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Diverse electron sources support denitrification under hypoxia in the obligate methanotroph Methylomicrobium album strain BG8.

Kits KD, Campbell DJ, Rosana AR, Stein LY - Front Microbiol (2015)

Bottom Line: Aerobic methane-oxidizing bacteria (MOB) are a diverse group of microorganisms that are ubiquitous in natural environments.Along with anaerobic MOB and archaea, aerobic methanotrophs are critical for attenuating emission of methane to the atmosphere.Our results suggest that expression of denitrification genes, found widely within genomes of aerobic methanotrophs, allow the coupling of substrate oxidation to the reduction of nitrogen oxide terminal electron acceptors under oxygen limitation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Faculty of Science, University of Alberta Edmonton, AB, Canada.

ABSTRACT
Aerobic methane-oxidizing bacteria (MOB) are a diverse group of microorganisms that are ubiquitous in natural environments. Along with anaerobic MOB and archaea, aerobic methanotrophs are critical for attenuating emission of methane to the atmosphere. Clearly, nitrogen availability in the form of ammonium and nitrite have strong effects on methanotrophic activity and their natural community structures. Previous findings show that nitrite amendment inhibits the activity of some cultivated methanotrophs; however, the physiological pathways that allow some strains to transform nitrite, expression of gene inventories, as well as the electron sources that support this activity remain largely uncharacterized. Here we show that Methylomicrobium album strain BG8 utilizes methane, methanol, formaldehyde, formate, ethane, ethanol, and ammonia to support denitrification activity under hypoxia only in the presence of nitrite. We also demonstrate that transcript abundance of putative denitrification genes, nirS and one of two norB genes, increased in response to nitrite. Furthermore, we found that transcript abundance of pxmA, encoding the alpha subunit of a putative copper-containing monooxygenase, increased in response to both nitrite and hypoxia. Our results suggest that expression of denitrification genes, found widely within genomes of aerobic methanotrophs, allow the coupling of substrate oxidation to the reduction of nitrogen oxide terminal electron acceptors under oxygen limitation. The present study expands current knowledge of the metabolic flexibility of methanotrophs by revealing that a diverse array of electron donors support nitrite reduction to nitrous oxide under hypoxia.

No MeSH data available.


Related in: MedlinePlus

The instantaneous coupling of CH4 oxidation to NO2- reduction in Methylomicrobium album strain BG8 under hypoxia. Experiments were performed in a closed 10 mL micro-respiratory chamber outfitted with an O2 and N2O microsensor and logged with Sensor Trace Basic software. O2 (black diamonds) and N2O (gray circles) were measured using microsensors. Cells of M. album strain BG8 were harvested as described in the materials and methods and resuspended in nitrogen free mineral salts medium. Arrows mark the addition of CH4 (∼300 μM) and NaNO2 (1 mM) to the micro-respiratory chamber in all panels. There is no measureable denitrification activity in the absence of NO2-(A); denitrification activity is dependent on CH4 and NO2-(B).
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Figure 2: The instantaneous coupling of CH4 oxidation to NO2- reduction in Methylomicrobium album strain BG8 under hypoxia. Experiments were performed in a closed 10 mL micro-respiratory chamber outfitted with an O2 and N2O microsensor and logged with Sensor Trace Basic software. O2 (black diamonds) and N2O (gray circles) were measured using microsensors. Cells of M. album strain BG8 were harvested as described in the materials and methods and resuspended in nitrogen free mineral salts medium. Arrows mark the addition of CH4 (∼300 μM) and NaNO2 (1 mM) to the micro-respiratory chamber in all panels. There is no measureable denitrification activity in the absence of NO2-(A); denitrification activity is dependent on CH4 and NO2-(B).

Mentions: Methylomicrobium album strain BG8 was grown in NMS + NO2- medium as described above. At 96 h of growth, when denitrification activity was highly evident, 4 × 1010 cells were harvested using a filtration manifold onto 0.2 μm filters (Supor 200, 47 mm, Pall Corporation). The biomass was washed three times with sterile, nitrogen-free mineral salts medium – identical to the mineral salts medium used for cultivation but devoid of NH4Cl, KNO3, or NaNO2. For data presented in Figures 2 and 4, the washed biomass was resuspended in the same nitrogen-free medium and transferred to a gastight 10 mL micro-respiration chamber equipped with an OX-MR O2 micro-sensor (Unisense) and an N2O-500 N2O micro-sensor (Unisense). For data presented in Figure 3, biomass was resuspended in mineral salts medium amended with 100 μM NaNO2. Data was logged using SensorTrace Basic software. CH4 gas, 0.001% CH3OH (HPLC grade methanol, Fisher Scientific), 0.01% CH2O (Methanol free 16% formaldehyde, Life technologies), 10 mM HCO2H, C2H6 gas (99.999%), 0.01% C2H6O (Methanol free 95% ethyl alcohol, Commercial Alcohols), 200 mM NH4Cl, and/or 1 M NO2- was injected directly into the chamber through the needle injection port with a gas-tight syringe (SGE Analytical Science). In Figures 3B–E, the dissolved O2 was decreased to <100 μmol/L (Figure 3B) and <25 μmol/L (Figures 3C–E), respectively, with additions of CH4 (Figure 3A), CH3OH (Figure 3B), CH2O (Figure 3C), HCO2H (Figure 3D), C2H6 (Figure 3E), C2H6O (Figure 3F) before data logging was enabled to limit the traces to <100 min and to reduce the number of sampling points. NO2- concentration was determined using a colorimetric method (Bollmann et al., 2011). Experiments were performed 3–4 times to demonstrate reproducibility of results and a single representative experiment was selected for presentation.


