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Genomic insights into Photobacterium damselae subsp. damselae strain KC-Na-1, isolated from the finless porpoise (Neophocaena asiaeorientalis)
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Contents lists available at ScienceDirect
Marine Genomics journal homepage: www.elsevier.com/locate/margen
Genomic insights into Photobacterium damselae subsp. damselae strain KCNa-1, isolated from the finless porpoise (Neophocaena asiaeorientalis) Kyunglee Leea,1, Hye Kwon Kimb,1, Hawsun Sohna, Yuna Choa, Young-Min Choia, Dae Gwin Jeongb,⁎, Ji Hyung Kimb,⁎ a b
Cetacean Research Institute (CRI), National Institute of Fisheries Science (NIFS), Ulsan 44780, Republic of Korea Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords: Photobacterium damselae ssp. damselae KC-Na-1 Cetaceans Pathogens
Photobacterium damselae subsp. damselae (PDD) is a marine bacterium that can infect a variety of marine animals and humans. Although this bacterium has been isolated from several stranded dolphins and whales, its pathogenic role in cetaceans is still unclear. In this study, we report the complete genome of PDD strain KC-Na-1 isolated from a finless porpoise (Neophocaena asiaeorientalis) rescued from the South Sea (Republic of Korea). The sequenced genome comprised two chromosomes and four plasmids. Among the recently identified major virulence factors in PDD, only phospholipase (plpV) was found in strain KC-Na-1. Interestingly, two genes homologous to Vibrio thermostable direct hemolysin (tdh) and its transcriptional regulator toxR, which are known virulence factors associated with Vibrio parahaemolyticus, were encoded on the plasmid pPDD-Na-1-3. Based on these results, strain KC-Na-1 may have potential pathogenicity in humans and other marine animals and also could act as a potential virulent strain. To the best of our knowledge, this is the first report of the complete genome sequence of P. damselae.
1. Introduction The genus Photobacterium comprises 23 valid species in Vibrionaceae (Proteobacteria: Gammaproteobacteria) and is ubiquitous in coastal, open-ocean, and deep-sea environments (Moi et al., 2017). Members of the genus are typically found in seawater and in association with marine animals as saprophytes and enteric commensals; several luminescent species form highly specific bacterial endosymbionts with fish and squid (Urbanczyk et al., 2011). However, two subspecies of P. damselae (ssp. damselae and ssp. piscicida) have been found to be associated with mortality in fish, and P. damselae ssp. damselae (hereinafter referred as PDD), which was originally described as Vibrio damsela (Love et al., 1981), is now considered a bacterial pathogen that can cause infections in a variety of marine ectotherms, including fish, sea turtles, mollusks, and crustaceans (Moi et al., 2017). Furthermore, PDD has been isolated as the causative agent of human infections, including some fatal cases (Rivas et al., 2013a, 2013b). Several bacterial species, including Brucella spp., Mycobacterium spp., and Erysipelothrix rhusiopathiae, were recently recognized as
causative agents of emerging infectious diseases in cetaceans (Van Bressem et al., 2009). PDD has also been isolated from stranded dolphins and whales (Fujioka et al., 1988; Buck et al., 1991; Casalone et al., 2014; Di Francesco et al., 2016); however, the pathogenic role of PDD in cetaceans still remains unclear due to the limitations of postmortem analyses of stranded individuals (Casalone et al., 2014). Moreover, no studies of porpoises have described PDD isolation or detection, so far. Although the mechanisms of infection and virulence in PDD have not been thoroughly investigated in marine animals (particularly cetaceans and chelonians), a recent genotyping approach revealed that a clonal PDD group might be related to the mortality events among cetaceans (Alba et al., 2016). Since 2016, we have investigated the bacterial diversity in cetacean species present in coastal waters in the Republic of Korea in order to identify the potential pathogens that can colonize and establish infection in marine mammals for conservation. In this study, we present the complete genome of PDD strain KC-Na-1, which was isolated from a finless porpoise (Neophocaena asiaeorientalis) found bycaught in 2016 along the South Sea (Republic of Korea). We aimed to provide insights
Abbreviations: PDD, Photobacterium damselae subsp. damselae; COG, Clusters of Orthologous Groups; plpV, phospholipase; colP, collagenase; nusB, N utilization substance protein B; tdh, thermostable direct hemolysin ⁎ Corresponding authors. E-mail addresses: [email protected] (D.G. Jeong), [email protected] (J.H. Kim). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.margen.2017.09.004 Received 1 August 2017; Received in revised form 12 September 2017; Accepted 13 September 2017 1874-7787/ © 2017 Published by Elsevier B.V.
