# Complete genome sequence of *Hyphomicrobium/sp* . strain 1Nfss2.1 from marine sediments of the Berre lagoon, France

Manon Bartoli, Nathalie Pradel, Stéphanie Fouteau, Zoé Rouy, Corinne Cruaud, Thierry Nadalig, David Vallenet, Pedro H Oliveira, Agnès Hirschler-Réa

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Manon Bartoli, Nathalie Pradel, Stéphanie Fouteau, Zoé Rouy, Corinne Cruaud, et al.. Complete genome sequence of *Hyphomicrobium/sp*. strain 1Nfss2.1 from marine sediments of the Berre lagoon, France. Microbiology Resource Announcements, 2025, 10.1128/mra.00254-25. cea-05163110

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Submitted on 15 Jul 2025

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# Complete genome sequence of *Hyphomicrobium* sp. strain 1Nfss2.1 from marine sediments of the Berre lagoon, France

Manon Bartoli,¹ Nathalie Pradel,¹ Stéphanie Fouteau,² Zoé Rouy,² Corinne Cruaud,³ Thierry Nadalig,⁴ David Vallenet,² Pedro H. Oliveira,² Agnès Hirschler-Réa¹

**AUTHOR AFFILIATIONS** See affiliation list on p. 2.

**ABSTRACT** We report the complete genome sequence of *Hyphomicrobium* sp. strain 1Nfss2.1, isolated from coastal marine sediments of the Berre lagoon, France. The genome consists of a 4,097,693-bp circular chromosome. It contains the genes involved in the Cmu chloromethane degradation pathway.

**KEYWORDS** bacterial genome, *Hyphomicrobium*, chloromethane, nitrate

The Berre lagoon, gathering steel industries and oil refineries, is open onto the Mediterranean Sea (1). The genome sequence of a *Hyphomicrobium* sp. isolated from this environment provides a resource to understand the adaptation mechanisms of microorganisms to marine anthropogenically disturbed environments.

Isolation of strain 1Nfss2.1 was performed using the Hungate technique (2) from 5 mL of sediments collected with a piston. The medium contained 0.1 g.L⁻¹ KH₂PO₄, 0.1 g.L⁻¹ K₂HPO₄, 0.2 g.L⁻¹ NH₄Cl, 7 g.L⁻¹ NaCl, 1.3 g.L⁻¹ MgCl₂·6H₂O, 0.1 g.L⁻¹ KCl, 0.05 g.L⁻¹ CaCl₂·2H₂O, 0.1% trace elements solution (DSMZ 320), 0.1% Se-Ni-Mo-W solution (DSMZ 385), 1% Balch's vitamins (3), 0.1 g.L⁻¹ B12 vitamin, and 20 mM KNO₃, under N₂ atm. Chloromethane was added at 4 mM, and pH was 7.5. Agar was added at 1.5% if needed. Three sub-cultures of 50 mL were performed at 30°C, without shaking, prior to serial dilutions on solid medium using roll tubes (4). Colonies developed after three weeks. From a colony, 10⁻² serial dilutions were repeated in liquid medium until extinction. DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega) from 5 mL of culture in the same medium.

Genome sequencing was carried out using Illumina and Nanopore Technologies. DNA was quantified using the Qubit 2.0 Fluorometer (Thermo Fisher). For Illumina, 250 ng DNA was sonicated to a mean size of 380 bp, using a Covaris E210, and profiles were visualized on an Agilent Bioanalyzer DNA chip. The fragments were end-repaired, 3'-adenylated, and ligated to NEXTflex barcodes (Bioo Scientific Corporation) (5). The ligated product was amplified by 12 PCR cycles using the KAPA HiFi Library Amplification kit (KAPABIOSYSTEMS) and purified with 0.6x AMPure XP (Beckman Coulter). The library was sequenced on an Illumina MiSeq with a MiSeq Reagent Kit-v2 (2 x 150 bp). A total of 1,313,850 paired-end reads were obtained. The Illumina reads were trimmed by removing low-quality nucleotides (Q < 20), adaptors, and sequences <30 nt, using `fastx_toolkit-v0.0.14` (http://hannonlab.cshl.edu/fastx_toolkit).

