新加坡珊瑚Pachyseris speciosa中三种弧菌的分离及其致病相关基因组特征分析

《Microbiology Resource Announcements》：Three Vibrio isolates from the coral Pachyseris speciosa as draft genome resources

　　

摘要

Vibrio coralliilyticus SCP4, Vibrio harveyi SCP5, and Vibrio sp. SCP2 were isolated from the coral Pachyseris speciosa in Singapore, adding to reports of Vibrio in tropical corals. Their draft genomes variably encode types II, III, and VI secretion systems, toxR, and vcpA, genes linked to pathogenic potential in Vibrio species.

公告

Vibrio species are well-recognized coral pathogens, prevalent in coral-associated bacterial communities in reef ecosystems (1). Isolation from equatorial coral reefs adds to the growing catalog of local environmental Vibrio isolates (2) and their potential for opportunistic pathogenicity (3).

Pachyseris speciosa samples were collected from the seawall at the northern side of Kusu Island, Singapore (1.2120°N, 103.8347°E) in August 2021, and maintained in an outdoor (0.8 m³) aquarium tank with seawater flow. Bacterial isolation methods were performed as described by Loh et al. (4). Briefly, coral tissue was separated from the skeleton by hydro-blasting, collected, and treated with proteinase K (Qiagen) in sterile artificial seawater, vortexed with mixed-sized (0.2–0.7 mm) glass beads in a bench top vortex (~200–2,700 rpm), and plated on marine broth agar (Difco 2216, BD). SCP2 and SCP4 were isolated at 33°C, and SCP5 at 28°C (Table 1). Colonies were subcultured under the same respective culturing conditions until pure isolates were obtained, then a single colony of each isolate was inoculated into marine broth and grown aerobically overnight.

TABLE 1

TABLE 1 Genome features and GenBank accession numbers of the three Vibrio spp. isolates^a

Isolate	Vibrio sp. SCP2	Vibrio sp. SCP4	Vibrio sp. SCP5
Source	Tissue	Tissue	Tissue
Growth temperature (°C)	33	33	28
Genome size (bp)	4,801,857	5,280,947	5,709,137
No. of CDS	4,375	4,761	5,079
No. of rRNAs	29	22	25
No. of tRNAs	99	102	123
G + C content (%)	44.9	45.4	44.9
No. of contigs	56	56	90
N50 (bp)	488,764	931,604	635,040
L50	3	2	3
Assembly accession no.	JCAFVV000000000	JCAFVU000000000	JCAFVT000000000
SRA accession no.	SRR39408556	SRR39408555	SRR39408554
No. of reads	9,433,035	11,343,331	14,057,343

^a

CDS, coding DNA sequences.

Genomic DNA was extracted with the DNeasy PowerSoil Pro Kit (Qiagen, Hilden, Germany), using a Qubit fluorometer for quantification and quality control (ThermoFisher, USA). DNA library preparation and sequencing were performed by the SCELSE Sequencing Facility, NTU. DNA library preparation used the Accel-NGS 2S Plus Low Input DNA Library Prep Kit (Swift Biosciences, Ann Arbor, USA). Sequencing was performed using an Illumina HiSeq X Ten platform, version 2.5 (Illumina, San Diego, CA, USA), generating 150-bp paired-end reads.

Reads were quality-filtered and adaptor-trimmed using Trimmomatic version 0.39 (5). Default parameters were used unless specified. Draft genomes were assembled using the Shovill pipeline version 1.1.0 (https://github.com/tseemann/shovill) and the St. Petersburg genome Assembler version 3.15.5 (6). Annotations of the draft genomes were carried out with the Prokaryotic Genome Annotation Pipeline (PGAP) version 6.11 (7). No plasmids were detected by PlasmidFinder version 2.0.1 (8) in any of the assemblies, whose genome features are listed in Table 1.

Taxonomic classification using the Genome Taxonomy Database Toolkit v1.7.0 (9) identified V. tubiashii (GCF000772105), V. coralliilyticus (GCF000176135), and V. harveyi (GCF001591145) as the closest reference genomes to SCP2, SCP4, and SCP5, respectively, based on topology and average nucleotide identity (ANI). Phylogenetic relatedness between each draft genome and all publicly available reference genomes was assessed using the Orthologous Average Nucleotide Identity Tool, version 0.93.1 (10), and the Genome-To-Genome Distance Calculator, version 3.0 (11), to calculate the ANI and digital DNA-DNA hybridization (dDDH) scores, respectively. All draft genomes were <95% ANI and <70% DDH to each other, with SCP4 and SCP5 showing >95% ANI and >70% dDDH to their respective reference genomes. SCP2 showed >95% ANI but <70% dDDH to its reference genome, below the species threshold (12).

Dimethylsulfoniopropionate (DMSP) demethylation (dmdA) or cleavage (ddd lyase) genes, despite being important in marine systems (13), were absent from the PGAP annotation of all three draft genomes. Type II and type VI secretion systems are found in all three isolates (14, 15), with a type III secretion system, additionally identified in SCP5 (16). Despite lacking luxI-luxR quorum sensing, all isolates encode toxR homologue, a master transcriptional regulator for virulence (17), and vcpA homologue, a metalloprotease capable of coral tissue degradation (18), identified by NCBI online web BLASTp (version 2.17.0) with query sequences against our draft genomes.

ACKNOWLEDGMENTS

This research/project is supported by the National Research Foundation, Singapore, and the National Parks Board, Singapore, under its Marine Climate Change Science Programme (MCCS Award NRF-MCCS21-1-5-0001). The Singapore Centre for Environmental Life Sciences Engineering (SCELSE) is funded by the Ministry of Education, Singapore, the National Research Foundation of Singapore, Nanyang Technological University (NTU), and the National University of Singapore (NUS).

The authors would like to acknowledge St. John’s island National Marine Laboratory (SJINML) for providing the facility necessary for conducting the research. The laboratory is a National Research Infrastructure under the National Research Foundation Singapore. We would also like to thank the Coral team at Tropical Marine Science Institute (TMSI) and SJINML for collecting the coral fragments used in this research.