Three nitrate reductase activities in Alcaligenes eutrophus

Warnecke-Eberz, Ute; Friedrich, Bärbel

doi:10.1007/bf00288585

Cited by 56 publications

(37 citation statements)

References 17 publications

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“…2, lane 1). The nitrate reductase activity measured is exclusively due to Nap and not due to the respiratory membrane-bound nitrate reductase, which is known to be expressed anaerobically only when nitrate serves as the alternative electron acceptor (42). Figure 2B shows that a Nap-negative variant of TF93 formed no anti-NapA reacting material, and in consequence, no Nap activity was trapped in the membrane of the aerobically grown cells.…”

Section: Resultsmentioning

confidence: 95%

“…The PCR product was cut by NdeI and XbaI and cloned downstream of the strong R. eutropha promoter of the soluble hydrogenase into the broad-host-range plasmid pEDY309 as previously described (23) (12). Synthetic media for aerobic heterotrophic growth, in order to optimally express Nap, contained 0.4% (wt/vol) gluconate or 0.4% (wt/vol) fructose and SL6 as described previously (42). Anaerobic heterotrophic growth, at the expense of 0.2% (vol/vol) nitrate (denitrification), was performed in medium containing fructose as previously described (30).…”

Section: Methodsmentioning

confidence: 99%

“…One unit of hydrogenase activity was the amount of enzyme which catalyzed the consumption of 1 mol of substrate per min. Nap reductase activity was determined in subcellular fractions obtained from aerobically grown cells (42) exploiting the formate-dependent nitrate reduction (35). One unit of Nap activity was the amount of enzyme which catalyzed the formation of 1 mol of nitrite per min.…”

Section: Methodsmentioning

confidence: 99%

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Ralstonia eutropha TF93 Is Blocked in Tat-Mediated Protein Export

2000

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Ralstonia eutropha (formerly Alcaligenes eutrophus) TF93 is pleiotropically affected in the translocation of redox enzymes synthesized with an N-terminal signal peptide bearing a twin arginine (S/T-R-R-X-F-L-K) motif. Immunoblot analyses showed that the catalytic subunits of the membrane-bound [NiFe] hydrogenase (MBH) and the molybdenum cofactor-binding periplasmic nitrate reductase (Nap) are mislocalized to the cytoplasm and to the inner membrane, respectively. Moreover, physiological studies showed that the coppercontaining nitrous oxide reductase (NosZ) was also not translocated to the periplasm in strain TF93. The cellular localization of enzymes exported by the general secretion system was unaffected. The translocationarrested MBH and Nap proteins were enzymatically active, suggesting that twin-arginine signal peptidedependent redox enzymes may have their cofactors inserted prior to transmembrane export. The periplasmic destination of MBH, Nap, and NosZ was restored by heterologous expression of Azotobacter chroococcum tatA mobilized into TF93. tatA encodes a bacterial Hcf106-like protein, a component of a novel protein transport system that has been characterized in thylakoids and shown to translocate folded proteins across the membrane.Periplasmic enzymes binding redox cofactors play a central role in alternative energy metabolism of gram-negative bacteria. In contrast to cytochrome c-type proteins, various periplasmic enzymes binding constituents, such as the molybdenum cofactor, a [NiFe] site, copper centers, or iron-sulfur clusters, contain a conserved, positively charged -S/T-RRXFLK-(twinarginine) element within their N-terminal signal peptides, pointing to a special translocation pathway (2). Very recently, a system responsible for the membrane targeting and translocation of [NiFe] hydrogenases, as well as the molybdenum enzymes dimethylsulfoxide reductase, trimethylamine N-oxide reductase, and periplasmic nitrate reductase, has been characterized for Escherichia coli (32,43). Mutant analyses suggested that the translocation of the twin-arginine signal peptide-bearing enzymes proceeds independently of the general secretion machinery of the cell (Sec), presumably via intermediates which have their cofactors inserted (31).Ralstonia eutropha (formerly Alcaligenes eutrophus [7]) is the host of at least three periplasmic cofactor-containing enzymes synthesized with an N-terminal twin-arginine signal peptide: the membrane-bound hydrogenase (MBH) (involved in energy generation from H 2 ), the periplasmic nitrate reductase (Nap) (reduces nitrate to nitrite), and the nitrous oxide reductase (NosZ) (a component of the denitrification pathway). All three enzymes are encoded in R. eutropha H16 by megaplasmidborne genes.The MBH of R. eutropha is a member of the [NiFe] hydrogenases (15) composed of a heterodimer (HoxKG) attached to the periplasmic surface of the inner membrane by a cytochrome b-type anchor protein (HoxZ) (4, 13). Hydrogen is activated at the [NiFe] site of HoxG (62 kDa), and the electrons are tra...

