Halomonas campisalis sp. nov., a denitrifying, moderately haloalkaliphilic bacterium

The isolation and characterization of a denitrifying bacterium that is both moderately halophilic and alkaliphilic is described. The organism was isolated for use in the development of a bioprocess that could potentially reduce the costs of ion exchange resin regenerant disposal. The process of ion exchange, after resin regeneration, produces a briny, alkaline waste that is difficult and expensive to dispose. The biological removal of nitrate and subsequent reuse of these brines can potentially provide a cost-saving alternative to disposing of this waste product. To achieve our objective, a moderately halophilic, alkaliphilic bacterium was isolated from sediment samples taken from the salt plain of Alkali Lake in Washington State (USA). The haloalkaliphilic bacterium, designated strain 4A, is motile with rod-shaped cells that are 3 to 5 microm long and 1 microm wide. Electron acceptors used include oxygen, nitrate, and nitrite. In addition, it has similar specific nitrate reduction rates and biomass yields as non-halophilic denitrifying bacteria. It is capable of using a variety of electron donors. This organism can grow at NaCl concentrations ranging from 0.2 to 4.5 M with optimum growth occurring at 1.5 M and pH values ranging from 6 to 12 with 9.5 being the optimum pH. The temperature range for growth of strain 4A is 4-50 degrees C with optimal growth occurring at 30 degrees C. The G + C content is 66 mol%. Phylogenetic analyses based upon 16S rDNA gene sequence placed isolate 4A in the genus Halomonas. In addition, DNA-DNA hybridization experiments clearly indicate that it is a unique species. Phenotypic and phylogenetic studies indicate that isolate 4A represents a new species. We propose the name Halomonas campisalis for this species and strain 4A (ATCC 700597) as the type strain. Due to its denitrification ability, broad carbon utilization range and its high salinity and pH tolerance this organism, and similar ones, hold promise for the treatment of saline, alkaline waste.     

Heterotrophic nitrification and aerobic denitrification by a novel Halomonas campisalis

A novel halophilic strain that could carry out heterotrophic nitrification and aerobic denitrification was isolated and named as Halomonas campisalis ha3. It removed inorganic nitrogen compounds (e.g. NO₃ ^?, NO₂ ^? and NH₄ ⁺) simultaneously, and grew well in the medium containing up to 20 % (w/v) NaCl. PCR revealed four genes in the genome of ha3 related to aerobic denitrification: napA, nirS, norB and nosZ. The optimal conditions for aerobic denitrification were pH 9.0, at 37 °C, with 4 % (w/v) NaCl and sodium succinate as carbon source. The nitrogen removal rate was 87.5 mg NO₃ ^?–N l^?1 h^?1. Therefore, this strain is a potential aerobic denitrifier for the treatment of saline wastewater.     

Properties of dissimilatory nitrate reductase purified from the denitrifier Pseudomonas aeruginosa

Dissimilatory nitrate reductase was purified to homogeneity from anaerobic cultures of the denitrifying bacterium Pseudomonas aeruginosa. The following procedures were used in the rapid isolation of this unstable enzyme: induction by nitrate in semianaerobic cell suspension, heat-stimulated activation and solubilization from the membrane fraction, and purification by hydrophobic interaction chromatography. The molecular weight of the purified enzyme was estimated by nondenaturing polyacrylamide gel electrophoresis, sucrose density gradient sedimentation, and gel filtration chromatography. Subunit molecular weights were estimated by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The active enzyme monomer, with a molecular weight of 176,000 to 260,000 (depending upon the method of determination), was composed of subunits with molecular weights of approximately 64,000 and 118,000. The monomer aggregated to form an inactive tetramer of about 800,000 molecular weight. Purified enzyme exhibited a broad pH optimum, between 6.5 and 7.5. Kinetic studies showed that the apparent Km was 0.30 mM for nitrate, and 2.2 to 2.9 microM for dithionite-reduced benzyl viologen. Azide was an effective inhibitor: the concentration required for half-maximal inhibition was 21 to 24 microM. Azide inhibition was competitive with nitrate (Ki = 2.0 microM) but uncompetitive with reduced benzyl viologen (Ki = 25 microM). Based upon spectral evidence, the purified molybdo-enzyme had no associated cytochromes but did contain nonhaem iron that responded to dithionite reduction and nitrate oxidation. The enzyme that was purified after being heat solubilized from membranes had properties essentially identical to those of the enzyme that was purified after deoxycholate solubilization.     

