After 2 y of enrichment a pure culture of a MOB from a landfill soil inoculum was obtained. The complete genome sequence of strain MG08 was determined from 94,080 long reads (Pacific Biosciences) which assembled into a single circular chromosome with 3,326,440 bp and a GC content of 58.94% (SI Appendix, Table S2). All reads mapped to the genome confirming that no contaminating organisms were present in the culture. The genome encoded 46 tRNA genes, a single 16S, 23S, and 5S rRNA gene operon, a single pmoCAB operon, and one orphan pmoC gene.

Screening of all publicly available environmental 16S rRNA gene amplicon datasets identified 194,764 sequences in 1,537 datasets (0.8% of all screened datasets) that were identical or very similar (>97% identity) to the 16S rRNA gene of M. gorgona MG08. These datasets originated almost exclusively from terrestrial environments (primarily from soil) spanning six continents, with latitudes ranging from the high arctic Svalbard over the tropics to southern Australia and New Zealand ( Fig. 3 ), demonstrating that microbes identical to or highly related to our isolate are globally distributed in terrestrial ecosystems.

The identification of M. gorgona as a member of the USCα group was confirmed by phylogenetic analysis based on a concatenated alignment of 34 conserved taxonomic marker genes ( Fig. 2B ). However, we did not recover strong support for a monophyletic clade of Methylocapsa, regardless of whether we considered USCα as part of the genus or not. This lack of resolution was due to the inclusion of two strains of Methylocella silvestris in our phylogenetic reconstruction. These strains share 64–65% AAI with recognized species of Methylocapsa, which is lower than the 67–72% AAI shared within recognized Methylocapsa but similar to the 63–69% AAI shared between recognized species of Methylocapsa and USCα genomes ( Fig. 2A ). Together, Methylocapsa, USCα, and Methylocella all form a single strongly supported clade. Although AAI values and phylogenetic arguments taken together support the assignment of M. gorgona MG08 and other USCα as members of the Methyolcapsa, our data do not resolve the relationship between Methylocella and lineages of Methylocapsa. We hope that the isolation and genome sequencing of additional Methylocapsa strains in the future will help resolve the branching order and relationships within this clade.

Average nucleotide and amino acid identities, phylogenetic relationship, and central metabolism comparison between M. gorgona MG08 and its genome sequenced relatives. (A) Symmetrical matrix of pairwise gANI and AAIs between all strains and MAGs and ordered as in B. ANI is presented in the Lower Left triangle and values ≥74 are provided. AAI is presented in the Upper Right triangle and values ≥60 are provided. M. gorgona MG08 and Ca. M. lahnbergensis (AAI, 71.3; ANI, 78.1), MAG USC1 (AAI, 70.1; ANI, 78.0), MAG USC2 (AAI, 70.0; ANI, 77.3), M. aurea KYG T (AAI, 69.3; ANI, 72.5), M. acidiphila B2 (AAI, 67.4; ANI, 74.75), M. palsarum NE2 (AAI, 66.1; ANI, 67.9) and M. silvestris BL2 (AAI, 62.5; ANI, 69.9) are below the species threshold of 96.6 ANI (3) and 95 AAI (4). (B) The phylogenomic tree was calculated with 10 independent chains of 11,000 generations under the LG model with four rate categories, using an input alignment of 34 concatenated marker genes ( Materials and Methods ). A total of 6,000 generations of each chain were discarded as burn-in, the remainder were subsampled every five trees (bpcomp -x 6000 5 11000) and pooled together for calculation of the reported 50% consensus tree and bipartition posterior probabilities (maxdiff = 0.814, meandiff = 0.010076). The model and number of rate categories was identified using ModelFinder ( Materials and Methods ). (C) Distribution of functional complexes presented in Fig. 4 and SI Appendix, Table S1 were determined using blast (5), OrthoFinder (6), and manual examination of trees. Presence of a complete complex is indicated by a solid square. Complexes that are incomplete are indicated with an embedded diamond. Abbreviations for functional complexes: aca, carbonic anhydrase; acc, acetyl-CoA carboxylase; atp, ATP synthase; cox, carbon monoxide dehydrogenase; cyd, terminal cytochrome oxidase; eno, enolase; fae, formaldehyde activating enzyme; fdh, formate dehydrogenase; fdx, ferredoxin, 2Fe-2S; fhc, formyltransferase/hydrolase complex; fhs, formate–tetrahydrofolateligase; FNR, ferredoxin-NADP+ oxidoreductase; fol, bifunctional 5,10-methylene-tetrahydrofolatedehydrogenase, and 5,10-methylene-tetrahydrofolatecyclohydrolase; gck, 2-glycerate kinase; gcv, glycine cleavage complex; gly, serine hydroxymethyltransferase; hhy, [NiFe] hydrogenase; hpr, hydroxypyruvate reductase; mch, methenyl tetrahydromethanopterin cyclohydrolase; mcl, malyl-CoA lyase; mdh, malate dehydrogenase; mtd, NAD(P)-dependent methylene tetrahydromethanopterin dehydrogenase; mtk, malate thiokinase; mxa, methanol dehydrogenase; nif, nitrogenase; nuo, NADH-quinone oxidoreductase; pet, ubiquinol-cytochrome c reductase; pmo, particulate methane monooxygenase; ppc, phosphoenolpyruvate carboxylase; and sga, serine–glyoxylate aminotransferase.

