Properties and genomic analysis of Lactococcus garvieae lysogenic bacteriophage PLgT-1, a new member of Siphoviridae, with homology to Lactococcus lactis phages

Accepted Manuscript Title: Properties and genomic analysis of Lactococcus garvieae lysogenic bacteriophage PLgT-1, a new member of Siphoviridae, with homology to Lactococcus lactis phages Author: Truong Dinh Hoai Issei Nishiki Terutoyo Yoshida PII: DOI: Reference:

S0168-1702(16)30203-9 http://dx.doi.org/doi:10.1016/j.virusres.2016.05.021 VIRUS 96887

To appear in:

Virus Research

Received date: Revised date: Accepted date:

30-3-2016 20-5-2016 20-5-2016

Please cite this article as: Hoai, Truong Dinh, Nishiki, Issei, Yoshida, Terutoyo, Properties and genomic analysis of Lactococcus garvieae lysogenic bacteriophage PLgT-1, a new member of Siphoviridae, with homology to Lactococcus lactis phages.Virus Research http://dx.doi.org/10.1016/j.virusres.2016.05.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Properties and genomic analysis of Lactococcus garvieae lysogenic bacteriophage PLgT-1, a new member of Siphoviridae, with homology to Lactococcus lactis phages

Truong Dinh Hoaia,c, Issei Nishikib*, Terutoyo Yoshidaa a

Faculty of Agriculture, University of Miyazaki, Gakuen kibanadai nishi 1-1, Miyazaki 8892192, Japan b National Research Institute of Fisheries Science, Fisheries Research Agency, 2-12-4 Fukuura, Kanazawa, Yokohama 236-8648, Japan c Faculty of Fisheries, Vietnam National University of Agriculture, Hanoi, Vietnam

*Corresponding author Email: [email protected] Running title: Properties and genomic analysis of phage PLgT-1

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Highlights    

We characterized to genome level of a novel dsDNA L. garvieae phage, named PLgT-1. PLgT-1 is a new member of the family Siphoviridae, with homology to L. lactis phages. PLgT-1 could be released frequently during cell division. Integration of PLgT-1 via transduction has generated the diversification of L. garvieae.

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Abstract The lysogenic phage PLgT-1 is highly prevalent in Lactococcus garvieae, which is a serious bacterial pathogen in marine fish. Therefore, information regarding this phage is one of the key factors to predict the evolution of this bacterium. However, many properties of this phage, its complete genome sequence, and its relationship with other viral communities has not been investigated to date. Here, we demonstrated that the phage PLgT-1 was not only induced by an induction agent (Mitomycin C), but could be released frequently during cell division in a nutrient-rich environment or in natural seawater. Integration of PLgT-1 into non-lysogenic bacteria via transduction changed the genotype, resulting in the diversification of L. garvieae. The complete DNA sequence of PLgT-1 was also determined. This phage has a dsDNA genome of 40,273 bp with 66 open reading frames (ORFs). Of these, the biological functions of 24 ORFs could be predicted but those of 42 ORFs are unknown. Thus, PLgT-1 is a novel phage with several novel proteins encoded in its genome. The strict MegaBLAST search program for the PLgT-1 genome revealed that this phage had no similarities with other previously investigated phages specific to L. garvieae (WP-2 and GE1). Notably, PLgT-1 was relatively homologous with several phages of L. lactis and 17 of the 24 predicted proteins encoded in PLgT-1 were homologous with the deduced proteins of various phages from these dairy bacteria. Comparative genome analysis revealed that the L. garvieae phage PLgT-1 was most closely related to the L. lactis phage TP712. However, they differed from each other in genome size and gene arrangement. The results obtained in this study suggest that the lysogenic phage PLgT-1 is a new member of the family Siphoviridae and has been involved in horizontal gene exchange with microbial communities, especially with L. lactis and its phages. Keywords: properties, genomic analysis, phage PLgT-1, Lactococcus garvieae, marine fish.

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1. Introduction Bacteriophages (commonly called phages) are bacterial viruses and are considered to be the most abundant forms of life on earth; up to 70% of marine bacteria may be infected by phages (Prescott et al., 1993; Wommack & Colwell, 2000). Phages interact with bacteria through lytic and lysogenic cycles, and some phages are capable of carrying out both. New phage variants have been emerging and the number of multidrug resistant bacteria has been increasing over time. Scientists have been positively investigating lytic phages to develop alternative treatments for bacterial infection because of their destructive effect on host organisms (Nobrega et al., 2015). Although lysogenic phages, which can integrate into their bacterial host genomes, play little role in phage therapy, they are highly useful tools for investigating bacterial biology and for developing vectors for recombinant DNA technology for manipulating their bacterial hosts (Murray, 2006). These viruses also play a vital role in introducing novel important genes, such as virulence genes encoding drug resistance proteins and toxins into their bacterial hosts through horizontal gene transfer among bacterial communities via the transduction process (Canchaya et al., 2003). Therefore, they additionally play a vital role in bacterial evolution.

Lactococcus garvieae is a widespread and economically important pathogen in aquatic animals worldwide and is considered an emerging zoonotic pathogen in agriculture, wild life, and humans (Devriese et al., 1999; Vendrell et al., 2006; Wang et al., 2007). Several phages specific to fish-infecting L. garvieae have been documented. Of these, lytic phages are dominant (Eraclio et al., 2015; Ghasemi et al., 2014; Nakai et al., 1999; Park et al., 1997) and only one lysogenic phage has been isolated (Hoai & Yoshida, 2015). These lytic phages were isolated to be mainly used for phage therapy and bacterial genotyping. The properties and complete

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genomes of both lytic and lysogenic phages specific to these bacteria, which could also assist in bacterial evolution, were limited in availability. To our knowledge, only two virulent phages of L. garvieae have been investigated at the genomic level, including the phage WP-2, which was isolated from a rainbow trout farm water sample (Podoviridae family, 18,899 bp), and the phage GE1, which was isolated from a compost sample (Siphoviridae family, 24,847 bp) (Eraclio et al., 2015; Ghasemi et al., 2014). Thus, recent studies provide some insights into the genetic relationships between L. garvieae and its phages.

Recently, the first lysogenic phage of L. garvieae was isolated from a diseased marine fish species and was demonstrated to be widely distributed within this bacterial community. This phage belonged to the family Siphoviridae containing an isometric head and a non-contractile tail, and was estimated to integrate into the genomes of approximately 75% strains of L. garvieae that infected marine fish in Japan (Hoai & Yoshida, 2015). Here, we describe the properties of PLgT-1, such as induction capacity, and evaluate the role of the PLgT-1 prophage in generating genotypic diversity in L. garvieae via transduction. We also extensively analyzed the entire DNA sequence of the PLgT-1 genome in order to understand the evolutionary characteristics of this phage and to gain an insight into the ecological and evolutionary relationships between this phage and microbial communities via horizontal gene exchange.

2. Materials and Methods

2.1. Bacterial strains and culture conditions The chromosomal DNA samples of 427 L. garvieae strains isolated from several marine fish species between 1974 and 2012 were digested with SmaI, and the fragments were separated 5

using biased sinusoidal field gel electrophoresis (BSFGE) and classified into bacterial genotypes as described in our previous study (Nishiki et al., 2011). Of those, 12/16 genotypes were determined as lysogenic genotypes (Hoai & Yoshida, 2015). In this study, representative L. garvieae strains with several genotypes were selected and used in this study for different targets (Table 1). All the strains were preserved at -80°C. A liquid culture medium and a solid medium (Bacto Todd Hewitt Broth [THB] and THB with agar [THA], respectively; Difco, Sparks, MD, USA) were used to propagate each strain at 25°C under aerobic conditions.

