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# Complete genome sequences of Methylococcus capsulatus (Norfolk) and Methylocaldum szegediense (Norfolk) isolated from a landfill methane biofilter

David Pearce, Elliot Brooks, Charles Wright, Daniel Rankin, Andrew T Crombie, J. Colin Murrell

► To cite this version:

David Pearce, Elliot Brooks, Charles Wright, Daniel Rankin, Andrew T Crombie, et al. Complete genome sequences of Methylococcus capsulatus (Norfolk) and Methylocaldum szegediense (Norfolk) isolated from a landfill methane biofilter. Microbiology Resource Announcements, 2024, 13 (2), 10.1128/mra.00675-23. cea-04529297

HAL Id: cea-04529297

https://cea.hal.science/cea-04529297v1

Submitted on 2 Apr 2024

**HAL** is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

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Distributed under a Creative Commons Attribution 4.0 International License
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# Complete genome sequences of *Methylococcus capsulatus* (Norfolk) and *Methylocaldum szegediense* (Norfolk) isolated from a landfill methane biofilter

David Pearce,¹ Elliot Brooks,¹ Charles Wright,² Daniel Rankin,² Andrew T. Crombie,¹ J. Colin Murrell¹

**AUTHOR AFFILIATIONS** See affiliation list on p. 3.

**ABSTRACT** Here we report the complete genome sequence of two moderately thermophilic methanotrophs isolated from a landfill methane biofilter, *Methylococcus capsulatus* (Norfolk) and *Methylocaldum szegediense* (Norfolk).

**KEYWORDS** methanotrophs, genomes, landfill, biofilter

The Strumpshaw closed landfill features a biofilter for the mitigation of the climate active gas methane, generated by the anaerobic breakdown of organic waste. This biofilter harnesses methanotrophic bacteria in a soil matrix for methane bio-oxidation. Two methanotrophs, *Methylococcus capsulatus* (Norfolk) and *Methylocaldum szegediense* (Norfolk), were isolated from this system. Biofilter soil was used to inoculate vials containing nitrate mineral salt (NMS) medium (1) and supplied with 20% (vol/ vol) methane. Isolates were obtained from enrichment cultures by serial dilution and plating onto NMS agar plates, incubated in gas-tight containers supplied with 50% (vol/ vol) methane. Optimal growth temperatures of the *Methylococcus* and *Methylocaldum* isolates were 45°C and 50°C, respectively. *M. capsulatus* (Norfolk) also grew on methanol (1%–5% vol/vol) as did *Methylococcus* strain MIR (2).

DNA extraction, sequencing, and genome assembly were done using a combined long- and short-read sequencing service at MicrobesNG (Birmingham, UK) as described in Fig. 1. This pipeline was used to construct genomes for *M. capsulatus* (Norfolk) and *M. szegediense* (Norfolk), producing a closed genome in both cases.

MicroScope v.3.16.0 (3) was used for automated annotation and taxonomic assignment of assembled genomes before further manual curation. Genome assembly and sequencing read summaries are shown in Table 1.

The Norfolk isolates were assigned to the *Methylococcus capsulatus* and *Methylocaldum szegediense* spp. first described by Foster and Davis (4) and Bodrossy et al. (5). Based on average nucleotide identity (ANI) scores generated using CJ Bioscience's online ANI calculator (6), the sequenced genomes with the highest similarity to *Methylococcus capsulatus* (Norfolk) and *Methylocaldum szegediense* (Norfolk) are *Methylococcus capsulatus* (Texas) (99.56%) and *Methylocaldum szegediense* (O-12) (99.64%), respectively (GenBank accession numbers GCA_000297615.1 and GCA_000427385.1).

Both genomes contain genes encoding a full methane oxidation pathway. Two *pmoCAB* clusters encoding particulate methane monooxygenase were found in each genome (7), and the *Methylococcus capsulatus* (Norfolk) genome also possesses a single soluble methane monooxygenase *mmoXYBZDCGQSR* cluster (8) and a putative copper chaperone (*mopE*) gene (9). Calcium-dependent (*mxaFJGIRSAckLD*) and lanthanide-dependent (*xoxFJ*) methanol dehydrogenase gene clusters (10, 11) were found in these genomes, with a clade 5 *xoxF* gene present in each and an additional clade 3 *xoxF* in *Methylocaldum szegediense* (Norfolk) (12). Both genomes feature complete

**Editor** Elinne Becket, California State University San Marcos, USA

Address correspondence to David Pearce,
David.Pearce@uea.ac.uk.

The authors declare no conflict of interest.

See the funding table on p. 4.

Received 23 July 2023

Accepted 10 December 2023

Published 18 January 2024

Copyright © 2024 Pearce et al. This is an open-access
article distributed under the terms of the Creative
Commons Attribution 4.0 International license.
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## DNA extraction

NMS media was inoculated with a single isolate colony and incubated with methane. After growth, 4-6x10⁹ cells (estimated by OD₆₀₀ measurement) were pelleted and resuspended in 500 µl of DNA/RNA shield (Zymo Research). 5-40 µl of **cell suspension lysed** with 120 µl of 0.1 mg/ml **lysozyme** and **RNase A** in TE buffer for 25 min at 37°C, then incubated for 5 min at 65°C with Proteinase K (0.1 mg/ml) and SDS (0.5% v/v). **Genomic DNA was purified** using an equal volume of solid-phase reversible immobilisation (SPRI) beads and resuspended in 10 mM Tris-HCl (pH 8.0).

