The genus Methylocystis belongs to the class Alphaproteobacteria, the family Methylocystaceae, and encompasses aerobic meth- anotrophic bacteria with the serine pathway of carbon assimilation. All Methylocystis species are able to ﬁx dinitrogen and several members of this genus are also capable of using acetate or ethanol in the absence of methane, which explains their wide distribution in various habitats. One additional trait that enables their survival in the environment is possession of two methane- oxidizing isozymes, the conventional particulate methane monooxygenase (pMMO) with low-afﬁnity to substrate (pMMO1) and the high-afﬁnity enzyme (pMMO2). Here, we report the ﬁnished genome sequence of Methylocystis bryophila S285, a pMMO2-possessing methanotroph from a Sphagnum-dominated wetland, and compare it to the genome of Methylocystis sp. strain SC2, which is the ﬁrst methanotroph with conﬁrmed high-afﬁnity methane oxidation potential. The complete genome of Methylocystis bryophila S285 consists of a 4.53 Mb chromosome and one plasmid, 175 kb in size. The genome encodes two types of particulate MMO (pMMO1 and pMMO2), soluble MMO and, in addition, contains a pxmABC-like gene cluster similar to that present in some gammaproteobacterial methanotrophs. The full set of genes related to the serine pathway, the tricarboxylic acid cycle as well as the ethylmalonyl-CoA pathway is present. In contrast to most described methanotrophs including Methylocystis sp. strain SC2, two different types of nitrogenases, that is, molybdenum–iron and vanadium–iron types, are encoded in the genome of strain S285. This unique combination of genome-based traits makes Methylocystis bryophila well adaptedtothe ﬂuctuation of carbonand nitrogen sources in wetlands. Key words: methanotrophs, Methylocystis, ﬁnished genome, comparative genomics, methane monooxygenase, nitrogenase. Introduction MOB, respectively. Members of these two groups differ in Aerobic methanotrophs (methane-oxidizing bacteria, MOB) their cellular ultrastructure, C1-utilization pathway, fatty are a unique subset of methylotrophic bacteria that can utilize acid composition, and other physiological and biochemical methane (CH ) as their sole source of energy. They use meth- characteristics. ane monooxygenase (MMO) enzymes to oxidize methane to The genus Methylocystis is one of the ﬁrst described and methanol (Hanson and Hanson 1996; Trotsenko and Murrell historically recognized genera of aerobic methanotrophic bac- 2008). Methanotrophic capabilities relying on MMO activity teria (Whittenbury et al. 1970). It belongs to the class are currently recognized in members of the bacterial phyla Alphaproteobacteria, the family Methylocystaceae,and Proteobacteria, Verrucomicrobia, and the candidate division encompasses obligate and restricted facultative methanotro- NC10 (Stein et al. 2012). Of these, methanotrophic phic bacteria with the serine pathway of carbon assimilation Proteobacteria are represented by the greatest number of (Belova et al. 2013). All members of this genus possess a characterized isolates. Belonging to the classes Gamma-and membrane-bound or particulate methane monooxygenase Alphaproteobacteria, they are classiﬁed as type I and type II (pMMO), whereas some also contain a soluble form of this The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non- commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact firstname.lastname@example.org Genome Biol. Evol. 10(2):623–628. doi:10.1093/gbe/evy025 Advance Access publication January 30, 2018 623 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/623/4830106 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Han et al. GBE enzyme (sMMO). Representatives of this genus inhabit a wide genome analysis, cells of strain S285 were grown in liquid variety of terrestrial and aquatic ecosystems and display a medium M2 with 20% methane and harvested after 2 weeks number of environmental adaptations. Thus, in the absence of incubation at 24 C. of methane, some species of this genus are capable of slow growth on acetate and ethanol (Belova et al. 2011; Im et al. Genome Sequencing and Assembly 2011; Belova et al. 2013). Another ecologically important ad- Genome sequencing of Methylocystis bryophila S285 was aptation of these methanotrophs is their ability to produce performed at the Max Planck Genome Centre Cologne two pMMO isozymes, the conventional form with low afﬁnity (MP-GCC), using the PacBio RSII platform (Paciﬁc to methane (pMMO1), and the high-afﬁnity enzyme Biosciences, Menlo