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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

Expression of pmoA, pxmA, nirS, norB1, and norB2 in Methylomicrobium album strain BG8 cultivated in NMS or NMS media amended with 1 mM NaNO2. Total RNA was extracted from Methylomicrobium album strain BG8 at 24, 48, and 72 h of growth (see Figure 1) from three separate cultures, converted to cDNA, and the abundance of pmoA, pxmA, nirS, norB1, and norB2 transcripts was determined using quantitative PCR. The transcript abundance of each gene of interest was normalized to that of 16s rRNA. The n-fold change in transcript abundance of the NO2- amended (1 mM NaNO2) NMS cultures relative to the unamended NMS cultures at 24 h of growth (light gray), 48 h of growth (diagonal white/gray), and at 72 h of growth (black). Error bars represent the SD calculated for triplicate qPCR reactions performed on each of the three biological replicates for each treatment. The (∗) above the bars designates a statistical significance (P < 0.05) as determined by t-test between NMS only and NMS + NO2- for each time point.
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Figure 5: Expression of pmoA, pxmA, nirS, norB1, and norB2 in Methylomicrobium album strain BG8 cultivated in NMS or NMS media amended with 1 mM NaNO2. Total RNA was extracted from Methylomicrobium album strain BG8 at 24, 48, and 72 h of growth (see Figure 1) from three separate cultures, converted to cDNA, and the abundance of pmoA, pxmA, nirS, norB1, and norB2 transcripts was determined using quantitative PCR. The transcript abundance of each gene of interest was normalized to that of 16s rRNA. The n-fold change in transcript abundance of the NO2- amended (1 mM NaNO2) NMS cultures relative to the unamended NMS cultures at 24 h of growth (light gray), 48 h of growth (diagonal white/gray), and at 72 h of growth (black). Error bars represent the SD calculated for triplicate qPCR reactions performed on each of the three biological replicates for each treatment. The (∗) above the bars designates a statistical significance (P < 0.05) as determined by t-test between NMS only and NMS + NO2- for each time point.

Mentions: To assess the effect of NO2- amendment on gene expression, we used cultures grown in NMS alone as the control. The O2 concentration in the headspace of NMS and NMS + NO2- cultures after 24 h growth was ca. 17.2 and 16.9%, respectively (Figure 1B). The transcript levels of pmoA, pxmA, nirS, and norB1 were significantly higher at the 24 and 48 h time points in the NO2- amended cultures when compared to the NMS alone (Figure 5). At the 72 h time point, levels of pmoA and nirS transcript levels remained significantly elevated in the NMS + NO2- relative to the NMS only cultures, whereas expression of norB1 was no longer significantly elevated (Figure 5). Most interestingly, the transcript abundance of pxmA at 72 h was 19.8-fold higher in NMS + NO2- relative to NMS only cultures (Figure 5). The second copy of norB (norB2) was unresponsive (below twofold) to NO2- amendment at all time points sampled.


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)

Expression of pmoA, pxmA, nirS, norB1, and norB2 in Methylomicrobium album strain BG8 cultivated in NMS or NMS media amended with 1 mM NaNO2. Total RNA was extracted from Methylomicrobium album strain BG8 at 24, 48, and 72 h of growth (see Figure 1) from three separate cultures, converted to cDNA, and the abundance of pmoA, pxmA, nirS, norB1, and norB2 transcripts was determined using quantitative PCR. The transcript abundance of each gene of interest was normalized to that of 16s rRNA. The n-fold change in transcript abundance of the NO2- amended (1 mM NaNO2) NMS cultures relative to the unamended NMS cultures at 24 h of growth (light gray), 48 h of growth (diagonal white/gray), and at 72 h of growth (black). Error bars represent the SD calculated for triplicate qPCR reactions performed on each of the three biological replicates for each treatment. The (∗) above the bars designates a statistical significance (P < 0.05) as determined by t-test between NMS only and NMS + NO2- for each time point.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Expression of pmoA, pxmA, nirS, norB1, and norB2 in Methylomicrobium album strain BG8 cultivated in NMS or NMS media amended with 1 mM NaNO2. Total RNA was extracted from Methylomicrobium album strain BG8 at 24, 48, and 72 h of growth (see Figure 1) from three separate cultures, converted to cDNA, and the abundance of pmoA, pxmA, nirS, norB1, and norB2 transcripts was determined using quantitative PCR. The transcript abundance of each gene of interest was normalized to that of 16s rRNA. The n-fold change in transcript abundance of the NO2- amended (1 mM NaNO2) NMS cultures relative to the unamended NMS cultures at 24 h of growth (light gray), 48 h of growth (diagonal white/gray), and at 72 h of growth (black). Error bars represent the SD calculated for triplicate qPCR reactions performed on each of the three biological replicates for each treatment. The (∗) above the bars designates a statistical significance (P < 0.05) as determined by t-test between NMS only and NMS + NO2- for each time point.
Mentions: To assess the effect of NO2- amendment on gene expression, we used cultures grown in NMS alone as the control. The O2 concentration in the headspace of NMS and NMS + NO2- cultures after 24 h growth was ca. 17.2 and 16.9%, respectively (Figure 1B). The transcript levels of pmoA, pxmA, nirS, and norB1 were significantly higher at the 24 and 48 h time points in the NO2- amended cultures when compared to the NMS alone (Figure 5). At the 72 h time point, levels of pmoA and nirS transcript levels remained significantly elevated in the NMS + NO2- relative to the NMS only cultures, whereas expression of norB1 was no longer significantly elevated (Figure 5). Most interestingly, the transcript abundance of pxmA at 72 h was 19.8-fold higher in NMS + NO2- relative to NMS only cultures (Figure 5). The second copy of norB (norB2) was unresponsive (below twofold) to NO2- amendment at all time points sampled.

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