Phylogenetic relationship of the PmoA and electron micrographs of M. gorgona MG08. (A) The unrooted maximum-likelihood tree based on 155-aa positions was computed using the Jones Taylor Thornton matrix-based model of amino acid substitution. Env. Seq., environmental sequence (clone or DGGE sequence). The PmoA of M. gorgona MG08 clusters with other uncultured MOB within the USCα JR1/cluster 5 (Methylocella species does not carry the genes that encode pMMO and is therefore not represented in the tree). Bootstrap values are presented at branch points (1,000 data resamplings). Bar, 0.05-aa substitutions per site. (B) Scanning electron micrograph of M. gorgona MG08 cells grown at 21 °C in liquid culture with 20% CH 4 headspace without shaking. (C) Transmission electron micrograph of cells grown under the same conditions showing intracytoplasmic membranes of type III and inclusions that resemble PHB granules.

The inferred PmoA sequence of the newly cultured MOB grouped within the previously described USCα Jasper Ridge 1/cluster 5 ( 32 ) ( Fig. 1A ) and had up to 87.7% amino acid identity with environmentally retrieved sequences (FR720169) within this cluster. Consistently, 16S rRNA gene-based phylogenetic analyses showed that this MOB clusters with the 16S rRNA gene sequence (97.8% identity) of the uncultured USCα Candidatus Methyloaffinis lahnbergensis ( 25 ) ( SI Appendix, Fig. S1 ). Two additional USCα genomes that were recently assembled from subarctic mire metagenomes did not contain 16S rRNA genes ( 31 ). The average amino acid identity (AAI) between our isolate and previously published USCα metagenome-assembled genomes (MAGs) ranges from 70% to 72% ( Fig. 2A ) confirming that our isolate and existing USCα MAGs likely belong to the same genus. Interestingly, the AAI between our isolate and characterized Methylocapsa species ranges from 67% to 69%. This is comparable to the AAI shared within validly published Methylocapsa species (67–72%), suggesting that our isolate and, by extension, other USCα MAGs are all members of the genus Methylocapsa. Genomic average nucleotide identity (gANI) shared between our isolate and other members of the Methylocapsa genus, including the USCα MAGs, ranges from 74% to 79%, demonstrating that the isolate constitutes a species within the genus Methylocapsa ( Fig. 2A ), which we named M. gorgona MG08; gor.go’na. L. fem. n. gorgona (from Gr. fem. n. gorgonè) a vicious female monster from Greek mythology with sharp fangs and hair of living, venomous snakes in reference to the hair-like structures produced by the type of strain ( Fig. 1B ).

Proteomic analyses of M. gorgona MG08 grown in liquid culture under an atmosphere containing 20% CH 4 (but at ambient concentrations of CO and H 2 ) in the presence of 10 mM nitrate confirmed that all proteins shown in Fig. 4 except nitrogenase and a cbb3 type cytochrome c oxidase were expressed ( SI Appendix, Table S1 ). Among the detected proteins were those of the reductive glycine pathway, including its CO 2 reductase, the high-affinity hydrogenase, and carbon monoxide dehydrogenase.