Please cite this article as: Lee, K., Marine Genomics (2017), http://dx.doi.org/10.1016/j.margen.2017.09.004
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SMRTbell template library and with paired-end Illumina short read data using a HiSeq 2000 instrument (Illumina, USA). The PacBio long read data (1,464,887,777 bp, 167,668 reads) were de novo assembled by the Hierarchical Genome Assembly Process program (ver. 3.0), and the Illumina pair end reads (1,532,556,320 bp, 15,183,058 reads) were mapped to the assembled contigs to improve the accuracy of the genome sequences. Genome annotation was carried out using the NCBI's Prokaryotic Genome Annotation Pipeline (http://www.ncbi. nlm.nih.gov/books/NBK174280/). Bacterial tRNAs and rRNAs were analyzed using tRNAscan-SE 1.21 (Lowe and Eddy, 1997) and RNAmmer 1.2 (Lagesen et al., 2007), respectively. Functional categories of open reading frames were analyzed by BLASTP search against Clusters of Orthologous Groups (COG) database (Tatusov et al., 2001) with an E-value cutoff of 1E-4 and an identity cutoff of 20%. The fully assembled and closed PDD strain KC-Na-1 genome contained 4,544,586 bp consisting of two chromosomes, designated Chr I (3,134,662 bp) and Chr II (1,105,401 bp), and a total of four plasmids, designated pPDD-Na-1-1 (105,771 bp), pPDD-Na-1-2 (81,002 bp), pPDD-Na-1-3 (72,321 bp), and pPDD-Na-1-4 (45,429 bp), as shown in Fig. 1a. The two chromosomes showed similar G + C contents (41.6% and 39.3%) and percentages of coding regions (86.2% and 84.9%). Moreover, most of the predicted tRNAs (n = 182), rRNA (n = 47), and ncRNA (n = 4) genes were encoded on Chr I, except 11 and one tRNA genes on Chr II and pPDD-Na-1-4, respectively (Table 2). The COG functional category analysis of PDD strain KC-Na-1 revealed that Chr I had higher percentages of genes related to basic cellular functions than Chr II (Supplementary Fig. 1a). Functional genes encoded on Chr I were mainly involved in COG categories of J (translation, ribosomal structure, and biogenesis), L (replication, recombination, and repair), D (cell cycle control, cell division, and chromosome partitioning), T (signal transduction mechanisms), M (cell wall/membrane/envelope biogenesis), N (cell motility), U (intracellular trafficking, secretion, and vesicular transport), O (post-translational modification, protein turnover, chaperones), C (energy production and conversion), E (amino acid transport and metabolism), F (nucleotide transport and metabolism), H (coenzyme transport and metabolism), and I (lipid transport and metabolism). In contrast, Chr II possessed higher percentages of genes involved in K (transcription), V (defense), X (mobilome: prophages, transposons), G (carbohydrate transport and metabolism), P (inorganic ion transport and metabolism), and Q (secondary metabolites biosynthesis, transport, and catabolism). However, both chromosomes contained genes involved in S (function unknown in COG database), and 6.8% and 15.1% of the predicted genes on Chr I and Chr II, respectively, failed to find a match in the database. As expected, most of the functional genes encoded on plasmids did not have matches in the COG database, and the remaining genes were mainly involved in K, L, U, and X (Supplementary Fig. 1b). Currently, the complete genome of P. damselae is not available in the GenBank database, except that for strain KC-Na-1. Therefore, the OrthoANI algorithm (Lee et al., 2016) was used to assess overall genome similarity between P. damselae and other related strains in the family Vibrionaceae. OrthoANI values were obtained, and a phylogenetic tree was constructed based on OrthoANI analysis of the three species of Photobacterium (P. damselae, P. profundum, and P. gaetbulicola) and the other seven related Vibrio species using the orthologous average nucleotide identity tool. The resulting phylogenetic trees based on OrthoANI values for strain KC-Na-1 and other related strains indicated that strain KC-Na-1 was most related to P. profundum and that Vibrio species were close relatives of the bacterium. However, the other species in the genus Photobacterium, i.e., P. gaetbulicola strain Group47, showed the lowest ANI value, thus suggesting that its taxonomical position in Vibrionaceae needs to be re-examined (Fig. 1b). Members of Photobacterium species, including P. angustum, P. aquimaris, P. kishitanii, P. leiognathi, P. mandapamensis, and P. phosphoreum, are known to produce luminescence, and certain strains of P. damselae are also reported to be bioluminescent (Urbanczyk et al., 2011).
Table 1 General features of PDD strain KC-Na-1 and MIGS mandatory information. Items
Description
Classification
Domain Bacteria Phylum Proteobacteria Order Vibrionales Family Vibrionaceae Genus Photobacterium Species damselae Subspecies damselae Strain: KC-Na-1
General features Gram stain Cell shape Motility Temperature Pigmentation MIGS data Investigation_type Project_name
Gram negative Curved rod Motile with polar flagella 4–42 °C Non-pigmented
Lat_lon Geo_loc_name Collection_date Env_biome Env_feature Env_material Num_replicons Extrachrom_elements Estimated_size Ref_biomaterial Source_mat_id biotic_relationship Host Rel_to_oxygen Isol_growth_condt Seq_meth Annot_source Finishing_strategy
Bacteria_archaea Genome sequence of P. damselae subsp. damselae KCNa-01 34.5 N, 128.4 E South Korea: South sea Jan-2017 Ocean [ENVO:00,000,015] Environmental material [ENVO:00,010,483] Body fluid [ENVO:02,000,019] 6 4 4,544,586 None KCTC 52975 Commensal (or Infectious) Finless porpoise (Neophocaena asiaeorientalis) Facultative anaerobic PMID: 21875966 Illumina Hiseq 2000, PacBio RSII sequencing GenBank Complete; 218 × coverage, 6 contigs
Genome assembly data Assembly method Assembly name Genome coverage Sequencing technology
HGAP HGAP algorithm ver. 3 218 × Illumina; PacBio
into the biodiversity of the genus Photobacterium and obtain useful information for the study of potential virulence factors and antibiotic resistance in PDD.
