Nitrosopumilus maritimus genome reveals unique mechanisms for nitrification and autotrophy in globally distributed marine crenarchaea.

Ammonia-oxidizing archaea are ubiquitous in marine and terrestrial environments and now thought to be significant contributors to carbon and nitrogen cycling. The isolation of Candidatus "Nitrosopumilus maritimus" strain SCM1 provided the opportunity for linking its chemolithotrophic physiology with a genomic inventory of the globally distributed archaea. Here we report the 1,645,259-bp closed genome of strain SCM1, revealing highly copper-dependent systems for ammonia oxidation and electron transport that are distinctly different from known ammonia-oxidizing bacteria. Consistent with in situ isotopic studies of marine archaea, the genome sequence indicates N. maritimus grows autotrophically using a variant of the 3-hydroxypropionate/4-hydroxybutryrate pathway for carbon assimilation, while maintaining limited capacity for assimilation of organic carbon. This unique instance of archaeal biosynthesis of the osmoprotectant ectoine and an unprecedented enrichment of multicopper oxidases, thioredoxin-like proteins, and transcriptional regulators points to an organism responsive to environmental cues and adapted to handling reactive copper and nitrogen species that likely derive from its distinctive biochemistry. The conservation of N. maritimus gene content and organization within marine metagenomes indicates that the unique physiology of these specialized oligophiles may play a significant role in the biogeochemical cycles of carbon and nitrogen.

Cell-free nitrogenase and hydrogenase from actinorhizal root nodules.

Field-grown alder (Alnus glutinosa) root nodules were disrupted in liquid nitrogen to release the actinomycete endophytes. The endophytes were broken by mild sonic oscillation and yielded a cell-free nitrogenase preparation capable of reducing acetylene and protons. In addition, the preparation carried a cell-free uptake hydrogenase.

Evidence for involvement of copper ions and redox state in regulation of butane monooxygenase in Pseudomonas butanovora.

Pseudomonas butanovora possesses an alcohol-inducible alkane monooxygenase, butane monooxygenase (BMO), that initiates growth on C(2)-C(9) alkanes. A lacZ transcriptional reporter strain, P. butanovora bmoX::lacZ, in which the BMO promoter controls the expression of beta-galactosidase activity, was used to show that 1-butanol induced the BMO promoter in the presence or absence of O(2) when lactate-grown, BMO-repressed cells were washed free of lactate and incubated in NH(4)Cl-KNa phosphate buffer. In contrast, when lactate-grown cells of the reporter strain were incubated in phosphate buffer containing the mineral salts of standard growth medium, 1-butanol-dependent induction was significantly repressed at low O(2) (1 to 2% [vol/vol]) and totally repressed under anoxic conditions. The repressive effect of the mineral salts was traced to its copper content. In cells exposed to 1% (vol/vol) O(2), CuSO(4) (0.5 microM) repressed 1-butanol-dependent induction of beta-galactosidase activity. Under oxic conditions (20% O(2) [vol/vol]), significantly higher concentrations of CuSO(4) (2 microM) were required for almost complete repression of induction in lactate-grown cells. A combination of the Cu(2+) reducing agent Na ascorbate (100 microM) and CuSO(4) (0.5 microM) repressed the induction of beta-galactosidase activity under oxic conditions to the same extent that 0.5 microM CuSO(4) alone repressed it under anoxic conditions. Under oxic conditions, 2 microM CuSO(4) repressed induction of the BMO promoter less effectively in butyrate-grown cells of the bmoX::lacZ strain and of an R8-bmoX::lacZ mutant reporter strain with a putative BMO regulator, BmoR, inactivated. Under anoxic conditions, CuSO(4) repression remained highly effective, regardless of the growth substrate, in both BmoR-positive and -negative reporter strains.

Purification and properties of the particulate hydrogenase from the bacteroids of soybean root nodules.

The uptake hydrogenase (hydrogen:ferricytochrome c3 oxidoreductase, EC 1.12.2.1) from the bacteroids of soybean root nodules infected with Rhizobium japonicum 110 has been purified and characterized. Bacteroids were prepared, then broken by sonication. The particulate enzyme was solubilized by treatment with Triton X-100 and further purified by polyethylene glycol fractionation, DEAE-cellulose and Sephadex G-100 chromatography. The specific activity has been increased 196-fold to 19.6 units/mg protein. The molecular weight is 63 300 as determined by gel filtration and 65 300 as determined by SDS-polyacrylamide gel electrophoresis, indicating that the enzyme is a monomer. The enzyme is O2 sensitive, with a half-life of 70 min when exposed to air. The pH optimum of the solubilized enzyme is near 5.5; the Km for H2 is 1.4 microM. Suitable electron acceptors are methylene blue, ferricyanide, 2,6-dichlorophenolindophenol, and cytochrome c. Benzyl viologen is reduced slowly; methyl viologen, NAD(P)+, FAD, FMN, and O2 are not reduced. The optimum temperature for activity is 65-70 degrees C with an activation energy of 9.2 kcal. H2 evolution by the enzyme has been demonstrated. The hydrogenase is well-suited to function in an environment where all the available H2 is generated in situ.

