Capacity for nitrate respiration as a new aspect of metabolism of the filamentous sulfur bacteria of the genus Thiothrix

Capacity of Thiothrix species (T. lacustris strains AS and BL^T, T. caldifontis G1^T, T. unzii A1^T, and T. eikelboomii AR3^T) for anaerobic respiration in the presence of nitrate was discovered. The dynamics of nitrate reduction to nitrite was studied and the coupling of this process to thiosulfate oxidation was shown. The investigated Thiothrix representatives performed anaerobic thiosulfate-dependent reduction of nitrate only to nitrite. The presence of the narG gene, encoding the α-subunit of respiratory nitrate reductase NarGHI, was revealed in the cells. The induction of this gene expression was shown for the T. lacustris strain AS under anaerobic conditions of growth. The activity of several enzymes involved in the conversion of reduced sulfur compounds was determined.     

Stoichiometry and kinetics of microbial toluene degradation under denitrifying conditions

Batch experiments were carried out to investigate the stoichiometry and kinetics of microbial degradation of toluene under denitrifying conditions. The inoculum originated from a mixture of sludges from sewage treatment plants with alternating nitrification and denitrification. The culture was able to degrade toluene under anaerobic conditions in the presence of nitrate, nitrite, nitric oxide, or nitrous oxide. No degradation occurred in the absence of Noxides. The culture was also able to use oxygen, but ferric iron could not be used as an electron acceptor. In experiments with¹⁴C-labeled toluene, 34%±8% of the carbon was incorporated into the biomass, while 53%±10% was recovered as¹⁴CO₂, and 6%±2% remained in the medium as nonvolatile water soluble products. The average consumption of nitrate in experiments, where all the reduced nitrate was recovered as nitrite, was 1.3±0.2 mg of nitrate-N per mg of toluene. This nitrate reduction accounted for 70% of the electrons donated during the oxidation of toluene. When nitrate was reduced to nitrogen gas, the consumption was 0.7±0.2 mg per mg of toluene, accounting for 97% of the donated electrons. Since the ammonia concentration decreased during degradation, dissimilatory reduction of nitrate to ammonia was not the reductive process. The degradation of toluene was modelled by classical Monod kinetics. The maximum specific rate of degradation, k, was estimated to be 0.71 mg toluene per mg of protein per hour, and the Monod saturation constant, K _s , to be 0.2 mg toluene/l. The maximum specific growth rate, _max , was estimated to be 0.1 per hour, and the yield coefficient, Y, was 0.14 mg protein per mg toluene.Abbreviations NVWP Non Volatile Water-soluble Products     

NarK enhances nitrate uptake and nitrite excretion in Escherichia coli.

narK mutants of Escherichia coli produce wild-type levels of nitrate reductase but, unlike the wild-type strain, do not accumulate nitrite when grown anaerobically on a glucose-nitrate medium. Comparison of the rates of nitrate and nitrite metabolism in cultures growing anaerobically on glucose-nitrate medium revealed that a narK mutant reduced nitrate at a rate only slightly slower than that in the NarK+ parental strain. Although the specific activities of nitrate reductase and nitrite reductase were similar in the two strains, the parental strain accumulated nitrite in the medium in almost stoichiometric amounts before it was further reduced, while the narK mutant did not accumulate nitrite in the medium but apparently reduced it as rapidly as it was formed. Under conditions in which nitrite reductase was not produced, the narK mutant excreted the nitrite formed from nitrate into the medium; however, the rate of reduction of nitrate to nitrite was significantly slower than that of the parental strain or that which occurred when nitrite reductase was present. These results demonstrate that E. coli is capable of taking up nitrate and excreting nitrite in the absence of a functional NarK protein; however, in growing cells, a functional NarK promotes a more rapid rate of anaerobic nitrate reduction and the continuous excretion of the nitrite formed. Based on the kinetics of nitrate reduction and of nitrite reduction and excretion in growing cultures and in washed cell suspensions, it is proposed that the narK gene encodes a nitrate/nitrite antiporter which facilitates anaerobic nitrate respiration by coupling the excretion of nitrite to nitrate uptake. The failure of nitrate to suppress the reduction of trimethylamine N-oxide in narK mutants was not due to a change in the level of trimethylamine N-oxide reductase but apparently resulted from a relative decrease in the rate of anaerobic nitrate reduction caused by the loss of the antiporter system.     