2.2. Induction assay and phage production

Mitomycin C (MMC) was demonstrated as power agent and has been used frequently for for phage induction. However, other factors could stimulate phage production in bacterial population such as nutrient (Williamson & Paul, 2004) or calcium supplementation which was demonstrated to affect to phage yield because it is not only involved in adsorption, penetration and propagation of phage (Lu et al., 2003), but it is also necessary for completion of some intracellular stage of prophage development such as phage transcription, replication and bacterial cell lysis (Landry & Zsigray, 1980). In this study, the lysogenic bacterial strain Lg2 (S1 genotype) was chosen to evaluate the inducible abilities of several induction agents. A 10 mL culture of strain Lg2 was grown in THB at 25°C until the exponential phase (OD600 = 0.8). The culture was then centrifuged at 4500 x g for 20 min using a high-speed refrigerated microcentrifuge (Kitman-T24; Tomy, Tokyo, Japan). The bacterial pellet was washed several times and was then resuspended in 10 mL of sterile saline solution (0.9 % NaCl). To evaluate the phage induction capacity, 100 µL of the resuspended solution was added into each of 10 mL of

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fresh THB, THB containing 500 ng mL-1of freshly-prepared MMC (Wako, Osaka, Japan), THB supplemented with CaCl2 (0.01 M), distilled water (DW) supplemented with CaCl2 (0.01 M), and natural seawater. The resuspended solution was directly used as the control treatment. All cultures were incubated at 25°C for 7 h. To obtain a phage-induced solution and to ensure the accuracy of phage production, filtration was not performed. Alternatively, 100 µL of chloroform was added to the incubated cultures followed by centrifugation at 4000 x g for 20 min at 4°C (Allegra X-30R, Beckman, California, USA), and the supernatant was collected carefully. This step was repeated twice to ensure that no bacterial debris remained in the induced phage solutions. The bacterial strain EH6704 was chosen as an indicator to quantify plaque-forming units (PFU). The induced phage production using each agent was then evaluated via the plaque assay using double-layer agar technique (Adams, 1959) and average phage production was determined by performing the experiment thrice.

2.3.Phage isolation and purification

Lysogenic bacterial strains Lg2 (S1 genotype) and KYS 9303 (S2 genotype) were used to induce PLgT-1. The PLgT-1 was isolated and concentrated using double-layer agar and the plate lysis method as described previously (Hoai & Yoshida, 2015).

2.4. Bacterial genotypes after transduction

During the lysis period, a proportion of the phage was demonstrated to switch to the lysogenic cycle instead of the lytic cycle and the recipient bacteria then became phage-

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transduced strains (transductants) (Moon et al., 2015). In a previous study, The PLgT-1 was demonstrated to be highly distributed in 12 of 16 tested genotypes isolated from marine fish (S1‒ S16), of which two genotypes (S7 and S16) were non-lysogenic strains and could be used as indicators for this phage (Hoai & Yoshida, 2015). To test the role of PLgT-1 in genotype diversification of L. garvieae via transduction, a spotting assay was used to transduce PLgT-1 (induced from Lg2) into two genotypes of L. garvieae, strains KG32 (genotype S7) and EH6704 (genotype S16), which are representative indicators. The solution of the induced and purified PLgT-1 was spotted onto the lawns of both recipient strains and incubated at 25°C for 24 h. The colonies that grew in the lysis zone on the bacterial lawns were suspected to be transduced colonies or transductants. Three representative colonies were randomly collected from each bacterial lawn and re-isolated thrice on THA. The genomic DNA of all the transductants and both original indicator bacteria was extracted using InstaGene Matrix (Bio-Rad Laboratories, USA) according to the manufacturer’s protocol. Consequently, a set of primers for the PCR template (5 ′ -GGATTGAAGCGATCTGAACC-3 ′

(forward primer) and 5 ′ -

TGGCGAGGTAAAGGTTATGC-3 ′ (reverse primer)) specific to PLgT-1 detection in the bacterial genome as described previously (Hoai & Yoshida, 2015), was used to confirm the success of transduction. The chromosomal DNA of each transductant was then digested with the restriction enzyme SmaI, and the DNA fragment was separated using BSFGE as described previously (Nishiki et al., 2011). The DNA fingerprints of the transductants were then compared with those of L. garvieae genotypes isolated from marine fish from 1974 to 2012 (Nishiki et al., 2011) to determine the new genotypes.

2.5. Phage DNA extraction

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Highly concentrated phage samples induced from two bacterial strains, Lg2 (genotype S1) and KYS9303 (genotype S2), were used to extract phage DNA by chemical lysis and the phenol/chloroform/isoamyl alcohol (25:24:1) extraction method as described in a previous study (Ghasemi et al., 2014). Briefly, 30 µL/mL of 1 M Tris-HCl (pH 7.0), 125 units/mL of deoxyribonuclease I (DNase I, Wako, Tokyo, Japan), and 3 µg/mL of ribonuclease (RNAse, Wako, Tokyo, Japan), were added to 10 mL of a concentrated solution of isolated and purified phage (1010 PFU/mL). The solution was then incubated at 25°C for 5 h to remove bacterial DNA and RNA. Phage particles were concentrated by ultracentrifugation at 200,000 x g at 15°C for 5 h using a preparative ultracentrifuge (Optima TLX; Beckman, California, USA). The supernatant was decanted, the phage pellet was suspended in 300 µl of lysis buffer (25 mM Tris-HCl, pH 8; 10 mM EDTA pH 8; 10 mM NaCl; and 1% SDS) containing proteinase K (1 mg/mL), followed by incubation at 37°C for 15 min and 60°C for 10 min. The phage DNA was then extracted twice by adding an equal volume of phenol/chloroform/isoamyl alcohol solution (Wako, Tokyo, Japan). The phage DNA was precipitated by adding an equal volume of 2-propanol (Sigma, USA) and was washed several times with ethanol (70%) before being re-suspended in sterile distilled water and stored at -80°C.

2.6. Phage DNA sequencing and analysis

A PLgT-1 DNA fragment library was prepared for whole genome shotgun sequencing using the Ion Xpress Plus gDNA Fragment Library kit and the Ion PGM Template OT2 400 kit (Life Technologies, Carlsbad, CA, USA) following the manufacturer’s protocol. Sequencing was

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carried out on an Ion PGM platform using the Ion PGM Sequencing 400 kit and the Ion 318 Chip kit v2 (Life Technologies). Data from the PGM run were processed using the Ion Torrent Suite 3.4.2 software for generating sequence reads and adapter trimming. De novo assembly of sequence reads was performed using Newbler version 2.8. Homology search of the PLgT-1 sequence with other sequences in the public database was performed using the MegaBLAST strict search program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Open reading frames (ORFs) were identified with a start codon (ATG, GTG, or TTG) and a stop codon (TAA, TGA, or TAG) using the

gene

prediction

software

GeneMarkS

version

4.28

(http://exon.gatech.edu/GeneMark/genemarks.cgi) (Besemer et al., 2001). The amino acid sequences were then comparatively analyzed with those of other viral sequences (tax id: 10239 viruses) based on a homology search using the Basic Local Alignment Search Tool (BLAST) at the NCBI website with a threshold E value of 10-4. The ExPAsy compute pI/Mw tool (http://web.expasy.org/compute_pi/) was used to determine the isoelectric pH and molecular weight of the predicted ORFs (Gasteiger et al., 2005). Transfer RNA (tRNA) genes were predicted using tRNA scan-SE (http://lowelab.ucsc.edu/tRNAscan-SE/) (Schattner et al., 2005). A comparison of the genome among PLgT-1 and other related phages was performed and was visualized using the Genome Matcher version 2.03 program (Ohtsubo et al., 2008).

2.7. Phylogenetic analysis To determine the phylogenetic position of PLgT-1, two representative genes encoding the major capsid protein (MCP) and the terminase protein large subunit (TerL) were used for phylogenetic analysis. The amino acid sequences of MCP and TerL in PLgT-1 and in other lactococcal phages were aligned using ClustalW (Thompson et al., 1994), and phylogenetic trees

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were then constructed using the neighbor-joining method (Saitou & Nei, 1987) in Mega 6 software (Tamura et al., 2013).