The central carbon and energy metabolism of M. gorgona MG08 as predicted from its genome and confirmed by proteomics. H 4 MPT, tetrahydromethanopterin. Dashed black arrows indicate passive diffusion across the cell membrane. Numbers for the metabolic steps in the figure refer to the following enzyme names: (1) particulate methane monooxygenase, (2) methanol dehydrogenase and corresponding cytochrome c, (3) formaldehyde activating enzyme, (4) NAD(P)-dependent methylene tetrahydromethanopterin dehydrogenase, (5) methenyl tetrahydromethanopterin cyclohydrolase, (6) formyltransferase/hydrolase, (7A) NAD-dependent formate dehydrogenase, (7B) molybdopterin binding reversible formate dehydrogenase/CO 2 reductase, (8) formate-tetrahydrofolate ligase, (9) bifunctional 5,10-methylene-tetrahydrofolate dehydrogenase and 5,10-methylene-tetrahydrofolate cyclohydrolase, (10) glycine cleavage system, (11) serine hydroxymethyltransferase, (12) serine–glyoxylate aminotransferase, (13) hydroxypyruvate reductase, (14) 2-glycerate kinase, (15) enolase, (16) phosphoenolpyruvate carboxylase, (17) malate dehydrogenase, (18) malate thiokinase, (19) malyl-CoA lyase, (20) carbonic anhydrase, (21) acetyl-CoA carboxylase, (22) [MuCo] class I carbon monoxide dehydrogenase, (23) cytochrome c reductase and corresponding cytochrome c, (24) cytochrome c/d/o terminal oxidases (cytochrome d oxidase—cydAB, cytochrome o ubiquinol oxidase—cyoABCD, heme-copper cytochrome c oxidase type A1 – ctaAEGBC, heme-copper cytochrome type C cbb 3 oxidase—ccoNOQP and heme-copper cytochrome c oxidase type A1—coxCAB). (25) [NiFe] group 1h hydrogenase, (26) NADH dehydrogenase, (27) ATP synthase, (28) nitrogenase, (29) ferredoxin, 2Fe-2S, and (30) ferredoxin-NADP + oxidoreductase. All enzymes and electron carrier proteins were also detected in the proteome, with the exception of the nitrogenase and the cbb 3 oxidase. A full list of the proteins, corresponding genes, and genome entries are found in SI Appendix, Table S1 .

Physiology and Morphology.

M. gorgona MG08 phylogenetically belongs to the clade of putative high-affinity alphaproteobacterial USCα MOB. We therefore performed experiments to test whether this isolate can grow on ambient air containing an atmospheric CH 4 concentration using a microcolony cultivation technique of filters floating on liquid nitrate mineral salt medium without any added energy or carbon source. These experiments unequivocally demonstrated that M. gorgona MG08 grew in ambient air (∼1.86 p.p.m.v. CH 4 , ∼0.2 p.p.m.v. CO, and ∼0.5 p.p.m.v. H 2 ) and higher (20 and 1,000 p.p.m.v.) CH 4 concentrations (Fig. 5). After up to 3 wk of incubation the largest colonies were observed under 1,000 p.p.m.v. CH 4 while smaller colonies formed under 20 p.p.m.v., similar to those in unamended atmospheric air, confirming that increased concentrations of CH 4 stimulated growth. Continued growth of M. gorgona MG08 in unamended air was confirmed after 5 and 12 mo (Fig. 5), reaching more than 10 times its initial population size after 5 mo and continuing to grow and form spherical colonies observable after 12 mo (Fig. 5). In comparison, starving Sinorhizobium meliloti were able to triple their initial population size using intracellular polyhydroxybutyrate (PHB) as a carbon and energy source and maintain its population for 5 mo before it declined to a level below its initial size (51). A species more closely related to M. gorgona MG08, the MOB Methylocystis parvus OBBP, did not replicate at all using stored PHB and depended on the access to CH 4 for co-oxidation of the two substrates for growth (52). Considering this, we find it implausible that M. gorgona MG08 would depend entirely upon PHB for its growth over the course of 12 mo, but it is possible that any existing PHB storages were tapped during the incubation period and used as a carbon and energy supplement to its main diet.