2. Data description The general features and MIxS mandatory information for PDD strain KC-Na-1 are summarized in Table 1. The bacterial strain was originally isolated from an anal swab of the rescued juvenile male finless porpoise (N. asiaeorientalis, voucher no. CRI007079) found bycaught from net fisheries in December 2016 along the South Sea (Republic of Korea). The non-luminescent, gram-negative, rod-shaped isolate was oxidase and catalase positive and showed weak β-hemolysis on 5% sheep blood agar (Hanil Komed, Republic of Korea) after 24 h of incubation at 37 °C. The 16S rRNA of the isolate (MF099892) showed a match of > 99% with other P. damselae strains in the GenBank database, and due to its growth ability at 37 °C (a temperature inhibitory for ssp. piscicida) and hemolysis on sheep blood agar (Rivas et al., 2013a,b), the isolate was classified in the subspecies damselae and finally designated as PDD strain KC-Na-1. Genomic DNA was isolated using a DNeasy blood and tissue kit (Qiagen Korea Ltd., South Korea) following the manufacturer's protocols. Sequencing of the strain KC-Na-1 was performed at Macrogen Inc. (South Korea) using the hybrid approach (Koren et al., 2012) with a PacBio RS II system (Pacific Biosciences, USA) by constructing a 20-kb 2
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(a)
Chr I 3,134,662 bp
pPDD-Na-1-1 105,771 bp
Chr II 1,105,401 bp
pPDD-Na-1-2 81,002 bp
pPDD-Na-1-4 45,429 bp
pPDD-Na-1-3 72,321 bp
(b) Chr I
Chr II PDD KC-Na-1 (This study, CP021151)
PDD KC-Na-1 (This study, CP021152)
Photobacterium profundum SS9 (CR354531)
Photobacterium profundum SS9 (CR354532)
Vibrio alginolyticus ATCC 17749 (CP006718)
Photobacterium gaetbulicola Gung47 (CP005974)
Vibrio parahaemolyticus ATCC 17802 (CP014046)
Vibrio alginolyticus ATCC 17749 (CP006719)
Vibrio harveyi ATCC 33843 (CP009467)
Vibrio parahaemolyticus ATCC 17802 (CP014047)
Vibrio vulnificus CMCP6 (AE016795)
Vibrio harveyi ATCC 33843 (CP009468)
Vibrio coralliilyticus 58 (CP016556)
Vibrio vulnificus CMCP6 (AE016796)
Vibrio tubiashii ATCC 19109 (CP009354)
Vibrio tubiashii ATCC 19109 (CP009355)
Vibrio cholerae NCTC5395 (CP013317)
Vibrio coralliilyticus 58 (CP016557)
Photobacterium gaetbulicola Gung47 (CP005973)
Vibrio cholerae NCTC5395 (CP013318)
Fig. 1. (a) Circular maps of the PDD strain KC-Na-1 genome. Marked characteristics are shown from outside to the center: CDS on forward strand, CDS on reverse strand, tRNA, rRNA, GC content, and GC skew. (b) Phylogenetic trees based on OrthoANI values calculated with available complete genomes of PDD, P. profundum, P. gaetbulicola, and other related Vibrio species. The results between two strains are given in the junction point of the diagonals departing from each strain, i.e., the OrthoANI value between PDD strain KC-Na-1 (CP021151) and P. profundum SS9 (CR354531) was 74.0%. (two-column fitting image).
(Rivas et al., 2013a,b, 2014) and hemolysin genes dly and hlyApl on plasmid pPHDD1 (Rivas et al., 2011); these genes play major roles in virulence and cell toxicity. Recently, phospholipase (plpV) and collagenase (colP) have been shown to act as ubiquitous chromosomal virulence gene markers in PDD (Vences et al., 2017). Although the PDD strain KC-Na-1 showed weak β-hemolysis on sheep blood agar, the three hemolysin genes (hlyAch, hlyApl, and dly) were not detected, and a total of five other putative hemolysin genes were found to be encoded on Chr I (n = 4) and Chr II (n = 1) in the sequenced genome. Among the two recently identified virulence-related genes, only the plpV gene was encoded on Chr I, and the colP gene was not detected. Additionally, the presence of other potential virulence genes was identified by
Although strain KC-Na-1 was not luminous, the lux-rib operon, which was involved in bacterial light production (Urbanczyk et al., 2011), was screened in the genome. As expected, the lux operon was not found, whereas the rib operon ribEBHA, which was involved in the synthesis of riboflavin, was encoded on Chr I. The rib operon in strain KC-Na-1 was almost identical (> 99%) to that in P. damselae subsp. piscicida strain 91–197 (AP018045) and distinct (< 75%) from other Photobacterium species in the GenBank database. Additionally, the N utilization substance protein B (nusB) gene, which was downstream of the rib operon, also showed relatively high similarity (> 83%) with Photobacterium species compared with other Vibrio species. PDD is known to contain the hemolysin gene hlyAch on chromosome 3
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Table 2 General features of the PDD KC-Na-1 genome. Attribute
Size (bp) Coding regions (%) G + C content Total genes tRNA genes rRNA genes ncRNA genes Protein-coding genes Pseudogenes
Value Chromosome I
Chromosome II
Plasmid pPDD-Na-1-1
Plasmid pPDD-Na-1-2
Plasmid pPDD-Na-1-3
Plasmid pPDD-Na-1-4
3,134,662 86.2 41.6 2964 182 47 4 2641 90
1,105,401 84.9 39.3 999 11 – – 900 88
105,771 76.2 38.6 113 – – – 91 22
81,002 81.9 40.3 81 – – – 72 9
72,321 75.2 40.4 73 – – – 64 9
45,429 78.6 37.2 50 1 – – 46 3
Acknowledgements
searching the Virulence Factor DataBase (http://www.mgc.ac.cn/VFs/ ); consequently, several virulence-related genes involved in pathogenic Vibrio species were detected in the KC-Na-1 genome (Supplementary Table S1). Interestingly, two genes homologous to Vibrio thermostable direct hemolysin (tdh, > 80% amino acid identity) and its transcriptional regulator toxR (> 75% amino acid identity), which are major virulence factors associated with V. parahaemolyticus hemolysis and cytotoxicity, causing illnesses in humans and marine animals (Letchumanan et al., 2014), were found to be encoded on the conjugative plasmid pPDD-Na-1-3. Antimicrobial-resistance genes in strain KC-Na-1 were detected by searching in the ARG-ANNOT database (http://en.mediterranee-infection.com/article.php?laref=283&titre= arg-annot-), and the genome was found to possess tet 34 and tet 35 homologs, which have recently been described in Vibrio spp. (Miranda et al., 2003) (Supplementary Table S1). However, acceptable phenotypical tetracycline resistance in strain KC-Na-1 was not observed (tetracycline 30 μg, minimum inhibitory concentration value: 2 μg/ mL). PDD is pathogenic in a variety of marine animals and in humans (Rivas et al., 2013a, 2013b). In cetaceans, this bacterium has been isolated from stranded dolphins and whales (Fujioka et al., 1988; Buck et al., 1991; Casalone et al., 2014; Di Francesco et al., 2016). However, the pathogenic role of PDD in cetaceans is still unclear (Casalone et al., 2014), and we cannot determine the specific position of PDD (i.e., whether it is commensal or pathogenic to marine mammals, particularly in N. asiaeorientalis) due to the lack of reports describing its isolation. Nevertheless, PDD strain KC-Na-1 may have potential pathogenicity in humans and other marine animals due to the presences of plpV, tdh and toxR homologs in the genome, making it a potentially virulent strain. Further studies are currently in progress to investigate the pathogenic correlations between PDD and marine mammals. To the best of our knowledge, this is the first report of the isolation of PDD from the finless porpoise and the first report of the complete genome sequence of PDD. The genome sequence and genomic data presented in this study provide important insights into the biodiversity of the genus Photobacterium and give valuable information for improving control strategies for this marine pathogen.