3. Results and discussion

3.1. Evaluation of phage induction capacity

The culture of lysogenic bacteria induced with MMC (a common phage induction agent) released the highest concentration of the PLgT-1 phage with more than 108 PFU/mL within 7 h. Surprisingly, this phage could also be released under the culture conditions of THB, THB supplemented with 0.01 M CaCl2, DW supplemented with 0.01 M CaCl2, and natural seawater as evaluated by the double layer agar method (Fig. 1). However, the amounts of the phage released under these culture conditions were smaller than the amount of the phage induced by MMC treatment (Fig. 2). The amount of phage produced in THB supplemented with 0.01 M CaCl2 was greater than that produced in THB alone. Thus, ionized calcium may contribute to phage induction. Natural seawater, in which the concentration of ionized calcium was similar to that in THB supplemented with 0.01 M CaCl2 (Dickson & Goyet, 1994) and DW supplemented with 0.01 M CaCl2, also affected the host bacteria resulting in release of the PLgT-1 phage. These results may explain why various phages are found in natural environments including the ocean (Wommack & Colwell, 2000). Calcium has been demonstrated to play a vital role in phage absorption, and is also necessary to accelerate cell lysis, to assist virulent phages in penetrating their hosts, and to improve plaque formation (Lu et al., 2003; Quiberoni et al., 2004). Therefore, calcium supplements in culture could be the reason for lysis of lysogenic bacterial cells, resulting

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in a large amount of phage release. Several antibiotics and chemical compounds have also been investigated for phage induction (Goerke et al., 2006; Mayer et al., 1969). However, only MMC and UV irradiation have been evaluated and commonly used. The amount of the phage produced and released was significantly larger in THB culture medium than in insufficient media (DW and natural seawater), suggesting that the PLgT-1 phage is probably released during the process of cell division.

3.2. The role of the PLgT-1 phage in bacterial genotype diversity via transduction

Cell-free phage lysates and lysogenic phages spontaneously released from lysogens were capable of transduction (Saye et al., 1987). As mentioned above, the probability of the PLgT-1 phage being released into the marine environment is high. Therefore, it is reasonable to assume that the PLgT-1 genome or its elements could be transduced into non-lysogenic bacterial strains, at least of its host bacteria, L. garvieae. During experimental transduction, a large number of host bacteria were resistant to PLgT-1 and grew in the lysis zone of a spot test. These bacteria were subsequently confirmed to carry the PLgT-1 prophage or its elements in their genomes upon random testing of three resistant colonies of each indicator strain with all of them being PCRpositive (Fig. 3). Jiang & Paul (1998) demonstrated that potential bacteriophage-mediated gene transfer might occur frequently in the marine environment, and that gene transfer by bacteriophage transduction probably played an important role in the mechanism underlying gene evolution and contributed to genetic diversification of marine microbial populations. This phenomenon was also observed for the PLgT-1 phage. The DNA fingerprints of transductants obviously differed from those of their original genotypes after PLgT-1 transduction. By

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comparing DNA fingerprint patterns of all genotypes of L. garvieae isolated from marine fish in Japan studied previously (Nishiki et al., 2011), the DNA fingerprints of the S7 and S16 genotype transductants by PLgT-1 were visibly consistent with those of the original S3 and S4 genotypes, respectively (Fig. 4). In addition, approximately 75% of L. garvieae isolated from diseased marine fish in Japan carried the PLgT-1 prophage (Hoai & Yoshida, 2015). These results suggest that the PLgT-1 phage probably plays a vital role in the mechanism underlying genetic evolution and contributes to the genetic diversification of L. garvieae. The significance of the widespread nature of the prophage is also a good indicator that there may not be significant diversity in the cell wall composition of these L.garvieae strains since the phage can infect high proportion of these bacteria or it may indicate that this phage employs a receptor that is common to these bacterial strains from marine fish. Epidemiological study on L. garvieae isolates from marine fish revealed that S1 genotype was prominent with 316/427 strains (Nishiki et al. 2011) and 100% strains (30/30) belong to this genotype contained integrated PLgT-1 prophage (Hoai & Yoshida, 2015). Moreover, Kawanishi et al. (2006) reported that several L. garvieae isolates from marine fish showed higher virulence in marine fish than do other strains isolated from the trout and terrestrial animals. Therefore, the lysogenic phage PLgT-1 may be involved in the transfer of a virulence factor into L. garvieae strains colonizing in marine fish.

3.3. General characteristics and comparative genomic analysis

The length of the complete genome sequence of the PLgT-1 phage isolated from each of two representative strains, Lg2 (genotype S1) and KYS9303 (genotype S2), was 40,273 bp. Alignment analysis of these two completed sequences revealed that their genome sizes were the

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same and differences in only 3 bp were detected between these two sequences (Data not shown). Thus, both PLgT-1 phages are highly homologous. In this study, a representative complete genome of the PLgT-1 phage isolated from the L. garvieae Lg2 strain, was used for genomic analysis, and was submitted to the GenBank database under the accession number KU892558. The PLgT-1 genome encodes 66 ORFs and consists of 40,273 bp with a G + C content of 35.4%, which is lower than that of its host bacteria, L. garvieae (38.8%) (Morita et al., 2011). In the PLgT-1 genome, two tRNA sequences were found. The presence of tRNA sequences has been reported in several other phage genomes and these sequences were considered as a factor to improve the efficiency of phage protein translation (Bailly-Bechet et al., 2007). MegaBLAST search revealed that the PLgT-1 genome sequence was only homologous with the genome sequences of several L. lactis subspecies, which are important bacteria extensively used to ferment dairy products, and their phages (except for PLgT-1 prophages that are part of the Lactococcus garvieae genome sequences available on a database within strain Lg2 and ATCC49156). The PLgT-1 sequence has considerable similarity to that of L. lactis ssp. lactis IO1 (83%, a match length of 41%) (Kato et al., 2012), L. lactis ssp. cremoris NZ9000, MG1363 (86%, a match length of 39%) (Linares et al., 2010), SK11 (85%, a match length of 34%) (Makarova et al., 2006), and to that of L. lactis strain AI06 (91%, a match length of 37%) (McCulloch et al., 2014). All the L. lactis phages showed similarity to PLgT-1 as shown in Table 2, and all these phages belong to the family Siphoviridae in the order Caudovirales. At the genome level, the sequence of L. lactis phage TP712 isolated from L. lactis ssp. cremoris MG1363, had the highest similarity to that of PLgT-1 (a match length of 38%). However, there was a limitation in the homology between PLgT-1 and other L. lactis phages (only sharing 1‒6% of the genome length). Two virulent phages specific to L. garvieae, WP-2

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(Picovirinae subfamily) (Ghasemi et al., 2014) and GE1 (Siphoviridae family) (Eraclio et al., 2015), showed no significant homology with PLgT-1. Thus, the L. garvieae lysogenic phage PLgT-1 is a new member of the family Siphoviridae in the order Caudovirales. The similarity between L. garvieae and L. lactis phages could be caused by genetic exchange between these two host bacteria because L. garvieae, a known fish pathogen, is also recognized as an opportunistic human pathogen (Chan et al., 2011) and is widespread in dairy products (milk, cheese), vegetables, and aquatic and terrestrial environments (Ferrario et al., 2012; Kawanishi et al., 2007). The activity of L. garvieae strains was believed to contribute to the sensory characteristics of some dairy products (Fernández et al., 2010) and the adaptation of this bacterium to dairy products was likely enabled by the acquisition of certain plasmids (Flórez & Mayo, 2015). Eraclio et al. (2015) also demonstrated that since most of the L. garvieae insertion sequences were substantially homologous to L. lactis elements, genes could be transferred and shared between these two species. Therefore, it is no surprise if L. garvieae phages show similarities to L. lactis phages.

