Complete Genome Sequence of the Siphovirus Roseophage RDJLΦ 2 Infecting Roseobacter Denitriﬁcans Och114

Marine Genomics 25 (2016) 17–19

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Marine Genomics

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Genomics/technical resources Complete genome sequence of the siphovirus Roseophage RDJLΦ 2 infecting Roseobacter denitriﬁcans OCh114

Yantao Liang a,c,1, Yongyu Zhang a,b,⁎,1,ChaoZhoua, Zhenghao Chen b,d, Suping Yang d, Changzhou Yan b,⁎⁎, Nianzhi Jiao c a Research Center for Marine Biology and Carbon Sequestration, Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, 266101, China b Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China c State Key Laboratory of Marine Environmental Science, Institute of Marine Microbes and Ecospheres, Xiamen University (Xiang'an), Xiamen 361102, China d Department of Bioengineering and Biotechnology, Huaqiao University, Xiamen 361021, China article info abstract

Article history: RDJLΦ2, a lytic phage that infects the marine bacterium Roseobacter denitriﬁcans OCh114, one of the model Received 26 August 2015 organisms of the Roseobacter clade, was isolated. Here we report the overall genome architecture of RDJLΦ2. Received in revised form 26 October 2015 Morphological and genome analysis revealed that RDJLΦ2 is a siphovirus with a 63.5 kb genome that contains Accepted 26 October 2015 76 putative gene products. Available online 2 November 2015 © 2015 Elsevier B.V. All rights reserved.

Keywords: Roseobacter Roseobacter denitriﬁcans OCh114 Siphovirus Genome

1. Introduction of roseophages specifically infecting the ubiquitous Roseobacter clade is of great ecological significance. However, only a small number of Viruses are the most abundant and diverse biological entities in ma- roseophages (currently 16 roseophages, Table 1) have been described. rine environments, and are mainly bacteriophages (viruses that infect Most of them were in Podoviridae family, and only one roseophage in bacteria) (Suttle, 2005). Bacteriophages harbor a vast genetic diversity, Siphoviridae family (RDJL Φ1 which infects R. denitrificans OCh114) and play an important role in marine biogeochemical cycles and driving was reported (Table 1, Zhang and Jiao, 2009; Huang et al., 2011). Very the evolution of bacteria (Weinbauer, 2004; Suttle, 2005). Roseobacter interestingly, four GTA (gene transfer agent) homologous genes were clade is one of the most abundant and metabolically diverse groups of found in the phage genome of RDJL Φ1. GTA is a bacteriophage-like el- bacteria in the oceans, and typically comprises 10–20% of marine bacte- ement produced by several bacteria that mediates horizontal gene rial communities (DeLong and Karl, 2005; Wagner-Dobler and Biebl, transfer. In the past, it is very seldom reported that GTA-like genes occu- 2006). More than 30 genomes of representative roseobacters have py a large proportion of a marine phage genome. Remarkably, the been sequenced (Newton et al., 2010), and the genomics studies genome of roseophage RDJLΦ1 provides important evidence into the showed that members of this clade are versatile in their metabolism, evolution of GTA in alphaproteobacteria. Here we present the whole employing diverse catalytic processes in the biogeochemical cycles, es- genome sequence of another roseophage RDJLΦ2, the second lytic pecially in the marine carbon, nitrogen and sulfur cycles (Moran et al., siphovirus that infects the same host bacteria of R. denitrificans 2007; Gulvik and Buchan, 2013). Roseobacter denitrificans OCh114 is OCh114 (Table 2). one of the model organisms of the Roseobacter clade and has been completely sequenced (Swingley et al., 2007). Studying the processes 2. Data description

⁎ Correspondence to: Y. Zhang, Research Center for Marine Biology and Carbon A summary of the genome project information was shown in Sequestration, Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute Table 2. Phage RDJLΦ2 was isolated from the surface coastal seawater of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, of Xiamen, China (118°10′ E, 24°31′ N) using standard virus enrichment China. and double-layer agar methods on May 2011. After being concentrated ⁎⁎ Corresponding author. ﬁ E-mail addresses: [email protected] (Y. Zhang), [email protected] (C. Yan). and puri ed by polyethylene glycol 8000, CsCl gradient centrifugation 1 These authors equally contributed to this work. and phenol-chloroform extraction, the genomic DNA of RDJLΦ2was

Table 1 Roseophages for which genome sequences are available.