Fig. 5. Microcolonies of M. gorgona MG08 cultivated at different CH 4 concentrations. Microcolonies were grown on polycarbonate filters floating on liquid nitrate mineral salt medium for the number of days specified on each picture, either in closed jars with air amended with different concentrations of CH 4 , or exposed to unamended air. For fixation, the filters were transferred to fresh-made 2% paraformaldehyde in 1× PBS in the refrigerator overnight. For staining, filters were transferred (side with bacteria up) on top of 200 µL droplets of 1,000× SYBRgreen and incubated for 10 min, washed, and air dried.

M. gorgona MG08 encodes only a single copy of the particulate methane monooxygenase which was also detected in its proteome. This shows that the same enzyme is responsible for catalyzing CH 4 oxidation at both high and low CH 4 concentrations. To confirm that M. gorgona MG08 is able to oxidize atmospheric concentrations of CH 4 we performed a CH 4 oxidation and microcolony-growth experiment with cells on floating filters under atmopheric air (1.86 p.p.m.v. CH 4 ). The results clearly show that CH 4 was oxidized during the 120 d of incubation under atmospheric air (Fig. 6A). Inspection of the filters confirmed cell growth (Fig. 6B). M. gorgona MG08 also carries single copies of the high-affinity respiratory hydrogenase and carbon monoxide dehydrogenase and both were expressed under 20% CH 4 headspace concentrations, without added CO or H 2 . Thus, it seems likely that these proteins are constitutively expressed at different CH 4 concentrations and thus contribute to the energy harvest from air. However, it is not possible to conclude on this matter until we have successfully determined the proteome of M. gorgona MG08 grown on air, which was not yet achieved due to biomass limitations. Furthermore, due to the difficulty in preparing headspace atmospheres and media free of trace amounts of alternative carbon and energy sources such as CO (0.2 p.p.m.v. in air) and H 2 (0.5 p.p.m.v. in air), and EDTA (53) (1.1 µM in the 1/10 diluted nitrate mineral salt medium used), respectively, we cannot yet conclude whether growth is possible solely on atmospheric CH 4 . However, growth did occur on high-purity carbon-free alumina matrix Anodisc inorganic filters floating on nitrate mineral salt medium under atmospheric air (SI Appendix, Fig. S5). This confirms that the cells at least did not depend on bisphenyl A (BPA) leaking from the polycarbonate filters (54, 55) as an additional carbon and energy source.

Fig. 6. CH 4 oxidation by M. gorgona MG08 microcolonies incubated on floating filters under atmospheric air (A). Microcolony formation under atmospheric air (B). (A) Five 170-mL bottles with floating polycarbonate filters were incubated on 35 mL 1/10 diluted nitrate mineral salt media (Materials and Methods) under atmospheric air (135 mL 1 atm headspace, sealed with rubber stopper) for 120 d. An additional set of five bottles was incubated without filters as a negative control for CH 4 oxidation. A two-sample t test assuming equal variances confirmed that headspace CH 4 concentrations were significantly different in bottles with filters containing cells (P value < 0.001), compared with those without cells. (B) Filters from A were manually inspected to identify colony formation. (Top) Showing single cells before incubation. (Bottom) Colonies formed after 120-d incubation in one of the bottles from A. For staining in B, filters were transferred (side with bacteria up) on top of 200 µL droplets of 1,000× SYBRgreen and incubated for 10 min, washed, and air dried.

