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

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.

Keywords: Methylomicrobium album BG8; denitrification; hypoxia; methane monooxygenase; methanotroph; nitrite reduction; nitrous oxide.

FIGURE 1

Growth, CH₄ and O₂ consumption, and N₂O production by Methylomicrobium album strain BG8 cultivated on NMS and NMS plus 1 mM NaNO₂. Methylomicrobium album strain BG8 was cultivated for 5 days in 100 mL of NMS (black triangles) or NMS + 1 mM NO₂^- (gray dashed squares) media in 300 mL closed glass Wheaton bottles sealed with butyl rubber septum caps. The initial headspace gas-mixing ratio of CH₄ to O₂ was 1.6:1. Cell density (A) was measured using direct count with a Petroff–Hausser counting chamber and headspace gas concentrations of O₂ (B), CH₄ (C) and N₂O (D) were measured using GC-TCD. All data points represent the mean ± SD for six biological replicates (n = 6).

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).

FIGURE 3

NO₂^- reduction to N₂O by M. album strain BG8 is dependent on an energy source at <50 nM O₂. 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). Cells of M. album strain BG8 were harvested as described in the materials and methods and resuspended in mineral salts medium containing 100 μM NO₂^-. Arrows mark the addition of either CH₄ (A), CH₃OH (B), CH₂O (C), HCO₂H (D), C₂H₆ (E), C₂H₆O (F), in all panels. The right y-axis is identical in all panels. However, it should be noted that the left y-axis differs in all panels.

FIGURE 4

The coupling of NH₃ 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), N₂O (gray circles), NO₂^- (black dashed triangles). Cells of M. album strain BG8 were grown and harvested as described in the materials and methods and resuspended in nitrogen free mineral salts medium. Arrows mark the addition of NH₄⁺ (100 μM) to the closed micro-respiratory chamber. Traces (O₂ + N₂O) are single representatives of reproducible results from cultures grown on different days. NO₂^- was measured using a colorimetric method as described in the Section “Materials and Methods” and data points represent the mean ± SD for three technical replicates.

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.

FIGURE 6

Proposed model for NO₂^- respiration and central metabolism in Methylomicrobium album strain BG8. During hypoxia, M. album strain BG8 utilizes electrons from aerobic CH₄ oxidation to respire NO₂^-. Abbreviations: pMMO, particulate methane monooxygenase; mdh, methanol dehydrogenase; Cyt, cytochrome; nor, nitric oxide reductase; nir, nitrite reductase; ndh, NAD(P)H dehydrogenase complex; Q, coenzyme Q; bc₁, cytochrome bc₁ complex.