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Section: Resultsmentioning

confidence: 95%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Ralstonia eutropha TF93 Is Blocked in Tat-Mediated Protein Export

2000

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“…The group 3d [NiFe]-hydrogenase of Ralstonia eutropha, which grows through aerobic and nitrate respiration, transiently reverses directionality to couple NADH reoxidation to H 2 generation during anaerobiosis (42,43). In contrast to this system, the tight regulation and sustained activity of Hyd3 under oxygen limitation in M. smegmatis is consistent with Hyd3 being specifically selected to catalyze the fermentative production of H 2 during dormancy.…”

Section: Discussionmentioning

confidence: 98%

An obligately aerobic soil bacterium activates fermentative hydrogen production to survive reductive stress during hypoxia

Berney

Greening

Conrad

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

118

126

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Oxygen availability is a major factor and evolutionary force determining the metabolic strategy of bacteria colonizing an environmental niche. In the soil, conditions can switch rapidly between oxia and anoxia, forcing soil bacteria to remodel their energy metabolism accordingly. Mycobacterium is a dominant genus in the soil, and all its species are obligate aerobes. Here we show that an obligate aerobe, the soil actinomycete Mycobacterium smegmatis, adopts an anaerobe-type strategy by activating fermentative hydrogen production to adapt to hypoxia. This process is controlled by the two-component system DosR-DosS/DosT, an oxygen and redox sensor that is well conserved in mycobacteria. We show that DosR tightly regulates the two [NiFe]-hydrogenases: Hyd3 (MSMEG_3931-3928) and Hyd2 (MSMEG_2719-2718). Using genetic manipulation and high-sensitivity GC, we demonstrate that Hyd3 facilitates the evolution of H 2 when oxygen is depleted. Combined activity of Hyd2 and Hyd3 was necessary to maintain an optimal NAD + /NADH ratio and enhanced adaptation to and survival of hypoxia. We demonstrate that fermentativelyproduced hydrogen can be recycled when fumarate or oxygen become available, suggesting Mycobacterium smegmatis can switch between fermentation, anaerobic respiration, and aerobic respiration. Hydrogen metabolism enables this obligate aerobe to rapidly meet its energetic needs when switching between microoxic and anoxic conditions and provides a competitive advantage in low oxygen environments.

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“…The activity of membrane-bound NAR is ostensibly high during anaerobic growth, identifying NAR as a likely driver of the isotope enrichment during denitrification. The cytosolic assimilatory NAS, in contrast, is probably not important in fractionation, because NAS expression is repressed in the presence of ammonia (Warneckeeberz and Friedrich 1993); ammonia is a sizeable constituent bactopeptone and casein hydrolysate in our growth media, and is further generated by catabolism of amino acids during bacterial growth. The activity of the periplasmic NAP during anaerobic growth, which will be addressed below, is probably considerably lower than that of the respiratory NAR, because NAP is not associated with energy production.…”

Section: Discussionmentioning

confidence: 98%

Nitrogen and oxygen isotope fractionation during dissimilatory nitrate reduction by denitrifying bacteria

Granger

Sigman

Lehmann

et al. 2008

Limnology & Oceanography

397

449

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We report the first measurements of coupled nitrogen (N) and oxygen (O) isotope fractionation of nitrate by laboratory cultures of denitrifying bacteria. Two seawater strains (Pseudomonas stutzeri, Ochrobactrum sp.) and three freshwater strains (Paracoccus denitrificans, Pseudomonas chlororaphis, Rhodobacter sphaeroides) were examined. Among four strains of facultative anaerobic denitrifiers, N and O isotope effects were variable, ranging from 5% to 25%, with evidence for a drop in the isotope effects as nitrate concentrations approached the halfsaturation constant for nitrate transport. O isotope effects were similar to their corresponding N isotope effect, such that the progressive increase in 1] 3 1000), yielded slopes of 0.86 to 1.02, with a mean value of 0.96. R. sphaeroides, a photo-heterotroph that possesses only a periplasmic (nonrespiring) dissimilatory nitrate reductase, showed less variability in nitrate N isotope effects, between 13% and 20%, with a modal value of ,15%. In contrast to the respiratory denitrifiers, R. sphaeroides consistently showed a distinct ratio of d 18 O to d 15 N change of ,0.62. We hypothesize that heavy N and O isotope discrimination during respiratory denitrification occurs during the intracellular reduction of nitrate by the respiratory nitrate reductase, and the observed magnitude of fractionation is likely regulated by the ratio of cellular nitrate efflux relative to uptake. The data for R. sphaeroides are consistent with isotope discrimination directly reflecting the N and O isotope effects of the periplasmic nitrate reductase NAP, without modification by nitrate uptake and efflux.

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Three nitrate reductase activities in Alcaligenes eutrophus

Cited by 56 publications

References 17 publications

Ralstonia eutropha TF93 Is Blocked in Tat-Mediated Protein Export

Ralstonia eutropha TF93 Is Blocked in Tat-Mediated Protein Export

An obligately aerobic soil bacterium activates fermentative hydrogen production to survive reductive stress during hypoxia

Nitrogen and oxygen isotope fractionation during dissimilatory nitrate reduction by denitrifying bacteria

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