Park, California). De novo assembly was (pMMO2) (Baani and Liesack 2008). The isozymes are done using the hierarchical genome-assembly process encoded by pmoCAB1 and pmoCAB2, respectively. To date, (HGAP2) via the SMRT Portal v.2.0 offered by Paciﬁc the complete genome sequence has been reported for only a Biosciences. single pMMO2-possessing member of the genus Methylocystis, Methylocystis sp. strain SC2 (Dam et al. 2012). Genome Annotation In this study, we obtained the complete genome sequence of another pMMO2-possessing methanotroph, Methylocystis With default parameters, the genome was annotated using bryophila S285. The species Methylocystis bryophila RAST v. 2.0 (Rapid Annotation using Subsystem Technology) accommodates facultative methanotrophs, which were iso- (Overbeek et al. 2014) and BASys (Bacterial Annotation lated from acidic Sphagnum-dominated wetlands and are System) (Van Domselaar et al. 2005). Furtherly, functional capable of slow growth on acetate in the absence of methane roles of protein-encoding genes were analyzed using SEED (Belova et al. 2011). Members of this species account for 20– Subsystems (Overbeek et al. 2014). 50% of all the methanotroph cells detectable in acidic peat by ﬂuorescence in situ hybridization (Belova et al. 2011). The tax- Genome Comparison onomic description ofMethylocystisbryophila was based on the We thoroughly compared the genome of Methylocystis bryo- characterization of two isolates, strains H2s and S284 (Belova phila S285 with that of the obligate methanotroph et al. 2013). One additional representative of this species, strain Methylocystis sp. SC2. To date, these two strains are the S285, was later obtained from the same peat sample as strain only members of the Methylocystaceae for which complete S284. These two isolates share identical 16S rRNA gene sequen- genome sequences are available. For reference, we included ces, which also match the 16S rRNA gene sequence of strain in our comparison the genome of Methylocella silvestris BL2. H2s , the type strain of Methylocystis bryophila (GenBank ac- This facultative sMMO-containing methanotroph is a member cession number FN422003). Our further analysis of pMMO- of the family Beijerinckiaceae. The map of all three ﬁnished encoding genes revealed that, in contrast to strain S284, only genomes was constructed using BRIG (BLAST Ring Image the pmoA2 gene fragments could be PCR-ampliﬁed from DNA Generator), a prokaryote genome comparison software of strain S285 by using the primer combination A189f–A682b (Alikhan et al. 2011). To determine the overall genome sim- (Holmes et al. 1995). These primers are routinely employed for ilarities among methanotrophs and the distribution of partic- pmoA1 detection in methanotrophs. To conclusively verify the ular genomic traits, we expanded this comparison to 15 absence or presence of pMMO1 and to get an insight into methanotrophs encompassing the genus Methylocystis (seven genome-encoded features of a pMMO2-possessing methano- genomes), other type II MOB (one genome each for the gen- troph from wetlands, we have determined and analyzed the era Methylosinus, Methylocella, Methylocapsa,and complete genome sequence of Methylocystis bryophila S285. Methyloferula), type I MOB (one genome each for the genera Methylococcus, Methylomonas,and Methylomicrobium), and Materials and Methods one genome representing Verrucomicrobia-like methano- trophs (“Methylacidiphilum”). Their genomic similarities Bacterial Strain Isolation and Characterization were estimated using the average nucleotide identity calcula- Strain S285 was isolated from a methanotrophic enrichment tor JSpeciesWS (Richter et al. 2016). culture that was established from an acidic peat soil (pH 3.8) sampled at a depth of 10 cm from Sphagnum peat bog Phylogenetic Analysis 0 0 Staroselsky moss (56 34 N, 32 46 E), Tver region, Russia, The relationships between the deduced amino acid sequences in August 2008. The enrichment culture was obtained from of pmoA1, pmoA2,and pxmA of methanotrophic bacteria as this peat sample using liquid mineral medium M2 of pH 5.0 (Belova et al. 2013) and 30% (vol/vol) methane in the head- well as those of their symporter genes were calculated using MEGA7 (Molecular Evolutionary Genetics Analysis version space. Strain S285 was isolated from this enrichment culture 7.0) (Kumaretal. 