An additional CH 4 oxidation experiment, using cells in liquid culture under a range of CH 4 concentrations, was performed to estimate the half saturation constant [K m(app) ] for M. gorgona MG08 (SI Appendix, Fig. S6). We show that it has a K m(app) of 4.905 µM, similar to that observed for Methylocapsa acidiphila B2, M. parvus, Methylocystis sp. SC2, and many other MOB strains (23). In contrast to M. gorgona MG08, neither of these strains was previously found to grow at atmopheric CH 4 concentrations. The specific affinity [a0 s = V max(app) /K m(app) ] was suggested as a better measure for oligotrophy (6) as Methylocystis sp. LR1 displayed high-affinity activity (K m of ∼100 nM) at low CH 4 concentrations (<275 p.p.m.v.) and low-affinity activity (K m of ∼1 µM) when CH 4 concentrations were higher while its specific affinity remained constant (56). With a V max(app) of 9.54 × 10−10 µmol⋅cell−1⋅h−1, the specific affinity of M. gorgona MG08 is ∼195 (× 10−12 L⋅cell−1⋅h−1), which is ∼17 times higher than the corresponding value of M. acidiphila B2 (12 × 10−12 L⋅cell−1⋅h−1), ∼10 times higher than for Methylocystis sp. LR1 (20 × 10−12 L⋅cell−1⋅h−1), and ∼6 times higher than for Methylocystis sp. SC2 (34 × 10−12 L⋅cell−1⋅h−1), formerly the MOB with the highest known a0 s (23). The high-affinity MOB is thus probably better defined as a high-specific affinity MOB as suggested by Dunfield (6) more than 10 y ago, but a confirmation of this must await standardized comparisons between USCα and other MOB strains.

Despite having lower specific affinities (a0 s ) than M. gorgona MG08, we decided to test whether other Methylocapsa species are able to grow at atmospheric CH 4 concentrations. Surprisingly, both M. acidiphila and Methylocapsa aurea were able to also grow on floating filters incubated under atmospheric air (SI Appendix, Fig. S7). This suggests that the ability of MOB to grow on filters floating on nitrate mineral salt medium under unamended air is not a unique capability of species within the USCα, and not dependent on a high a0 s for CH 4 . However, the apparent success of USCα in many upland soils may still be a result of having the highest a0 s .

Consistent with the physiological and genomic predictions, NanoSIMS experiments performed on microcolonies on polycarbonate filters qualitatively showed that M. gorgona MG08 incorporated 13C-labeled carbon into its biomass when grown in a closed jar containing 20 p.p.m.v. 13CH 4 and unlabeled CO 2 (Fig. 7A). Incorporation of 13C was also observed in the presence of 13CO 2 at 20 p.p.m.v. and unlabeled CH 4 (Fig. 7B), as expected from CO 2 /HCO 3 − incorporation via the carboxylation reaction of the serine cycle (enzyme no. 16, Fig. 4). There is also the possibility that CO 2 could be assimilated via the reductive glycine pathway, but with the current data the different possible entry points of carbon assimilated from CO 2 cannot be distinguished. Accumulation of 13CO 2 in the headspace of the cultures during incubation with 20 p.p.m.v. and 1,000 p.p.m.v. 13CH 4 confirmed that the organism is able to oxidize CH 4 completely to CO 2 and subsequently release CO 2 from the cell (SI Appendix, Fig. S8).

Fig. 7. Carbon fixation in M. gorgona MG08. NanoSIMS visualization of the 13C isotope label incorporation in M. gorgona MG08 cells grown on polycarbonate filters in atmospheres containing 20 p.p.m.v. CH 4 and 1,000 p.p.m.v. CO 2 . Two incubations are compared: 13CH 4 in combination with 12CO 2 (A) and 12CH 4 in combination with 13CO 2 (B). 13C/(12C + 13C) isotope fraction values, given in at%, are displayed on a false color scale ranging from 0.9 at% (dark blue) to 2.5 at% (red). Cells grown in isotopically unlabeled methane and carbon dioxide showed a 13C content of 1.09 ± 0,01 at% (SD, n = 60).

Additional physiological characterizations of M. gorgona MG08 were carried out in liquid cultures grown at a high CH 4 concentration. Efficient growth was observed when N 2 was offered as the sole nitrogen source under fully aerobic conditions (SI Appendix, Fig. S9). M. gorgona MG08 shares this ability with the closely related M. acidiphila B2T, and nonmethanotrophic members of the genus Beijerinckia, while Methylocystis and Methylosinus species require a somewhat reduced oxygen tension and Methylococcus capsulatus (Bath) is highly sensitive to oxygen (39, 57, 58). Almost all tested nitrogen containing compounds including N 2 , NO 3 −, and NH 4 + acted as good nitrogen sources for M. gorgona MG08 (SI Appendix, Fig. S9), while histidine and glycine inhibited growth as shown previously for M. capsulatus (only histidine) (59), Thiobacillus neapolitanus (only histidine) (60), and methylotrophic bacteria (only glycine) (61).

