Complete genome sequence of Methanoculleus bourgensis strain MAB1, the syntrophic partner of mesophilic acetate-oxidising bacteria (SAOB)

Methanoculleus bourgensis strain MAB1 has been identified as the hydrogenotrophic partner of mesophilic acetate-oxidising bacteria, a syntrophic relationship operating close to the thermodynamic equilibrium and of considerable importance in ammonia-rich engineered biogas processes. Methanoculleus bourgensis strain MAB1 belongs to the order Methanomicrobiales, family Methanomicrobiaceae, within the phylum Euryarchaeota. The genome shows a total size of 2,859,299 bp encoding 3450 predicted protein-encoding genes, of which only 1472 (43 %) have been assigned tentative functions. The genome encodes further 44 tRNA genes and three rRNA genes (5S, 16S and 23S rRNA). This study presents assembling and annotation features as well as genomic traits related to ammonia tolerance and methanogenesis. Electronic supplementary material The online version of this article (doi:10.1186/s40793-016-0199-x) contains supplementary material, which is available to authorized users.


Introduction
In anaerobic digestion processes, syntrophy is a particular important interspecies relationship that is of benefit to all contributing partners and is essential for the methanogenesis of organic matter [1,2]. Syntrophic interaction operates close to the thermodynamic equilibrium, whereby both partners have to share the limited energy released in the overall reactions [2]. Syntrophic acetateoxidation (SAO) releases a very small amount of energy (ΔG o`= −35 kJ per mol rct), just enough to support microbial growth. The two-step reaction starts with the oxidation of acetate to CO 2 and hydrogen/formate performed by so-called syntrophic acetate-oxidising bacteria. This can only proceed when, in a second step, the hydrogen/ formate is immediately consumed by a hydrogenotrophic methanogenic archaea reducing CO 2 to methane, which makes the overall acetate oxidation thermodynamically favourable [1]. In a mesophilic co-culture, the hydrogen partial pressure has been observed to be as low as 1.6-6.8 Pa [3] and in thermophilic co-cultures as low as 10-50 Pa [4].
This study reports the genome sequencing, assembly and annotation of the methanogenic SAOB partner Methanoculleus bourgensis strain MAB1, a key organism in methane production from ammonia-rich feed stocks in anaerobic digestion processes.

Classification and features
Methanoculleus bourgensis MAB1 is an obligate anaerobic archaea that has been isolated from a mesophilic methanogenic reactor operating with swine manure at 6 g NH4 + −N/L and a pH of 7.5. The isolated cells were between 1.0 and 3.0 μm in diameter, irregular and coccoid in shape ( Fig. 1) and surrounded by a protein S-layer [11]. The strain forms methane from H 2 /CO 2 , formate, 2propanol and 1,2-propanol, but not from acetate, which is required for growth. A more detailed description can be found in [11]. Although isolated from mesophilic reactors, the optimal methane production rate has been observed at hyper-mesophilic temperatures of between 44 and 45°C [23]. It can probably tolerate ammonia concentrations up to 1 g/L [12]. Minimum information about the genome sequence (MIGS) of M. bourgensis strain MAB1 is given in Table 1 and Table S1 (Additional file 1).
Phylogenetic analysis of the single 16 s rRNA gene copy affiliates M. bourgensis MAB1 to the Methanomicrobia class within the phylum Euryarchaeota and therein to the family Methanomicrobiaceae (RDP Naive Bayesian rRNA Classifier Version 2.10, October 2014). The comparison of the 16S rRNA gene with the latest available databases from GenBank (2016-01-29) using BLAST under default settings have revealed Methanoculleus marisnigri JR1 (NC_009051.1) to be the closest current relative, sharing 97 % identity (Fig. 2). The type strain is Methanoculleus bourgensis MS2 (T), whose 16 s rRNA gene is 99 % identical to strain MAB1 and which was isolated from a tannery by-product enrichment culture inoculated with sewage sludge [24]. Methanoculleus olentangyi and Methanoculleus oldenburgensis are subjective synonyms [25]. Cells of M. bourgensis strain MAB1 show a polyamine pattern that is distinctly different from the type strain MS2 [11].

