Arch Microbiol (2014) 196:401–409 DOI 10.1007/s00203-014-0980-4
Original Paper
Genetic diversity of Microcystis cyanophages in two different freshwater environments Ginji Nakamura · Shigeko Kimura · Yoshihiko Sako · Takashi Yoshida
Received: 28 February 2014 / Revised: 13 March 2014 / Accepted: 15 March 2014 / Published online: 27 March 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Bacteriophages rapidly diversify their genes through co-evolution with their hosts. We hypothesize that gene diversification of phages leads to locality in phages genome. To test this hypothesis, we investigated the genetic diversity and composition of Microcystis cyanophages using 104 sequences of Ma-LMM01-type cyanophages from two geographically distant sampling sites. The intergenetic region between the ribonucleotide reductase genes nrdA and nrdB was used as the genetic marker. This region contains the host-derived auxiliary metabolic genes nblA, an unknown function gene g04, and RNA ligase gene g03. The sequences obtained were conserved in the Ma-LMM01 gene order and contents. Although the genetic diversity of the sequences was high, it varied by gene. The genetic diversity of nblA was the lowest, suggesting that nblA is a highly significant gene that does not allow mutation. In contrast, g03 sequences had many point mutations. RNA ligase is involved in the counter-host’s phage defense mechanism, suggesting that phage defense also plays an important role for rapid gene diversification. The maximum parsimony network and phylogenic analysis showed the sequences from the two sampling sites were distinct. These findings suggest Ma-LMM01-type phages rapidly diversify their genomes through co-evolution with hosts in each location and eventually provided locality of their genomes.
Communicated by Erko Stackebrandt. G. Nakamura · S. Kimura · Y. Sako · T. Yoshida (*) Laboratory of Marine Microbiology, Graduate School of Agriculture, Kyoto University, Kitashirakawa‑Oiwake, Sakyo‑ku, Kyoto 606‑8502, Japan e-mail: [email protected]‑u.ac.jp
Keywords Cyanobacteria · Microcystis aeruginosa · Ma-LMM01 · Cyanophage · Co-evolution
Introduction Bacteriophages (phages) are abundant in marine and freshwater ecosystems (Suttle 2005). They infect bacterial hosts and eventually contribute to biogeochemical cycles (Suttle 2005, 2007) and may be directly involved in the genetic diversity of bacteria mediating horizontal gene transfer (Lindell et al. 2004). In addition, phages also affect genetic diversity of hosts via two ways: (1) frequency-dependent selection; host-selective phage infection influences and maintains bacterial diversity through a mechanism where phage infection checks bacterial populations that become dominant in populations (e.g., constant-diversity dynamics) and thereby enables coexistence of multiple bacterial host and phage populations (Rodriguez-Valera et al. 2009); (2) co-evolution (the continual evolution of host defense system and phages counter-defense); a hosts have various phage defense systems, such as the clustered regularly interspaced short palindromic repeats (CRISPR)–Cas (CRISPR-associated genes) systems, the toxin–antitoxin (TA) systems, the classical restriction–modification (RM) systems and the abortive infection (Abi) (Labrie et al. 2010; Marston et al. 2012). These defense systems are thought to promote the high evolution rate of phage genes through overcoming of defense systems by phages (Labrie et al. 2010). Among defense systems, the CRISPR–Cas system provides adaptive immunity in prokaryotes against phages and plasmids via incorporation of short sequences called spacers (Kuno et al. 2012; Labrie et al. 2010). A single mutation the origin of the spacers (protospacers) can abolish CRISPR-mediated host immunity against phages (Kuno
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et al. 2012; Labrie et al. 2010). Thus, phages rapidly diversify their genes for the continual host-phage co-evolution in the presence of CRISPR–Cas system (Iranzo et al. 2013). Microcystis aeruginosa is a bloom-forming cyanobacterium from freshwater environments worldwide. Some of this species produce potent hepatotoxins called microcystins that are involved in many cases of animal and human poisonings (Jochimsen et al. 1998; Codd et al. 2005). Many studies show each M. aeruginosa bloom consist of genetically distinct populations that undergo temporal changes in genetic composition (Yoshida et al. 2008a; Moisander et al. 2009; Sabart et al. 2009; Gaevsky et al. 2011). Further, the