3.4. Functional modules and comparative proteomic analysis of the phage PLgT-1

Of the 66 ORFs encoded in the PLgT-1 genome, 58 ORFs exhibited a leftward strand and 8 ORFs exhibited a rightward strand. All the predicted ORFs began with ATG except for ORF5 (GTG), ORF9 (TTG), and ORF34 (TTG). As shown in Table 3, the specific functions of only 24 ORFs (36.3%) in the PLgT-1 genome could be predicted and assigned based on sequence similarity to other phage proteins through BLASTP searches in the GenBank database. The proteins encoded by 42 remaining ORFs showed no significant evidence of homology with any

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other phage proteins, or they showed considerable similarity with proteins whose biological functions have not been assigned, confirming the novelty of PLgT-1 (Fig. 5, Table 3). These results indicate that PLgT-1 is a novel phage.

To achieve successful infection, a bacteriophage needs the machinery for gene expression, gene regulation, DNA replication, phage capsid formation, and release of new phage particles from the infected host. The genes in most phages that encode the above basic functions are clustered according to biological function and are divided into functional modules (Botstein, 1980; Casjens et al., 1992). Inspection of the identified functions encoded in the PLgT-1 ORFs reveals that genome the can be divided into “functional modules” including cell lysis, tail structural components and assembly, head structural components and assembly, DNA packaging, late gene control, DNA replication, early gene control, and site-specific recombination (Fig. 5).

The lysis module: The lysis module in most bacteriophages consists of holin and lysis proteins. The PLgT-1 phage consists of two holin proteins, ORF8 and ORF9, but no lysis protein could be predicted. The products of ORF8 and ORF9 were proposed as phage holins because they showed 57% and 64% similarity to holins encoded in Lactococcus phage r1t and Lactococcus phage ul36.k1t1, respectively. Holins are a group of membrane proteins produced by phages for phageinduced lysis that collapse the membrane of an infected cell and permeabilize the bacterial membrane allowing the lytic enzyme to access the cell wall prior to injecting the genome into host bacterial cells (Young, 1992).

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Tail structural components and assembly: The functional module for tail structural components and assembly is proposed to cover ORF12 to ORF19. ORF12 was found to exhibit significant similarity (52% overall identity) to the PbIB-like tail protein of Enterococcus phage phiFL3A (Yasmin et al., 2010), and other minor tail proteins of various phages. The protein specified by ORF14 shows high similarity (73% overall identity) to the unknown structural protein of the Lactococcus phage TP712 (Roces et al., 2013). However, this encoded protein in PLgT-1 exhibits considerable similarity to the tape-measure protein (TMP) of various phages. For example, this protein shares 32% sequence identity to the TMP of the Oenococcus phage phi9805 (Jaomanjaka et al., 2013). In addition, PLgT-1 is a long tailed phage (Figure 1; Hoai & Yoshida, 2015), and all long-tailed phages possess a large gene encoding a TMP for precisely determining the tail length (Pell et al., 2009). Thus, based on observed similarities, ORF14 was probably responsible for functioning as a tail length tape-measure protein in PLgT-1. The deduced protein sequence of ORF17 was identified as the major tail protein of PLgT-1. It displayed 42%‒92% resemblance to the major tail protein of the Lactococcus phage TP712 (Roces et al., 2013), Enterococcus phage phiFL3A (Yasmin et al., 2010), and the Oenococcus phages phiS13 and phi8905 (Jaomanjaka et al., 2013). In almost all phages, the genes located between the major tail and head proteins are involved in the formation and connection of head and tail structures and DNA packaging. This is consistent with the function of ORF19 located between the tail and head proteins of PLgT-1 and shared resemblance (34%) to the neck protein of Streptococcus phage 040922 (Croucher et al., 2011). Thus, the protein encoded by ORF19 could be identified as a neck protein, and the region encoded by ORF18 to ORF21 could be the head-tail joining region of PLgT-1.

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Head structural components and assembly: The module for the head structural components and assembly of the PLgT-1 phage involves ORF22 to ORF26 because the functions of three ORFs in this region (ORF22, ORF23, and ORF26) were identified based on their resemblance to the head protein of other phages. ORF22 was predicted to encode the major capsid protein based on sequence similarity to that of Listeria phage A500 (59%) (Dorscht et al., 2009) and many other Staphylococcus phages (Staphylococcus phages PH15, PhiB557, StauST398-1, and SA13) with considerable resemblance (46–48%). The predicted product of ORF23 was identified as a scaffold protein because it displayed 58‒62% similarity to various Enterococcus faecalis lysogenic phages (Yasmin et al., 2010). The deduced product of ORF26 could be assigned as a head protein of PLgT-1 based on its considerable similarity to the head protein of Enterococcus phage phiFL3A (68%) (Yasmin et al., 2010). This product also shared similarity (40%) with the minor head protein of Oenococcus phages (Jaomanjaka et al., 2013) and the putative head morphogenesis protein of various Streptococcus phages (Croucher et al., 2011).

DNA packaging: Packaging of the bacteriophage double-stranded DNA into the viral capsid relies on a molecular “motor” consisting of several proteins: the portal pore, which is located at one vertex of the capsid, and the terminases. The predicted products of ORF27 and ORF29 encoded by PLgT-1 were identified as the ribosomal and portal proteins of PLgT-1, respectively. These two proteins were identical to the putative ribosomal and portal proteins of Enterococcus phage phiFL3A (47% and 71% overall identity, respectively). During DNA packaging, the portal protein associates with the terminase protein (Black, 1989; Rao & Feiss, 2008). The terminase is a component of the molecular motor that translocates genomic DNA into an empty capsid during DNA packaging (Black, 1989). In the PLgT-1 genome, terminases constituted by large and small

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subunits were found to be encoded by ORF30 and ORF31, respectively, as they are highly homologous (98% and 79% overall identity) with those of the proteins encoded by Lactococcus phage TP712. Thus, the DNA packaging module of PLgT-1 contains two terminase subunits and one portal protein.

Late gene control: The predicted product of ORF33 was identified as a transcriptional regulator which was highly homologous with the late transcriptional regulator of the Lactococcus phage TP901-1 (91% overall identity) (Brøndsted et al., 2001) and that of Lactococcus phage P335 (91% overall identity) (Labrie et al., 2008).

DNA replication: We proposed that the replication module of PLgT-1 was covered by ORF36 to ORF54 in its genome since several functions related to DNA replication were located within this region. ORF36 was determined as DNA methylase as it shared 41% similarity with that of Lactobacillus phage Lv-1, while ORF48 showed considerable homology (60% overall identity) with RusA resolvase of Lactococcus phage P335, which was proposed to play an important role in DNA repair and recombination of Lactococcus phages such as r1t, P335, and TP901-1. ORF51 showed 66% similarity to a replication protein of Lactococcus phage BK5-T (Desiere et al., 2001). An endonuclease protein (ORF52) was found to share 53% homology with HNH endonuclease of Burkholderia phage G068, and this endonuclease protein is demonstrated to have diverse physiological roles in replication, recombination, repair, maturation, and packaging of phage DNA (Crutz-Le Coq et al., 2002). ORF53 and ORF54 are highly homologous with proteins encoded by Lactococcus phage TP712 and Streptococcus phage Alq132, and could be

19

assigned as single stranded DNA binding and DNA binding proteins of PLgT-1, respectively. The functions of ORF37‒ORF47, ORF49, and ORF50 are currently unknown.

