Methanotrophic archaea possessing diverging methane-oxidizing and electron-transporting pathways

Abstract

Anaerobic oxidation of methane (AOM) is a crucial process limiting the flux of methane from marine environments to the atmosphere. The process is thought to be mediated by three groups of uncultivated methane-oxidizing archaea (ANME-1, 2 and 3). Although the responsible microbes have been intensively studied for more than a decade, central mechanistic details remain unresolved. On the basis of an integrated analysis of both environmental metatranscriptome and single-aggregate genome of a highly active AOM enrichment dominated by ANME-2a, we provide evidence for a complete and functioning AOM pathway in ANME-2a. All genes required for performing the seven steps of methanogenesis from CO2 were found present and actively expressed. Meanwhile, genes for energy conservation and electron transportation including those encoding F420H2 dehydrogenase (Fpo), the cytoplasmic and membrane-associated Coenzyme B-Coenzyme M heterodisulfide (CoB-S-SCoM) reductase (HdrABC, HdrDE), cytochrome C and the Rhodobacter nitrogen fixation (Rnf) complex were identified and expressed, whereas genes encoding for hydrogenases were absent. Thus, ANME-2a is likely performing AOM through a complete reversal of methanogenesis from CO2 reduction without involvement of canonical hydrogenase. ANME-2a is demonstrated to possess versatile electron transfer pathways that would provide the organism with more flexibility in substrate utilization and capacity for rapid adjustment to fluctuating environments. This work lays the foundation for understanding the environmental niche differentiation, physiology and evolution of different ANME subgroups.

Figure 1

The proposed methane-oxidizing pathway and energy-converting mechanisms in ANME-2a. Only positive gene identifications were displayed (in boxes). The carbon flow was demonstrated with black arrows; the electron flow was indicated with grey arrows. Detailed information of methanogenesis-associated genes is displayed in Table 1, and the names of genes involved in electron transport are displayed in Supplementary Table S3.

Figure 2

The maximum likelihood tree showing the deduced amino acid sequences of mer-1 and mer-2 genes to the selected reference sequences. A total of 25 full-length amino acid sequences of mer were aligned. Bootstrap values were based on 1000 replicates and shown at the nodes. Mer-1 and Mer-2 from M25 identified in the single-aggregate genome were highlighted, with their detailed information displayed in Table 1.