Nanopore sequencing was performed according to Oxford Nanopore Technologies (Oxford, UK). The library was prepared with 400 ng DNA following the Native Barcoding Kit 24-v14 protocol, with the SQK-NBD114.24 ligation kit. The library was sequenced using an R10.4.1 flow cell and the GridION device with the MinKNOW-v6.0.14 and Guppy-v7.4.14+d47971cbb software. A total of 97,336 reads were obtained with an N50 of 18 Kb. Assembly was performed with Flye-v2.9.5 (6) on long reads filtered with Filtlong-v0.2.1 (min length 6,000; keep_percent 90; https://github.com/rrwick/Filtlong),

**Editor** Frank J. Stewart, Montana State University, Bozeman, Montana, USA

Address correspondence to Nathalie Pradel, nathalie.pradel@ird.fr, or Agnès Hirschler-Réa, agnes.hirschler@univ-amu.fr.

The authors declare no conflict of interest.

Received 28 April 2025

Accepted 19 June 2025

Published 14 July 2025

Copyright © 2025 Bartoli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
resulting in an assembly in a single loop-edge representing a circular chromosome with a 102x mean coverage. Polishing was done with long reads using Medaka-v2.0.1 (https://github.com/nanoporetech/medaka), and with Pypolca-v0.3.1 (7). Completeness (99.20%) and contamination (0.89%) were estimated using CheckM (8). Default settings were used for all software. Annotation was performed at the Microscope platform-v3.17.3 (9).

The whole genome consisted of a 4,097,693 bp circular chromosome with an average G+C content of 62.95%. A total of 4,187 coding DNA sequences were predicted. NCBI blastn analysis indicated 97.9% identity over 98% length of the 16S rDNA sequence of *Hyphomicrobium album* XQ2¹, isolated from soil, in China (10). The average nucleotide identity value between the two genomes was only 77.6%, suggesting that strain 1Nfss2.1 may represent a new species (EZ BioCloud ANI Calculator, 21 April 2025). It is the first genome of *Hyphomicrobium sp.* that possesses both genes of the chloromethane degradation pathway and genes for the complete anaerobic nitrate respiratory chain.

## ACKNOWLEDGMENTS

The research was supported by the MAHABIO 2022-2026 (ANR-21-CE02-0029-02) Project. The LABGeM (CEA/Genoscope & CNRS UMR8030), the France Génomique and French Bioinformatics Institute national infrastructures (funded as part of the Investissement d'Avenir program managed by the Agence Nationale pour la Recherche, contracts ANR-10-INBS-09, ANR-11-INBS-0013, and ANR-21-ESRE-0048) are acknowledged for support within the MicroScope annotation platform.

## AUTHOR AFFILIATIONS

## AUTHOR ORCIDs

Nathalie Pradel ID http://orcid.org/0000-0002-6223-7420

Agnès Hirschler-Réa ID http://orcid.org/0000-0001-5004-0981

## DATA AVAILABILITY

This whole genome project and the raw data have been deposited in ENA under the numbers PRJEB86990 and ERA32774194, respectively. The chromosome sequence is available at ENA under the accession no. OZ251603.

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<footnote n="1">¹Aix Marseille Univ, Université de Toulon, CNRS, IRD, MIO, Marseille, France</footnote>

<footnote n="2">²Génomique Métabolique, Genoscope, Institut François Jacob, Commissariat à l'Energie Atomique (CEA), CNRS, Université Evry, Université Paris-Saclay, Gif-sur-Yvette, France</footnote>

<footnote n="3">³Genoscope, Institut François Jacob, CEA, Université Paris-Saclay, Evry, France</footnote>

<footnote n="4">⁴Génétique Moléculaire, Génomique, Microbiologie (GMGM), Université de Strasbourg, UMR 7156 CNRS, Strasbourg, France</footnote>
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