Effect of light and darkness on nitrate assimilation by Rhodopseudomonas capsulata E1F1

The photosynthetic nonsulfur purple bacterium Rhodopseudomonas capsulata strain E1F1 assimilated nitrate or nitrite only in illuminated cultures under anaerobic conditions. The bacterial cells grew aerobically in the dark only when ammonia or other forms of reduced nitrogen were present in the medium. However, nitrate reductase was detected either in light-anaerobic or in dark-aerobic conditions upon addition of nitrate to the media. Changes from light-anaerobic to dark-aerobic conditions and vice versa markedly influenced growth, nitrate uptake and the nitrate reductase levels. Growth on nitrate in the light and nitrate reductase activity were dependent on the presence of molybdenum in the medium whereas the addition of tungstate inhibited both growth and enzyme activity.     

Nitrogenase activity and nitrate respiration inAzospirillum spp.

The interaction between nitrate respiration and nitrogen fixation inAzospirillum lipoferum andA. brasilense was studied. All strains examined were capable of nitrogen fixation (acetylene reduction) under conditions of severe oxygen limitation in the presence of nitrate. A lag phase of about 1 h was observed for both nitrate reduction and nitrogenase activity corresponding to the period of induction of the dissimilatory nitrate reductase. Nitrogenase activity ceased when nitrate was exhausted suggesting that the reduction of nitrate to nitrite, rather than denitrification (the further reduction of nitrite to gas) is coupled to nitrogen fixation. The addition of nitrate to nitrate reductase negative mutants (nr^-) ofAzospirillum did not stimulate nitrogenase activity. Under oxygen-limited conditionsA. brasilense andA. lipoferum were also shown to reduce nitrate to ammonia, which accumulated in the medium. Both species, including strains ofA. brasilense which do not possess a dissimilatory nitrite reductase (nir^-) were also capable of reducing nitrous oxide to N₂.     

Light and Dark Controls of Nitrate Reduction in Wheat (Triticum aestivum L.) Protoplasts

Protoplasts were isolated from the leaves of nitrate-cultured wheat (Triticum aestivum L. var. Frederick) seedlings. When incubated in the dark, protoplasts accumulated nitrite under anaerobic, but not under aerobic, conditions. The assimilation of [¹⁵N]nitrite by protoplasts was strictly light-dependent, and no loss of nitrite from the assay medium was observed under dark aerobic conditions. Therefore, the absence of nitrite accumulation under dark aerobic conditions was the result of an O₂ inhibition of nitrate reduction and not a stimulation of nitrite reduction. In the presence of antimycin A, protoplasts accumulated nitrite under dark aerobic conditions. The oxygen inhibition of nitrate reduction was apparently due to a competition between nitrate reduction and dark respiration for cytoplasmic-reducing equivalents.     

Anaerobic co-reduction of chromate and nitrate by bacterial cultures of Staphylococcus epidermidis L-02

