Detection, isolation, and characterization of acidophilic methanotrophs from Sphagnum mosses

Abstract

Sphagnum peatlands are important ecosystems in the methane cycle. Methane-oxidizing bacteria in these ecosystems serve as a methane filter and limit methane emissions. Yet little is known about the diversity and identity of the methanotrophs present in and on Sphagnum mosses of peatlands, and only a few isolates are known. The methanotrophic community in Sphagnum mosses, originating from a Dutch peat bog, was investigated using a pmoA microarray. A high biodiversity of both gamma- and alphaproteobacterial methanotrophs was found. With Sphagnum mosses as the inoculum, alpha- and gammaproteobacterial acidophilic methanotrophs were isolated using established and newly designed media. The 16S rRNA, pmoA, pxmA, and mmoX gene sequences showed that the alphaproteobacterial isolates belonged to the Methylocystis and Methylosinus genera. The Methylosinus species isolated are the first acid-tolerant members of this genus. Of the acidophilic gammaproteobacterial strains isolated, strain M5 was affiliated with the Methylomonas genus, and the other strain, M200, may represent a novel genus, most closely related to the genera Methylosoma and Methylovulum. So far, no acidophilic or acid-tolerant methanotrophs in the Gammaproteobacteria class are known. All strains showed the typical features of either type I or II methanotrophs and are, to the best of our knowledge, the first isolated (acidophilic or acid-tolerant) methanotrophs from Sphagnum mosses.

Fig. 1.

Results for the pmoA-based microbial methanotrophic community analysis microarray of two Sphagnum moss samples from the Mariapeel. The color-coding bar on the left represents the achievable signal for an individual probe (1 indicates maximum signal obtained, 0.1 indicates 10% signal, i.e., only 10% hybridization to that probe, and 0 indicates no signal). Mp = Mariapeel; NL = The Netherlands. Mp1.1A and Mp1.2 are two samples taken in the same peat bog. A list of probes in their respective order is given in Table S2 in the supplemental material.

Fig. 2.

16S rRNA gene neighbor-joining tree showing the relationship of all the isolated strains to selected methanotrophs (>500 bootstrap replicates). Bootstrap values of >60% are indicated at the nodes of the branches with black dots. The scale bar represents the number of base substitutions per site.

Fig. 3.

pmoA and pxmA gene neighbor-joining tree based on DNA showing the relationship of all the isolated strains to selected methanotrophs (>500 bootstrap replicates). Bootstrap values of >60% are indicated at the nodes of the branches with black dots. The scale bar represents the number of base substitutions per site. *, GenBank accession no. EF591086, EF591085, FJ462788, FJ462789, FJ462791, and CP000975; **, GenBank accession no. EF591087, FJ462790, and CP000975.

Fig. 4.

mmoX gene neighbor-joining tree based on DNA showing the relationship of all the isolated strains (>500 bootstrap replicates). Bootstrap values of >60% are indicated at the nodes of the branches with black dots. The scale bar represents the number of base substitutions per site.

Fig. 5.

(A) Scanning electron micrograph of actively growing cells of strain 29. Bar, 5 μm. (B) Transmission electron micrograph of a cell of strain 29. Bar, 5 μm. (C) Transmission electron micrograph of a cell of strain M175. (D) Transmission electron micrograph of a cell of strain M242.

Fig. 6.

Transmission electron micrographs of high-pressure frozen, freeze-substituted, and Epon-embedded cells (A, B) and negatively stained cells (C, D) of strain M5. (A) Whole cell with typical intracytoplasmic membranes. (B) Whole cell with a putative storage particle (white). (C) Negative staining of several single cells of strain M5. (D) Negative staining of one/two cells of strain M5. The putative flagellum is visible at the right side.

Fig. 7.

Transmission electron micrographs of high-pressure frozen, freeze-substituted, and Epon-embedded cells of strain M200. (A) Whole cell with typical intracytoplasmic membranes. (B) Whole cell with a putative storage particle (white). (C) Cell periphery with the Gram-negative cell wall and the slime layer around the cell. (D) Several single cells of strain M200.