No growth was observed in liquid batch culture controls provided with acetate, ethanol, formate, galactose, glucose, methanol, oxalate, pyruvate, sucrose, succinate, or urea without added CH 4 (SI Appendix, Fig. S10A). To evaluate potential concentration effects of methanol, we attempted growth on 5%, 0.5%, 0.01%, 0.005%, and 0.001% CH 3 OH, but observed only minimal increases in the optical density of the cultures (SI Appendix, Fig. S10B). Considering that M. gorgona MG08 carries and expresses the genes for methanol oxidation, its inability to grow on methanol was surprising but it is in line with the observations that M. aurea, M. acidiphila, and Methylocapsa palsarum grow poorly on methanol (39, 62, 63). Interestingly, we observed a concentration-dependent growth inhibition of M. gorgona MG08 in the presence of methanol concentrations ≥0.01% when growing on CH 4 (20% CH 4 in headspace). Thus, toxicity alone could not explain its inability to use methanol. Perhaps methanol dehydrogenase, which requires oxidized cytochrome c for accepting electrons, thus depends on simultaneous CH 4 oxidation (Fig. 4) due to tightly coupled CH 4 and methanol oxidation reactions ocurring within a supercomplex. If so, the redox interactions would be different from intracytoplasmic membrane assemblies of pMMO and methanol dehydrogenase (MDH) in the methanol and CH 4 utilizing M. capsulatus (Bath) (64).

The incapability of M. gorgona MG08 to grow on acetate was also surprising, considering that it carries all of the genes required for aerobic metabolization of acetate, i.e., acetate-coA ligase, acetate kinase, phosphotransacetylase, and a complete TCA cycle. Similarly, M. acidiphila B2T also possesses the full set of enzymes for acetate metabolism (65), but is unable to grow on acetate as a sole carbon source (39). Dedysh et al. (66) reported similar findings for the obligate methanotroph Methyloferula stellata AR4T and suggested that the lack of ability to utilize acetate might be due to an absence of a specific acetate/glycolate transporter gene, actP, rather than the quantity of membrane transporter genes (67, 68). In support of this hypothesis, the actP gene encoding acetate permease is absent both in strain M. gorgona MG08 and M. acidiphila B2T.

The cells of M. gorgona MG08 are Gram-negative, nonmotile coccoids, or thick rods that occur singly or in conglomerates. Cells are 0.6–0.8 µm wide and 0.8–1.5 µm long and show numerous hair-like structures (Fig. 1B). Growth on surfaces occurs by formation of microcolonies of a circular form. It reproduces by normal cell division and does not form rosettes. Cells contain a well-developed intracytoplasmic membrane system of type III arrangement, which appears as stacks of membrane vesicles packed in parallel on only one side of the cell membrane (Fig. 1C). This arrangement has been shown to be characteristic for members of the Methylocapsa genus (39, 62, 63). Inclusions resembling PHB granules were present (Fig. 1C) but did not exhibit the bipolar arrangement of refractile PHB granules characteristic of M. aurea KYGT and members of the Methylocella genus. Optimal growth was observed between 15 °C and 27 °C, while some growth still occurred at the extremes tested, 7 °C and 37 °C (SI Appendix, Fig. S11A). The optimum growth temperature range was similar to but wider than M. aurea (25–30 °C), and higher/wider than that of M. acidiphila (20 °C) and M. palsarum (18–25 °C) (39, 62, 63). M. gorgona MG08 is thus far the only Methylocapsa strain capable of growth at 37 °C. The optimum pH for growth of M. gorgona MG08 was in the range of 6.5 to 7 (SI Appendix, Fig. S11B), considerably higher than for M. acidiphila (5.0–5.5), M. aurea (6.0–6.2), and M. palsarum (5.2–6.5), but in line with the frequent detection of USCα in neutral and slightly acidic soils (8, 16). NaCl was shown to inhibit growth at 0.5% (wt/vol) and above (SI Appendix, Fig. S11C), similarly to the three other Methylocapsa strains (39, 62, 63).