This study was supported by grants from the KRIBB Initiative Program [KGM4691612] and the Global Frontier Program [2015M3A6B2063544] funded by the Ministry of Science, ICT & Future Planning, and a grant from the National Institute of Fisheries Science [R2017028] funded by the Ministry of Oceans and Fisheries in Republic of Korea. Conflicts of interest The authors declare that they have no conflict of interest. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.margen.2017.09.004. References Alba, P., Caprioli, A., Cocumelli, C., Ianzano, A., Donati, V., Scholl, F., Sorbara, L., Terracciano, G., Fichi, G., Di Nocera, F., Franco, A., Battisti, A., 2016. A new multilocus sequence typing scheme and its application for the characterization of Photobacterium damselae subsp. damselae associated with mortality in cetaceans. Front. Microbiol. 7. Buck, J.D., Overstrom, N.A., Patton, G.W., Anderson, H.F., Gorzelany, J.F., 1991. Bacteria associated with stranded cetaceans from the North-east USA and Southwest Florida Gulf coasts. Dis. Aquat. Org. 10, 147–152. Casalone, C., Mazzariol, S., Pautasso, A., Di Guardo, G., Di Nocera, F., Lucifora, G., Ligios, C., Franco, A., Fichi, G., Cocumelli, C., Cersini, A., Guercio, A., Puleio, R., Goria, M., Podestà, M., Marsili, L., Pavan, G., Pintore, A., De Carlo, E., Eleni, C., Caracappa, S., 2014. Cetacean strandings in Italy: an unusual mortality event along the Tyrrhenian Sea coast in 2013. Dis. Aquat. Org. 109, 81–86. Di Francesco, G., Cammà, C., Curini, V., Mazzariol, S., Proietto, U., Di Francesco, C.E., Ferri, N., Di Provvido, A., Di Guardo, G., 2016. Coinfection by Ureaplasma spp., Photobacterium damselae and an Actinomyces-like microorganism in a bottlenose dolphin (Tursiops truncatus) with pleuropneumonia stranded along the Adriatic coast of Italy. Res. Vet. Sci. 105, 111–114. Fujioka, R.S., Greco, S.B., Cates, M.B., Schroeder, J.P., 1988. Vibrio damsela from wounds in bottlenose dolphins Tursiops truncatus. Dis. Aquat. Org 4, 1–8. Koren, S., Schatz, M.C., Walenz, B.P., Martin, J., Howard, J.T., Ganapathy, G., Wang, Z., Rasko, D.A., McCombie, W.R., Jarvis, E.D., Phillippy, A.M., 2012. Hybrid error correction and de novo assembly of single-molecule sequencing reads. Nat. Biotechnol. 30, 693–700. Lagesen, K., Hallin, P., Rødland, E.A., Stærfeldt, H.-H., Rognes, T., Ussery, D.W., 2007. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 35, 3100–3108. Lee, I., Kim, O.Y., Park, S.C., Chun, J., 2016. OrthoANI: an improved algorithm and software for calculating average nucleotide identity. Int. J. Syst. Evol. Microbiol. 66, 1100–1103. Letchumanan, V., Chan, K.G., Lee, L.H., 2014. Vibrio parahaemolyticus: a review on the pathogenesis, prevalence, and advance molecular identification techniques. Front. Microbiol. 5, 1–13. Love, M., Teebken-Fisher, D., Hose, J.E., Farmer, J.J., Hickman III, F.W., Fanning, G.R., 1981. Vibrio damsela, a marine bacterium, causes skin ulcers on the damselfish Chromis punctipinnis. Science 214, 1139–1140. Lowe, T.M., Eddy, S.R., 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 25, 955–964. Miranda, C.D., Kehrenberg, C., Ulep, C., Schwarz, S., Roberts, M.C., 2003. Diversity of tetracycline resistance genes in bacteria from Chilean salmon farms. Antimicrob. Agents Chemother. 47, 883–888. Moi, I.M., Roslan, N.N., Leow, A.T.C., Ali, M.S.M., Rahman, R.N.Z., Rahimpour, A., Sabri, S., 2017. The biology and the importance of Photobacterium species. Appl. Microbiol.
3. Culture deposition and nucleotide sequence accession numbers PDD strain KC-Na-1 was deposited in the Korean Collection for Type Cultures (KCTC) under KCTC 52975. The partial 16S rRNA and complete genome sequences of PDD strain KC-Na-1 have been deposited in GenBank under accession numbers MF099892 (16S rRNA), CP021151 (chromosome I), CP021152 (chromosome II), CP021153 (pPDD-Na-11), CP021154 (pPDD-Na-1-2), CP021155 (pPDD-Na-1-3), and CP021156 (pPDD-Na-1-4).
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B.S., Kiryutin, B., Galperin, M.Y., Fedorova, N.D., Koonin, E.V., 2001. The COG database: new developments in phylogenetic classification of proteins from complete genomes. Nucleic Acids Res. 29, 22–28. Urbanczyk, H., Ast, J.C., Dunlap, P.V., 2011. Phylogeny, genomics, and symbiosis of Photobacterium. FEMS Microbiol. Rev. 35, 324–342. Van Bressem, M.F., Raga, J.A., Di Guardo, G., Jepson, P.D., Duignan, P.J., Siebert, U., Barrett, T., Santos, M.C.D., Moreno, I.B., Siciliano, S., Aguilar, A., Van Waerebeek, K., 2009. Emerging infectious diseases in cetaceans worldwide and the possible role of environmental stressors. Dis. Aquat. Org. 86, 143–157. Vences, A., Rivas, A.J., Lemos, M.L., Husmann, M., Osorio, C.R., 2017. Chromosomeencoded hemolysin, phospholipase and collagenase contribute to virulence for fish in plasmidless isolates of Photobacterium damselae subsp. damselae. Appl. Environ. Microbiol. 83, e00401–17.