Early gene control: The ORF60‒ORF65 region is part of the early gene control module of PLgT-1. The anti-repressor protein was encoded by ORF60, which showed significant similarity to this type of protein in the genome of Lactococcus phage BM13 (Mahony et al., 2015). The deduced products of ORF63 could be assigned as a repressor protein that showed a considerable similarity (54%) to Lactococcus phage TP712. ORF65 was proposed to encode an immunity region in PLgT-1 although it showed a limited similarity (41%) to this type of region encoded by the Streptococcus phage Sfi21 (Desiere et al., 1998). The immunity region is one of the factors responsible for maintaining the lysogenic state and affects immunity against superinfecting phages (Botstein et al., 1975).

The site-specific recombination: The site-specific recombination module of PLgT-1 was located in the last ORF in the genome. ORF66 was identified as the integrase of PLgT-1 based on its homology with integrase and site specific recombination of various lactococcal and streptococcal phages such as Lactococcus phage bIL286 (Chopin et al., 2001), ul36 (Labrie & Moineau, 2002) (74–75% overall identity), and Streptococcus phage T12 (58% overall identity) (McShan et al., 1997).

3.5. Phylogenetic position of PLgT-1 phage and its comparison with related phages.

20

Although the homology between PLgT-1 phage and L. lactis phages was not significantly observed at the genome level, comparative proteomic analysis showed that the proteins encoded by PLgT-1 genes showed similarities to proteins coded by various phages. Of these, 17 out of 24 predicted proteins shared the best homology with those of various L. lactis phages (Table 3) (11 ORFs were used to predict PLgT-1 proteins and six other ORFs had the highest homology with L. lactis phage TP712, but were not used for gene prediction because these products are currently unknown). These results suggested that the genes in PLgT-1 were involved in horizontal genetic exchanges with other microbial communities, resulting in acquisition of biological functions and modules in its genome, especially from L. lactis and its phage community. The sequences of some “core” genes encoding terminase, portal, major capsid, and DNA polymerase protein were demonstrated to be useful for determining the phylogenetic positions of various phages (Casjens et al., 2005; Ghasemi et al., 2014; Yoshida et al., 2015). To determine the phylogenetic position of PLgT-1 and to compare PLgT-1 with other Lactococcus phages, phylogenetic analysis was conducted for two representative phage core genes, MCP and TerL. The results showed that the MCP and TerL genes of PLgT-1 formed a cluster with various L. lactis phages, but were distant from two other virulent phages of L. garvieae, WP-2 and GE1 (Fig 6). When PLgT-1 was compared with related phages with respect to phylogenetic position, no significant similarity was observed in the genome sequence between PLgT-1 and the L. lactis phage P335 at the genomic level (4% length identity). Synteny analysis revealed that the L. lactis phage TP712 was the closest to PLgT-1 at both genome (38% homology, Table 2) and protein levels (12/24 ORFs, Table 3), and the similarity was significantly observed in the middle region

21

of the genome, which encodes the head structure proteins and DNA packaging module of PLgT1 (ORF22‒ORF31). However, the genome organization of PLgT-1 differed from that of TP712. For example, the majority of PLgT-1 ORFs were located on the negative strand while those of TP712 are located on the positive strand; additionally, the position of the functional modules in the PLgT-1 genome differed from that of TP712 in arrangement (Figs. 5 and 7). Thus, even though the phylogenetic position of PLgT-1 was close to that of TP712, they differed from each other in other factors.

4.

Conclusions

In this study, the PLgT-1 phage, a lysogenic phage widely distributed in L. garvieae isolated from diseased marine fish in Japan, was investigated at the genome level. This phage is easily released during cell division under high nutrient conditions or in seawater, which may promote genetic exchange with other microbial communities or among L. garvieae bacterial genotypes via transduction. Thus, this phage has played an important role in the diversification of L. garvieae genotypes. PLgT-1 genomic analysis revealed that this phage was a novel phage belonging to the family Siphoviridae in the order Caudovirales, and the genes encoded by the PLgT-1 genome showed homology with various phage proteins, suggesting that they had access to a large common genetic pool through horizontal exchange. Many genes in PLgT-1 were also homologous with genes from various L. lactis phages. This may be due to high interaction between their hosts. In particular, the adaptation of L. garvieae to the dairy environment has been demonstrated.

22

Acknowledgements This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Sports, Japan (15K07554). The authors are grateful to the Government of Japan for funding a scholarship to Truong Dinh Hoai for his research.

References Adams, M. H., 1959. Bacteriophages. New York: Interscience Publishers Inc. Bailly-Bechet, M., Vergassola M., & Rocha E., 2007. Causes for the intriguing presence of tRNAs in phages. Genome Research. 17, 1486-1495. Besemer, J., Lomsadze A., & Borodovsky M., 2001. GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions. Nucleic Acids Research. 29, 2607-2618. Black, L. W., 1989. DNA packaging in dsDNA bacteriophages. Annual Reviews in Microbiology. 43, 267-292. Botstein, D., 1980. A theory of modular evolution for bacteriophages. Annals of the New York Academy of Sciences. 354, 484-491. Botstein, D., Lew K. K., Jarvik V., & Swanson C. A., 1975. Role of antirepressor in the bipartite control of repression and immunity by bacteriophage P22. Journal of Molecular Biology. 91, 439-462. Brøndsted, L., Østergaard S., Pedersen M., Hammer K., & Vogensen F. K., 2001. Analysis of the complete DNA sequence of the temperate bacteriophage TP901-1: evolution, structure, and genome organization of lactococcal bacteriophages. Virology. 283, 93-109. Canchaya, C., Fournous G., Chibani-Chennoufi S., Dillmann M.-L., & Brüssow H., 2003. Phage as agents of lateral gene transfer. Current Opinion in Microbiology. 6, 417-424. Casjens, S. R., Gilcrease E. B., Winn-Stapley D. A., Schicklmaier P., Schmieger H., Pedulla M. L., et al., 2005. The generalized transducing Salmonella bacteriophage ES18: complete genome sequence and DNA packaging strategy. Journal of Bacteriology. 187, 1091-1104. Casjens, S., Hatfull G., & Hendrix R., 1992. Evolution of dsDNA tailed-bacteriophage genomes. Seminars in Virology. 3, 383–397. Chan, J., Woo P., Teng J., Lau S., Leung S., Tam F., et al., 2011. Primary infective spondylodiscitis caused by Lactococcus garvieae and a review of human L. garvieae infections. Infection. 39, 259-264. Chopin, A., Bolotin A., Sorokin A., Ehrlich S. D., & Chopin M.-C., 2001. Analysis of six prophages in Lactococcus lactis IL1403: different genetic structure of temperate and virulent phage populations. Nucleic Acids Research. 29, 644-651. Croucher, N. J., Harris S. R., Fraser C., Quail M. A., Burton J., van der Linden M., et al., 2011. Rapid pneumococcal evolution in response to clinical interventions. Science. 331, 430-434. Crutz-Le Coq, A.-M., Cesselin B., Commissaire J., & Anba J., 2002. Sequence analysis of the lactococcal bacteriophage bIL170: insights into structural proteins and HNH endonucleases in dairy phages. Microbiology. 148, 985-1001. 23