2016). by means of surface plating onto the agar medium M2. For 624 Genome Biol. Evol. 10(2):623–628 doi:10.1093/gbe/evy025 Advance Access publication January 30, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/623/4830106 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Complete Genome of Methylocystis bryophila S285 GBE Table 1 Results and Discussion General Genomic Features of Methylocystis bryophila S285, Methylocystis Finished Genome of Methylocystis bryophila S285 sp. SC2, and Methylocella silvestris BL2 Features S285 SC2 BL2 A total of 50,623 PacBio reads were obtained with a mean length of 3,228 bp. These reads enabled the assembly of the Accession number CP019948 HE956757 CP001280 Size (Mb) 4.53 3.77 4.3 complete genome sequence of Methylocystis bryophila S285. GþC (%) 63 63 63 The ﬁnished genome consists of one circular chromosome of Genes (total) 4,444 3,677 4,014 4,532,950 bp and a single plasmid of 175,021 bp. The chro- CDS (total) 4,387 3,623 3,956 mosome contains two identical rrn operon copies (16S-23S- Genes (coding) 4,285 3,583 3,875 5S rRNA), a full complement of 47 tRNA genes and 4,387 Pseudogenes 102 40 81 CDS (table 1). The distribution of protein-coding genes into Genes (RNA) 57 54 58 SEED subsystem is shown in supplementary table S1, rRNAs (5S,16S,23S) 2,2,2 1, 1, 1 2,2, 2 Supplementary Material online. tRNAs 474748 ncRNAs 4 4 4 pmoCAB1 operon 2 2 Absent Genome Alignment pmoCAB2 operon 1 1 Absent The complete genome map (ﬁg. 1) of the three alphaproteo- Monocistronic pmoC 2 3 (1 in plasmid) Absent bacterial methanotrophs suggests that the genomes of pxmABC operon 1 Absent Absent Methylocystis bryophila S285 and Methylocystis sp. SC2 dis- sMMO operon 1 Absent 1 play high synteny. In fact, the comparison of average nucle- Serine pathway genes Present Present Present otide identities (ANIs) between the seven Methylocystis RuMP pathway genes Absent Absent Absent genomes and the eight genomes from other methanotrophic Plasmid(s) 1 2 NR taxa revealed that Methylocystis bryophila S285 shares highest ncRNAs, noncoding RNAs; NR, not reported. ANI values with the genomes of the other six Methylocystis spp. and Methylosinus trichosporium OB3b (72–74%) (sup- plementary table S2, Supplementary Material online). The fur- pmoA1 (G–G mismatch in primer position 14 from 3 -end) ther comparison of particular gene traits between and pmoA2 (C-T mismatch in position 13 from 3 -end). In Methylocystis bryophila S285 and the other six Methylocystis addition, adjacent primer positions are deﬁned by N (position genomes provided additional evidence for high genomic sim- 0 0 12 from 3 -end) andS (position15from3 -end). Thus, our ilarity between Methylocystis bryophila S285 and experimental results indicate that the G–G mismatch (pmoA1) Methylocystis sp. SC2. These two type II MOB share the larg- in primer position 14 is more detrimental for efﬁcient pmoA estnumberofcommon features (88.2%) and differin the least ampliﬁcation than the C–T mismatch (pmoA2) inposition13. number of unique traits (5.2%) among the seven Methylocystis Notably, Radajewski et al. (2002) also detected only genomes (supplementary table S3, Supplementary Material pmoA2 but not pmoA1 in their study on metabolically active online). By contrast, Methylocella silvestris BL2 shows low sim- methanotrophs in an acidic forest soil. This group of research- ilarity to Methylocystis bryophila S285 and Methylocystis sp. ers also used the primer set A189f–A682b for their analysis. SC2, with regard to both complete genome (ﬁg. 1) and partic- The inferred peptide sequences of pmoA clones retrieved ular gene traits (table 1 and supplementary tables S1 and S4, by means of stable isotope probing (SIP) technique (accession Supplementary Material online). numbers AY080950, AY080958, AY080959) showed high similarity (97.7–98.3%) to PmoA2 from Methylocystis Diverse Methane Monooxygenase Genes bryophila S285, whereas no pmoA1 fragments were obtained Despite the inability to detect the pMMO1-encoding genes in from Methylocystis bryophila-like methanotrophs (Radajewski Methylocystis bryophila S285 by PCR, our genomic analysis et al. 2002). revealed that this strain possesses both types of pmoCAB:two In addition to pMMO, strain S285 is able to produce the copies of pmoCAB1 and one copy of pmoCAB2 (table 1 and soluble form of MMO. Although pMMO is encoded by three supplementary ﬁg. S1, Supplementary Material online). The genes (pmoCAB), the sMMO operon encompasses ﬁve con- reason for the failure to detect pmoA1 is not fully clear but secutive genes (mmoYZXBC). Two monocistronic pmoC may be due to the fact that, if using primer set A189f–A682b, genes were also identiﬁed (table 1). Based on our survey of pmoA2 of strain S285 is