Wide Distribution of a Novel pmoA -Like Gene Copy among Type II Methanotrophs, and Its Expression in Methylocystis Strain SC2

Abstract

Experiments were conducted to determine if a novel pmoA-like gene (pmoA2) recently discovered in the methane-oxidizing bacterium Methylocystis strain SC2 (P. F. Dunfield, M. Tchawa Yimga, S. D. Dedysh, U. Berger, W. Liesack, and J. Heyer, FEMS Microbiol. Ecol. 41:17-26, 2002) is present in other methane-oxidizing bacteria (MOB), and if it is expressed. A newly developed primer combination (pmoA206f-pmoA703b) allowed a differential detection of pmoA1 and pmoA2. By using this primer combination, we identified pmoA2 in a wide range of type II MOB of the Methylosinus-Methylocystis group. However, screening by PCR and by Southern hybridization using a newly developed pmoA2-specific oligonucleotide probe also showed that closely related type II MOB, exhibiting 16S rRNA gene sequence identities of higher than 97%, may or may not harbor pmoA2. No pmoA2 was detected in five type I MOB tested: Methylococcus capsulatus strain Bath, Methylocaldum strain E10A, Methylobacter luteus, Methylomicrobium album, and Methylomonas strain D1a. In comparative sequence analyses, all pmoA2-like sequences formed a coherent cluster clearly distinct from pmoA1 sequences of type I and type II MOB, and from amoA sequences of the Nitrosomonas-Nitrosospira group. Phylogenetic analysis using the paml model suggested that pmoA2 is subject to strong purifying selection and therefore has an important cellular function. We probed total RNA extracts of Methylocystis strain SC2 for gene expression of pmoA. A strong signal was observed for pmoA1 in Northern hybridization, while the results obtained for pmoA2 were ambiguous. However, reverse transcription-PCR confirmed that pmoA2 was expressed, albeit at lower level than pmoA1. This provided experimental evidence that the gene product of pmoA2 may be a functionally active enzyme.Methane-oxidizing bacteria (MOB) are able to utilize methane (CH 4 ) as their sole source of carbon and energy for growth (13). Their ability to oxidize CH 4 released at the interface of methanogenic environments and to act as sink for atmospheric CH 4 makes these organisms key players in balancing the global CH 4 budget and mitigating global warming due to CH 4 (4, 28). Phylogenies based on 16S rRNA genes show that MOB form distinct lineages in the Gammaproteobacteria (type I MOB) and Alphaproteobacteria (type II MOB) (2,5,6,13,16).The first step in CH 4 oxidation, the conversion of methane to methanol, is carried out by a methane monooxygenase (MMO). This enzyme exists in two forms, a particulate, membrane-associated form (pMMO) and a soluble form (sMMO). The two forms of enzyme differ in their structures, kinetic properties, and ranges of substrates they utilize (26). Only a restricted number of MOB species harbor sMMO, while almost all MOB possess pMMO. The only MOB lacking pMMO is Methylocella palustris (5).Cloning and sequence analysis of genes encoding pMMO revealed three consecutive open reading frames (pmoC, pmoA, and pmoB) in both type I (30, 31) and type II MOB (11). The pmoA gene, which encodes the 27-kDa subunit (Pm...

Development of a "genome-proxy" microarray for profiling marine microbial communities, and its application to a time series in Monterey Bay, California

“…(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).…”

“…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).…”

“…Based on alignments of the predicted peptide sequences of the ␣ subunits of 112 particulate methane monooxygenases (PmoAs) and 349 ammonia monooxygenases (AmoAs), Tukhvatullin et al (52) identified residues common to both proteins. Ricke et al (42) extended this analysis to include the second PmoA gene copy, PmoA2, present in many Type II methanotrophs (50). The inferred translation of the region amplified by the primers used in our study spans 16 of these highly conserved residues ( Table 2).…”

First Genome Data from Uncultured Upland Soil Cluster Alpha Methanotrophs Provide Further Evidence for a Close Phylogenetic Relationship toMethylocapsa acidiphilaB2 and for High-Affinity Methanotrophy Involving Particulate Methane Monooxygenase

“…In PmoA-based phylogenies, USC␣ and M. acidiphila B2 formed a common branch, together with the conventional PmoA1 sequences of the Methylosinus/Methylocystis group. The novel PmoA2 sequences formed a separate cluster (55). In contrast to the phylogeny of partial PmoA sequences, the concatenated PmoCAB sequences of USC␣ and M. acidiphila B2 branch clearly separately from a lineage defined, on the one hand, by PmoCAB1 of type II MB, and on the other hand, by PmoCAB2 of Methylocystis sp.…”

“…In general, pmoA and 16S rRNA phylogenies show good correlation (33,35). Despite this, the evolutionary identity of USC␣ is still uncertain because (i) the pmoA gene enables the inference of putative relationships only among MB and (ii) the recent detection of multiple, diverse pmoA-like genes in single genospecies of MB implies that pmoA phylogenies have to be interpreted with caution (55). Moreover, the apparent affinity for methane exhibited by M. acidiphila B2 was 1 to 2 M, which is similar to values measured in other cultured type I and type II MB (10).…”