Complete genome sequence of “Candidatus Dehalogenimonas loeffleri” strain W, a highly salt-tolerant chlorinated alkane-dechlorinating bacterium isolated from estuarine sediments

Bioremediation of chlorinated contaminants in saline environments is challenging due to the inability of most organohalide-respiring bacteria (OHRB) to thrive under elevated salinity conditions (1–4), highlighting our limited understanding of OHRB diversity and survival mechanisms in saline habitats. To date, only four species within Dehalogenimonas have been identified, with a variety of isolates originating from diverse terrestrial habitats (2–5). “Candidatus Dehalogenimonas loeffleri” strain W exhibits 1,2-dichloroethane (1,2-DCA) dechlorinating activity under high salinity conditions (e.g., 5.1% NaCl), a trait that has not been previously reported in Dehalogenimonas species. Strain W was enriched from estuarine sediments collected in June 2018 where the River Wuli meets the Bohai Sea in Huludao City, Liaoning Province, China (40°44′44″N, 120°59′11″E), and isolated using a dilution-to-extinction approach in bicarbonate-buffered (30 mM) basal mineral salt medium (6), supplemented with 1 g liter⁻¹ ampicillin, 0.1 g liter⁻¹ vancomycin, 5 mM acetate (carbon source), 413 µmol hydrogen (electron donor), 1,2-DCA (56.3 µmol, electron acceptor), and Wolin vitamin mix. Incubation was at 30°C in the dark without agitation.

Genomic DNA from strain W was extracted using the cetyltrimethylammonium bromide method (7). For PacBio sequencing, genomic DNA was sheared into 10-kb fragments using g-TUBEs (Covaris, USA) to prepare a long-insert library without size selection. These fragments were then ligated with universal hairpin adapters using the SMRTbell Express Template Prep Kit version 2.0 (Pacific Biosciences, USA) (8). Sequencing was subsequently performed using the Sequel II System. Raw read quality control, error correction, and adapter trimming were performed using the SMRT Link version 10.1. For Illumina sequencing, a library with an average insert size of 350 bp was constructed using the NEBNext Ultra DNA Library Prep Kit (New England Biolabs, USA), followed by paired-end sequencing (2 × 150 bp) on an Illumina NovaSeq 6000 instrument (Illumina Inc., USA). The quality-control filtering was conducted using fastp version 0.23.4 (9) for short Illumina reads and Filtlong version 0.2.1 (10) for long PacBio reads, respectively. Assembly integrating PacBio-filtered long reads with a read N50 of 10,710 bp (coverage, 478×) and Illumina-filtered short reads (coverage, 852×) was accomplished using Unicycler version 0.47 (Table 1) (11). Sequencing data were analyzed on the Galaxy web platform (https://usegalaxy.org.au) (12). Default parameters were used unless otherwise noted. The complete genome of strain W, assembled as a single circular chromosome of 1,772,240 bp with a G + C content of 52.5%, was annotated using the NCBI Prokaryotic Genome Annotation Pipeline version 6.6 for gene prediction and functional assignment (13). This analysis revealed 1,763 predicted protein-coding genes, 47 tRNAs, and a single copy each of the 5S rRNA, 16S rRNA, and 23S rRNA genes. The genome harbors 28 nonidentical reductive dehalogenase (rdh) subunit A genes, including one encoding a putative dihaloelimination RDase (V8247_04055, DdeA) that shares a remarkable 91.4%–94.9% amino acid identity with DcpA, previously identified in Dehalococcoides mccartyi strains RC and KS (14), Dehalogenimonas formicexedens strain NSZ-14^T (3), and Dehalogenimonas lykanthroporepellens strain BL-DC-9^T (14). Average nucleotide identity (ANI) analysis using JSpeciesWS version 4.1.1 (15) demonstrated that strain W shares 69.1%–73.7% ANI with Dehalogenimonas strains BL-DC-9^T (CP002084.1), WBC-2 (CP011392.1), IP3-3^T (LFDV00000000.1), NSZ-14^T (CP018258.1), and GP^T (CP058566.2), suggesting that strain W may represent a new species within the genus Dehalogenimonas. The genome of strain W expands the Dehalogenimonas pangenome and provides a foundation for elucidating the molecular mechanisms underpinning its remarkable ability to perform reductive dechlorination under elevated salinity conditions.

This work was supported by the National Key Research and Development Program of China (grant no. 2023YFE0122000), National Natural Science Foundation of China (grant nos. 42177220 and 42377133), Key Research Program of Frontier Sciences, Chinese Academy of Sciences (CAS) (grant no. ZDBS-LY-DQC038), and Galaxy Australia, a service provided by the Australian Biocommons and its partners. The service receives NCRIS funding through Bioplatforms Australia and the Australian Research Data Commons (https://doi.org/10.47486/PL105) as well as The University of Melbourne and Queensland Government RICF funding.