Diverse electron sources support denitrification under hypoxia in the obligate methanotroph Methylomicrobium album strain BG8.

Kits KD, Campbell DJ, Rosana AR, Stein LY - Front Microbiol (2015)

The instantaneous coupling of CH4 oxidation to NO2- reduction in Methylomicrobium album strain BG8 under hypoxia. Experiments were performed in a closed 10 mL micro-respiratory chamber outfitted with an O2 and N2O microsensor and logged with Sensor Trace Basic software. O2 (black diamonds) and N2O (gray circles) were measured using microsensors. Cells of M. album strain BG8 were harvested as described in the materials and methods and resuspended in nitrogen free mineral salts medium. Arrows mark the addition of CH4 (∼300 μM) and NaNO2 (1 mM) to the micro-respiratory chamber in all panels. There is no measureable denitrification activity in the absence of NO2-(A); denitrification activity is dependent on CH4 and NO2-(B).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4594100&req=5

Figure 2: The instantaneous coupling of CH4 oxidation to NO2- reduction in Methylomicrobium album strain BG8 under hypoxia. Experiments were performed in a closed 10 mL micro-respiratory chamber outfitted with an O2 and N2O microsensor and logged with Sensor Trace Basic software. O2 (black diamonds) and N2O (gray circles) were measured using microsensors. Cells of M. album strain BG8 were harvested as described in the materials and methods and resuspended in nitrogen free mineral salts medium. Arrows mark the addition of CH4 (∼300 μM) and NaNO2 (1 mM) to the micro-respiratory chamber in all panels. There is no measureable denitrification activity in the absence of NO2-(A); denitrification activity is dependent on CH4 and NO2-(B).
Mentions: Methylomicrobium album strain BG8 was grown in NMS + NO2- medium as described above. At 96 h of growth, when denitrification activity was highly evident, 4 × 1010 cells were harvested using a filtration manifold onto 0.2 μm filters (Supor 200, 47 mm, Pall Corporation). The biomass was washed three times with sterile, nitrogen-free mineral salts medium – identical to the mineral salts medium used for cultivation but devoid of NH4Cl, KNO3, or NaNO2. For data presented in Figures 2 and 4, the washed biomass was resuspended in the same nitrogen-free medium and transferred to a gastight 10 mL micro-respiration chamber equipped with an OX-MR O2 micro-sensor (Unisense) and an N2O-500 N2O micro-sensor (Unisense). For data presented in Figure 3, biomass was resuspended in mineral salts medium amended with 100 μM NaNO2. Data was logged using SensorTrace Basic software. CH4 gas, 0.001% CH3OH (HPLC grade methanol, Fisher Scientific), 0.01% CH2O (Methanol free 16% formaldehyde, Life technologies), 10 mM HCO2H, C2H6 gas (99.999%), 0.01% C2H6O (Methanol free 95% ethyl alcohol, Commercial Alcohols), 200 mM NH4Cl, and/or 1 M NO2- was injected directly into the chamber through the needle injection port with a gas-tight syringe (SGE Analytical Science). In Figures 3B–E, the dissolved O2 was decreased to <100 μmol/L (Figure 3B) and <25 μmol/L (Figures 3C–E), respectively, with additions of CH4 (Figure 3A), CH3OH (Figure 3B), CH2O (Figure 3C), HCO2H (Figure 3D), C2H6 (Figure 3E), C2H6O (Figure 3F) before data logging was enabled to limit the traces to <100 min and to reduce the number of sampling points. NO2- concentration was determined using a colorimetric method (Bollmann et al., 2011). Experiments were performed 3–4 times to demonstrate reproducibility of results and a single representative experiment was selected for presentation.

Bottom Line: Aerobic methane-oxidizing bacteria (MOB) are a diverse group of microorganisms that are ubiquitous in natural environments.Along with anaerobic MOB and archaea, aerobic methanotrophs are critical for attenuating emission of methane to the atmosphere.Our results suggest that expression of denitrification genes, found widely within genomes of aerobic methanotrophs, allow the coupling of substrate oxidation to the reduction of nitrogen oxide terminal electron acceptors under oxygen limitation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Faculty of Science, University of Alberta Edmonton, AB, Canada.

ABSTRACT
Aerobic methane-oxidizing bacteria (MOB) are a diverse group of microorganisms that are ubiquitous in natural environments. Along with anaerobic MOB and archaea, aerobic methanotrophs are critical for attenuating emission of methane to the atmosphere. Clearly, nitrogen availability in the form of ammonium and nitrite have strong effects on methanotrophic activity and their natural community structures. Previous findings show that nitrite amendment inhibits the activity of some cultivated methanotrophs; however, the physiological pathways that allow some strains to transform nitrite, expression of gene inventories, as well as the electron sources that support this activity remain largely uncharacterized. Here we show that Methylomicrobium album strain BG8 utilizes methane, methanol, formaldehyde, formate, ethane, ethanol, and ammonia to support denitrification activity under hypoxia only in the presence of nitrite. We also demonstrate that transcript abundance of putative denitrification genes, nirS and one of two norB genes, increased in response to nitrite. Furthermore, we found that transcript abundance of pxmA, encoding the alpha subunit of a putative copper-containing monooxygenase, increased in response to both nitrite and hypoxia. Our results suggest that expression of denitrification genes, found widely within genomes of aerobic methanotrophs, allow the coupling of substrate oxidation to the reduction of nitrogen oxide terminal electron acceptors under oxygen limitation. The present study expands current knowledge of the metabolic flexibility of methanotrophs by revealing that a diverse array of electron donors support nitrite reduction to nitrous oxide under hypoxia.

No MeSH data available.


Related in: MedlinePlus