Biotechnol. 101, 4371–4385. Rivas, A.J., Balado, M., Lemos, M.L., Osorio, C.R., 2011. The Photobacterium damselae subsp. damselae hemolysins damselysin and HlyA are encoded within a new virulence plasmid. Infect. Immun. 79, 4617–4627. Rivas, A.J., Balado, M., Lemos, M.L., Osorio, C.R., 2013a. Synergistic and additive effects of chromosomal and plasmid-encoded hemolysins contribute to hemolysis and virulence in Photobacterium damselae subsp. damselae. Infect. Immun. 81, 3287–3299. Rivas, A.J., Lemos, M.L., Osorio, C.R., 2013b. Photobacterium damselae subsp. damselae, a bacterium pathogenic for marine animals and humans. Front. Microbiol. 4, 283. Rivas, A.J., Labella, A., Borrego, J.J., Lemos, M.L., Osorio, C.R., 2014. Evidences for horizontal gene transfer, gene duplication and genetic variation as driving forces of the diversity of haemolytic phenotypes in Photobacterium damselae subsp. damselae. FEMS Microbiol. Lett. 355, 152–162. Tatusov, R.L., Natale, D.A., Garkavtsev, I.V., Tatusova, T.A., Shankavaram, U.T., Rao,
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Marine Genomics journal homepage: www.elsevier.com/locate/margen
Genomic insights into Photobacterium damselae subsp. damselae strain KCNa-1, isolated from the finless porpoise (Neophocaena asiaeorientalis) Kyunglee Leea,1, Hye Kwon Kimb,1, Hawsun Sohna, Yuna Choa, Young-Min Choia, Dae Gwin Jeongb,⁎, Ji Hyung Kimb,⁎ a b
Cetacean Research Institute (CRI), National Institute of Fisheries Science (NIFS), Ulsan 44780, Republic of Korea Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords: Photobacterium damselae ssp. damselae KC-Na-1 Cetaceans Pathogens
Photobacterium damselae subsp. damselae (PDD) is a marine bacterium that can infect a variety of marine animals and humans. Although this bacterium has been isolated from several stranded dolphins and whales, its pathogenic role in cetaceans is still unclear. In this study, we report the complete genome of PDD strain KC-Na-1 isolated from a finless porpoise (Neophocaena asiaeorientalis) rescued from the South Sea (Republic of Korea). The sequenced genome comprised two chromosomes and four plasmids. Among the recently identified major virulence factors in PDD, only phospholipase (plpV) was found in strain KC-Na-1. Interestingly, two genes homologous to Vibrio thermostable direct hemolysin (tdh) and its transcriptional regulator toxR, which are known virulence factors associated with Vibrio parahaemolyticus, were encoded on the plasmid pPDD-Na-1-3. Based on these results, strain KC-Na-1 may have potential pathogenicity in humans and other marine animals and also could act as a potential virulent strain. To the best of our knowledge, this is the first report of the complete genome sequence of P. damselae.
1. Introduction The genus Photobacterium comprises 23 valid species in Vibrionaceae (Proteobacteria: Gammaproteobacteria) and is ubiquitous in coastal, open-ocean, and deep-sea environments (Moi et al., 2017). Members of the genus are typically found in seawater and in association with marine animals as saprophytes and enteric commensals; several luminescent species form highly specific bacterial endosymbionts with fish and squid (Urbanczyk et al., 2011). However, two subspecies of P. damselae (ssp. damselae and ssp. piscicida) have been found to be associated with mortality in fish, and P. damselae ssp. damselae (hereinafter referred as PDD), which was originally described as Vibrio damsela (Love et al., 1981), is now considered a bacterial pathogen that can cause infections in a variety of marine ectotherms, including fish, sea turtles, mollusks, and crustaceans (Moi et al., 2017). Furthermore, PDD has been isolated as the causative agent of human infections, including some fatal cases (Rivas et al., 2013a, 2013b). Several bacterial species, including Brucella spp., Mycobacterium spp., and Erysipelothrix rhusiopathiae, were recently recognized as
causative agents of emerging infectious diseases in cetaceans (Van Bressem et al., 2009). PDD has also been isolated from stranded dolphins and whales (Fujioka et al., 1988; Buck et al., 1991; Casalone et al., 2014; Di Francesco et al., 2016); however, the pathogenic role of PDD in cetaceans still remains unclear due to the limitations of postmortem analyses of stranded individuals (Casalone et al., 2014). Moreover, no studies of porpoises have described PDD isolation or detection, so far. Although the mechanisms of infection and virulence in PDD have not been thoroughly investigated in marine animals (particularly cetaceans and chelonians), a recent genotyping approach revealed that a clonal PDD group might be related to the mortality events among cetaceans (Alba et al., 2016). Since 2016, we have investigated the bacterial diversity in cetacean species present in coastal waters in the Republic of Korea in order to identify the potential pathogens that can colonize and establish infection in marine mammals for conservation. In this study, we present the complete genome of PDD strain KC-Na-1, which was isolated from a finless porpoise (Neophocaena asiaeorientalis) found bycaught in 2016 along the South Sea (Republic of Korea). We aimed to provide insights
Abbreviations: PDD, Photobacterium damselae subsp. damselae; COG, Clusters of Orthologous Groups; plpV, phospholipase; colP, collagenase; nusB, N utilization substance protein B; tdh, thermostable direct hemolysin ⁎ Corresponding authors. E-mail addresses: [email protected] (D.G. Jeong), [email protected] (J.H. Kim). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.margen.2017.09.004 Received 1 August 2017; Received in revised form 12 September 2017; Accepted 13 September 2017 1874-7787/ © 2017 Published by Elsevier B.V.