Kinetic analysis of the interaction of nitric oxide with the membrane-associated, nickel and iron-sulfur-containing hydrogenase from Azotobacter vinelandii.

The effects of nitric oxide (NO) on the membrane-associated form of the nickel and iron-sulfur-containing hydrogenase from Azotobacter vinelandii have been investigated. In the presence of H2 and an electron acceptor (turnover conditions), NO acts as a noncompetitive inhibitor vs. methylene blue (Ki = 12 microM). There is no element of competition between NO and H2, implying that the site of NO action is not the H2-activating site of the hydrogenase. When the membrane-associated hydrogenase is incubated under non-turnover conditions, the enzyme is irreversibly inactivated by NO in a time-dependent process. The inactivation is a non-saturable, pseudo-first-order process which is consistent with a direct chemical reaction between NO and the hydrogenase. Kinetic evidence is presented which is compatible with an interaction between NO and a redox-active component other than the H2-activating site on the enzyme. The complex inhibition pattern of NO has been interpreted in terms of two distinct interactions of NO with iron-sulfur centers of the hydrogenase.

Comparison of isotope exchange, H2 evolution, and H2 oxidation activities of Azotobacter vinelandii hydrogenase.

Azotobacter vinelandii hydrogenase was purified aerobically with a 35% yield. The purified enzyme catalyzed H2 oxidation at much greater velocity than H2 evolution. There was a large difference in activation energy for the two reactions. EA was 10 kcal/mol for H2 oxidation and 22 kcal/mol for evolution. This difference in activation energies between the two reactions means that the ratio of oxidation velocity to evolution velocity drops from 70 at 33 degrees C to 8 at 48 degrees C. With D2 and H2O as substrates, both membranes and purified enzyme produced only H2 and no HD in the isotope exchange reaction. The velocity of isotope exchange was equal to the velocity of H2 evolution from reduced methyl viologen, indicating that the two reactions share the same rate-limiting step. D2 and H2 inhibited H2 evolution, but D2 did not inhibit isotope exchange. We conclude that H2 and D2 do not inhibit H2 evolution by competing with H+ for the active site of the reduced enzyme. The Km for D2 in isotope exchange is 40-times greater than its Km in D2 oxidation. The difference in Km cannot be accounted for by differences in kcat. We propose that redox environment regulates hydrogenase's affinity for D2 (and likely H2 as well).

Aerobic, inactive forms of Azotobacter vinelandii hydrogenase: activation kinetics and insensitivity to C2H2 inhibition.

Azotobacter vinelandii hydrogenase (EC class 1.12), either purified or membrane-associated, was obtained aerobically in an inactive state. The kinetics of activation by treatment with a reductant (H2 or dithionite) were determined. Three distinct phases of the activation were observed. Aerobically prepared, inactive hydrogenase was insensitive to acetylene inhibition, but could be rendered acetylene-sensitive by reduction with dithionite. These findings indicate that acetylene inhibition of hydrogenase requires catalytically active enzyme.

Kinetic characterization of the inactivation of ammonia monooxygenase in Nitrosomonas europaea by alkyne, aniline and cyclopropane derivatives.

The kinetic mechanisms of seven inactivators of ammonia oxidation activity in cells of the nitrifying bacterium, Nitrosomonas europaea were investigated. The effects of the inactivators were specific for ammonia monooxygenase (AMO) which oxidizes ammonia to hydroxylamine. The aniline derivatives, 1,3-phenylenediamine and p-anisidine, were potent inactivators of AMO while other derivatives were ineffective as inactivators. Two cyclopropane derivatives, 1, 2-dimethylcyclopropane and cyclopropyl bromide, were inactivators while cyclopropane was not an inactivator. The mechanisms of three alkynes, 1-hexyne, 3-hexyne, and acetylene, were also examined. For all seven compounds, the inactivation of AMO was irreversible, time-dependent, first-order, and dependent on catalytic turnover. Saturation of the rate of inactivation was indicated for p-anisidine (kinact=2.85 min-1; KI=1.0 mM) and cyclopropyl bromide (kinact=4.4 min-1; KI=97 microM), but not for any of the remaining five inactivators, including acetylene. Ammonia slowed the rate of inactivation for acetylene and cyclopropyl bromide, but enhanced the rate of inactivation for the remaining inactivators. All seven compounds appear to be mechanism-based inactivators of AMO.

