Nitric and nitrous oxide reductases are active under aerobic conditions in cells of <i>Thiosphaera pantotropha</i>

Bell, Louise C.; Ferguson, Stuart J.

doi:10.1042/bj2730423

Cited by 87 publications

(34 citation statements)

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“…The low level of denitrification activity observed in the presence of oxygen may thus be attributable to the residual activity of Anr. It is unknown, however, whether aerobic denitrification in T. pantotropha also depends on an Fnr-like regulator (4,24). a Strains of P. aeruginosa were grown in 10 ml of tryptic soy broth in 15-ml culture tubes with an inverted Durham tube inside.…”

Section: Figmentioning

confidence: 99%

“…Oxygen-limiting conditions (Ͻ10 M O 2 ) are usually necessary for denitrification (33). However, some bacteria such as Thiosphaera pantotropha are able to denitrify in the presence of higher oxygen concentrations (4,24). Although Escherichia coli and other enteric bacteria do not denitrify, they can respire nitrate and nitrite under anaerobic conditions and thus offer insight into how denitrification might be regulated by oxygen limitation.…”

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Anaerobic activation of the entire denitrification pathway in Pseudomonas aeruginosa requires Anr, an analog of Fnr

Haas

et al. 1995

J Bacteriol

152

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The Pseudomonas aeruginosa gene anr, which encodes a structural and functional analog of the anaerobic regulator Fnr in Escherichia coli, was mapped to the SpeI fragment R, which is at about 59 min on the genomic map of P. aeruginosa PAO1. Wild-type P. aeruginosa PAO1 grew under anaerobic conditions with nitrate, nitrite, and nitrous oxide as alternative electron acceptors. An anr deletion mutant, PAO6261, was constructed. It was unable to grow with these alternative electron acceptors; however, its ability to denitrify was restored upon the introduction of the wild-type anr gene. In addition, the activities of two enzymes in the denitrification pathway, nitrite reductase and nitric oxide reductase, were not detectable under oxygen-limiting conditions in strain PAO6261 but were restored when complemented with the anr ؉ gene. These results indicate that the anr gene product plays a key role in anaerobically activating the entire denitrification pathway.

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confidence: 99%

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Anaerobic activation of the entire denitrification pathway in Pseudomonas aeruginosa requires Anr, an analog of Fnr

Haas

et al. 1995

J Bacteriol

152

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“…is reported to catalyse the complete series of denitrification reactions under aerobic conditions (Robertson & Kuenen, 1983Bell et al, 1990;Bell & Ferguson, 1991 ;Moir et al, 1993 ;Berks et al, 1993). The organism has recently been shown to possess two respiratory nitrate reductases, one in the periplasm and the other associated with the cytoplasmic membrane (Bell et al, 1990).…”

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confidence: 99%

Insertion of transposon Tn5 into a structural gene of the membrane-bound nitrate reductase of Thiosphaera pantotropha results in anaerobic overexpression of periplasmic nitrate reductase activity

Bell

Page

Berks

et al. 1993

Journal of General Microbiology

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Chlorate-resistant mutants of the denitrifying bacterium Thiosphaera pantotropha were generated by transposon Tn5 mutagenesis. One class was deficient in membrane-bound nitrate reductase activity but retained a periplasmic nitrate reductase activity. Using transposon marker rescue it was shown that in one such mutant, M-6, the transposon was inserted in the membrane-bound nitrate reductase p subunit structural gene (termed narH in order to be consistent with the nomenclature of the Escherichia coli major nitrate reductase operon). The translated sequence (total of 106 amino acids) from around the point of transposon insertion showed approximately 90% amino acid identity with the j9 subunits of the E. coli nitrate reductases. Under anaerobic growth conditions M-6 overproduced the periplasmic nitrate reductase activity allowing anaerobic growth with nitrate as electron acceptor. A regulatory link was inferred between the presence of the membrane-bound nitrate reductase and expression of the periplasmic nitrate reductase. This is the first demonstration of full denitrification in an organism possessing only a periplasmic nitrate reductase.