Please cite this article as: Lee, K., Marine Genomics (2017), http://dx.doi.org/10.1016/j.margen.2017.09.004
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SMRTbell template library and with paired-end Illumina short read data using a HiSeq 2000 instrument (Illumina, USA). The PacBio long read data (1,464,887,777 bp, 167,668 reads) were de novo assembled by the Hierarchical Genome Assembly Process program (ver. 3.0), and the Illumina pair end reads (1,532,556,320 bp, 15,183,058 reads) were mapped to the assembled contigs to improve the accuracy of the genome sequences. Genome annotation was carried out using the NCBI's Prokaryotic Genome Annotation Pipeline (http://www.ncbi. nlm.nih.gov/books/NBK174280/). Bacterial tRNAs and rRNAs were analyzed using tRNAscan-SE 1.21 (Lowe and Eddy, 1997) and RNAmmer 1.2 (Lagesen et al., 2007), respectively. Functional categories of open reading frames were analyzed by BLASTP search against Clusters of Orthologous Groups (COG) database (Tatusov et al., 2001) with an E-value cutoff of 1E-4 and an identity cutoff of 20%. The fully assembled and closed PDD strain KC-Na-1 genome contained 4,544,586 bp consisting of two chromosomes, designated Chr I (3,134,662 bp) and Chr II (1,105,401 bp), and a total of four plasmids, designated pPDD-Na-1-1 (105,771 bp), pPDD-Na-1-2 (81,002 bp), pPDD-Na-1-3 (72,321 bp), and pPDD-Na-1-4 (45,429 bp), as shown in Fig. 1a. The two chromosomes showed similar G + C contents (41.6% and 39.3%) and percentages of coding regions (86.2% and 84.9%). Moreover, most of the predicted tRNAs (n = 182), rRNA (n = 47), and ncRNA (n = 4) genes were encoded on Chr I, except 11 and one tRNA genes on Chr II and pPDD-Na-1-4, respectively (Table 2). The COG functional category analysis of PDD strain KC-Na-1 revealed that Chr I had higher percentages of genes related to basic cellular functions than Chr II (Supplementary Fig. 1a). Functional genes encoded on Chr I were mainly involved in COG categories of J (translation, ribosomal structure, and biogenesis), L (replication, recombination, and repair), D (cell cycle control, cell division, and chromosome partitioning), T (signal transduction mechanisms), M (cell wall/membrane/envelope biogenesis), N (cell motility), U (intracellular trafficking, secretion, and vesicular transport), O (post-translational modification, protein turnover, chaperones), C (energy production and conversion), E (amino acid transport and metabolism), F (nucleotide transport and metabolism), H (coenzyme transport and metabolism), and I (lipid transport and metabolism). In contrast, Chr II possessed higher percentages of genes involved in K (transcription), V (defense), X (mobilome: prophages, transposons), G (carbohydrate transport and metabolism), P (inorganic ion transport and metabolism), and Q (secondary metabolites biosynthesis, transport, and catabolism). However, both chromosomes contained genes involved in S (function unknown in COG database), and 6.8% and 15.1% of the predicted genes on Chr I and Chr II, respectively, failed to find a match in the database. As expected, most of the functional genes encoded on plasmids did not have matches in the COG database, and the remaining genes were mainly involved in K, L, U, and X (Supplementary Fig. 1b). Currently, the complete genome of P. damselae is not available in the GenBank database, except that for strain KC-Na-1. Therefore, the OrthoANI algorithm (Lee et al., 2016) was used to assess overall genome similarity between P. damselae and other related strains in the family Vibrionaceae. OrthoANI values were obtained, and a phylogenetic tree was constructed based on OrthoANI analysis of the three species of Photobacterium (P. damselae, P. profundum, and P. gaetbulicola) and the other seven related Vibrio species using the orthologous average nucleotide identity tool. The resulting phylogenetic trees based on OrthoANI values for strain KC-Na-1 and other related strains indicated that strain KC-Na-1 was most related to P. profundum and that Vibrio species were close relatives of the bacterium. However, the other species in the genus Photobacterium, i.e., P. gaetbulicola strain Group47, showed the lowest ANI value, thus suggesting that its taxonomical position in Vibrionaceae needs to be re-examined (Fig. 1b). Members of Photobacterium species, including P. angustum, P. aquimaris, P. kishitanii, P. leiognathi, P. mandapamensis, and P. phosphoreum, are known to produce luminescence, and certain strains of P. damselae are also reported to be bioluminescent (Urbanczyk et al., 2011).
Table 1 General features of PDD strain KC-Na-1 and MIGS mandatory information. Items
Description
Classification
Domain Bacteria Phylum Proteobacteria Order Vibrionales Family Vibrionaceae Genus Photobacterium Species damselae Subspecies damselae Strain: KC-Na-1
General features Gram stain Cell shape Motility Temperature Pigmentation MIGS data Investigation_type Project_name
Gram negative Curved rod Motile with polar flagella 4–42 °C Non-pigmented
Lat_lon Geo_loc_name Collection_date Env_biome Env_feature Env_material Num_replicons Extrachrom_elements Estimated_size Ref_biomaterial Source_mat_id biotic_relationship Host Rel_to_oxygen Isol_growth_condt Seq_meth Annot_source Finishing_strategy
Bacteria_archaea Genome sequence of P. damselae subsp. damselae KCNa-01 34.5 N, 128.4 E South Korea: South sea Jan-2017 Ocean [ENVO:00,000,015] Environmental material [ENVO:00,010,483] Body fluid [ENVO:02,000,019] 6 4 4,544,586 None KCTC 52975 Commensal (or Infectious) Finless porpoise (Neophocaena asiaeorientalis) Facultative anaerobic PMID: 21875966 Illumina Hiseq 2000, PacBio RSII sequencing GenBank Complete; 218 × coverage, 6 contigs
Genome assembly data Assembly method Assembly name Genome coverage Sequencing technology
HGAP HGAP algorithm ver. 3 218 × Illumina; PacBio
into the biodiversity of the genus Photobacterium and obtain useful information for the study of potential virulence factors and antibiotic resistance in PDD.