M. aeruginosa NIES-843 genome contains a large number of phage defense systems, the largest among the bacteria and archaea (Makarova et al. 2011). Our recent study shows CRISPR spacers in M. aeruginosa are diversified (Kuno et al. 2012). This suggests diverse viral communities infect to M. aeruginosa and co-evolve with their hosts. Several studies suggest M. aeruginosa is attacked by morphologically and genetically diverse cyanophages (Tucker and Pollard 2005; Honjo et al. 2006; Hargreaves et al. 2013; Ou et al. 2013). However, detailed genomic information of Microcystis cyanophages has not been reported except Ma-LMM01. Ma-LMM01 is assigned as a member of a new lineage in the Myoviridae family and is a lytic cyanophage that specifically infects a toxic strain of M. aeruginosa (Yoshida et al. 2006, 2008b; Lavigne et al. 2009; Carstens 2010). Recently, clone analysis of a phage g91 gene shows that high frequency of clone sequences almost always co-occurred with their variants (one to two nucleotides mutants) (Kimura et al. 2013). This suggests that increased contact frequency with a host–phage population promotes rapid co-evolution and generates phage diversity (Kimura et al. 2013). Several studies show coevolutionary interactions can readily lead to local adaptation in phages, where the phages often have high infectivity to hosts from the same location rather than hosts from different locations (Brockhurst et al. 2007; Vos et al. 2009; Koskella et al. 2011). We hypothesized the genetic composition and gene diversity of Ma-LMM01-type phages differs in each location. Ma-LMM01 genome harbors nrdA and nrdB coding for the alpha- and beta-subunits of ribonucleotide reductase, an essential enzyme that generates precursors of DNA by converting ribonucleotides to deoxyribonucleotides (Yoshida et al. 2006, 2008b). The nrdA and nrdB genes are often found in double-stranded DNA phages and thus are recognized as core genes (Sullivan et al. 2005; Ignacio-Espinoza and Sullivan 2012; Dwivedi et al. 2013). The intergenetic region between nrdA and nrdB in Ma-LMM01 contains nblA that is involved in degradation of the phycobilisome and is an example of host-derived auxiliary metabolic genes (AMGs) (Yoshida et al. 2006, 2008b; Sanmukh et al.
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2012). Here, to determine whether the gene diversification of phages leads to locality in phage genome, we investigated the genetic diversity of Ma-LMM01-type phages by sequencing the phage intergenetic region between nrdA and nrdB from two geographically distant natural freshwater environments.
Materials and methods Sampling sites and sample collection Surface water samples were collected from Hirosawanoike Pond (Kyoto, Japan) (35°26′N, 135°690′E) on Oct 21, 2009, Sep 13, 2010 and Sep 15, 2011. Also, a water sample was collected from Lake Shinji (Shimane, Japan) (35°28′N, 133°1′E) on Oct 31, 2011. Microcysits blooms have occurred in the lake since 2010. To concentrate the phage particles, 10 mL of samples water was filtered through a 0.2-μm polycarbonate filter and ultracentrifuged at 111,000 × g for 1.5 h at 4 °C as previously described (Takashima et al. 2007). The pellets was re-suspended in 200 μL sterilized milliQ water and stored at −80 °C. DNA extraction DNA was extracted from the phage pellets using the xanthogenate-SDS method as previously described (Takashima et al. 2007). To avoid contamination with dissolved DNA, the stored pellets were treated with mixture contained 10 U of Turbo DNase (Ambion, Austin, TX) and 1 × DNase buffer (final concentration) at 37 °C for 1 h before DNA extraction. Purified DNA was suspended in 20 μL sterilized milliQ water. Each DNA extract was used as a PCR template. Primer design, PCR, cloning and sequencing We designed a degenerate primer set (Ma-nrdA3/nrdB3) for this study (Fig. 1). As no strain closely related to MaLMM01 has been isolated, the degenerated primer set was designed based on Ma-LMM01 and 24 sequences (18 sequences from Hirosawanoike Pond and six sequences from Lake Shinji) obtained using PCR products with the primer sets Ma-nrdA1 (5′-CAGGCCAGTGTAGAACTAGCAGAG-3′) and Ma-nrdB1 (5′-GTCTACGCTTCTGTGGGCTATC-3′) designed based on corresponding environmental sequences of Ma-LMM01. This strategy is detailed in our previous paper (Kimura et al. 2013). PCR amplification with the degenerate primer sets ManrdA3 (5′-GGTCAGMRATAATAGCRCCGAC-3′) and Ma-nrdB3 (5′-GTARCGGTA ACACGCGGGTCT-3′) was performed using an i Cycler (Bio-Rad, Hercules, CA).
