We discovered a remarkably high diversity of
pmoA gene types in our study (Fig.
1), including those closely related to the
pmoA of known members of the class
Alphaproteobacteria as well as gene types distinct from known species forming hitherto undescribed
pmoA lineages. Within type II methanotrophs, we found sequences closely related to
M. parvus (clade JR4), as well as the recently characterized type II
pmoA gene copy (
50) of
Methylocystis sp. (clade JR5). Interestingly, the relative abundance of the T-RFs was consistently higher for JR4 than for JR5 in our T-RFLP profiles (data not shown), which agrees with the findings of Tchawa Yimga et al. (
50) that not all type II methanotrophs possess this additional gene copy. We also discovered the clade JR1, which forms a distinct subgroup of the “RA 14” clade, the clade that has been putatively identified as atmospheric methane consumers (
21,
25). This finding considerably expands the known depth of the “RA 14” clade and demonstrates that methanotrophs possessing this gene type are not restricted to forest soils. We did not detect the other putative atmospheric methane consumers, the “WB5FH-A” clade (
32), although we did discover two novel clades (JR2 and JR3) which are distantly related to the “WB5FH-A” clade.
There are several lines of evidence that suggest that the three novel
pmoA clades we discovered (JR1, JR2, and JR3) encode functional monooxygenases, with a primary substrate of methane rather than ammonia. All three of the novel clades had dN/dS ratios well below 1 (Fig.
2), evidence for purifying selection (
55,
56). If these genes were nonfunctional copies, a lack of selection would result in nonsynonymous changes occurring at the same rate as synonymous changes, pushing the overall dN/dS ratio towards 1; how closely it approached 1 would depend on the divergence time of these clades. The dN/dS of the branches leading to all three novel clades, however, are not statistically different from the “background” dN/dS in the rest of each respective half of the
pmoA phylogeny. In addition, the high number of synonymous changes along the branches leading to the three novel clades suggests that they did not diverge recently (see Results above), and thus their low dN/dS ratios suggest that their encoded proteins are expressed and functional.
The conservation of functionally diagnostic amino acid residues provides further evidence for retained function in the novel clades and for their substrate specificity for methane rather than ammonia. The novel sequences contain a very high percentage of those amino acid residues conserved in both methane and ammonia monooxygenases (
42,
52). These conserved residues include those proposed to bind metal ions within the active site and at secondary stabilization sites (
42,
52), as well as a majority of the previously identified PmoA-specific residues (
25). Among the mismatched residues, almost all are in the same amino acid similarity groups as the PmoA-specific residues. The novel
Alphaproteobacteria clade JR1 has the lowest number of perfect matches to putatively PmoA-specific residues (11 of 16) (Table
2) and has several putatively AmoA-diagnostic residues. However, two of these “AmoA-like” residues are, in fact, shared by several other PmoA clades. Furthermore, JR1 robustly clusters in the
Alphaproteobacteria, within which there are no known
amoA-containing members. Thus, the total evidence suggests that JR1 likely binds methane rather than ammonia. The novel
Gammaproteobacteria clades JR2 and JR3 did not contain any AmoA-diagnostic residues. However, this picture is complicated somewhat by the fact that the only known ammonia-oxidizing bacteria within the class
Gammaproteobacteria, the
N. oceani-like clade, also lack many of the AmoA-diagnostic residues, and they are the closest phylogenetic relatives of JR2 and JR3 (Fig.
1). However, based on protein and inferred-translation alignments, there appear to be six sites that distinguish the
N. oceani-like AmoA from the
Gammaproteobacteria PmoA (Table
2) and from the enzyme encoded by JR2 and JR3. At position 71, the
N. oceani-like clade contains an AmoA-diagnostic residue present in no known PmoAs. This residue is not present in JR2 or JR3. At five other sites, the
N. oceani-like clade contains conserved residues distinct from known PmoAs and AmoAs; two residues are at PmoA-/AmoA-diagnostic positions, and three others are at positions conserved in all other PmoAs and AmoAs examined (Table
2), strongly suggesting functional relevance. None of these residues is present in JR2 or JR3. Finally, hydrophobicity plots of the consensus protein sequence of JR2 and JR3 show four transmembrane domains at positions identical to those of the
Gammaproteobacteria PmoA consensus; in contrast, the fourth domain of the consensus for
N. oceani-like AmoA is shifted 12 residues towards the C terminus, exactly matching the position of the corresponding hydrophobic domain of AmoA found within the class
Betaproteobacteria (data not shown). Together, these sequence analyses suggest strongly that JR2 and JR3 are more likely to preferentially bind methane than ammonia.