Sequence of hcy, a gene encoding cytochrome c-554 from Nitrosomonas europaea.

Cytochrome c-554 (Cyt c-554) was purified from Nitrosomonas europaea. The N-terminal and internal amino acid sequences were determined. A synthetic oligodeoxyribonucleotide primer based on the N-terminal sequence was used to construct a PCR clone. This clone was used to identify genomic DNA fragments containing the gene encoding Cyt c-554. We determined the nucleotide sequence of this gene and named it hcy for hydroxylamine oxidoreductase-linked cytochrome.

Purification to homogeneity of Azotobacter vinelandii hydrogenase: a nickel and iron containing alpha beta dimer.

Azotobacter vinelandii hydrogenase has been purified to homogeneity from membranes. The enzyme was solubilized with Triton X-100 followed by ammonium sulfate-hexane extractions to remove lipids and detergent. The enzyme was then purified by carboxymethyl-Sepharose and octyl-Sepharose column chromatography. All purification steps were performed under anaerobic conditions in the presence of dithionite and dithiothreitol. The enzyme was purified 143-fold from membranes to a specific activity of 124 mumol of H2 uptake . min-1 . mg protein-1. Nondenaturing polyacrylamide gel electrophoresis of the hydrogenase revealed a single band which stained for both activity and protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands corresponding to peptides of 67,000 and 31,000 daltons. Densitometric scans of the SDS-gel indicated a molar ratio of the two bands of 1.07 +/- 0.05. The molecular weight of the native enzyme was determined by three different methods. While gel permeation gave a molecular weight of 53,000, sucrose density gradient centrifugation and native polyacrylamide gel electrophoresis gave molecular weights of 98,600 +/- 10,000 and 98,600 +/- 2,000, respectively. We conclude that the A. vinelandii hydrogenase is an alpha beta dimer (98,000 daltons) with subunits of 67,000 and 31,000 daltons. Analyses for nickel and iron indicated 0.68 +/- 0.01 mol Ni/mol hydrogenase and 6.6 +/- 0.5 mol Fe/mol hydrogenase. The isoelectric point of the enzyme was 6.1 +/- 0.01. In addition, several catalytic properties of the enzyme have been examined. The Km for H2 was 0.86 microM, and H2 evolution was observed in the presence of reduced methyl viologen. The pH profile of enzyme activity with methylene blue as the electron acceptor has been determined, along with the Km and Vmax for various electron acceptors.

Investigation of the H2-oxidizing activities of Alcaligenes eutrophus H16 membranes with artificial electron acceptors, respiratory inhibitors and redox-spectroscopic procedures.

Membrane particles, prepared from cells of Alcaligenes eutrophus H16 by lysozyme treatment and 100 000 X g centrifugation, catalyzed a H2-dependent reduction of methylene blue, menadione, 2,6-dichlorophenol-indophenol (DCPIP), and O2. While the reaction with methylene blue was not altered by 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO), the H2-dependent reductions of menadione, DCPIP and O2 were strongly inhibited, indicating that in these reaction components of the respiratory chain other than the membrane-bound hydrogenase were involved. The effect of pentane extraction of membranes on the H2-dependent reductions of methylene blue and menadione were different from those of DCPIP and O2. This suggested that ubiquinone might not be involved in the pathway of the electrons from H2 to methylene blue or menadione, while it might be involved in the pathway to DCPIP and O2. Because the H2-dependent reduction of menadione is sensitive to HQNO, it follows that HQNO might bind to a site upstream of ubiquinone. Further evidence for this hypothesis came from a new technique to record UV and visible redox-difference spectra of membranes under the conditions of a steady-state electron flow. HQNO did not increase the reduction level of ubiquinone relative to the cytochromes. Neither HQNO nor menadione had any influence on the redox difference patterns of the cytochromes as determined with low temperature and room temperature spectroscopy.

Isolation and characterization of alkane-utilizing Nocardioides sp. strain CF8.

A butane-utilizing bacterial strain CF8 was isolated and identified as a member of the genus Nocardioides from chemotaxonomic and 16S rDNA sequence analysis. Strain CF8 grew on alkanes ranging from C(2) to C(16) in addition to butane and various other substrates including primary alcohols, carboxylic acids, and phenol. Butane degradation by strain CF8 was inactivated by light, a specific inactivator of copper-containing monooxygenases. The unique thermal aggregation phenomenon of acetylene-binding polypeptides was also observed for strain CF8. These results suggest that butane monooxygenase in strain CF8 is a third example of the copper-containing monooxygenases previously described in ammonia oxidizers and methanotrophs.