Tandem and single genes of three membrane-bound nitrate transporters in the nar gene cluster of the moderately halophilic denitrifier, Halomonas halodenitrificans.

We identified the genes encoding the membrane-bound nitrate reductase (Nar) from the moderate halophile, Halomonas halodenitrificans, and examined the structure of the gene cluster. Screening of a H. halodenitrificans genomic DNA library in lambda EMBL3 phage by chromosome walking revealed that the region adjacent to the nor gene cluster encoding nitric oxide (NO) reductase contains three nitrate transporters: tandem narK2 and narK1.1 genes and a single narK1.2 gene encoded in opposite directions. NarK1.1 and NarK1.2 proteins, which have 12 putative membrane-spanning helices, were classified as type I NarK, whereas NarK2, which has 14 putative membrane-spanning helices, was classified as a type II NarK. NarK1.1 and NarK2 proteins were considered to be functionally and structurally linked in the cytoplasmic membrane. The systems regulating the expression of the tandem narK2K1.1 gene and the single narK1.2 gene were found to be different. Further, binding sites for NarL and Fnr-like proteins are present in the promoter region of the narK2 gene.     

Tungstate, a molybdate analog inactivating nitrate reductase, deregulates the expression of the nitrate reductase structural gene

Nitrate reductase (NR, EC 1.6.6.1) from higher plants is a homodimeric enzyme carrying a molybdenum cofactor at the catalytic site. Tungsten can be substituted for molybdenum in the cofactor structure, resulting in an inactive enzyme. When nitratefed Nicotiana tabacum plants were grown on a nutrient solution in which tungstate was substituted for molybdate, NR activity in the leaves decreased to a very low level within 24 hours while NR protein accumulated progressively to a level severalfold higher than the control after 6 days. NR mRNA level in molybdate-grown plants exhibited a considerable day-night fluctuation. However, when plants were treated with tungstate, NR mRNA level remained very high. NR activity and protein increased over a 24-hour period when nitrate was added back to N-starved molybdate-grown plants. NR mRNA level increased markedly during the first 2 hours and then decreased. In the presence of tungstate, however, the induction of NR activity by nitrate was totally abolished while high levels of NR protein and mRNA were both induced, and the high level of NR mRNA was maintained over a 10-hour period. These results suggest that the substitution of tungsten for molybdenum in NR complex leads to an overexpression of the NR structural gene. Possible mechanisms involved in this deregulation are discussed.     

Effects of freezing on purified nitrite reductase from a denitrifier, Alcaligenes sp. NCIB 11015

The effects of freezing on Alcaligenes sp. nitrite reductase [nitric-oxide: ferricytochrome c oxidoreductase, EC 1.7.2.1] dissolved in sodium phosphate (pH 7.2) were investigated. The nitrite reductase was gradually activated with time in the frozen state, resulting in an increase in its activity of 2.5-4.5 times. The final freezing temperature influenced the enzyme activation, maximal activation being observed at around -20 degrees C. All the enzymatic activities that the nitrite reductase is known to catalyze were enhanced by freeze-thawing. The activation was followed by neither association-dissociation nor any gross conformational change of the enzyme molecule, but was accompanied by an increase in the fluorescence intensity of 2-p-toluidinonaphthalene-6-sulfonate used as a hydrophobic probe. The results are consistent with the hypothesis that the activation of the NiR is due to a limited conformational change of the enzyme molecule, particularly in the hydrophobic region. The mechanism of the activation of NiR by freeze-thawing is discussed, in comparison with the mechanisms of inactivation by freeze-thawing of many enzymes reported by previous workers.     