more efﬁciently PCR-ampliﬁed than currently available genomes (including complete and draft its pmoA1 counterpart. Although primer A189f is perfectly genomes), Methylocystis bryophila S285 and Methylocystis matching to its target site in either pmoA1 or pmoA2,primer sp. strain LW5 are the only type II MOB which harbor all three A682b has one mismatch in its target site of both pmoA1 and types of MMO: pMMO1, pMMO2, and sMMO. Strain SC2 pmoA2 (supplementary table S5, Supplementary Material on- does not produce sMMO (table 1 and supplementary table line). However, this mismatch position differs between S4, Supplementary Material online). Genome Biol. Evol. 10(2):623–628 doi:10.1093/gbe/evy025 Advance Access publication January 30, 2018 625 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/623/4830106 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Han et al. GBE FIG.1.—Genomic map of Methylocystis bryophila S285 in comparison to those of Methylocystis sp. SC2 and Methylocella silvestris BL2. The concentric circles denote the following features (from inside to outside): genome of Methylocystis bryophila S285 with coordinates (chromosome region, black circle), the GC content, GC skew (green and red indicate that the nucleotides Guanine and Cytosine are over-respectively underrepresented) and the genome alignment of Methylocystis sp. SC2 and Methylocella silvestris BL2 against Methylocystis bryophila S285, where color indicates a BLAST match of nucleotide sequence identity of 70–100% (based on BLASTn) between central genome (Methylocystis bryophila S285) and comparative genomes, Methylocystis sp. SC2 (brown) and Methylocella silvestris BL2 (blue). A particular trait of strain S285 is the presence of a gammaproteobacterial type I MOB, including strains of the pxmABC-like gene cluster. The pxmABC operon is predicted genera Methylomonas, Methylobacter,and to encode a member of the copper-containing membrane- Methylomicrobium (Tavormina et al. 2011). Among type II bound monooxygenase (Cu-MMO) protein family, pXMO. Its MOB, the pxmABC-like gene cluster has previously been function and substrate are not yet known (Tavormina et al. detected only in Methylocystis rosea SV97 and Methylocystis 2011). Recent evidence for pxmABC expression in response sp. strain SB2 (Knief 2015)(supplementary table S4, to hypoxia suggests that pXMO is important for survival of Supplementary Material online). Thus, Methylocystis bryophila methanotrophs under O limitation (Kits et al. 2015). The S285 is the only third type II MOB shown to possess pxmABC pxmABC-like gene clusters are widely distributed among genes. Notably, its PxmA fragment clusters together with a 626 Genome Biol. Evol. 10(2):623–628 doi:10.1093/gbe/evy025 Advance Access publication January 30, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/623/4830106 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Complete Genome of Methylocystis bryophila S285 GBE large group of environmental pxmA transcripts obtained from substrate for growth of Methylocella spp. In addition, M. sil- a subarctic peatland (AFY11631.1 and AFY11641.1 in sup- vestris BL2 also grows on ethanol, pyruvate, succinate, malate, plementary ﬁg. S1, Supplementary Material online; Liebner propanol, propanediol, acetone, methyl acetate, acetol, glyc- and Svenning 2013) as well as with PxmA fragments from erol, propionate, tetrahydrofuran, gluconate, ethane, and two type I MOB, Methylomonas sp. strainM5and propane (Dedysh and Dunﬁeld 2016). Methylococcaceae bacterium M200. Like Methylocystis bryo- The repertoire of membrane transporters encoded by the phila S285, these two strains were isolated from a Sphagnum S285 genome also includes acetate permease ActP (cation: wetland (Kip et al. 2011). This wide array of different MMO acetate symporter; gene locus: B1812_15125). ActP has types, including MMO-like enzymes, may ensure survival of proved function for acetate transportation in E. coli strain S285-like methanotrophs in environments with ﬂuctu- (Gimenez et al. 2003). However, the mere genomic presence ating methane concentrations as well as under copper or O of actP is not a valid indicator for facultative methanotrophy. limitation. Genes encoding putative ActP are also present in Methylocystis spp. hitherto characterized as obligate metha- notrophs such as, for example, Methylocystis sp. strain SC2, Multiple Carbon Assimilation Pathways Methylocystis rosea SV97, and Methylocystis sp. strain All the genes required for the serine pathway are present in Rockwell (supplementary