Methane-oxidizing bacteria in a California upland grassland soil: diversity and response to simulated global change

Abstract

We investigated the diversity of methane-oxidizing bacteria (i.e., methanotrophs) in an annual upland grassland in northern California, using comparative sequence analysis of the pmoA gene. In addition to identifying type II methanotrophs commonly found in soils, we discovered three novel pmoA lineages for which no cultivated members have been previously reported. These novel pmoA clades clustered together either with clone sequences related to "RA 14" or "WB5FH-A," which both represent clusters of environmentally retrieved sequences of putative atmospheric methane oxidizers. Conservation of amino acid residues and rates of nonsynonymous versus synonymous nucleotide substitution in these novel lineages suggests that the pmoA genes in these clades code for functionally active methane monooxygenases. The novel clades responded to simulated global changes differently than the type II methanotrophs. We observed that the relative abundance of type II methanotrophs declined in response to increased precipitation and increased atmospheric temperature, with a significant antagonistic interaction between these factors such that the effect of both together was less than that expected from their individual effects. Two of the novel clades were not observed to respond significantly to these environmental changes, while one of the novel clades had an opposite response, increasing in relative abundance in response to increased precipitation and atmospheric temperature, with a significant antagonistic interaction between these factors.

FIG. 1.

Phylogenetic relationships among pmoA gene types identified in the Jasper Ridge Global Change Experiment and pmoA and amoA gene types available in public databases (2, 3, 6-9, 17, 21, 25, 28-30, 32, 35, 37, 45, 53). Sequences obtained in this study are shown in boldface type with the prefix “JR” and are designated clades JR1 to JR6. The environmental pmoA sequences used for reference were retrieved from various habitats, as follows: forest soils (AF148527, AF148521 [25], AF148522 [25], AF200727 [21], AY500134, AY372360 [29]), rice fields (AJ299961 [28]), peat soil (AF358043, AF358046 [35], AY236518 [9]), and upland grassland soils (AJ579670, AJ579669, AJ579668, AJ579667 [32]). The scale bar corresponds to 0.1 substitutions per nucleotide. The tree was calculated using 475 nucleotide positions and the neighbor joining approach (with the Felsenstein correction), via the ARB program package (33). The tree topology was confirmed using the maximum likelihood approach. Bootstrap values were calculated using 1,000 replications. AOB, ammonia-oxidizing bacteria.

FIG. 2.

The dN/dS values of the major lineages of pmoA as estimated using the codeml executable of the PAML program. The numbers at each branch are the dN/dS ratios estimated by the program under the freely varying model, which allowed the dN/dS of each major branch and clade (as denoted with dashed circles) to vary simultaneously. (The asterisk at the branch connecting the type I and “WB5FH-A” clades to the other Alphaproteobacteria clades indicates a noncomputable dN/dS ratio, where dN = 0.03 and dS = 0). Novel clades are shown in boldface type, as are the dN/dS ratios of the branches leading to these clades. The dN/dS ratios within each clade are not shown. Analyses were run on each half of the pmoA tree independently; the split between the Alphaproteobacteria and Gammaproteobacteria sections is indicated by a dashed line. MOB, methane-oxidizing bacteria.

FIG. 3.

Representative T-RFLP profile of the methanotroph community and the assignment (arrows) of the T-RFs to known methanotrophs-sublineages and to pmoA gene types determined in this study. The phylogenetic tree was graphically modified from Fig. 1. Arrows with dashed lines indicate the existence of multiple sequence types that potentially can produce the respective T-RFs according to the sequence information of the pmoA database (i.e., T-RFs of 80 bp, 440 bp, 503 bp, and 511 bp). AOB, ammonia-oxidizing bacteria.

FIG. 4.

Effect of temperature and precipitation on pmoA clade JR4 (type II methanotrophs) in the JRGCE. The mean relative abundance of JR4 is depicted for all samples, grouped by temperature and precipitation treatments. For example, the first bar depicts the mean relative abundance of JR4 from all experimental plots under ambient temperature and precipitation, including those under both ambient and elevated CO₂ and ambient and elevated nitrogen treatments (n = 32). Error bars are 95% confidence limits. MOB, methane-oxidizing bacteria.

FIG. 5.

Effect of temperature and precipitation on novel pmoA clade JR2 in the JRGCE. The mean relative abundance of JR2 is depicted for all samples, grouped by temperature and precipitation treatments. For example, the first bar depicts the mean relative abundance of JR2 from all experimental plots under ambient temperature and precipitation, including those under both ambient and elevated CO₂ and ambient and elevated nitrogen treatments (n = 32). Error bars are 95% confidence limits. MOB, methane-oxidizing bacteria.