Differential regulation of amoA and amoB gene copies in Nitrosomonas europaea.

Nitrosomonas europaea contains two nearly identical copies of the operon, amoCAB, which encodes the ammonia monooxygenase (AMO) enzyme. Cells of N. europaea containing single mutations in either amoA or amoB gene copies were incubated in ammonium both prior to and after exposure to acetylene or light. For each strain, the O(2) consumption rates and amounts of AmoA polypeptide, the active site-containing subunit of AMO, produced in each strain were determined. Strains carrying a mutation in either the amoA(2) or amoB(2) genes responded similarly to wild-type cells, but the strains carrying mutations in the amoA(1) or amoB(1) genes responded differently from the wild-type, or from each other. These results suggest that the copies of amoA and amoB are differentially regulated upon exposure to different external stimuli.

Transcription of the amoC, amoA and amoB genes in Nitrosomonas europaea and Nitrosospira sp. NpAV.

Nitrifying bacteria such as Nitrosomonas europaea and Nitrosospira sp. NpAV use ammonia monooxygenase (AMO) for oxidation of their primary growth substrate, ammonia. Two polypeptides of AMO are coded for by contiguous genes, amoA and amoB, which are preceded by a third gene, amoC. The amoCAB clusters are present in multiple copies in nitrifying bacteria of the beta subdivision. These bacteria also have one amoC copy that is not adjacent to a copy of amoAB. The seven known amoC genes in different nitrifiers code for similar polypeptides (> 68%). Reverse transcriptase-polymerase chain reactions and Northern blots indicated that amoC from the amoCAB cluster is contained on a transcript with amoAB. Two other transcripts were detected with amo probes and may be a product of processing of the amoCAB mRNA or independent transcripts.

Propionate inactivation of butane monooxygenase activity in 'Pseudomonas butanovora': biochemical and physiological implications.

Butane monooxygenase (BMO) catalyses the oxidation of alkanes to alcohols in the alkane-utilizing bacterium 'Pseudomonas butanovora'. Incubation of alkane-grown 'P. butanovora' with butyrate or propionate led to irreversible time- and O2-dependent loss of BMO activity. In contrast, BMO activity was unaffected by incubation with lactate or acetate. Chloramphenicol inhibited the synthesis of new BMO, but did not change the kinetics of propionate-dependent BMO inactivation, suggesting that the propionate effect was not simply due to it acting as a repressor of BMO transcription. BMO was protected from propionate-dependent inactivation by the presence of its natural substrate, butane. Although both the time and O2 dependency of propionate inactivation of BMO imply that propionate might be a suicide substrate, no evidence was obtained for BMO-dependent propionate consumption, or 14C labelling of BMO polypeptides by [2-(14)C]propionate during inactivation. Propionate-dependent BMO inactivation was also explored in mutant strains of 'P. butanovora' containing single amino acid substitutions in the alpha-subunit of the BMO hydroxylase. Propionate-dependent BMO inactivation in two mutant strains with amino acid substitutions close to the catalytic site differed from wild-type (one was more sensitive and the other less), providing further evidence that propionate-dependent inactivation involves interaction with the BMO catalytic site. A putative model is presented that might explain propionate-dependent inactivation of BMO when framed within the context of the catalytic cycle of the closely related enzyme, soluble methane monooxygenase.

Product repression of alkane monooxygenase expression in Pseudomonas butanovora.

Physiological and regulatory mechanisms that allow the alkane-oxidizing bacterium Pseudomonas butanovora to consume C2 to C8 alkane substrates via butane monooxygenase (BMO) were examined. Striking differences were observed in response to even- versus odd-chain-length alkanes. Propionate, the downstream product of propane oxidation and of the oxidation of other odd-chain-length alkanes following beta-oxidation, was a potent repressor of BMO expression. The transcriptional activity of the BMO promoter was reduced with as little as 10 microM propionate, even in the presence of appropriate inducers. Propionate accumulated stoichiometrically when 1-propanol and propionaldehyde were added to butane- and ethane-grown cells, indicating that propionate catabolism was inactive during growth on even-chain-length alkanes. In contrast, propionate consumption was induced (about 80 nmol propionate consumed.min(-1).mg protein(-1)) following growth on the odd-chain-length alkanes, propane and pentane. The induction of propionate consumption could be brought on by the addition of propionate or pentanoate to the growth medium. In a reporter strain of P. butanovora in which the BMO promoter controls beta-galactosidase expression, only even-chain-length alcohols (C2 to C8) induced beta-galactosidase following growth on acetate or butyrate. In contrast, both even- and odd-chain-length alcohols (C3 to C7) were able to induce beta-galactosidase following the induction of propionate consumption by propionate or pentanoate.

