Characterization of methanotrophic bacterial populations in soils showing atmospheric methane uptake

Abstract

The global methane cycle includes both terrestrial and atmospheric processes and may contribute to feedback regulation of the climate. Most oxic soils are a net sink for methane, and these soils consume approximately 20 to 60 Tg of methane per year. The soil sink for atmospheric methane is microbially mediated and sensitive to disturbance. A decrease in the capacity of this sink may have contributed to the approximately 1%. year(-1) increase in the atmospheric methane level in this century. The organisms responsible for methane uptake by soils (the atmospheric methane sink) are not known, and factors that influence the activity of these organisms are poorly understood. In this study the soil methane-oxidizing population was characterized by both labelling soil microbiota with (14)CH(4) and analyzing a total soil monooxygenase gene library. Comparative analyses of [(14)C]phospholipid ester-linked fatty acid profiles performed with representative methane-oxidizing bacteria revealed that the soil sink for atmospheric methane consists of an unknown group of methanotrophic bacteria that exhibit some similarity to type II methanotrophs. An analysis of monooxygenase gene libraries from the same soil samples indicated that an unknown group of bacteria belonging to the alpha subclass of the class Proteobacteria was present; these organisms were only distantly related to extant methane-oxidizing strains. Studies on factors that affect the activity, population dynamics, and contribution to global methane flux of "atmospheric methane oxidizers" should be greatly facilitated by use of biomarkers identified in this study.

FIG. 1

¹⁴C-PLFA fingerprints for MOB. (A) Unknown methanotrophs that metabolized atmospheric methane in two forest soils in Denmark (Rold) and the United States (Maine). (B) Type I methanotroph Methylomicrobium album BG8 and type II methanotroph Methylosinus trichosporium OB3b. Each bar represents the amount of radiolabel in a fraction as a percentage of the radioactivity in all fractions. The following reference standard PLFAs coeluted with the ¹⁴C-PLFAs: 14:0, i15:1, and a15:1 in fraction 2; 16:1w9 and 16:1w8 in fraction 4; 16:1w7, 16:1w5, and 16:0 in fraction 5; i17:0, a17:0, and 17:1w8 in fraction 7; and 18:1w9, 18:1w8, and 18:1w7 in fraction 11.

FIG. 2

Alignment of predicted peptide sequences of PmoA and AmoA from representative methanotrophic and nitrifying bacteria with soil clone RA14 and RA21 sequences. Residues which are universally conserved in extant MOB are highlighted with black. Putative MMO signatures are highlighted with dots, and putative AMO signatures are highlighted with grey. The horizontal lines indicate predicted hydrophilic domains conserved in all of the peptides (15). The sequences shown (and their accession numbers) are as follows: Ms.tri, Methylosinus trichosporium (U31650); Mc. M, Methylocystis sp. strain M (U81596); Mc.par, Methylocystis parvus (U31651); Nc.oce, Nitrosococcus oceanus (U31652); Mb.alb, Methylomicrobium album BG8 (U31654); Mm.met, Methylomonas methanica S1 (U31653); LK6, Methylocaldum tepidum LK6 (U89304); Mc.cap, Methylococcus capsulatus Bath (L40804); Nm.eur, Nitrosomonas europaea (L08050); Nl.mul, Nitrosolobus multiformis (U31649); AHB1, Nitrosospira sp. strain AHB1 (X90821); Nspira, Nitrosospira sp. strain Np22 (U31655).

FIG. 3

Unrooted phylogenetic trees showing relationships of RA14 and RA21 to representative methanotrophs and ammonia-oxidizing bacteria. (A) Tree constructed by using a 171-amino-acid segment of the 27-kDa subunit of MMO (PmoA or AmoA). (B) Tree constructed by using a restricted sequence alignment (125 amino acids) in which putative signature residues (see Fig. 2) were omitted from the analysis. On the basis of the bacterial phylogeny derived from rRNA (10, 26, 42), the root of the trees is predicted to lie on the branch indicated by a dot. The numbers at the nodes indicate the numbers of times the groups occurred in 100 bootstrap replicates. Approximate K_m values for CH₄ (K_m CH₄) of the holoenzymes are indicated as micromolar concentrations (Table 1). For the sequence accession numbers see the legend to Fig. 2. Abbreviations: Mb, Methylomicrobium; Mm., Methylomonas; Nc., Nitrosococcus; Mc., Methylocystis; Ms., Methylosinus; Nm., Nitrosomonas; Nl., Nitrosolobus; Nspira, Nitrosospira.

FIG. 4

Phylogenetic analysis of the derived amino acid sequences encoded by the pmoA genes of methanotrophs, nitrifiers, and Rold, Maine, and Pantanal soil DNA samples. The dendrogram shows the results of an analysis in which PROTDIST was used. Bootstrap values greater than 50% derived from 100 replicates are also shown. The bar represents 10% sequence divergence, as determined by measuring the lengths of the horizontal lines connecting any two species.