Genome sequencing information
Genome project history Methanoculleus bourgensis MAB1 was sequenced and annotated by the SLU-Global Bioinformatics Centre at the Swedish University of Agricultural Sciences, Uppsala, Sweden. The genome project is deposited in the Genomes OnLine Database [26] with GOLD id Gb0126792, and the complete genome is deposited in the European Nucleotide Archive database with accession number ERS1044365. This methanogenic partner of SAOB was selected for sequencing on the basis of environmental relevance to issues in global carbon cycling, alternative energy production and geochemical importance. Table 2 contains a summary of the project information.

Growth conditions and genomic DNA preparation
The strain had been stored as liquid cultures since its isolation in the laboratory. For DNA isolation, batch cultures were grown in basal medium as described by Zehnder et al. [27] and modified by Schnürer et al., [28] supplemented with 5 mM acetate and 0.3 M NH 4 Cl 2 .
The headspace was filled with H 2 /CO 2 (80:20, v/v). Cells were grown over 2 months at 37°C without shaking, and harvested at 5000 X g. DNA was isolated using the Blood & Tissue Kit from Qiagen (Hilden, Germany) according to the standard protocol, but omitting the lysozyme step. The quality was visualised by agarose gel electrophoresis and the quantity determined by fluorometric measurements using Qubit (Thermo Fisher Scientific, Waltham, MA, USA).

Genome sequencing and assembly
The genome of Methanoculleus bourgensis strain MAB1 was sequenced at the SciLifeLab Uppsala, Sweden using Ion Torrent PM systems with a mean length of 206 bp, a longest read length 392 bp and a total of final library reads of 2,985,963 for single end reads. General aspects about the sequencing performed can be found on the SciLifeLab website [29]. The FastQC software package [30] was used for read quality assessment. After preassembly quality checking, the reads were assembled with MIRA 4.0 and Newbler 2.8 assemblers. Possible missassemblies were corrected manually using Tablet, a graphical viewer for visualisation of assemblies and read mappings [31]. Whole-genome assembly of the M. bourgensis strain MAB1 genome was accomplished using a comparative genome assembly method [32], which combines de novo and mapping assemblies. The filtered reads were fed into MIRA version 4.0 [33] for both mapping and de novo assembly, and the same read data were also provided to Newbler 2.8 de novo assembler. Mapping assembly was undertaken against the available genome of Methanoculleus marisnigri JR1 (accession no. NC_009051.1). Contigs produced through de novo assembly of read data from both assemblers were sorted and oriented along the reference genome and then aligned to the mapping assembly using Mauve genome alignment software [34]. Alignment of contigs to mapping assembly indels covered all the gaps in the genome. These covered gaps were all verified through PCR amplification using a Hot Start High-fidelity DNA polymerase (Phusion, Thermo Fisher Scientific, Waltham, MA, USA) and subsequent Sanger sequencing (Macrogen Corporation, Geumcheon District, South Korea). The complete genome sequence of Methanoculleus bourgensis strain MAB1 contained 2,859,299 bp based on the analysis performed using the tools summarised above.

Genome annotation
Automated gene modelling was completed by MaGe [35], a bacterial genome annotation system. Genes were identified using Prodigal [36] and AMIGene [37] as part of the MaGe genome annotation pipeline. The predicted CDSs were translated and used to search the NCBI non-redundant database and UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG and InterPro databases using BLASTP. Predicted coding sequences were subjected to manual analysis using the MaGe web-based platform, which also provides functional information about proteins and was used to assess and correct genes predicted through the automated pipeline. The predicted functions were also further analysed by the MaGe annotation system (Fig. 4). Evidence codes -IDA Inferred from Direct Assay, TAS Traceable Author Statement (i.e., a direct report exists in the literature); NAS Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence) These evidence codes are from the Gene Ontology project [52] Genome properties The complete genome comprised a single contig with a total size of 2,859,299 bp and a calculated GC content of 60.26 %. The genome showed a protein coding density of 84.59 % with an average intergenic length of 162.92 bp. The genome encoded a further 44 tRNA genes and three rRNA genes (5S, 16S and 23S rRNA) ( Table 3, Fig. 3).
The genome of Methanoculleus bourgensis strain MAB1 genome contained 3450 predicted proteinencoding genes, of which 1472 (43 %) have been assigned tentative functions. The remaining 1978 ORFs were hypothetical/unknown proteins. 2323 (app. 66 %) of all predicted protein-encoding genes could Fig. 2 Phylogentic tree. Maximum likelihood tree highlighting the phylogenetic position of Methanoculleus bourgensis strain MAB1 within the family Methanomicrobiacaea. The 16S rRNA-based alignment was carried out using MUSCLE [53] and the phylogenetic tree was inferred from 1521 aligned characteristics of the 16S rRNA gene sequence using the maximum-likelihood (ML) algorithm [54] with MEGA 6.06 [55,56]. Bootstrap analysis [57] with 100 replicates was performed to assess the support of the clusters

