Table 1.
PCR Primers sets used in this study.
Figure 1.
Congruency tests between mxaF and 16S rRNA gene nucleotide sequences of methanotrophs from GenBank database.
Phylogenetic trees for congruency tests based on maximum likelihood (ML) analysis of mxaF (∼513 bp) and 16S rRNA gene nucleotide sequences (∼1471 bp) from methanotrophs in GenBank, including the mxaF nucleotide sequences of Methylomonas rubra and Methylobacter luteus sequenced in this study. The ADH gene of Solibacter usitatus Ellin 6076 was used as outgroup. Methanotrophs (in the Methylococcaceae, Methylocystaceae and Beijerinckiaceae) are indicated by shaded clusters. Accession numbers of mxaF and 16S rRNA gene sequences downloaded from GenBank are indicated in parentheses. Bootstrap values from 1,000 replicates are indicated at the nodes of branches (if >50). The scale bar represents the number of nucleotide changes.
Figure 2.
Simplified phylogenetic tree of methanotrophs and their close relatives based on mxaF nucleotide sequences from GenBank database.
Unrooted phylogenetic tree based on maximum parsimony (MP) analysis of known proteobacterial partial mxaF and xoxF/xoxF-like nucleotide sequences (∼513 bp) from GenBank and the mxaF nucleotide sequences (in bold) of Methylomonas rubra and Methylobacter luteus sequenced in this study. The ADH gene of Solibacter usitatus Ellin 6076 was used as outgroup. Accession numbers of sequences downloaded from GenBank are indicated in parentheses. Bootstrap values from 1,000 replicates are indicated at the nodes of branches (if >50). The three bacterial families containing methanotrophs (Methylococcaceae, Methylocystaceae and methanotrophic members of the Beijerinckiaceae) are indicated by shaded clusters and the other alphaproteobacterial and betaproteobacterial methylotrophs are delineated by lines. The identity of mxaF and mxaF-like sequences from the “Methylobacterium cluster (within cluster 2)”, “Mainly Hyphomicrobium (Cluster 3)”, “β-proteobacterial methylotrophs (Cluster 5)”, and “xoxF/xoxF-like genes” is shown in Table 2. The scale bar represents the number of nucleotide changes. The complete phylogenetic tree of methanotrophs and their close relatives based on mxaF nucleotide sequences is shown in Supplement S1.
Table 2.
Taxa included (but not shown) in the phylogenetic analyses for Fig. 2.
Figure 3.
Phylogenetic tree of environmental mxaF gene sequences detected in this study.
Phylogenetic tree based on MP analyses of environmental partial mxaF nucleotide sequences (∼513 bp) detected in this study (in bold) in comparison with their close relatives, with Solibacter usitatus Ellin6076 as outgroup. All mxaF sequences were obtained using primer pair F1003 and R1561, except for the 13 putative symbiont mxaF genes from Bathymodiolus azoricus and B. puteoserpentis, which were obtained using primer pair F1003degen and R1561degen. Accession numbers of sequences downloaded from GenBank are indicated in parentheses. Bootstrap values from 1,000 replicates are indicated at the nodes of branches (if >50). Clone sequences are labeled P_C, pine soil (control); P_F, pine soil (fertilized); H_F, hardwood soil (fertilized); Sphag, Sphagnum moss; HBHA, Halls Brook Holding Area; RB, Rainbow; LS, Lucky Strike; LO, Logatchev, followed by clone number (#).Methanotrophs found in the three bacterial families (Methylococcaceae, Methylcystaceae and Beijerinckiaceae) are shaded. Bootstrap values from 1,000 replicates are indicated at the nodes of branches (if >50). The scale bar represents the number of nucleotide changes.