Desiere, F., Lucchini S., & Brüssow H., 1998. Evolution of Streptococcus thermophilus Bacteriophage Genomes by Modular Exchanges Followed by Point Mutations and Small Deletions and Insertions. Virology. 241, 345-356. Desiere, F., Mahanivong C., Hillier A. J., Chandry P. S., Davidson B. E., & Brüssow H., 2001. Comparative genomics of lactococcal phages: insight from the complete genome sequence of Lactococcus lactis phage BK5-T. Virology. 283, 240-252. Devriese, L., Hommez J., Laevens H., Pot B., Vandamme P., & Haesebrouck F., 1999. Identification of aesculin-hydrolyzing streptococci, lactococci, aerococci and enterococci from subclinical intramammary infections in dairy cows. Veterinary Microbiology. 70, 87-94. Dickson, A. G., & Goyet C., 1994. Handbook of methods for the analysis of the various parameters of the carbon dioxide system in sea water: Version 2 (ORNL/CDIAC-74), US Department of Energy, Washington, DC (1994). Dorscht, J., Klumpp J., Bielmann R., Schmelcher M., Born Y., Zimmer M., et al., 2009. Comparative genome analysis of Listeria bacteriophages reveals extensive mosaicism, programmed translational frameshifting, and a novel prophage insertion site. Journal of Bacteriology. 191, 7206-7215. Eraclio, G., Ricci G., & Fortina M. G., 2015. Insertion sequence elements in Lactococcus garvieae. Gene. 555, 291-296. Eraclio, G., Tremblay D. M., Lacelle-Côté A., Labrie S. J., Fortina M. G., & Moineau S., 2015. A new virulent phage infecting Lactococcus garvieae, with homology to Lactococcus lactis phages. Applied and Environmental Microbiology. 81, 02603-02615. Fernández, E., Alegría Á., Delgado S., & Mayo B., 2010. Phenotypic, genetic and technological characterization of Lactococcus garvieae strains isolated from a raw milk cheese. International Dairy Journal. 20, 142-148. Ferrario, C., Ricci G., Borgo F., Rollando A., & Fortina M. G., 2012. Genetic investigation within Lactococcus garvieae revealed two genomic lineages. FEMS Microbiology Letters. 332, 153-161. Flórez, A. B., & Mayo B., 2015. The Plasmid Complement of the Cheese Isolate Lactococcus garvieae IPLA 31405 Revealed Adaptation to the Dairy Environment. PLoS One. 10 10.1371/journal.pone.0126101. Gasteiger, E., Hoogland C., Gattiker A., Wilkins M. R., Appel R. D., & Bairoch A., 2005. Protein identification and analysis tools on the ExPASy server: Humana Press. Ghasemi, S. M., Bouzari M., Yoon B. H., & Chang H. I., 2014. Comparative genomic analysis of Lactococcus garvieae phage WP-2, a new member of Picovirinae subfamily of Podoviridae. Gene. 551, 222-229. Goerke, C., Köller J., & Wolz C., 2006. Ciprofloxacin and trimethoprim cause phage induction and virulence modulation in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy. 50, 171-177. Hoai, T. D., & Yoshida T., 2015. Induction and characterization of a lysogenic bacteriophage of Lactococcus garvieae isolated from marine fish species. Journal of Fish Diseases. doi: 10.1111/jfd.12410 Jaomanjaka, F., Ballestra P., Dols-lafargue M., & Le Marrec C., 2013. Expanding the diversity of oenococcal bacteriophages: insights into a novel group based on the integrase sequence. International Journal of Food Microbiology. 166, 331-340. Jiang, S. C., & Paul J. H., 1998. Gene transfer by transduction in the marine environment. Applied and Environmental Microbiology. 64, 2780-2787. Kato, H., Shiwa Y., Oshima K., Machii M., Araya-Kojima T., Zendo T., et al., 2012. Complete genome sequence of Lactococcus lactis IO-1, a lactic acid bacterium that utilizes xylose and produces high levels of L-lactic acid. Journal of Bacteriology. 194, 2102-2103. 24

Kawanishi, M., Yoshida T., Kijima M., Yagyu K., Nakai T., Okada S., et al., 2007. Characterization of Lactococcus garvieae isolated from radish and broccoli sprouts that exhibited a KG+ phenotype, lack of virulence and absence of a capsule. Letters in Applied Microbiology. 44, 481-487. Kawanishi, M., Yoshida T., Yagashiro S., Kijima M., Yagyu K., Nakai T., et al., 2006. Differences between Lactococcus garvieae isolated from the genus Seriola in Japan and those isolated from other animals (trout, terrestrial animals from Europe) with regard to pathogenicity, phage susceptibility and genetic characterization. Journal of Applied Microbiology. 101, 496-504. Labrie, S. J., Josephsen J., Neve H., Vogensen F. K., & Moineau S., 2008. Morphology, genome sequence, and structural proteome of type phage P335 from Lactococcus lactis. Applied and Environmental Microbiology. 74, 4636-4644. Labrie, S., & Moineau S., 2002. Complete genomic sequence of bacteriophage ul36: demonstration of phage heterogeneity within the P335 quasi-species of lactococcal phages. Virology. 296, 308-320. Landry, E. F., & Zsigray R. M., 1980. Effects of calcium on the lytic cycle of Bacillus subtilis phage 41c. Journal of General Virology. 51, 125-135. Linares, D. M., Kok J., & Poolman B., 2010. Genome sequences of Lactococcus lactis MG1363 (revised) and NZ9000 and comparative physiological studies. Journal of Bacteriology. 192, 5806-5812. Lu, Z., Breidt F., Fleming H., Altermann E., & Klaenhammer T., 2003. Isolation and characterization of a Lactobacillus plantarum bacteriophage, ΦJL-1, from a cucumber fermentation. International Journal of Food Microbiology. 84, 225-235. Mahony, J., Randazzo W., Neve H., Settanni L., & van Sinderen D., 2015. Lactococcal 949 group phages recognize a carbohydrate receptor on the host cell surface. Applied and Environmental Microbiology. 81, 3299-3305. Makarova, K., Slesarev A., Wolf Y., Sorokin A., Mirkin B., Koonin E., et al., 2006. Comparative genomics of the lactic acid bacteria. Proceedings of the National Academy of Sciences. 103, 15611-15616. Mayer, V., Gabridge M., & Oswald E., 1969. Rapid plate test for evaluating phage induction capacity. Applied Microbiology. 18, 697-698. McCulloch, J. A., de Oliveira V. M., de Almeida Pina A. V., Pérez-Chaparro P. J., de Almeida L. M., de Vasconcelos J. M., et al., 2014. Complete genome sequence of Lactococcus lactis strain AI06, an endophyte of the Amazonian açaí palm. Genome Announcements. 2, e0122501214. McShan, W. M., Tang Y. F., & Ferretti J. J., 1997. Bacteriophage T12 of Streptococcus pyogenes integrates into the gene encoding a serine tRNA. Molecular Microbiology. 23, 719-728. Moon, B. Y., Park J. Y., Hwang S. Y., Robinson D. A., Thomas J. C., Fitzgerald J. R., et al., 2015. Phage-mediated horizontal transfer of a Staphylococcus aureus virulence-associated genomic island. Scientific Reports. 5. doi: 10.1038/srep09784. Morita, H., Toh H., Oshima K., Yoshizaki M., Kawanishi M., Nakaya K., et al., 2011. Complete genome sequence and comparative analysis of the fish pathogen Lactococcus garvieae. PLoS One. 6, e23184. Murray, N., 2006. Phage-based expression systems. The bacteriophages, 2nd ed. Plenum Publishing Corp., New York, NY, 674-685. Nakai, T., Sugimoto R., Park K., Matsuoka S., Mori K., Nishioka T., et al., 1999. Protective effects of bacteriophage on experimental Lactococcus garvieae infection in yellowtail. Diseases of Aquatic Organisms. 37, 33-41. Nishiki, I., Furukawa M., Matui S., Itami T., Nakai T., & Yoshida T., 2011. Epidemiological study on Lactococcus garvieae isolates from fish in Japan. Fisheries Science. 77, 367-373. 25