Insights from the genome sequence
Synteny-based analysis revealed that Methanoculleus bourgensis strain MAB1 had approximately 55 % of the total genome size in synteny with its closest relative Methanoculleus marisnigri JR1 (Fig. 4). The type strain Methanoculleus bourgensis strain MS2 had approximately 70 % of the total genome size in synteny Analysis of COGs revealed that 2323 (app. 66 %) of all predicted protein-encoding genes of M. bourgensis strain MAB1 could be allocated to the 22 functional COGs, which is slightly lower than that predicted for M. bourgensis strain MS2 (2072 genes; 69 %) and for M. marisnigri JR1 (2016 genes; 75 %), where the proteinencoding genes of both could be allocated to 23 functional GOGs.
The genome does not contain genes related to ammonium transport systems, which have been found to be encoded by the genome of its close relative Methanoculleus marisnigri (BlastP search using ammonium transporter of M. marisnigri as protein query), and might therefore be considered an adaptation to high osmolarity environments and to high ammonium levels in particular. The same genotype has also described for the type strain MS2 [39] and for its acetate-oxidising syntrophic partner organism "T. acetatoxydans" [40] and S. schinkii (unpublished). As also predicted for the type strain MS2 [39], a putative potassium ABC transport system (MMBA1_2581,MMAB1_2585) with an adjacent twocomponent regulatory system (MMAB1_2586, MMAB1_2587), two cation transporter (MMAB1_0409, MMAB1_1566), two potassium antiporter (MMBA1_1794, MMAB1_2374), a choline/carnitine/betaine transporter and one glycine/betaine ABC transport system might be involved in osmoregulation. The genes encoding the methanogenesis pathway from H 2 and CO 2 , including formylmethanofuran dehydrogenase (MMAB1_2225-MMAB1_2227), formylmethanofurantetrahydromethanopterin fromyltransferase (MMAB1_2217), methenyltetrahydromethanopterin cyclohydrolase (MMA-B1_2292), methylenetetrahydromethanopterin dehydrogenase (MMAB1_2159), methylenetetrahydromethanopterin reductase (MMAB1_2155), tetrahydromethanopterin Smethyltransferase (MMAB1_2236-MMAB1_2244), methyl-CoM reductase (MMAB1_2231-2235) and CoB-CoM heterodisulfide reductase (MMAB1_2220), were found clustering together. The genome further revealed duplicates in the case of formylmethanofuran dehydrogenase (MMAB1_1584-1586, MMAB1_1958-1962) and methyl-CoM reductase (MMAB1_1952-MMAB1_1956). The expression levels of the two methyl-CoM reductases encoded by the genome of M. thermoautotrophicus were shown to be dependent on syntrophic or autotrophic growth conditions [41].
Moreover, the genome of M. bourgensis strain MAB1 codes for three formate dehydrogenases (MMBA1_1689-1690, MMAB1_1105-1106, MMAB1_2913-2914), as has also been described for strain MS2 [39] as well as a putative formate/nitrite transporter (MMAB1_1101/2). These proteins might be involved in efficient uptake and utilisation of formate by donating electrons from formate via formate dehydrogenases to the heterodisulfide reductase, an electron-bifurcating mechanism recently suggested for Methanococcus maripaludis [40].

Conclusions
Methanoculleus bourgensis strain MAB1 has been identified as a syntrophic partner for acetate-oxidising bacteria in biogas processes operating with high ammonia levels. Initial genome surveillance indicates an adaption of strain MAB1 to the high osmolarity of this particular environment, as has also been observed for its syntrophic partner organisms. It reveals further gene sets likely to mediate efficient formate uptake and conversion, a possible end product of acetate oxidation. There is a remarkable discrepancy between Methanoculleus bourgensis strain MAB1 and the type strain Methanoculleus bourgensis strain MS2, as indicated by the number of orthologous and synteny percentages. Follow-up genome analysis and -omics approaches will investigate these differences further and elucidate what is specific about strain MAB1 that makes it the preferred partner organism of this particular syntrophy.