Dissimilatory nitrate reduction to nitrate, nitrous oxide, and ammonium by Pseudomonas putrefaciens

The influence of redox potential on dissimilatory nitrate reduction to ammonium was investigated on a marine bacterium, Pseudomonas putrefaciens. Nitrate was consumed (3.1 mmol liter-1), and ammonium was produced in cultures with glucose and without sodium thioglycolate. When sodium thioglycolate was added, nitrate was consumed at a lower rate (1.1 mmol liter-1), and no significant amounts of nitrite or ammonium were produced. No growth was detected in glucose media either with or without sodium thioglycolate. When grown on tryptic soy broth, the production of nitrous oxide paralleled growth. In the same medium, but with sodium thioglycolate, nitrous oxide was first produced during growth and then consumed. Acetylene caused the nitrous oxide to accumulate. These results and the mass balance calculations for different nitrogen components indicate that P. putrefaciens has the capacity to dissimilate nitrate to ammonium as well as to dinitrogen gas and nitrous oxide (denitrification). The dissimilatory pathway to ammonium dominates except when sodium thioglycolate is added to the medium.     

Identification of a copper protein as part of the nitrous oxide-reducing system in nitrite-respiring (denitrifying) pseudomonads

Copper is the essential transition element for nitrous oxide respiration in Pseudomonas perfectomarinus. Two novel kinds of copper proteins were detected in this organism. Their distribution was studied under different growth conditions and in other pseudomonads, as well as their association with N₂O reduction of intact cells. A low molecular mass copper protein (M _r 38,000) with a single absorption band at 340 nm (oxidized form), was found only in P. perfectomarinus and was not required for N₂O reduction. N₂O respiration was consistently associated with a high molecular mass copper protein (M _r 120,000) in P. perfectomarinus, Pseudomonas stutzeri, and in strains of Pseudomonas fluorescens that were capable of this type of respiration. The oxidized protein was violet to pink with absorption bands at 350, 480, 530, 620, and 780 nm. Pseudomonas chlororaphis and Pseudomonas aureofaciens which did not respire with N₂O as electron acceptor, did not contain the novel type of copper protein. Cytochrome patterns were compared in these denitrifying pseudomonads to search for the physiological electron carrier to N₂O reductase. The content and nature of the soluble c-type cytochromes depended strongly on the species and the particular growth condition.Abbreviations M _r relative molecular mass     

Response of Halomonas campisalis to saline stress: changes in growth kinetics, compatible solute production and membrane phospholipid fatty acid composition

The haloalkaliphile Halomonas campisalis, isolated near Soap Lake, Washington, was grown under both aerobic and denitrifying conditions from 0 to 260 g L(-1) NaCl, with optimal growth occurring at 20 and 30 g L(-1) NaCl, respectively. Halomonas campisalis was observed to produce high concentrations of compatible solutes, most notably ectoine (up to 500 mM within the cytoplasm), but hydroxyectoine and glycine betaine were also detected. The types and amounts of compatible solutes produced depended on salinity and specific growth rate, as well as on the terminal electron acceptor available (O(2) or NO(3) (-)). A decrease in ectoine production was observed with NO(3) (-) as compared with O(2) as the terminal electron acceptor. In addition, changes in the phospholipid fatty acid composition were measured with changing salinity. An increase in trans fatty acids was observed in the absence of salinity, and may be a response to membrane instability. Cyclic fatty acids were also observed to increase, both in the absence of salinity, and at very high salinities, indicating cell stress at these conditions.     

Temporary low oxygen conditions for the formation of nitrate reductase and nitrous oxide reductase by denitrifying Pseudomonas sp. G59

Formation of nitrate reductase (NaR) and nitrous oxide reductase (N2OR) by a Pseudomonas sp. G59 did not occur in aerobic or anaerobic conditions, but was observed in a microaerobic incubation in which an anaerobically grown culture was agitated in a sealed vessel initially containing 20 kPa oxygen in the headspace. During the microaerobic incubation, the oxygen concentration in the headspace decreased and dissolved oxygen reached 0.1-0.2 kPa. NaR activity was detected immediately and N2OR activity after 3 h of incubation irrespective of the presence or absence of NO3- or N2O. In the presence of NO3-, NO2- was accumulated as a major product, but N2O was observed in low concentrations only after N2OR appeared. After microaerobic incubation for 3 h, N2OR formation continued even anaerobically in an atmosphere of N2O. In contrast, Escherichia coli formed NaR not only microaerobically but also anaerobically. However, NaR formation by E. coli was inhibited by sodium fluoride under anaerobic, but not under microaerobic conditions. The Pseudomonas culture did not possess fermentative activity. It is suggested that the dependence on microaerobiosis for the formation of these reductases by the Pseudomonas culture was due to an inability to produce energy anaerobically until these anaerobic respiratory enzymes were formed.     