Industrial wastewater is often polluted by Cr(VI) compounds, presenting a serious environmental problem. This study addresses the removal of toxic, mutagenic Cr(VI) by means of microbial reduction to Cr(III), which can then be precipitated as oxides or hydroxides and extracted from the aquatic system. A strain of Staphylococcus epidermidis L-02 was isolated from a bacterial consortium used for the remediation of a chromate-contaminated constructed wetland system. This strain reduced Cr(VI) by using pyruvate as an electron donor under anaerobic conditions. The aims of the present study were to investigate the specific rate of Cr(VI) reduction by the strain L-02, the effects of chromate and nitrate (available as electron acceptors) on the strain, and the interference of chromate and nitrate reduction processes. The presence of Cr(VI) decreased the growth rate of the bacterium. Chromate and nitrate reduction did not occur under sterile conditions but was observed during tests with the strain L-02. The presence of nitrate increased both the specific Cr(VI) reduction rate and the cell number. Under denitrifying conditions, Cr(VI) reduction was not inhibited by nitrite, which was produced during nitrate reduction. The average specific rate of chromate reduction reached 4.4 μmol Cr 10¹⁰ cells⁻¹ h⁻¹, but was only 2.0 μmol Cr 10¹⁰ cells⁻¹ h⁻¹ at 20 °C. The maximum specific rate was as high as 8.8–9.8 μmol Cr 10¹⁰ cells⁻¹ h⁻¹. The role of nitrate in chromate reduction is discussed.     

Effects of non-steady-state iron limitation on nitrogen assimilatory enzymes in the marine diatom thalassiosira weissflogii (BACILLARIOPHYCEAE)

Since the recognition of iron‐limited high nitrate (or nutrient) low chlorophyll (HNLC) regions of the ocean, low iron availability has been hypothesized to limit the assimilation of nitrate by diatoms. To determine the influence of non‐steady‐state iron availability on nitrogen assimilatory enzymes, cultures of Thalassiosira weissflogii (Grunow) Fryxell et Hasle were grown under iron‐limited and iron‐replete conditions using artificial seawater medium. Iron‐limited cultures suffered from decreased efficiency of PSII as indicated by the DCMU‐induced variable fluorescence signal (F_v/F_m). Under iron‐replete conditions, in vitro nitrate reductase (NR) activity was rate limiting to nitrogen assimilation and in vitro nitrite reductase (NiR) activity was 50‐fold higher. Under iron limitation, cultures excreted up to 100 fmol NO₂^?·cell^?1·d^?1 (about 10% of incorporated N) and NiR activities declined by 50‐fold while internal NO₂^? pools remained relatively constant. Activities of both NR and NiR remained in excess of nitrogen incorporation rates throughout iron‐limited growth. One possible explanation is that the supply of photosynthetically derived reductant to NiR may be responsible for the limitation of nitrogen assimilation at the NO₂^? reduction step. Urease activity showed no response to iron limitation. Carbon:nitrogen ratios were equivalent in both iron conditions, indicating that, relative to carbon, nitrogen was assimilated at similar rates whether iron was limiting growth or not. We hypothesize that, diatoms in HNLC regions are not deficient in their ability to assimilate nitrate when they are iron limited. Rather, it appears that diatoms are limited in their ability to process photons within the photosynthetic electron transport chain which results in nitrite reduction becoming the rate‐limiting step in nitrogenassimilation.     

Anaerobic degradation of toluene in denitrifying Pseudomonas sp.: indication for toluene methylhydroxylation and benzoyl-CoA as central aromatic intermediate

The anaerobic degradation of toluene has been studied with whole cells and by measuring enzyme activities. Cultures of Pseudomonas strain K 172 were grown in mineral medium up to a cell density of 0.5 g of dry cells per liter in fed-batch culture with toluene and nitrate as the sole carbon and energy sources. A molar growth yield of 57 g of cell dry matter formed per mol toluene totally consumed was determined. The mean generation time was 24 h. The redox balance between toluene consumed (oxidation and cell material synthesis) and nitrate consumed (reduction to nitrogen gas and assimilation as NH₃) was 77% of expectation if toluene was completely oxidized; this indicated that the major amount of toluene was mineralized to CO₂. It was tested whether the initial reaction in anaerobic toluene degradation was a carboxylation or a dehydrogenation (anaerobic hydroxylation); the hypothetical carboxylated or hydroxylated intermediates were tested with whole cells applying the method of simultanous adaptation: cells pregrown on toluene degraded benzyl alcohol, benzaldehyde, and benzoic acid without lag, 4-hydroxybenzoate and p-cresol with a 90 min lag phase and phenylacetate after a 200 min lag phase. The cells were not at all adapted to degrade 2-methylbenzoate, 4-methylbenzoate, o-cresol, and m-cresol, nor did these compounds support growth within a few days after inoculation with cells grown on toluene. In extracts of cells anaerobically grown on toluene, benzyl alcohol dehydrogenase, benzaldehyde dehydrogenase, and benzoyl-CoA synthetase (AMP forming) activities were present. The data (1) conclusively show anaerobic growth of a pure culture on tolucne; (2) suggest that toluene is anaerobically degraded via benzoyl-CoA; (3) imply that water functions as the source of the hydroxyl group in a toluene methylhydroxylase reaction.     