Nobrega, F. L., Costa A. R., Kluskens L. D., & Azeredo J., 2015. Revisiting phage therapy: new applications for old resources. Trends in Microbiology. 23, 185-191. Ohtsubo, Y., Ikeda-Ohtsubo W., Nagata Y., & Tsuda M., 2008. GenomeMatcher: a graphical user interface for DNA sequence comparison. BMC Bioinformatics. 9, 376. Park, K. H., Matsuoka S., Nakai T., & Muroga K., 1997. A virulent bacteriophage of Lactococcus garvieae (formerly Enterococcus seriolicida) isolated from yellowtail Seriola quinqueradiata. Diseases of Aquatic Organisms. 29, 145-149. Pell, L. G., Kanelis V., Donaldson L. W., Howell P. L., & Davidson A. R., 2009. The phage λ major tail protein structure reveals a common evolution for long-tailed phages and the type VI bacterial secretion system. Proceedings of the National Academy of Sciences. 106, 41604165. Prescott, L., Harley J. P., & Klein D. (1993). Microbiology, Wm.C.: Brown Publishers, Chicago, London. Quiberoni, A., Guglielmotti D., Binetti A., & Reinheimer J., 2004. Characterization of three Lactobacillus delbrueckii subsp. bulgaricus phages and the physicochemical analysis of phage adsorption. Journal of Applied Microbiology. 96, 340-351. Rao, V. B., & Feiss M., 2008. The bacteriophage DNA packaging motor. Annual Review of Genetics. 42, 647-681. Roces, C., Wegmann U., Campelo A. B., García P., Rodríguez A., & Martínez B., 2013. Lack of the host membrane protease FtsH hinders release of the Lactococcus lactis bacteriophage TP712. Journal of General Virology. 94, 2814-2818. Saitou, N., & Nei M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution. 4, 406-425. Saye, D. J., Ogunseitan O., Sayler G., & Miller R. V., 1987. Potential for transduction of plasmids in a natural freshwater environment: effect of plasmid donor concentration and a natural microbial community on transduction in Pseudomonas aeruginosa. Applied and Environmental Microbiology. 53, 987-995. Schattner, P., Brooks A. N., & Lowe T. M., 2005. The tRNAscan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs. Nucleic Acids Research. 33, W686-W689. Tamura, K., Stecher G., Peterson D., Filipski A., & Kumar S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution. 30, 2725-2729. Thompson, J. D., Higgins D. G., & Gibson T. J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research. 22, 4673-4680. Vendrell, D., Balcázar J. L., Ruiz-Zarzuela I., De Blas I., Gironés O., & Múzquiz J. L., 2006. Lactococcus garvieae in fish: a review. Comparative Immunology, Microbiology and Infectious Diseases. 29, 177-198. Wang, C. Y. C., Shie H. S., Chen S. C., Huang J. P., Hsieh I. C., Wen M. S., et al., 2007. Lactococcus garvieae infections in humans: possible association with aquaculture outbreaks. International Journal of Clinical Practice. 61, 68-73. Williamson, S., & Paul J. H., 2004. Nutrient stimulation of lytic phage production in bacterial populations of the Gulf of Mexico. Aquatic Microbial Ecology. 36, 9-17. Wommack, K. E., & Colwell R. R., 2000. Virioplankton: viruses in aquatic ecosystems. Microbiology and Molecular Biology Reviews. 64, 69-114. Yasmin, A., Kenny J. G., Shankar J., Darby A. C., Hall N., Edwards C., et al., 2010. Comparative genomics and transduction potential of Enterococcus faecalis temperate bacteriophages. Journal of Bacteriology. 192, 1122-1130.

26

Yoshida, M., Yoshida-Takashima Y., Nunoura T., & Takai K., 2015. Genomic characterization of a temperate phage of the psychrotolerant deep-sea bacterium Aurantimonas sp. Extremophiles. 19, 49-58. Young, R., 1992. Bacteriophage lysis: mechanism and regulation. Microbiological Reviews. 56, 430481.

27

Figure legends

Figure 1. Lactococcus garvieae lysogenic phage PLgT-1 A: PLgT-1 form plaques on the lawn of indicator bacteria by the double layer agar method. B: PLgT-1 morphology photographed by TEM with a scale bar =100 nm.

Figure 2. Phage production induced or released by different agents after 7 h of treatment. All data presented are the average of triplicate assays and phage titers were expressed as the mean ± standard error.

Figure 3. Confirmation of PLgT-1 transduction into the genomes of two representative indicator strains KG32 (genotype S7) and EH6704 (genotype S16) by PCR. Lane M: Molecular weight standard. Lane 1: Lg2 (positive control). Lanes 2 and 6: Genomic DNA of KG32 and EH704, respectively (negative control). Lanes 3‒5 and 7‒9: Genomic DNA of three isolates from KG32 and EH6704, respectively, into which PLgT-1 was transduced.

Figure 4. Genotype changes due to transduction of the PLgT-1 phage. M: Molecular weight standard. Lanes 1 and 6: Two original representative indicators of KG32 (genotype S7) and EH6704 (genotype S16). Lanes 2‒4 and 7‒9: Transductants of genotypes S7 and S16, respectively, into which the PLgT-1 phage is transduced. Lanes 5 and 10: Representative strains of KS9402 (genotype S3) and NA8706 (genotype S4), respectively.

28

Figure 5. PLgT-1 genome organization. The arrows represent predicted genes and coding directions. The colors indicate the functions of the gene products and clusters according to their biological functions (dark thin light); gray, unknown.

Figure 6. Neighbor-joining phylogenetic tree based on amino acid sequences of the major capsid protein (A) and the terminase large subunit (B) among Lactococcus phages. Bootstrap analysis was performed with 2000 replicates. The Staphylococcus phage A5W major capsid and terminate large subunit proteins were used as outgroups. The numbers on the lines represent the supporting rates.

Figure 7. Comparison of gene homology and genome arrangement between PLgT-1 and TP712. The gene homology between PLgT-1 and TP712 was visualized by the degree of sequence identity between these phages.

29

Tables:

Table 1. Bacteria strains and target uses in this study Strain

Genotype

Status

Target uses

Year isolated

Lg2

S1

lysogenic

Phage induction

2002

KYS9303

S2

lysogenic

Phage induction

1993

KG32

S7

Non-lysogenic

Indicator, phage transduction

1980

EH6704

S16

Non-lysogenic

Indicator, phage transduction

2006

KS9402

S3

lysogenic

Genotype comparison

1994

NA8706

S4

lysogenic

Genotype comparison

1987

30

Table 2. Comparison of genomic properties of the PLgT-1 and other related phages Siphoviridae (accession number)

Genome length (bp)

GC content

Total ORF

Lactococcus phage PLgT-1 (KU892558)

40273

35.4%

Lactococcus phage TP712 (AY766464.1)

42073

Lactococcus phage ul36.k1 (DQ394806.1)

Significant alignments with the PLgT-1 Coverage

Identity

E value

66

-

-

-

35.1%

59

86%

38%

0.0

37131

35.7%

58

93%

6%

0.0

Lactococcus phage ul36 (AF349457.1)

36798

35.8%

59

93%

6%

0.0

Lactococcus phage phismq86 (DQ394810.1)

33641

36.0%

50

93%

2%

0.0

Lactococcus phage ul36.t1k1 (DQ394809.1)

34897

35.8%

49

93%

4%

0.0

Lactococcus phage ul36.t1 (DQ394808.1)

35992

36.0%

49

93%

5%

0.0

Lactococcus phage ul36.k1t1 (DQ394809.1)

35594

35.7%

50

93%

4%

0.0

Lactococcus phage BM13 (JX567312.1)

30910

36.0%

55

93%

3%

0.0

Lactococcus phage 4268 (AF489521.1)

36596

35.4%

48

94%

4%

0.0

Lactococcus phage TP901-1 (AF304433.1)

37667

35.4%

56

88%

3%

0.0

Lactococcus phage phi15 (KM091442.1)

31945

34.6%

53

90%

1%

0.0

Lactococcus phage bIL286 (AF323669.1)

41834

35.3%

61

86%

3%

0.0

Lactococcus phage bIL309 (AF323670.1)

36949

35.7%

56

88%

2%

0.0

Lactococcus phage P335 (DQ838728.1)

33613

35.5%

50

88%

4%

0.0

Lactococcus phage phi LC3 (AF242738.3)

32172

35.5%

50

86%

2%

0.0

Lactococcus phage Tuc2009 (AF109874.2)