Illumina short-read
sequencing

<figure><img src="image_2.png" /></figure>

**DNA libraries** were generated with a modified **Nextera XT Library Prep Kit** protocol (input DNA doubled, and PCR elongation time was increased to 45 seconds).

<figure><img src="image_3.png" /></figure>

Sequenced using an **Illumina NovaSeq6000** to produce 250bp paired-end reads.

<figure><img src="image_4.png" /></figure>

**Trimmomatic** version 0.30 was used for Nextera adapter removal and read trimming with a **Q15 sliding window cut-off**.

<figure><img src="image_5.png" /></figure>

### Hybrid genome assembly

**Unicycler** version 0.4.0 was used for hybrid genome assembly, circularisation and rotation. Assembled contigs were rotated in between rounds of polishing, the final assembly was rotated to an appropriate start gene (*dnaA*).

Note - Default parameters were used except where otherwise noted

<figure><img src="image_6.png" /><figcaption>FIG 1 Sequencing and assembly pipeline.</figcaption></figure>

### Oxford Nanopore
long-read sequencing

**DNA libraries** were generated with **SQK-LSK109** Oxford Nanopore kits and native barcoding expansions **EXP-NBD104** or **EXP-NBD114** using 400-500 ng of HMW DNA (no shearing/size selection).

<figure><img src="image_7.png" /></figure>

Sequenced using an **Oxford Nanopore GridION** platform and **FLO-MIN106 (R.9.4.1)** flow cell.

<figure><img src="image_8.png" /></figure>

BaseCALLing (**high accuracy baseCALLing model**) and barcode trimming were performed using the **GridION** deployment of **Guppy** ver3.2.8+bd67289** (with no further filtering).

<figure><img src="image_9.png" /></figure>
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<table><caption>TABLE 1 *Methylocaldum szegediense* (Norfolk) and *Methylococcus capsulatus* (Norfolk) genome summaries</caption><thead><tr><th colspan="9">DNA sequencing reads</th></tr><tr><th>Isolate</th><th>Illumina total reads</th><th>Illumina read length (bp)</th><th>Nanopore total reads</th><th>Nanopore N<sub>50</sub> (bp)</th><th>Illumina reads ENA accession no.</th><th>Nanopore reads ENA accession no.</th></tr></thead><tbody><tr><td><em>Methylocaldum</em></td><td>936,436</td><td>250</td><td>184,537</td><td>4,370</td><td>ERR11151912</td><td>ERR11151913</td></tr><tr><td><em>Methylococcus</em></td><td>891,006</td><td>250</td><td>15,738</td><td>13,497</td><td>ERR11151914</td><td>ERR11151915</td></tr><tr><th colspan="8">Methylocaldum szegediense (Norfolk) assembly</th></tr><tr><th>Replicon</th><th>Sequence length (bp)</th><th>Assembly coverage</th><th>% GC</th><th>No. of CDS</th><th>Ribosomal RNA genes 16S rRNA</th><th>Ribosomal RNA genes 23S rRNA</th><th>Ribosomal RNA genes 5S rRNA</th></tr><tr><td>Chromosome</td><td>4,869,648</td><td>173x</td><td>57</td><td>5,038</td><td>2</td><td>2</td><td>2</td></tr><tr><td>Plasmid</td><td>25,724</td><td>481x</td><td>58</td><td>38</td><td>0</td><td>0</td><td>0</td></tr><tr><th colspan="8">Methylococcus capsulatus (Norfolk) assembly</th></tr><tr><th>Replicon</th><th>Sequence length (bp)</th><th>Assembly coverage</th><th>% GC</th><th>No. of CDS</th><th>Ribosomal RNA genes 16S rRNA</th><th>Ribosomal RNA genes 23S rRNA</th><th>Ribosomal RNA genes 5S rRNA</th></tr><tr><td>Chromosome</td><td>3,398,174</td><td>85x</td><td>63.5</td><td>3318</td><td>2</td><td>2</td><td>2</td></tr></tbody></table>

gene inventories for tetrahydromethanopterin and tetrahydrofolate-linked formaldehyde oxidation, in addition to formate dehydrogenase genes (13). Carbon is presumed to be assimilated primarily via the ribulose monophosphate pathway as in *Methylococcus capsulatus* (Bath), although genes for a partial serine cycle and complete Calvin-Benson-Bassham pathway were detected (14). Alanine dehydrogenase and GS/GOGAT cycle genes for ammonia assimilation were present (15).

In addition to the 4.87 Mbp chromosome, *Methylocaldum szegediense* (Norfolk) also contained a ~25-kbp plasmid, encoding a plasmid replication initiator protein (TrfA), replication protein (RepA) and a toxin anti-toxin plasmid retention mechanism. A gene encoding a putative siphovirus Gp157 protein was also found, which may confer increased bacteriophage resistance (16, 17).