2. Data description The general features and MIxS mandatory information for PDD strain KC-Na-1 are summarized in Table 1. The bacterial strain was originally isolated from an anal swab of the rescued juvenile male finless porpoise (N. asiaeorientalis, voucher no. CRI007079) found bycaught from net fisheries in December 2016 along the South Sea (Republic of Korea). The non-luminescent, gram-negative, rod-shaped isolate was oxidase and catalase positive and showed weak β-hemolysis on 5% sheep blood agar (Hanil Komed, Republic of Korea) after 24 h of incubation at 37 °C. The 16S rRNA of the isolate (MF099892) showed a match of > 99% with other P. damselae strains in the GenBank database, and due to its growth ability at 37 °C (a temperature inhibitory for ssp. piscicida) and hemolysis on sheep blood agar (Rivas et al., 2013a,b), the isolate was classified in the subspecies damselae and finally designated as PDD strain KC-Na-1. Genomic DNA was isolated using a DNeasy blood and tissue kit (Qiagen Korea Ltd., South Korea) following the manufacturer's protocols. Sequencing of the strain KC-Na-1 was performed at Macrogen Inc. (South Korea) using the hybrid approach (Koren et al., 2012) with a PacBio RS II system (Pacific Biosciences, USA) by constructing a 20-kb 2
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(a)
Chr I 3,134,662 bp
pPDD-Na-1-1 105,771 bp
Chr II 1,105,401 bp
pPDD-Na-1-2 81,002 bp
pPDD-Na-1-4 45,429 bp
pPDD-Na-1-3 72,321 bp
(b) Chr I
Chr II PDD KC-Na-1 (This study, CP021151)
PDD KC-Na-1 (This study, CP021152)
Photobacterium profundum SS9 (CR354531)
Photobacterium profundum SS9 (CR354532)
Vibrio alginolyticus ATCC 17749 (CP006718)
Photobacterium gaetbulicola Gung47 (CP005974)
Vibrio parahaemolyticus ATCC 17802 (CP014046)
Vibrio alginolyticus ATCC 17749 (CP006719)
Vibrio harveyi ATCC 33843 (CP009467)
Vibrio parahaemolyticus ATCC 17802 (CP014047)
Vibrio vulnificus CMCP6 (AE016795)
Vibrio harveyi ATCC 33843 (CP009468)
Vibrio coralliilyticus 58 (CP016556)
Vibrio vulnificus CMCP6 (AE016796)
Vibrio tubiashii ATCC 19109 (CP009354)
Vibrio tubiashii ATCC 19109 (CP009355)
Vibrio cholerae NCTC5395 (CP013317)
Vibrio coralliilyticus 58 (CP016557)
Photobacterium gaetbulicola Gung47 (CP005973)
Vibrio cholerae NCTC5395 (CP013318)
Fig. 1. (a) Circular maps of the PDD strain KC-Na-1 genome. Marked characteristics are shown from outside to the center: CDS on forward strand, CDS on reverse strand, tRNA, rRNA, GC content, and GC skew. (b) Phylogenetic trees based on OrthoANI values calculated with available complete genomes of PDD, P. profundum, P. gaetbulicola, and other related Vibrio species. The results between two strains are given in the junction point of the diagonals departing from each strain, i.e., the OrthoANI value between PDD strain KC-Na-1 (CP021151) and P. profundum SS9 (CR354531) was 74.0%. (two-column fitting image).
(Rivas et al., 2013a,b, 2014) and hemolysin genes dly and hlyApl on plasmid pPHDD1 (Rivas et al., 2011); these genes play major roles in virulence and cell toxicity. Recently, phospholipase (plpV) and collagenase (colP) have been shown to act as ubiquitous chromosomal virulence gene markers in PDD (Vences et al., 2017). Although the PDD strain KC-Na-1 showed weak β-hemolysis on sheep blood agar, the three hemolysin genes (hlyAch, hlyApl, and dly) were not detected, and a total of five other putative hemolysin genes were found to be encoded on Chr I (n = 4) and Chr II (n = 1) in the sequenced genome. Among the two recently identified virulence-related genes, only the plpV gene was encoded on Chr I, and the colP gene was not detected. Additionally, the presence of other potential virulence genes was identified by
Although strain KC-Na-1 was not luminous, the lux-rib operon, which was involved in bacterial light production (Urbanczyk et al., 2011), was screened in the genome. As expected, the lux operon was not found, whereas the rib operon ribEBHA, which was involved in the synthesis of riboflavin, was encoded on Chr I. The rib operon in strain KC-Na-1 was almost identical (> 99%) to that in P. damselae subsp. piscicida strain 91–197 (AP018045) and distinct (< 75%) from other Photobacterium species in the GenBank database. Additionally, the N utilization substance protein B (nusB) gene, which was downstream of the rib operon, also showed relatively high similarity (> 83%) with Photobacterium species compared with other Vibrio species. PDD is known to contain the hemolysin gene hlyAch on chromosome 3
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Table 2 General features of the PDD KC-Na-1 genome. Attribute
Size (bp) Coding regions (%) G + C content Total genes tRNA genes rRNA genes ncRNA genes Protein-coding genes Pseudogenes
Value Chromosome I
Chromosome II
Plasmid pPDD-Na-1-1
Plasmid pPDD-Na-1-2
Plasmid pPDD-Na-1-3
Plasmid pPDD-Na-1-4
3,134,662 86.2 41.6 2964 182 47 4 2641 90
1,105,401 84.9 39.3 999 11 – – 900 88
105,771 76.2 38.6 113 – – – 91 22
81,002 81.9 40.3 81 – – – 72 9
72,321 75.2 40.4 73 – – – 64 9
45,429 78.6 37.2 50 1 – – 46 3
Acknowledgements
searching the Virulence Factor DataBase (http://www.mgc.ac.cn/VFs/ ); consequently, several virulence-related genes involved in pathogenic Vibrio species were detected in the KC-Na-1 genome (Supplementary Table S1). Interestingly, two genes homologous to Vibrio thermostable direct hemolysin (tdh, > 80% amino acid identity) and its transcriptional regulator toxR (> 75% amino acid identity), which are major virulence factors associated with V. parahaemolyticus hemolysis and cytotoxicity, causing illnesses in humans and marine animals (Letchumanan et al., 2014), were found to be encoded on the conjugative plasmid pPDD-Na-1-3. Antimicrobial-resistance genes in strain KC-Na-1 were detected by searching in the ARG-ANNOT database (http://en.mediterranee-infection.com/article.php?laref=283&titre= arg-annot-), and the genome was found to possess tet 34 and tet 35 homologs, which have recently been described in Vibrio spp. (Miranda et al., 2003) (Supplementary Table S1). However, acceptable phenotypical tetracycline resistance in strain KC-Na-1 was not observed (tetracycline 30 μg, minimum inhibitory concentration value: 2 μg/ mL). PDD is pathogenic in a variety of marine animals and in humans (Rivas et al., 2013a, 2013b). In cetaceans, this bacterium has been isolated from stranded dolphins and whales (Fujioka et al., 1988; Buck et al., 1991; Casalone et al., 2014; Di Francesco et al., 2016). However, the pathogenic role of PDD in cetaceans is still unclear (Casalone et al., 2014), and we cannot determine the specific position of PDD (i.e., whether it is commensal or pathogenic to marine mammals, particularly in N. asiaeorientalis) due to the lack of reports describing its isolation. Nevertheless, PDD strain KC-Na-1 may have potential pathogenicity in humans and other marine animals due to the presences of plpV, tdh and toxR homologs in the genome, making it a potentially virulent strain. Further studies are currently in progress to investigate the pathogenic correlations between PDD and marine mammals. To the best of our knowledge, this is the first report of the isolation of PDD from the finless porpoise and the first report of the complete genome sequence of PDD. The genome sequence and genomic data presented in this study provide important insights into the biodiversity of the genus Photobacterium and give valuable information for improving control strategies for this marine pathogen.