Sample Genome size Accession Phage Host Isolation location Family/Order References collection time (kb) number

The pier at Scripps Institution of SIO1 Roseobacter SIO67 03-11-1989 Podoviridae 39.9 AF189021 Rohwer et al. (2000) Oceanography, USA The pier at Scripps Institution of SIO1 Roseobacter SIO67 2001 Podoviridae 39.4 FJ867910 Angly et al. (2009) Oceanography, USA SIO1 Roseobacter SIO67 Solana Beach, California, USA 2001 Podoviridae 38.1 FJ867912 Angly et al. (2009) SIO1 Roseobacter SIO67 Oceanside, California, USA 2001 Podoviridae 38.2 FJ867913 Angly et al. (2009) SIO1 Roseobacter SIO67 Mission Bay, California, USA 2001 Podoviridae 38.2 FJ867914 Angly et al. (2009) DSS3Φ2 Ruegeria pomeroyi DSS3 Baltimore Inner Harbor water, USA 24-01-2007 Podoviridae 74.6 FJ591093 Zhao et al. (2009) ESS36Φ1 Sulfitobacter sp. EE36 Baltimore Inner Harbor water, USA 24-01-2007 Podoviridae 73.3 FJ591094 Zhao et al. (2009) P12053L Celeribacter sp. strain IMCC12053 The coast of the Yellow Sea, South Korea 16-03-2010 Caudovirales 35.9 JQ809650 Kang et al. (2012) ΦCB2047-B Sulfitobacter sp. strain 2047 Mesocosm study, Raunefjorden, Norway 06-2008 Podoviridae 74.5 HQ317387 Ankrah et al. (2014a) ΦCB2047-A Sulfitobacter sp. strain 2047 Mesocosm study, Raunefjorden, Norway 06-2008 Podoviridae 40.9 HQ332142 Ankrah et al. (2014b) ΦCB2047-C Sulfitobacter sp. strain 2047 Mesocosm study, Raunefjorden, Norway 06-2008 Podoviridae 40.9 HQ317384 Ankrah et al. (2014b) RLP1 Roseovarius sp. 217 Langstone Harbor, Hampshire, UK 17-09-2005 Podoviridae 74.6 FR682616 Chan et al. (2014) RPP1 Roseovarius nubinhibens L4 sampling station, Plymouth, UK 24-11-1998 Podoviridae 74.7 FR719956 Chan et al. (2014) R1 Dinoroseobacter shibae DFL12T Baicheng Harbor, Xiamen, China 22-05-2012 Podoviridae 75.0 KJ621082 Ji et al. (2015) R2C Dinoroseobacter shibae DFL12T Huangcuo station, Xiamen, China 16-10-2012 Podoviridae 74.8 KJ803031 Cai et al. (2015) RDJLΦ1 Roseobacter denitrificans OCh114 South China Sea surface seawater 09-2007 Siphoviridae 62.7 HM151342 Huang et al. (2011) RDJLΦ2 Roseobacter denitrificans OCh114 Wuyuan Bay, Xiamen, China 2010 Siphoviridae 63.5 KT266805 This study

