Genome sequences of four novel Endozoicomonas strains associated with a tropical octocoral in a long-term aquarium facility

The bacterial genus Endozoicomonas (Pseudomonadota, Endozoicomonadaceae) is a subject of increasing research interest owing to its widespread association with marine animals, particularly corals (1–4). However, Endozoicomonas spp. are typically difficult to cultivate and maintain in the laboratory (3, 4).

We report the genomes of four Endozoicomonas strains isolated from two Litophyton sp. specimens kept in a 19-m³ tropical exhibition aquarium at the Oceanário de Lisboa, Portugal. Host-derived microbial cell suspensions were retrieved as described previously (2). One gram of coral tissue was homogenized in 9 mL of sterile Ca²⁺- and Mg²⁺-free artificial seawater (2). The homogenate was serially diluted, plated separately on marine agar diluted 1:2 and R2A diluted 1:10 media, and incubated at 21°C for 4 weeks. Genomic DNA of single colonies was extracted from cultures freshly grown in 1:2 marine broth using the Wizard Genomic DNA Purification kit (Promega, USA). Purity was confirmed by Sanger sequencing of 16S rRNA genes amplified from genomic DNA using universal primers (F27 and R1492). Taxonomy assignment was performed with the SILVA Alignment, Classification, and Tree Service (v1.2.12) and database (v138.1). The same genomic DNA samples were used for genome sequencing at the DOE Joint Genome Institute (JGI) using PacBio sequencing technology (5). For each sample, genomic DNA was sheared to 6–10 kb, treated using SMRTbell Express Template Prep Kit 3.0, and purified with SMRTbell cleanup beads (PacBio). The purified product was enriched using barcoded amplification oligos (IDT) and SMRTbell gDNA Sample Amplification Kit (PacBio). A 10-kb PacBio SMRTbell library was constructed and sequenced on the PacBio Revio system using HiFi chemistry. Raw reads were quality-filtered as per the JGI standard operating practice (SOP) protocol 1061 using BBTools v.38.86 (http://bbtools.jgi.doe.gov). Filtered reads >5 kb were assembled using Flye v2.8.3 (6). Organism and project metadata were deposited in the Genomes OnLine database (7). Contigs were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP v.6.7) (8) and the DOE-JGI Microbial Genome Annotation Pipeline (MGAP v.4) (9) coupled to the Integrated Microbial Genomes and Microbiomes system v7 (IMG/M) for comparative analyses (10). Genome completeness and contamination were assessed with CheckM (v1.2.3) (11). AntiSMASH v7.1 (12) was used to identify secondary metabolite biosynthetic gene clusters (SM-BGCs). Default parameters were used for all software, unless otherwise specified.

Sequencing statistics and genome features are shown in Table 1. Average nucleotide identities (ANIs), calculated with FastANI v0.1.3 on KBase (13, 14), among strains NE35, NE40, NE41, and NE43, were above 99.9% in all pairwise comparisons. All four strains shared approximately 89.3% ANI with their closest relative, as determined by phylogenomics, including all Endozoicomonas-type strains with a publicly available genome: Endozoicomonas gorgoniicola PS125^T (GCA_025562715), also isolated from an octocoral (15).

All four genomes encode several glycoside hydrolases, featuring chitinase, polysaccharide deacetylase, N-acetylglucosaminidase, and beta-galactosidase-encoding genes, congruent with the emerging view of complex carbon metabolism among Endozoicomonadaceae spp. associated with marine invertebrates (16–18). Multiple protein domains underlying Type II, III, IV, and VI secretion systems were predicted to be encoded in all genomes. Additionally, three CRISPR–Cas antiviral defense systems, several eukaryotic-like repeat protein motifs, and the potential to synthesize putatively novel ribosomally synthesized and post-translationally modified peptides, among other natural products, were encoded (Fig. 1).

This study was financed by the “Blue Bioeconomy Pact” (Project N°. C644915664-00000026), co-funded by Next Generation EU European Fund, under the incentive line “Agendas for Business Innovation” within Funding Scheme 5-Capitalization and Business Innovation of the Portuguese Recovery and Resilience Plan (RRP). Further support was provided by the Portuguese Foundation for Science and Technology (FCT) through the projects UIDB/04565/2020 and UIDP/04565/2020 of iBB and the project LA/P/0140/2020 of i4HB. Sequencing, assembly, and annotation of the four Endozoicomonas sp. genomes were performed at the Joint Genome Institute (JGI) as part of the Genomic Encyclopedia of Type Strains, Phase V (KMG-V): Genome sequencing to study the core and pangenomes of soil and plant-associated prokaryotes. The work (proposal: 10.46936/10.25585/60001079) conducted by the U.S. Department of Energy Joint Genome Institute (https://ror.org/04xm1d337), a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy operated under Contract No. DE-AC02-05CH11231. M.M. is the recipient of a PhD scholarship conceded by FCT through the MIT Portugal program (10.54499/SFRH/BD/151376/2021). D.M.G.D.S. is the recipient of a MSc grant conceded by the “Blue Bioeconomy Pact” project. T.K.-C. is the recipient of a Research Scientist contract conceded by FCT (CEECIND/00788/2017).