## ACKNOWLEDGMENTS

Genome sequencing was provided by MicrobesNG (http://www.microbesng.com). This work was funded by the EnvEast DTP (NERC NE/L002582/1) and Norfolk County Council. The LABGeM (CEA/Genoscope and CNRS UMR8030), the France Génomique, and French Bioinformatics Institute National Infrastructures (funded as part of Investissement d'Avenir program managed by Agence Nationale pour la Recherche, contracts ANR-10-INBS-09 and ANR-11-INBS-0013) are acknowledged for support within the MicroScope annotation platform.

## AUTHOR AFFILIATIONS

## PRESENT ADDRESS

David Pearce, School of Biological Sciences, University of East Anglia, Norwich, United Kingdom

Elliot Brooks, The Department of Infection, Immunity and Cardiovascular Disease, and the Bateson Centre, University of Sheffield, Sheffield, United Kingdom

## AUTHOR ORCIDs

David Pearce http://orcid.org/0000-0002-7378-2707

<footnote n="1">¹School of Environmental Sciences, University of East Anglia, Norwich, United Kingdom</footnote>

<footnote n="2">²Norfolk County Council, Norfolk, United Kingdom</footnote>
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<table><thead><tr><td>Funder</td><td>Grant(s)</td><td>Author(s)</td></tr></thead><tbody><tr><td>UK Research and Innovation (UKRI)</td><td>NE/L002582/1</td><td>David Pearce</td></tr><tr><td>Government of the United Kingdom (UK Government)</td><td></td><td>David Pearce</td></tr></tbody></table>

DATA AVAILABILITY

Genome assembly and raw read accession numbers are listed in Table 1.

REFERENCES

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2. Oshkin IY, Suleimanov RZ, Khmelenina VN, Mardanov AV, Pimenov NV, Dedysh SN. 2022. Complete genome sequence of *Methylococcus capsulatus* MIR, a methanotroph capable of growth on methanol. Microbiol Resour Announc 11:e0054222. https://doi.org/10.1128/mra.00542-22

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5. Bodrossy L, Holmes EM, Holmes AJ, Kovács KL, Murrell JC. 1997. Analysis of 16S rRNA and methane Monooxygenase gene sequences reveals a novel group of Thermotolerant and Thermophilic Methanotrophs, *Methylocaldum* gen. nov.. Arch Microbiol 168:493-503. https://doi.org/10.1007/s002030050527

6. Yoon SH, Ha SM, Lim J, Kwon S, Chun J. 2017. A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie Van Leeuwenhoek 110:1281-1286. https://doi.org/10.1007/s10482-017-0844-4

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*Methylococcus capsulatus* (Bath) secreted protein, MopE*, binds both reduced and oxidized copper. PLoS One 7:e43146. https://doi.org/10.1371/journal.pone.0043146

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11. Oshkin IY, Danilova OV, But SY, Miroshnikov KK, Suleimanov RZ, Belova SE, Tikhonova EN, Kuznetsov NN, Khmelenina VN, Pimenov NV, Dedysh SN. 2021. Expanding characterized diversity and the pool of complete genome sequences of *Methylococcus* species, the bacteria of high environmental and biotechnological relevance. Front Microbiol 12:756830. https://doi.org/10.3389/fmicb.2021.756830

12. Keltjens JT, Pol A, Reimann J, Op den Camp HJM. 2014. PQQ-dependent methanol dehydrogenases: rare-earth elements make a difference. Appl Microbiol Biotechnol 98:6163-6183. https://doi.org/10.1007/s00253-014-5766-8

13. Trotsenko YA, Murrell JC. 2008. Metabolic aspects of aerobic obligate methanotrophy. Adv Appl Microbiol 63:183-229. https://doi.org/10.1016/S0065-2164(07)00005-6

14. Ward N, Larsen O, Sakwa J, Bruseth L, Khouri H, Durkin AS, Dimitrov G, Jiang L, Scanlan D, Kang KH, et al. 2004. Genomic insights into methanotrophy: the complete genome sequence of *Methylococcus capsulatus* (Bath). PLoS Biol 2:e303. https://doi.org/10.1371/journal.pbio.0020303

15. Murrell JC, Dalton H. 1983. Ammonia assimilation in *Methylococcus capsulatus* (Bath) and other obligate methanotrophs. Microbiology 129:1197-1206. https://doi.org/10.1099/00221287-129-4-1197

16. Zhong KX, Suttle CA, Baudoux A-C, Derelle E, Colombet J, Cho A, Caleta J, Six C, Jacquet S. 2018. A new freshwater cyanosiphovirus harboring integrase. Front Microbiol 9:2204. https://doi.org/10.3389/fmicb.2018.02204

17. Foley S, Lucchini S, Zwahlen M-C, Brüssow H. 1998. A short noncoding viral DNA element showing characteristics of a replication origin confers bacteriophage resistance to *Streptococcus thermophilus*. Virology 250:377-387. https://doi.org/10.1006/viro.1998.9387
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