Expression of the 2,4-D degradative pathway of pJP4 in an alkaliphilic, moderately halophilic soda lake isolate, Halomonas sp. EF43

The broad host range plasmid pJP4, which carries genes for the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methyl-4-chlorophenoxyacetic acid, and 3-chlorobenzoic acid, was used in conjugation experiments with mixed cultures enriched from water and sediment samples from an alkaline pond in the area of Szegedi Fehértó, a soda lake in south Hungary. pJP4-encoded mercury resistance was used as a selection marker. One of the transconjugants, the alkaliphilic, moderately halophilic strain EF43, stably maintained the plasmid and was able to degrade 2,4-D and 3-chlorobenzoate under alkaline conditions in the presence of an additional carbon source such as pyruvate, benzoate, or alpha-ketoglutarate, indicating that the degradative genes of pJP4 were expressed in this strain. However, it was unable to grow on these chloroaromatic substrates when the substrate was the sole source of carbon and energy. Chemostat cultivation experiments revealed that the 2,4-D degradation rate during growth on benzoate or pyruvate was limited by the low activity of chlorocatechol-degrading enzymes, particularly chloromuconate cycloisomerase. Strain EF43 was identified as Halomonas sp. on the basis of 16S rRNA sequencing and additional taxonomic studies. 16S rRNA sequence analysis revealed that strain EF43 is closely related to typical soda lake isolates belonging to the genus Halomonas.     

Dissimilatory nitrate reduction to nitrate, nitrous oxide, and ammonium by Pseudomonas putrefaciens.

The influence of redox potential on dissimilatory nitrate reduction to ammonium was investigated on a marine bacterium, Pseudomonas putrefaciens. Nitrate was consumed (3.1 mmol liter-1), and ammonium was produced in cultures with glucose and without sodium thioglycolate. When sodium thioglycolate was added, nitrate was consumed at a lower rate (1.1 mmol liter-1), and no significant amounts of nitrite or ammonium were produced. No growth was detected in glucose media either with or without sodium thioglycolate. When grown on tryptic soy broth, the production of nitrous oxide paralleled growth. In the same medium, but with sodium thioglycolate, nitrous oxide was first produced during growth and then consumed. Acetylene caused the nitrous oxide to accumulate. These results and the mass balance calculations for different nitrogen components indicate that P. putrefaciens has the capacity to dissimilate nitrate to ammonium as well as to dinitrogen gas and nitrous oxide (denitrification). The dissimilatory pathway to ammonium dominates except when sodium thioglycolate is added to the medium.     

The catalytic center in nitrous oxide reductase, CuZ, is a copper-sulfide cluster

The crystal structure of nitrous oxide reductase, the enzyme catalyzing the final step of bacterial denitrification in which nitrous oxide is reduced to dinitrogen, exhibits a novel catalytic site, called Cu(Z). This comprises a cluster of four copper ions bound by seven histidines and three other ligands modeled in the X-ray structure as OH(-) or H(2)O. However, elemental analyses and resonance Raman spectroscopy of isotopically labeled enzyme conclusively demonstrate that Cu(Z) has one acid-labile sulfur ligand. Thus, nitrous oxide reductase contains the first reported biological copper-sulfide cluster.     

Characterization of nitrite reductase from a denitrifier, Alcaligenes sp. NCIB 11015. A novel copper protein

Covalent cross-linking reaction between SH1 and SH2 groups in myosin subfragment-1 (S-1) by N,N'-p-phenylenedimaleimide (pPDM) was followed by the degree of inactivation of NH4+-EDTA ATPase activity. The rate of the cross-linking reaction decreased to less than a 20th in the presence of F-actin. The inhibitory effect of F-actin was not observed in the presence of MgATP. Binding of F-actin to S-1 was measured using ultracentrifugation. S-1 whose SH1 and SH2 were covalently cross-linked by pPDM or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) did not bind F-actin. After the DTNB-cross-linked S-1 is reduced by dithiothreitol, the ability to bind F-actin is recovered. These results suggest that S-1 has a binding site for F-actin in the region between SH1 and SH2. This site appears to determine the high affinity of acto-S-1 complex at the rigor while decreasing the affinity more than 10(2) times in the presence of MgATP.     