This study was supported by grants from the KRIBB Initiative Program [KGM4691612] and the Global Frontier Program [2015M3A6B2063544] funded by the Ministry of Science, ICT & Future Planning, and a grant from the National Institute of Fisheries Science [R2017028] funded by the Ministry of Oceans and Fisheries in Republic of Korea. Conflicts of interest The authors declare that they have no conflict of interest. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.margen.2017.09.004. References Alba, P., Caprioli, A., Cocumelli, C., Ianzano, A., Donati, V., Scholl, F., Sorbara, L., Terracciano, G., Fichi, G., Di Nocera, F., Franco, A., Battisti, A., 2016. A new multilocus sequence typing scheme and its application for the characterization of Photobacterium damselae subsp. damselae associated with mortality in cetaceans. Front. Microbiol. 7. Buck, J.D., Overstrom, N.A., Patton, G.W., Anderson, H.F., Gorzelany, J.F., 1991. Bacteria associated with stranded cetaceans from the North-east USA and Southwest Florida Gulf coasts. Dis. Aquat. Org. 10, 147–152. Casalone, C., Mazzariol, S., Pautasso, A., Di Guardo, G., Di Nocera, F., Lucifora, G., Ligios, C., Franco, A., Fichi, G., Cocumelli, C., Cersini, A., Guercio, A., Puleio, R., Goria, M., Podestà, M., Marsili, L., Pavan, G., Pintore, A., De Carlo, E., Eleni, C., Caracappa, S., 2014. Cetacean strandings in Italy: an unusual mortality event along the Tyrrhenian Sea coast in 2013. Dis. Aquat. Org. 109, 81–86. Di Francesco, G., Cammà, C., Curini, V., Mazzariol, S., Proietto, U., Di Francesco, C.E., Ferri, N., Di Provvido, A., Di Guardo, G., 2016. Coinfection by Ureaplasma spp., Photobacterium damselae and an Actinomyces-like microorganism in a bottlenose dolphin (Tursiops truncatus) with pleuropneumonia stranded along the Adriatic coast of Italy. Res. Vet. Sci. 105, 111–114. Fujioka, R.S., Greco, S.B., Cates, M.B., Schroeder, J.P., 1988. Vibrio damsela from wounds in bottlenose dolphins Tursiops truncatus. Dis. Aquat. Org 4, 1–8. Koren, S., Schatz, M.C., Walenz, B.P., Martin, J., Howard, J.T., Ganapathy, G., Wang, Z., Rasko, D.A., McCombie, W.R., Jarvis, E.D., Phillippy, A.M., 2012. Hybrid error correction and de novo assembly of single-molecule sequencing reads. Nat. Biotechnol. 30, 693–700. Lagesen, K., Hallin, P., Rødland, E.A., Stærfeldt, H.-H., Rognes, T., Ussery, D.W., 2007. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 35, 3100–3108. Lee, I., Kim, O.Y., Park, S.C., Chun, J., 2016. OrthoANI: an improved algorithm and software for calculating average nucleotide identity. Int. J. Syst. Evol. Microbiol. 66, 1100–1103. Letchumanan, V., Chan, K.G., Lee, L.H., 2014. Vibrio parahaemolyticus: a review on the pathogenesis, prevalence, and advance molecular identification techniques. Front. Microbiol. 5, 1–13. Love, M., Teebken-Fisher, D., Hose, J.E., Farmer, J.J., Hickman III, F.W., Fanning, G.R., 1981. Vibrio damsela, a marine bacterium, causes skin ulcers on the damselfish Chromis punctipinnis. Science 214, 1139–1140. Lowe, T.M., Eddy, S.R., 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 25, 955–964. Miranda, C.D., Kehrenberg, C., Ulep, C., Schwarz, S., Roberts, M.C., 2003. Diversity of tetracycline resistance genes in bacteria from Chilean salmon farms. Antimicrob. Agents Chemother. 47, 883–888. Moi, I.M., Roslan, N.N., Leow, A.T.C., Ali, M.S.M., Rahman, R.N.Z., Rahimpour, A., Sabri, S., 2017. The biology and the importance of Photobacterium species. Appl. Microbiol.
3. Culture deposition and nucleotide sequence accession numbers PDD strain KC-Na-1 was deposited in the Korean Collection for Type Cultures (KCTC) under KCTC 52975. The partial 16S rRNA and complete genome sequences of PDD strain KC-Na-1 have been deposited in GenBank under accession numbers MF099892 (16S rRNA), CP021151 (chromosome I), CP021152 (chromosome II), CP021153 (pPDD-Na-11), CP021154 (pPDD-Na-1-2), CP021155 (pPDD-Na-1-3), and CP021156 (pPDD-Na-1-4).
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