerobic Hydrogen Production via Nitrogenase in Azotobacter vinelandii CA6

The diazotroph Azotobacter vinelandii possesses three distinct nitrogenase isoenzymes, all of which produce molecular hydrogen as a by-product. In batch cultures, A. vinelandii strain CA6, a mutant of strain CA, displays multiple phenotypes distinct from its parent: tolerance to tungstate, impaired growth and molybdate transport, and increased hydrogen evolution. Determining and comparing the genomic sequences of strains CA and CA6 revealed a large deletion in CA6''s genome, encompassing genes related to molybdate and iron transport and hydrogen reoxidation. A series of iron uptake analyses and chemostat culture experiments confirmed iron transport impairment and showed that the addition of fixed nitrogen (ammonia) resulted in cessation of hydrogen production. Additional chemostat experiments compared the hydrogen-producing parameters of different strains: in iron-sufficient, tungstate-free conditions, strain CA6''s yields were identical to those of a strain lacking only a single hydrogenase gene. However, in the presence of tungstate, CA6 produced several times more hydrogen. A. vinelandii may hold promise for developing a novel strategy for production of hydrogen as an energy compound.     

Structure of the molybdate/tungstate binding protein mop from Sporomusa ovata

BACKGROUND: Transport of molybdenum into bacteria involves a high-affinity ABC transporter system whose expression is controlled by a repressor protein called ModE. While molybdate transport is tightly coupled to utilization in some bacteria, other organisms have molybdenum storage proteins. One class of putative molybdate storage proteins is characterized by a sequence consisting of about 70 amino acids (Mop). A tandem repeat of Mop sequences also constitutes the molybdate binding domain of ModE. RESULTS: We have determined the crystal structure of the 7 kDa Mop protein from the methanol-utilizing anaerobic eubacterium Sporomusa ovata grown in the presence of molybdate and tungstate. The protein occurs as highly symmetric hexamers binding eight oxyanions. Each peptide assumes a so-called OB fold, which has previously also been observed in ModE. There are two types of oxyanion binding sites in Mo at the interface between two or three peptides. All oxyanion binding sites were found to be occupied by WO(4) rather than MoO(4). CONCLUSIONS: The biological function of proteins containing only Mop sequences is unknown, but they have been implicated in molybdate homeostasis and molybdopterin cofactor biosynthesis. While there are few indications that the S. ovata Mop binds pterin, the structure suggests that only the type-1 oxyanion binding sites would be sufficiently accessible to bind a cofactor. The observed occupation of the oxyanion binding sites by WO(4) indicates that Mop might also be involved in controlling intracellular tungstate levels.     

Sulfate inhibition of molybdenum-dependent nitrogen fixation by planktonic cyanobacteria under seawater conditions: a non-reversible effect

The trace element molybdenum is a central component of several enzymes essential to bacterial nitrogen metabolism, including nitrogen fixation. Despite reasonably high dissolved concentrations (for a trace metal) of molybdenum in seawater, evidence suggests that its biological reactivity and availability are lower in seawater than in freshwater. We have previously argued that this difference is related to an inhibition in the uptake of molybdate (the thermodynamically stable form of molybdenum in oxic natural waters) by sulfate, a stereochemically similar ion. Low molybdenum availability may slow the growth rate of nitrogen-fixing cyanobacteria, and in combination with an ecological control such as grazing by zooplankton, keep fixation rates very low in even strongly nitrogen-limited coastal marine ecosystems. Here we present results from a seawater mesocosm experiment where the molybdenum concentration was increased 10-fold under highly nitrogen-limited conditions. The observed effects on nitrogen-fixing cyanobacterial abundance and nitrogen-fixation inputs were much smaller than expected. A follow-up experiment with sulfate and molybdenum additions to freshwater microcosms showed that sulfate (at seawater concentrations) greatly reduced nitrogen fixation by cyanobacteria and that additions of molybdenum to the levels present in the seawater mesocosm experiment only slightly reversed this effect. In light of these results, we re-evaluated our previous work on the uptake of radio-labeled molybdenum by lake plankton and by cultures of heterocystic cyanobacteria. Our new interpretation indicates that sulfate at saline estuarine levels (>8–10 mM) up to seawater (28 mM) concentrations does inhibit molybdenum assimilation. However, the maximum molybdenum uptake rate (V _max) was a function of the sulfate concentration, with lower V _max values at higher sulfate levels. This indicates that this inhibition is not fully reversed at some saturating level of molybdenum, as assumed in a simple competitive inhibition model. A multi-enzyme, mixed kinetics model with two or more uptake enzyme systems activated in response to the environmental sulfate and molybdate conditions may better explain the repressive effect of sulfate on Mo-mediated processes such as nitrogen fixation.