The activation state of nitrate reductase is not always correlated with total nitrate reductase activity in leaves

The relation between nitrate reductase (NR; EC 1.6.6.1) activity, activation state and NR protein in leaves of barley (Hordeum vulgare L.) seedlings was investigated. Maximum NR activity (NRA_max) and NR protein content (Western blotting) were modified by growing plants hydroponically at low (0.3 mM) or high (10 mM) nitrate supply. In addition, plants were kept under short-day (8 h light/16 h dark) or long-day (16 h light/8 h dark) conditions in order to manipulate the concentration of nitrate stored in the leaves during the dark phase, and the concentrations of sugars and amino acids accumulated during the light phase, which are potential signalling compounds. Plants were also grown under phosphate deficiency in order to modify their glucose-6-phosphate content. In high-nitrate/long-day conditions, NRA_max and NR protein were almost constant during the whole light period. Low-nitrate/long-day plants had only about 30% of the NRA_max and NR protein of high-nitrate plants. In low-nitrate/long-day plants, NRA_max and NR protein decreased strongly during the second half of the light phase. The decrease was preceded by a strong decrease in the leaf nitrate content. Short daylength generally led to higher nitrate concentrations in leaves. Under short-day/low-nitrate conditions, NRA_max was slightly higher than under long-day conditions and remained almost constant during the day. This correlated with maintenance of higher nitrate concentrations during the short light period. The NR activation state in the light was very similar in high-nitrate and low-nitrate plants, but dark inactivation was twice as high in the high-nitrate plants. Thus, the low NRA_max in low-nitrate/long-day plants was slightly compensated by a higher activation state of NR. Such a partial compensation of a low NR_max by a higher dark activation state was not observed with phosphate-depleted plants. Total leaf concentrations of sugars, of glutamine and glutamate and of glucose-6-phosphate did not correlate with the NR activation state nor with NRA_max. Received: 24 March 1999 / Accepted: 31 May 1999     

Characterization of a chlorate-hypersensitive,high nitrate reductase Arabidopsis thaliana mutant

Summary A population of A. thaliana, produced by self-fertilization of ethylmethane sulfonate treated plants, was exposed to chlorate in the watering solution, and plants showing early susceptibility symptoms were rescued. Among the progeny lines of these plants five were shown to be repeatably chlorate-hypersusceptible. One of these lines (designated C-4) possessed elevated activity of nitrate reductase (NR). The NR activity of mutant C-4 was higher than that of normal plants throughout the life cycle. Nitrite reductase and glutamine synthetase activities of C-4 were normal, as were chlorate uptake rate and tissue nitrate content. The elevated NR activity apparently was responsible for the chlorate hypersusceptibility of C-4. Inheritance studies of NR indicated that the elevated activity of C-4 was probably controlled by a single recessive allele.     

Inheritance of nitrite reductase and regulation of nitrate reductase, nitrite reductase, and glutamine synthetase isozymes

Banding patterns of nitrate reductase (NR), nitrite reductase (NiR), and glutamine synthetase (GS) from leaves of diploid barley (Hordeum vulgare), tetraploid wheat (Triticum durum), hexaploid wheat (Triticum aestivum), and tetraploid wild oats (Avena barbata) were compared following starch gel electrophoresis. Two NR isozymes, which appeared to be under different regulatory control, were observed in each of the three species. The activity of the more slowly migrating nitrate reductase isozyme (NR1) was induced by NO3- in green seedlings and cycloheximide inhibited induction. However, the activity of the faster NR isozyme (NR2) was unaffected by addition of KNO3, and it was not affected by treatments of cycloheximide or chloramphenicol. Only a single isozyme of nitrite reductase was detected in surveys of three tetraploid and 18 hexaploid wheat, and 48 barley accessions; however, three isozymes associated with different ecotypes were detected in the wild oats. Inheritance patterns showed that two of the wild oat isozymes were governed by a single Mendelian locus with two codominant alleles; however, no variation was detected for the third isozyme. Treatment of excised barely and wild oat seedlings with cycloheximide and chloramphenicol showed that induction of NiR activity was greatly inhibited by cycloheximide, but only slightly by chloramphenicol. Only a single GS isozyme was detected in extracts of green leaves of wheat, barley, and wild oat seedlings. No electrophoretic variation was observed within or among any of these three species. Thus, this enzyme appears to be the most structurally conserved of the three enzymes.