A metagenomic insight into freshwater methane-utilizing communities and evidence for cooperation between the Methylococcaceae and the Methylophilaceae

Abstract

We investigated microbial communities active in methane oxidation in lake sediment at different oxygen tensions and their response to the addition of nitrate, via stable isotope probing combined with deep metagenomic sequencing. Communities from a total of four manipulated microcosms were analyzed, supplied with (13)C-methane in, respectively, ambient air, ambient air with the addition of nitrate, nitrogen atmosphere and nitrogen atmosphere with the addition of nitrate, and these were compared to the community from an unamended sediment sample. We found that the major group involved in methane oxidation in both aerobic and microaerobic conditions were members of the family Methylococcaceae, dominated by species of the genus Methylobacter, and these were stimulated by nitrate in aerobic but not microaerobic conditions. In aerobic conditions, we also noted a pronounced response to both methane and nitrate by members of the family Methylophilaceae that are non-methane-oxidizing methylotrophs, and predominantly by the members of the genus Methylotenera. The relevant abundances of the Methylococcaceae and the Methylophilaceae and their coordinated response to methane and nitrate suggest that these species may be engaged in cooperative behavior, the nature of which remains unknown.

Keywords: Lake sediment; Metagenomics; Methane; Methylococcaceae; Methylophilaceae; Methylotrophy; Microbial community; Nitrate.

Figure 1

Schematic of experimental setup shows workflow, duration of each enrichment and actual DNA samples separated into heavy and light fractions.

Figure 2

Taxonomic profiling of microcosms based on pyrotag analysis (A and B) and metagenome data analysis (C and D) shows high abundance of Methylococcaceae and Methylophilaceae in aerobic conditions. A. Distribution of pyrotag sequences among major phyla. Other, phyla making up less than 1% of total. B. Proportions of Methylococcaceae and Methylophilaceae sequences in pyrotag libraries. C. Distribution of sequences in metagenomes taxonomically classified at 90% identity level. D. Proportions of Methylococcaceae, Methylophilaceae and Methylocystaceae of total sequences taxonomically profiled at 90% identity level.

Figure 3

Abundances of the Methylococcaceae and the Methylophilaceae sequences as per cent of total taxonomically classified sequences show good correlation.

Figure 4

Relative abundance of nitrate metabolism genes ascribed to Methylococcaceae (blue), Methylophilaceae (red) and Methylocystaceae (green). Other (purple) represents a variety of phylotypes, including methylotrophs of other families, present at low abundances. See Supplemental Tables 4–8 for statistics.