Methane oxidation at 55 degrees C and pH 2 by a thermoacidophilic bacterium belonging to the Verrucomicrobia phylum

Abstract

Methanotrophic bacteria constitute a ubiquitous group of microorganisms playing an important role in the biogeochemical carbon cycle and in control of global warming through natural reduction of methane emission. These bacteria share the unique ability of using methane as a sole carbon and energy source and have been found in a great variety of habitats. Phylogenetically, known methanotrophs constitute a rather limited group and have so far only been affiliated with the Proteobacteria. Here, we report the isolation and initial characterization of a nonproteobacterial obligately methanotrophic bacterium. The isolate, designated Kam1, was recovered from an acidic hot spring in Kamchatka, Russia, and is more thermoacidophilic than any other known methanotroph, with optimal growth at approximately 55 degrees C and pH 3.5. Kam1 is only distantly related to all previously known methanotrophs and belongs to the Verrucomicrobia lineage of evolution. Genes for methane monooxygenases, essential for initiation of methane oxidation, could not be detected by using standard primers in PCR amplification and Southern blot analysis, suggesting the presence of a different methane oxidation enzyme. Kam1 also lacks the well developed intracellular membrane systems typical for other methanotrophs. The isolate represents a previously unrecognized biological methane sink, and, due to its unusual phylogenetic affiliation, it will shed important light on the origin, evolution, and diversity of biological methane oxidation and on the adaptation of this process to extreme habitats. Furthermore, Kam1 will add to our knowledge of the metabolic traits and biogeochemical roles of the widespread but poorly understood Verrucomicrobia phylum.

Fig. 1.

Photomicrographs of the methanotrophic nonproteobacterial strain Kam1. (A) Phase-contrast image of Kam1 cells at 1,000× magnification. (Scale bar, 5 μm.) (B and C) Fluorescent in situ hybridization images of Kam1 cells visualized with DAPI staining and the Cy3-labeled 16S rRNA gene probe Kam1_964 (5′-CTGTGCCGTTCGCCCTTGC-3′), specifically designed for the Verrucomicrobia thermoacidophilic methanotroph (VTAM) cluster, respectively. (Scale bar, 5 μm.) (D) Transmission electron micrograph of a thin section of a Kam1 cell. (Scale bar, 200 nm.) (E) Magnified part of the Kam1 cell in D, highlighting the polyhedral organelles. (Scale bar, 200 nm.)

Fig. 2.

Phylogenetic tree based on 16S rRNA gene sequences from Kam1 and representatives from the Verrucomicrobia phylum. Sequences were retrieved from the Ribosome Database (http://rdp.cme.msu.edu), and the Kam1 sequence was added to this alignment. The tree was constructed by using the neighbor-joining algorithm with the Kimura two-parameter correction based on 1,211 positions. Nodes supported by bootstrap values >50% after 100 resamplings of neighbor-joining, and maximum-likelihood (in brackets) analyses are indicated. The 16S rRNA gene sequence of Methylococcus capsulatus was used as an outgroup. Subdivisions are indicated by encircled numbers. The scale bar represents 0.1 nucleotide changes per position.

Fig. 3.

Growth and corresponding methane consumption of Kam1. Averages ± standard error are shown for triplicate 60-ml serum bottles shaken at 55°C. The pH was 3.5. No decrease in headspace methane was observed from three parallel bottles with only medium (data not shown). Black circles indicate cell numbers; open circles indicate the methane level.