Effects of dichloroethene isomers on the induction and activity of butane monooxygenase in the alkane-oxidizing bacterium "Pseudomonas butanovora".

We examined cooxidation of three different dichloroethenes (1,1-DCE, 1,2-trans DCE, and 1,2-cis DCE) by butane monooxygenase (BMO) in the butane-utilizing bacterium "Pseudomonas butanovora." Different organic acids were tested as exogenous reductant sources for this process. In addition, we determined if DCEs could serve as surrogate inducers of BMO gene expression. Lactic acid supported greater rates of oxidation of the three DCEs than the other organic acids tested. The impacts of lactic acid-supported DCE oxidation on BMO activity differed among the isomers. In intact cells, 50% of BMO activity was irreversibly lost after consumption of approximately 20 nmol mg protein(-1) of 1,1-DCE and 1,2-trans DCE in 0.5 and 5 min, respectively. In contrast, a comparable loss of activity required the oxidation of 120 nmol 1,2-cis DCE mg protein(-1). Oxidation of similar amounts of each DCE isomer ( approximately 20 nmol mg protein(-1)) produced different negative effects on lactic acid-dependent respiration. Despite 1,1-DCE being consumed 10 times faster than 1,2,-trans DCE, respiration declined at similar rates, suggesting that the product(s) of oxidation of 1,2-trans DCE was more toxic to respiration than 1,1-DCE. Lactate-grown "P. butanovora" did not express BMO activity but gained activity after exposure to butane, ethene, 1,2-cis DCE, or 1,2-trans DCE. The products of BMO activity, ethene oxide and 1-butanol, induced lacZ in a reporter strain containing lacZ fused to the BMO promoter, whereas butane, ethene, and 1,2-cis DCE did not. 1,2-trans DCE was unique among the BMO substrates tested in its ability to induce lacZ expression.

Rhizobium japonicum hydrogenase: purification to homogeneity from soybean nodules, and molecular characterization.

Rhizobium japonicum hydrogenase was purified to homogeneity from soybean root nodules by four column chromatography steps after solubilization from membranes by treatment with a nonionic detergent. The specific activity was from 40 to 65 mumol H2 oxidized min-1 mg protein-1 and was increased 450-fold relative to that in bacteroids. The yield of activity was from 7 to 12%. The molecular weight of the native enzyme was 104,000 as determined by sucrose density gradient centrifugation. Electrophoresis in the presence of sodium dodecyl sulfate revealed two subunits with molecular weights of 64,000 and 35,000, indicating an alpha beta subunit structure. The amino acid content of the protein indicated 20 cysteine residues. Analysis of the metal content indicated 0.59 +/- 0.06 mol Ni/mol hydrogenase and 6.5 +/- 1.2 mol Fe/mol hydrogenase. Antisera prepared to the hydrogenase cross-reacted with the enzyme in bacteroid extracts at all stages of the purification but did not cross-react with extracts of Alcaligenes eutrophus grown under chemolithotrophic conditions. The similarity of rhizobial hydrogenase to the particulate hydrogenases of A. eutrophus and A. latus is discussed.

Effects of alcohols on the reactivity and stability of Azotobacter vinelandii hydrogenase.

The effects of alcohols on the reactivity of Azotobacter vinelandii hydrogenase were investigated. Hydrogenase catalyzed H2 oxidation coupled to methylene blue, benzyl viologen, or phenazine methosulfate when in the presence of solvents containing 15 or 40% ethanol or 40% methanol or 2-propanol. In general, the Km's for the electron acceptors were increased substantially by the presence of the alcohols, while the Km for H2 was not altered in a solvent containing 40% ethanol. Calculation of the apparent maximum velocities for H2 oxidation in the presence of alcohols indicated that the maximum velocity was not decreased in most cases. In contrast, the rates of both H2 evolution and isotope exchange by hydrogenase were substantially decreased when solvent containing alcohol. Hydrogenase was inactivated by 100% ethanol with a half-life of 17 s. Hydrogenase from A. vinelandii was stable when stored in alcohol/buffer solvents at 20 degrees C or below. However, the thermal stability of hydrogenase was greatly decreased by inclusion of an alcohol in the solvent. When incubated at 55 degrees C in a solvent containing 40% ethanol, activity decreased in a first-order process with a half-life of 7 min. When incubated at the same temperature in aqueous buffer, no loss of activity was observed over 30 min.