Effect of aerobic and anaerobic conditions on the in vivo nitrate reductase assay in spinach leaves

¹⁵N-labelled nitrate was used to show that nitrate reduction by leaf discs in darkness was suppressed by oxygen, whereas nitrite present within the cell could be reduced under aerobic dark conditions. In other experiments, unlabelled nitrite, allowed to accumulate in the tissue during the dark anaerobic reduction of nitrate was shown by chemical analysis to be metabolised during a subsequent dark aerobic period. Leaves of intact plants resembled incubated leaf discs in accumulating nitrite under anaerobic conditions. Nitrate, n-propanol and several respiratory inhibitors or uncouplers partly reversed the inhibitory effect of oxygen on nitrate reduction in leaf discs in the dark. Of these nitrate and propanol acted synergistically. Reversal was usually associated with inhibition of respiration but some concentrations of 2,4-dinitrophenol (DNP) and ioxynil reversed inhibition without affecting respiratory rates. Respiratory inhibitors and uncouplers stimulated nitrate reduction in the anaerobic in vivo assay i.e. in conditions where the respiratory process is non-functional. Freezing and thawing leaf discs diminished but did not eliminate the sensitivity of nitrate reduction to oxygen inhibition.Abbreviations DNP 2,4-dinitrophenol - HOQNO 8-hydroxyquinoline-N-oxide - DCPIP 2,6-dichlorophenolindophenol - CCCP Carbonyl cyanide m-chlorophenylhydrazone - TES N-tris(hydroxymethyl)methyl-2-amino ethanesulphonic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid     

Anaerobic mineralization of cholesterol by a novel type of denitrifying bacterium

A novel denitrifying bacterium, strain 72Chol, was enriched and isolated under strictly anoxic conditions on cholesterol as sole electron donor and carbon source. Strain 72Chol grew on cholesterol with oxygen or nitrate as electron acceptor. Strictly anaerobic growth in the absence of oxygen was demonstrated using chemically reduced culture media. During anaerobic growth, nitrate was initially reduced to nitrite. At low nitrate concentrations, nitrite was further reduced to nitrogen gas. Ammonia was assimilated. The degradation balance measured in cholesterol-limited cultures and the amounts of carbon dioxide, nitrite, and nitrogen gas formed during the microbial process indicated a complete oxidation of cholesterol to carbon dioxide. A phylogenetic comparison based on total 16S rDNA sequence analysis indicated that the isolated micro-organism, strain 72Chol, belongs to the β2-subgroup in the Proteobacteria and is related to Rhodocyclus, Thauera, and Azoarcus species. Received: 16 July 1996 / Accepted: 5 December 1996     

Control of nitrate and nitrite assimilation by carbohydrate reserves,adenosine nucleotides and pyridine nucleotides in leaves of Zea mays L. under dark conditions

The rate of in-vivo nitrate reduction by leaf segments of Zea mays L. was found to decline during the second hour of dark anaerobic treatment. On transfer to oxygen the capacity to reduce nitrate under dark conditions was restored. These observations led to the proposal that nitrate reductase is a regulatory enzyme with ADP acting as a negative effector. The effect of ADP on the invitro activity of nitrate reductase and the changes in the in-vivo adenylate pool under dark-N₂ and dark-O₂ were investigated. It was found that ADP inhibited the activity of partially purified nitrate reductase. Similarly, the in-vivo anaerobic inhibition of nitrate reduction was associated with a build-up of ADP in the leaf tissue. Under anaerobic conditions nitrite accumulated and on transfer to oxygen the accumulated nitrite was reduced. To explain this phenomenon the following hypothesis was proposed and tested. Under anaerobic conditions the supply of reducing equivalents for nitrite reduction in the plastid becomes restricted and nitrite accumulates as a consequence. On transfer to oxygen this restriction is removed and nitrite disappears. This capacity to reduce accumulated nitrite was found to be dependent on the carbohydrate status of the leaf tissue.     