ﬁg. S2 and table S4, thegenomeof Methylocystis bryophila S285 and those of two Supplementary Material online). Alternatively, it may be that key enzymes of the ribulose monophosphate cycle (RuMP) are these methanotrophs can uptake and utilize acetate for absent. The serine pathway is the main carbon assimilation growth but that appropriate growth conditions have yet to pathway of type IIMOB. Inaddition tothe complete tricar- be identiﬁed. The genome of M. silvestris BL2 also encodes boxylic acid (TCA) cycle, the genomes of Methylocystis bryo- ActP, but its sequence clusters on a branch separate from phila S285 and Methylocystis sp. strain SC2 encode the those of the Methylocystaceae spp. (supplementary ﬁg. S2, complete ethylmalonyl-CoA (EMC) pathway. This pathway Supplementary Material online). is also present in Methylocystis sp. strain SB2, which has proven ability to grow on acetate or ethanol (Im et al. Nitrogen Fixation 2011; Vorobev et al. 2014). We also identiﬁed the genes encoding alcohol dehydrogenase and aldehyde dehydroge- Nitrogenase is a metalloprotein complex that comprises two nase in the S285 genome. These two enzymes convert etha- components, a nitrogenase iron protein and a dinitrogenase nol to acetate. Thus, presence of the ethanol-converting reductase (McGlynn et al. 2012). At least three genetically enzymes coupled with EMC pathway provides the possibility distinct but homologous nitrogenase systems have been iden- for growth of Methylocystis bryophila S285 on ethanol or tiﬁed until now (Hu and Ribbe 2015). These O -sensitive nitro- acetate. Strain S285 may convert ethanol or acetate via genases are primarily distinguished by the metal composition acetyl-coenzyme A synthetase (gene locus: B1812_19345) of their active-site metallocluster: conventional molybdenum– to acetyl-CoA, which is then funneled into the TCA cycle iron nitrogenase and the alternative vanadium–iron type, and for energy generation or incorporated into biomass via the iron-only nitrogenase (Eady 1996; Hu and Ribbe 2015). Genes EMC pathway. Utilization of these two-carbon compounds encoding the molybdenum–iron (Mo) and vanadium–iron (V) was conﬁrmed for strains H2s and S284, the two taxonom- types of nitrogenase were identiﬁed in the S285 genome ically characterized—but not genome-sequenced—represen- (supplementary table S4, Supplementary Material online). tatives of Methylocystis bryophila (Belova et al. 2013). Thus, The Mo-nitrogenase is the most universally distributed nitro- facultative methanotrophy represents an important alterna- genase in nature. The V-nitrogenase is present only in a lim- tive strategy for life in peatlands, allowing survival if no meth- ited number of microorganisms such as, for example, aneis available(Belova et al. 2011). Azotobacteriaceae. The V-nitrogenase, encoded along with Even to date, the exact metabolic basis for facultative the Mo-nitrogenase, is expressed in the case of Mo- methanotrophy is not known, but it has been suggested deﬁciency and has therefore been considered an alternative that a key determinant of obligate methanotrophy is the re- or “back-up” system (Rehder 2000; Zhao et al. 2006). stricted ability to transport potential substrates across the However, despite the high structural similarity between the membrane (Tamas et al. 2014). Correspondingly, the total two nitrogenases, V-nitrogenase can also reduce CO (Hu et al. number of membrane transporters encoded in the genome 2012). The ability of V-nitrogenase to catalyze the reduction of strain S285 exceeds that in strain SC2. Notably, the ge- of both CO and N suggests a potential link between the nome of Methylocella silvestris BL2 even encodes a greater evolution of carbon and nitrogen cycles (Lee et al. 2010). repertoire of membrane transporters than the two We conducted a survey (August 10, 2017) on the distribution Methylocystis spp., strains S285 and SC2 (supplementary ta- of V-nitrogenase-coding gene clusters among all 49 public ble S1, Supplementary Material online). This ﬁnding corre- methanotroph genomes that are available in NCBI sponds well to the fact that acetate is the preferred GenBank. The survey showed that the genes encoding Genome Biol. Evol. 10(2):623–628 doi:10.1093/gbe/evy025 Advance Access publication January 30, 2018 627 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/623/4830106 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Han et al. GBE Hu Y, Ribbe MW. 2015. Nitrogenase and homologs. J Biol Inorg Chem. 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