sequenced on Roche 454 Genome Sequencer FLX+ platform using GS flexible, non-contractile tail (ca. 175 nm long and ca. 9 nm wide), and DNA Library Preparation kit combined with GS emPCR kit (Roche Ap- belongs to Siphoviridae family, Caudovirales order. The phage RDJLΦ2 plied Science, USA) to generate ~500 base long reads. The sequences encapsulated a linear dsDNA genome of 63,513 bp with 57.3% G + C were assembled using Newbler software (242× coverage). GeneMarkS content, a total of 76 ORFs and no tRNA sequences (using the online server and ORF Finder were used to identify putative open read- tRNAscan-SE program, Lowe and Eddy, 1997). Of the predicted ORFs, ing frames (ORFs). Gene annotation was achieved by the algorithms of 27 ORFs were sorted into known functional categories. The majority of BLAST search (NCBI) against the nonredundancy (nr) nucleotide data- genes to which a function could be assigned (14 of 27) were found to base with E-value ≤ 0.001. encode proteins related to DNA metabolism, replication and transcrip- Morphological analysis of the purified phage negatively stained with tion, including DNA polymerase A domain protein, ribonucleotide re- 2% uranyl acetate by transmission electron microscopy showed that ductase, cof hydrolase, helicase, exonuclease, transcriptional regulator RDJLΦ2 had an isometric head (ca. 74 nm in diameter) and a long, CtrA, ATPase, integrase, endonuclease V, large subunit of terminase, dihydrofolate reductase, thymidylate synthase, deoxycytidylate deami- nase and ribonuclease III. Six ORFs were predicted to encode proteins in- volved in the structure and assembly of virions. Two ORFs acted as Table 2 signal transduction and cell metabolism genes were predicted. One General features and genome sequencing project information for roseophage RDJLΦ2 according to the MIxS recommendations (Yilmaz et al., 2011). ORF was predicted as CRISPER/Cas associated protein. Interestingly, four GTA homologous genes were again found in the second roseophage Property Term in Siphoviridae family (RDJLΦ2), suggesting it is likely not occasional for Investigation type Virus the existence of GTA-like genes in the roseophages infecting Project name Relationships between algae, bacteria R. denitrificans Och114. Four ORFs (orfs 70 to 73) of RDJLΦ2 are homol- and virus Φ Collection date 05.2011 ogous to ORFs (81 to 84) of RDJL 1, which are highly homologous to Geographic location (latitude and 24.5225833317 N, 118.1787498659E GTA-like (Gene transfer agent produced by Rhodobacter capsulatus) longitude) genes 12, 13, 14 and 15, respectively (Lang and Beatty, 2007; Huang Geographic location (depth) 0.5 m et al., 2011). RDJLΦ2 exhibited 88% genome sequence similarity with Geographic location (country and/or sea, China, Xiamen, Wuyuan Bay its closest relative RDJLΦ1(Huang et al., 2011). The genome comparison region) Environment Marine waters between these two phages indicated that RDJLΦ2 was different from Pathogenicity Bacteria RDJLΦ1 in three additional ORFs (orfs 22, 51, 55) and the loss of four- Biotic relationship Free-living teen genes of RDJLΦ1 (orfs 05, 06, 09, 24, 27, 31, 45, 53, 57, 59, 60, 64, Specific host Roseobacter denitrificans OCh114 fi 65, 66). Most of the 17 genes had unknown function, and one orf of Host speci city or range NA Φ Φ Trophic level Parasitism RDJL 2 (orf 55) and two orfs of RDJL 1 (orfs 24, 64) encodes protein Relationship to oxygen Aerobe related to N-acyl-L-homoserine lactone synthetase, phosphohydrolase Nucleic acid extraction PMID: 19139231 and nitrate reductase, respectively (Fig. 1). The other analogous genes Sequencing method Pyrosequencing shared 51–100% identities at amino acid level between the RDJLΦ1 Number of replicons 1 and RDJLΦ2. The genome sequence of RDJLΦ2 may provide more in- Assembly Newbler v. 2.7 Finishing strategy Complete sights and information for the genetic characteristics and biological Estimated size 63.5 KB functions of roseophage, especially for the Siphoviridae family of Biome ENVO:01000046 roseophage. However, the majority of (49 of 76) predicted ORFs of Feature ENVO:02000049 RDJLΦ2 genome were annotated as hypothetical proteins, indicating Material ENVO:00002042 fi Propagation Lytic the insuf cient information about the functional genes of phage ge- Genome Coverage 242× nomes in the database. Further isolation, analyses and characterization Sequencing Technology 454 of more roseophages, in particular those infecting the ecologically NCBI project ID KT266805 important host bacteria of Roseobacter clade, are required to clarify the CGMCC No. CGMCC 7.210 genetic characteristic of this phage group. Y. Liang et al. / Marine Genomics 25 (2016) 17–19 19

Fig. 1. Genome maps of Roseophages RDJLΦ1andRDJLΦ2. ORFs are depicted by leftward or rightward oriented arrows according to the direction of transcription. Seventeen unique genes of RDJLΦ1 (orfs 05, 06, 09, 24, 27, 31, 45, 53, 57, 59, 60, 64, 65, 66) and RDJLΦ2 (orfs 22, 51, 55) are shaded in yellow and pointed out using arrows. Genes sharing amino acid similarity between these two phages are connected by shadowing: green indicates 68%–80%; white, 81%–97%; red, 98%–100%.

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