微生物介导硝酸盐还原耦合亚铁氧化过程的动力学及其影响因素

[目的]探究不同菌浓度和亚铁浓度条件下,Acidovorax sp.strain BoFeN1介导的厌氧亚铁氧化耦合硝酸盐还原过程的动力学和次生矿物.[方法]构建包含菌BoFeN1、硝酸盐、亚铁的厌氧培养体系,测试硝酸根、亚硝酸根、乙酸根、亚铁等浓度,并收集次生矿物,采用XRD、SEM进行矿物种类和形貌表征.[结果]在...     

Anaerobic degradation of toluene by a denitrifying bacterium

A denitrifying bacterium, designated strain T1, that grew with toluene as the sole source of carbon under anaerobic conditions was isolated. The type of agar used in solid media and the toxicity of toluene were determinative factors in the successful isolation of strain T1. Greater than 50% of the toluene carbon was oxidized to CO2, and 29% was assimilated into biomass. The oxidation of toluene to CO2 was stoichiometrically coupled to nitrate reduction and denitrification. Strain T1 was tolerant of and grew on 3 mM toluene after a lag phase. The rate of toluene degradation was 1.8 mumol min-1 liter-1 (56 nmol min-1 mg of protein-1) in a cell suspension. Strain T1 was distinct from other bacteria that oxidize toluene anaerobically, but it may utilize a similar biochemical pathway of oxidation. In addition, o-xylene was transformed to a metabolite in the presence of toluene but did not serve as the sole source of carbon for growth of strain T1. This transformation was dependent on the degradation of toluene.     

Anaerobic degradation of toluene by a denitrifying bacterium.

A denitrifying bacterium, designated strain T1, that grew with toluene as the sole source of carbon under anaerobic conditions was isolated. The type of agar used in solid media and the toxicity of toluene were determinative factors in the successful isolation of strain T1. Greater than 50% of the toluene carbon was oxidized to CO2, and 29% was assimilated into biomass. The oxidation of toluene to CO2 was stoichiometrically coupled to nitrate reduction and denitrification. Strain T1 was tolerant of and grew on 3 mM toluene after a lag phase. The rate of toluene degradation was 1.8 mumol min-1 liter-1 (56 nmol min-1 mg of protein-1) in a cell suspension. Strain T1 was distinct from other bacteria that oxidize toluene anaerobically, but it may utilize a similar biochemical pathway of oxidation. In addition, o-xylene was transformed to a metabolite in the presence of toluene but did not serve as the sole source of carbon for growth of strain T1. This transformation was dependent on the degradation of toluene.     

