Wetlands are important sources of methane emissions, and the impacts of these emissions can be mitigated by methanotrophic bacteria. The genomes of methanotrophs Methylomonas sp. strain LL1 and Methylosinus sp. strain H3A, as well as Methylocystis sp. strains H4A, H15, H62, and L43, were sequenced and are reported here.
ABSTRACT
Wetlands are important sources of methane emissions, and the impacts of these emissions can be mitigated by methanotrophic bacteria. The genomes of methanotrophs Methylomonas sp. strain LL1 and Methylosinus sp. strain H3A, as well as Methylocystis sp. strains H4A, H15, H62, and L43, were sequenced and are reported here.
ANNOUNCEMENT
Wetlands are among the largest source of the greenhouse gas methane (1). Methanotrophic bacteria are the only biological filter, mitigating emission to the atmosphere (1). Here, we report the complete genome sequence of Methylomonas sp. strain LL1 and the draft genome sequences of Methylosinus sp. strain H3A and Methylocystis sp. strains H4A, H15, H62, and L43. These bacteria were isolated as previously described (2) from the same riparian wetland where composition and functioning of the methanotrophic community were described (3–5).
Strains were grown as described previously (2) in a nitrate mineral salts (NMS) medium, at 25°C in an atmosphere of 30% CH4 in air, and were harvested in mid-exponential phase. Bacterial genomic DNA was extracted using either the GNOME DNA isolation kit (MP Biomedicals, USA) (all Methylocystis sp. strains) or the Genomic-tip 100/G kit (Qiagen Benelux BV, The Netherlands) (Methylomonas sp. strain LL1 and Methylosinus sp. strain H3A), according to the manufacturers' instructions. The sequencing libraries of the genomes of Methylomonas sp. strain LL1 and Methylosinus sp. strain H3A were prepared using the SMRTbell template prep kit 1.0 and sequenced in one single-molecule real-time (SMRT) cell using the PacBio RS II (Pacific Biosciences, Inc., USA) sequencing platform, which was executed by the Genomics Facility of the School of Medicine of the University of Maryland (Baltimore, USA). Quality control, raw read filtering, and genome assembly were performed with the help of SMRT analysis software v2.3.0 (Pacific Biosciences, Inc.) featuring Hierarchical Genome Assembly Process algorithm v3 (HGAP3) (6). The HGAP3 data processing pipeline comprised PreAssembler v1 for filtering, Celera assembler v8.1 for assembly (7), BLASR v1 (8) for mapping, and Quiver v1 (6) for consensus polishing using only unambiguously mapped reads. HGAP3 defaults settings were applied, except for the genome size estimate parameter, set to 5.0 Mbp. The completeness of the Methylomonas sp. strain LL1 genome was assessed using the benchmarking universal single-copy orthologs (BUSCO; v4.14) software (https://busco.ezlab.org) (9) with the gammaproteobacteria_odb10 database (quality score, 98.9%; 366 total BUSCO groups searched). The genomes of all Methylocystis sp. strains were sequenced using an Illumina HiSeq 2000 instrument. Library preparation (10), sequencing, and sequence quality control and trimming using internally developed software SOAPnuke v1.4.0 and parameters -l 15 -q 0.2 -n 0.05 (11) were performed at BGI Tech Solutions (Hong Kong, China). The sequencing library was prepared according to BGI protocols. Briefly, 1 μg genomic DNA was randomly fragmented by a Covaris sonicator. DNA fragments were end repaired and A tailed. Next, Illumina adapters were ligated to the 3′-adenylated DNA, which was purified using AxyPrep Mag PCR clean up kit. The resulting fragments were size selected by agarose gel electrophoresis. The library quality and quantity were assessed using the Agilent Technologies 2100 bioanalyzer and ABI StepOnePlus real-time PCR system. The quality of all reads was checked by FastQC v0.11.9 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Clean reads were assembled using Ray v2.0.1 (12). Automatic gene prediction and annotation were performed by using NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (13). Default parameters were used for all software unless otherwise specified. The Methylomonas sp. LL1 genome includes a 0.12-Mb plasmid. General genome statistics and predicted metabolic pathways are detailed in Table 1.
TABLE 1.
General genome information
| Strain | Sequencing platform | Total size of assembly (Mb) | Assembler | No. of raw reads | Raw read N50 (bp) | Coverage (×) | Completion | G+C content (%) | No of CDSa | No of scaffolds | Scaffold N50 (bp) | Core metabolic pathwaysb | GenBank (SRA) accession no.c |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Methylomonas sp. LL1 | PacBio RS II | 4,923,893 | Celera v8.1 | 78,774 | 14,377 | 16 | Yes | 50.9 | 4,426 | 2 | 4,798,577 | pMMO, Mox, PQQ, sMMO, SC, PPP, RuMP, EDD, EMP, TCA | CP064653.1, CP064652.1 (SRR13259185) |
| Methylosinus sp. H3A | PacBio RS II | 5,384,582 | Celera v8.1 | 63,638 | 15,418 | 16 | No | 64.2 | 4,974 | 11 | 4,391,449 | pMMO, Mox, PQQ, sMMO, SC, EMP, TCA | JADNQW000000000 (SRR13259182) |
| Methylocystis sp. H15 | Illumina HiSeq 2000 | 4,053,493 | Ray 2.0.1 | 6,891,600 | 49.8 | No | 62.3 | 3,913 | 39 | 217,286 | pMMO, Mox, PQQ, sMMO, SC, EMP, TCA | JADNQX000000000 (SRR13259184) | |
| Methylocystis sp. H4A | Illumina HiSeq 2000 | 4,288,666 | Ray 2.0.1 | 6,990,370 | 49.8 | No | 62.3 | 4,214 | 38 | 238,602 | pMMO, Mox, PQQ, sMMO, SC, EDD, EMP, TCA | JADNQY000000000 (SRR13259180) | |
| Methylocystis sp. H62 | Illumina HiSeq 2000 | 4,540,521 | Ray 2.0.1 | 6,914,734 | 43.9 | No | 62.1 | 4,408 | 63 | 216,123 | pMMO, Mox, PQQ, sMMO, SC, EMP, TCA | JADNQZ000000000 (SRR13259181) | |
| Methylocystis sp. L43 | Illumina HiSeq 2000 | 4,061,225 | Ray 2.0.1 | 6,899,554 | 48.6 | No | 62.4 | 3,919 | 38 | 145,473 | pMMO, Mox, PQQ, sMMO, SC, EMP, TCA | JADNRA000000000 (SRR13259183) |
CDS, coding DNA sequences.
pMMO, membrane-bound methane monooxygenase; Mox, PQQ-linked methanol and formaldehyde dehydrogenases; PQQ, pyrroloquinoline quinone biosynthesis; sMMO, soluble methane monooxygenase; SC, serine cycle; PPP, pentose phosphate pathway; RuMP, assimilatory ribulose monophosphate pathway; EDD, Entner-Doudoroff pathway; EMP, Embden-Meyerhof-Parnas pathway; TCA, tricarboxylic acid cycle.
The BioProject number of the study is PRJNA674997.
Data availability.
GenBank and SRA (raw data) accession numbers are listed in Table 1.
ACKNOWLEDGMENTS
We thank Mattias de Hollander, Fleur Gahwens-Bruning, and Victor de Jager for bioinformatic support and the Royal Netherlands Academy of Arts and Sciences for the financial support.
Footnotes
This article is publication number 7151 of the Netherlands Institute of Ecology (NIOO-KNAW).
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
GenBank and SRA (raw data) accession numbers are listed in Table 1.