38347

36.2%

53

86%

3%

0.0

Lactococcus phage WRP3 (KM677185.1)

130008

32.4%

190

91%

1%

9E-138

Lactococcus phage bIL285 (AF323668.1)

35538

35.2%

61

88%

1%

1E-131

Lactococcus phage BK5-T (AF176025.1)

40003

35.0%

50

77%

1%

1E-97 31

Table 3: General features of the main putative ORFs of lysogenic phage PLgT-1 ORF

strand

Location

Size (aa)

GC Content (%)

Mw (kDa)

Pi

Product Product[Organism]

8

-

4607-4837

76

41.1

7.9

5.1

Holin

9

-

4850-5071

73

35.6

8.4

6.5

Holin

12

-

6125-8689

854

33.8

95.6

4.8

14

-

9069-13850

1593

37.7

172.5

9.1

17

-

14743-15168

141

38.7

14.8

5.0

19

-

15543-16091

182

39.2

20.4

7.8

22

-

16762- 17880

372

39.1

39.0

5.5

23

-

17893-18567

224

40.9

24.6

5.0

26

-

19545-20642

365

36.7

42.0

9.2

Head protein*

27

-

20648-20935

95

34.7

10.7

5.0

Ribosomal protein*

29

-

21054-22562

502

34.7

113.9

4.9

Portal protein*

30

-

22571-23806

411

34.8

47.4

6.3

31

-

23796-24308

170

36.1

19.0

5.5

33

-

24831-25253

140

33.8

16.7

6.0

36

-

26433-27125

230

36.6

26.3

4.7

DNA methylase

48

-

30785-31189

134

36.5

15.9

9.5

Resolvase

51

-

31548-32357

269

31.7

31.1

8.8

Replication protein

Tail protein Tail length tapemeasure protein Major tail protein Neck protein Major capsid protein* Scaffold protein*

Terminase large subunit Terminase small subunit Transcriptional regulator

Holin [Lactococcus phager1t] Holin [Lactococcus phage ul36.k1t1] PbIB-liketailprotein [Enterococcus phage phiFL3A] Tail length tape-measure protein [Oenococcus phage phi9805] Tailprotein [Enterococcus phage phiFL3A] Neck protein [Streptococcus phage 040922] Major capsid protein [Listeria phage A500] Scaffold protein [Enterococcus phage phiFL3A] Head protein [Enterococcus phage phiFL3A] Ribosomal protein [Enterococcus phage phiFL3A] Portal protein [Enterococcus phage phiFL3A] TerL [Lactococcus phage TP172] TerS [Lactococcus phage TP712] ALT [Lactococcus phage TP172] DNA methylase [Lactobbacillus phage Lv-1] RusA resolvase [Lactococcus phage P335] Replication protein [Lactococcus phage BK5-T]

Best match (NCBI database) Coverage Identity

E value

Accession no.

94%

57%

1E-20

NP_695076.1

100%

63%

1E-28

ABD63743.1

98%

52%

0.0

YP_003347620.1

100%

32%

0.0

YP_009005192.1

95%

73%

2E-67

YP_003347615.1

94%

34%

5e-18

CBW38981.1

72%

59%

6E-110

YP_001468392.1

86%

58%

6E-65

YP_003347609.1

98%

68%

0.0

YP_003347608.1

97%

47%

2E-18

YP_003347607.1

94%

71%

0.0

YP_003347606.1

100%

98%

0.0

AAX13218.1

100%

79%

3E-87

AAX13217.1

100%

91%

3E-87

NP_112692.1

89%

41%

7e-49

YP_002455835.1

96%

65%

2E-51

ABI54212.1

98%

66%

1E-109

NP_116541.1

32

52

-

32357-32695

112

31.9

13.1

9.7

53

-

32787-33293

168

39.8

18.5

4.9

54

-

33293-34009

238

36.4

26.9

6.8

60

-

36000-36719

239

34.4

27.9

6.4

63

+

37470-37814

114

34.8

13.0

5.2

65

+

38317-38895

192

34.9

20.4

8.7

66

+

39020-40102

360

34.5

41.5

9.6

Endonuclease Single stranded DNA binding protein DNA-binding protein* Antirepressor protein* Repressor protein Immunity region Integrase

HNH Endonuclease [Burkholderia phage JG068]

43%

53%

Single stranded binding protein [Lactococcus phage TP712]

100%

81%

6E-97

AAX13200.1

94%

64%

4E-103

YP_003344880.1

100%

86%

1E-149

YP_008320144.1

100%

54%

97%

41%

2e-37

NP_049991.1

100%

75%

0.0

NP_076635.1

Nucleoside triphosphate binding motifs protein [Streptococcus phage Alq132] Anti-repressor [Lactococcus phage BM13] Repressor protein [Lactococcus phage TP172] Immunity region [Streptococcus phage Sfi21] Integrase [Lactococcus phage bIL286]

2E-11

2E-41

YP_008853858.1

AAF12710.1

*ORFs of the PLgT-1 show homology with unknown gene of the Lactococcus lactis phage TP712 (% total identity): ORF22 (96%), ORF26 (91%), ORF27 (96%), ORF29 (97%), ORF54 (92%) and ORF60 (85%)

33

Figures: Figure 1.

1

Figure 2.

2

Figure 3.

3

Figure 4.

4

Figure 5.

5

Figure 6. Lactococcus phage asccphi28 32

65

Lactococcus phage Q33

58

100 100 99

Lactococcus phage SK1

Lactococcus phage ul36.k1 Lactococcus garvieae Phage PLgT-1

Lactococcus phage bIBB29

49

Lactococcus phage P680

Lactococcus phage SL4

Lactococcus phage P335 Lactococcus phage bIL286

36 76

100

100

37

Lactococcus Phage ASCC544

25

Lactococcus phage CB20

50

Lactococcus phage CaseusJM1

73 5

Lactococcus phage jm2 Lactococcus phage phi7

Lactococcus phage 4268 76

Lactococcus phage BK5-T

Lactococcus phage 936 Lactococcus phage c2 Lactococcus phage P078

51

30

Lactococcus phage 1706

100

Lactococcus phage phiLC3

Lactococcus phage F4-1

33 48 100

Lactococcus phage P008

83

18

Lactococcus phage bIL285

25

Lactococcus phage phi145

65

Lactococcus phage SL4

Lactococcus phage bIL309

47

Lactococcus phage 4268

100

Lactoccocus phage WP-2

Lactococcus phage bIL286

100 78

Lactococcus phage bIL309

100

Lactococcus phage BK5-T Lactococcus garvieae phage PLgT-1

Lactococcus phage bIL285

92

Lactococcus phage Q54

100

99 64 100

Lactococcus phage ul36.t1

Lactococcus phage eb1 Lactococcus phage c2

100

100

Lactococcus phage ul36.k1t1 Lactococcus phage ul36.t1k1

Lactococcus phage Q44

Lactococcus phage phismq86

Lactococcus phage GR6 95

Lactococcus phage CB17

100

90

Lactococcus lactis phage p2

51

35

Lactococcus phage ul36.k1t1

100

Lactococcus phage phismq86 Lactococcus phage ul36.t1k1

Staphylococcus phage A5W

A

Lactococcus phage ul36 Lactococcus phage ul36.k1

Lactococcus phage ul36.t1 100

Lactococcus phage BM13 Lactococcus phage Q33

Lactococcus phage SK1833

30

Lactococcus phage TP712

Lactococcus phage 1358

Lactococcus phage GE1

84

19

47

Lactococcus phage TP712

8

Lactococcus phage CB20 Lactococcus phage P008

Lactococcus phage ul36

99

Lactococcus phage fd13

Lactococcus phage Tuc2009

47

Lactococcus phage P335

23

Lactococcus phage TP901-1

Staphylococcus phage A5W

B

6

Figure 7.

7