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“…Paracoccus denitrificans ATCC 35512, formerly named Thiosphaera pantotropha GB17T, was isolated in 1983 by Robertson and Kuenen (21) and catalyzes successive reductions of nitrate, nitrite, nitric oxide (NO), and nitrous oxide (N 2 O) (1,2,20). Although nitrate reductase, nitrite reductase, nitrous oxide reductase, cytochrome c-550, and pseudoazurin have been isolated from the bacterium and their biochemical and molecular features have been well characterized (4,5,20,22), the NO reductase of the bacterium remains poorly characterized.…”

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Cytochrome cb-type nitric oxide reductase with cytochrome c oxidase activity from Paracoccus denitrificans ATCC 35512

1996

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A highly active nitric oxide reductase was purified from Paracoccus denitrificans ATCC 35512, formerly named Thiosphaera pantotropha, which was anaerobically cultivated in the presence of nitrate. The enzyme was composed of two subunits with molecular masses of 34 and 15 kDa and contained two hemes b and one heme c per molecule. Copper was not found in the enzyme. The spectral properties suggested that one of the two hemes b and heme c were in six-coordinated low-spin states and another heme b was in a five-coordinated high-spin state and reacted with carbon monoxide. The enzyme showed high cytochrome c-nitric oxide oxidoreductase activity and formed nitrous oxide from nitric oxide with the expected stoichiometry when P. denitrificans ATCC 35512 ferrocytochrome c-550 was used as the electron donor. The V max and K m values for nitric oxide were 84 mol of nitric oxide per min/mg of protein and 0.25 M, respectively. Furthermore, the enzyme showed ferrocytochrome c-550-O 2 oxidoreductase activity with a V max of 8.4 mol of O 2 per min/mg of protein and a K m value of 0.9 mM. Both activities were 50% inhibited by about 0.3 mM KCN. (4,5,20,22), the NO reductase of the bacterium remains poorly characterized. NO, which is one of the reaction intermediates in the denitrification process (15,29), is chemically active and toxic to living cells. However, the concentration of NO in denitrifiers is stabilized only at the nano-or subnanomolar level because of the active consumption by NO reductase (12,30). NO reductases have been purified from only two kinds of denitrifying bacteria, Pseudomonas stutzeri (14, 17) and P. denitrificans ATCC 19367 (NCIB 8944) (6, 9). Both enzyme molecules contain protoheme and heme c and catalyze the reduction of NO to N 2 O in artificial electron-donating assays using phenazine methosulfate-ascorbate or 2,3,5,6-tetramethylphenylenediamine-ascorbate-horse cytochrome c. However, the physiological electron pathway to NO has not been reconstituted in vitro. Paracoccus denitrificansWe describe here a simple purification of NO reductase from P. denitrificans ATCC 35512 by using sucrose monocaprate (SM-1080) as a solubilizing detergent. The purified enzyme showed a subunit structure and spectral properties similar to those of cb-type NO reductases. Furthermore, the enzyme was very stable in the presence of the detergent and oxygen and showed not only cytochrome c-NO reductase activity but also cytochrome c-O 2 reductase activity when P. denitrificans ATCC 35512 cytochrome c-550 was used as the electron donor. We succeeded in reconstituting the electron transport from physiological cytochrome c to NO catalyzed by NO reductase. MATERIALS AND METHODSOrganisms and cultivation. P. denitrificans ATCC 35512 was anaerobically cultivated as previously described by Robertson and Kuenen (21), with some modifications. The bacterium was grown at 37ЊC in a medium containing (per liter) 1.36 g of CH 3 COONa ⅐ 3H 2 O, 3.26 g of KNO 3 , 0.8 g of K 2 HPO 4 , 0.3 g of KH 2 PO 4 , and 0.4 g of MgSO 4 and a trace amount ...

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Nitric and nitrous oxide reductases are active under aerobic conditions in cells of Thiosphaera pantotropha

Cited by 87 publications

References 23 publications

Anaerobic activation of the entire denitrification pathway in Pseudomonas aeruginosa requires Anr, an analog of Fnr

Anaerobic activation of the entire denitrification pathway in Pseudomonas aeruginosa requires Anr, an analog of Fnr

Insertion of transposon Tn5 into a structural gene of the membrane-bound nitrate reductase of Thiosphaera pantotropha results in anaerobic overexpression of periplasmic nitrate reductase activity

Cytochrome cb-type nitric oxide reductase with cytochrome c oxidase activity from Paracoccus denitrificans ATCC 35512

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