Production and Consumption of Nitric Oxide by Three Methanotrophic Bacteria

We studied nitrogen oxide production and consumption by methanotrophs Methylobacter luteus (group I), Methylosinus trichosporium OB3b (group II), and an isolate from a hardwood swamp soil, here identified by 16S ribosomal DNA sequencing as Methylobacter sp. strain T20 (group I). All could consume nitric oxide (nitrogen monoxide, NO), and produce small amounts of nitrous oxide (N₂O). Only Methylobacter strain T20 produced large amounts of NO (>250 parts per million by volume [ppmv] in the headspace) at specific activities of up to 2.0 × 10⁻¹⁷ mol of NO cell⁻¹ day⁻¹, mostly after a culture became O₂ limited. Production of NO by strain T20 occurred mostly in nitrate-containing medium under anaerobic or nearly anaerobic conditions, was inhibited by chlorate, tungstate, and O₂, and required CH₄. Denitrification (methanol-supported N₂O production from nitrate in the presence of acetylene) could not be detected and thus did not appear to be involved in the production of NO. Furthermore, cd₁ and Cu nitrite reductases, NO reductase, and N₂O reductase could not be detected by PCR amplification of the nirS, nirK, norB, and nosZ genes, respectively. M. luteus and M. trichosporium produced some NO in ammonium-containing medium under aerobic conditions, likely as a result of methanotrophic nitrification and chemical decomposition of nitrite. For Methylobacter strain T20, arginine did not stimulate NO production under aerobiosis, suggesting that NO synthase was not involved. We conclude that strain T20 causes assimilatory reduction of nitrate to nitrite, which then decomposes chemically to NO. The production of NO by methanotrophs such as Methylobacter strain T20 could be of ecological significance in habitats near aerobic-anaerobic interfaces where fluctuating O₂ and nitrate availability occur.     

<Emphasis Type="Italic">Desulfurispirillum alkaliphilum</Emphasis> gen. nov. sp. nov., a novel obligately anaerobic sulfur- and dissimilatory nitrate-reducing bacterium from a full-scale sulfide-removing bioreactor

Strain SR 1^T was isolated under anaerobic conditions using elemental sulfur as electron acceptor and acetate as carbon and energy source from the Thiopaq bioreactor in Eerbeek (The Netherlands), which is removing H₂S from biogas by oxidation to elemental sulfur under oxygen-limiting and moderately haloalkaline conditions. The bacterium is obligately anaerobic, using elemental sulfur, nitrate and fumarate as electron acceptors. Elemental sulfur is reduced to sulfide through intermediate polysulfide, while nitrate is dissimilatory reduced to ammonium. Furthermore, in the presence of nitrate, strain SR 1^T was able to oxidize limited amounts of sulfide to elemental sulfur during anaerobic growth with acetate. The new isolate is mesophilic and belongs to moderate haloalkaliphiles, with a pH range for growth (on acetate and nitrate) from 7.5 to 10.25 (optimum 9.0), and a salt range from 0.1 to 2.5 M Na⁺ (optimum 0.4 M). According to phylogenetic analysis, SR 1^T is a member of a deep bacterial lineage, distantly related to Chrysiogenes arsenatis (Macy et al. 1996). On the basis of the phenotypic and genetic data, the novel isolate is placed into a new genus and species, Desulfurispirillum alkaliphilum (type strain SR^T = DSM 18275 = UNIQEM U250). Nucleotide sequence accession number: the GenBank/EMBL accession number of the 16S rRNA gene sequence of strain SR 1^T is DQ666683.     

New insights into the energy metabolism and taxonomy of Deferribacteres revealed by the characterization of a new isolate from a hypersaline microbial mat

Strain L21-Ace-BES^T, isolated from a lithifying cyanobacterial mat, could be assigned to a novel species and genus within the class Deferribacteres. It is an important model organism for the study of anaerobic acetate degradation under hypersaline conditions. The metabolism of strain L21-Ace-BES^T was characterized by biochemical studies, comparative genome analyses, and the evaluation of gene expression patterns. The central metabolic pathway is the citric acid cycle, which is mainly controlled by the enzyme succinyl-CoA:acetate-CoA transferase. The potential use of a reversed oxidative citric acid cycle to fix CO₂ has been revealed through genome analysis. However, no autotrophic growth was detected in this strain, whereas sulfide and H₂ can be used mixotrophically. Preferred electron acceptors for the anaerobic oxidation of acetate are nitrate, fumarate and dimethyl sulfoxide, while oxygen can be utilized only under microoxic conditions. Aerotolerant growth by fermentation was observed at higher oxygen concentrations. The redox cycling of sulfur/sulfide enables the generation of reducing power for the assimilation of acetate during growth and could prevent the over-reduction of cells in stationary phase. Extracellular electron transfer appears to be an essential component of the respiratory metabolism in this clade of Deferribacteres and may be involved in the reduction of nitrite to ammonium.