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403
4.9 kbp Ma-nrdA1
2.6 kbp
Ma-nrdA3
2.4 kbp
Ma-nrdB1 Ma-nrdB3
5’
3’ nrdA (2228)
nblA g04 (560) (239)
g03 (794)
nrdB (1046)
Fig. 1 Design of primer sets Ma-nrdA3 and Ma-nrdB3 for clonal analysis of the nrdA–nrdB intergenetic region of Ma-LMM01-type phages. Based on sequences obtained using a combination of Ma-
nrdA1 and Ma-nrdB1 PCR products from environmental samples. Gray shades represent non-coding regions. The numbers in parentheses indicate the number of base pairs
The reaction conditions were 2 min of initial denaturing at 94 °C followed by 30 cycles: 94 °C for 30 s, 60 °C for 30 s and 72 °C for 2.5 min. and a final extension at 72 °C for 7 min. The 25 μL reaction mixture contained 10 × Ex Taq Buffer (TaKaRa Bio Inc., Shiga, Japan), 200 μM each dNTP mix, 10 μM primers, 2.5 U of Ex Taq polymerase (TaKaRa Bio Inc.) and 1 μL of each DNA template (q.s. to 25 μl). The PCR products (25 μl) were electrophoresed on a 1.0 % (wt/vol) agarose gel in 1 × TAE buffer and stained with GelRed (Biotium, Hayward, CA). The gel image was captured and analyzed with the Gel Doc XR system (BioRad Laboratories). Visually confirmed bands were purified with a Wizard Miniprep Purification Kit (Promega, Madison, WI) according to the manufacturer’s instructions. The purified PCR products were cloned into the pTAC-1 vector (BioDynamics Laboratory Inc., Tokyo, Japan) and then transformed into E. coli DH5α-competent cells according to the manufacturer’s instructions. Positive clones containing an insert of the correct size from each clone library were verified using colony PCR. The plasmids were extracted using a Mini Plus™ Plasmid DNA Extraction System (VIOGENE, Taipei, Taiwan) from positive clones. Sequence templates were amplified by PCR using the commercial primers M13 BDFw and M13 BDRev for the pTAC-1 vector. The sequencing was performed using a 3130 Genetic Analyzer (Applied Biosystems) with a BigDye Terminator v3.1 Cycle Sequencing Kit according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). The sequences obtained were aligned using MEGA5 (Tamura et al. 2011) where the primer sequences were removed from all sequences.
the number of genotypes appearing only once). Maximum parsimony network analysis was performed using the statistical parsimony program TCS v1.21 (Clement et al. 2000). According to the estimated Ex Taq error rate (a single error in 5.0 × 103 nucleotides), approximately one in the 3.3 amplified nrdA–B sequences is expected to contain a single PCR error. However, phages can abolish CRISPRmediated immunity for a single mutation in the protospacers (Labrie et al. 2010). Thus, we evaluated the genetic diversity using 100 % nucleotide identity due to detection of a single mutation. Other studies also use same identity for genetic diversity analysis of phage or virus (Bellec et al. 2010; Kimura et al. 2013).
Sequence analysis The Shannon index and Chao1 index were estimated for the obtained sequences using EstimateS v8.2.0 (Colwell 2004). Coverage index (C) was calculated as C = (1 − n/N) × 100 (where N is the number of sequences in each sample, n is
Nucleotide sequence The nucleotide sequences determined in this study are deposited in the DDBJ/EMBL/GenBank database. The accession numbers are AB812896-AB812999 and AB818405-AB818452.
Results Characterization of nrdA–B intergenetic region sequences from natural freshwater environments In all, 104 nrdA–B intergenetic region sequences (approximately 2.4 kbp, 24–27 sequences from each sample) were obtained. The sequences obtained showed significant similarities to the corresponding regions of Ma-LMM01 when searched against the NCBI non-redundant protein sequence database using BLASTx (data not shown). The gene order and contents of Ma-LMM01 (Fig. 1: nblA, g04, g03 and two non-coding regions) were conserved in all sequences. The nblA from those sequences possessed highly conserved motifs. LRLEQ (N terminus) is involved in the attachment to ClpC which is an HSP100 chaperone partner of
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Fig. 2 Alignment of deduced amino acid sequences on the gene encoding the nblA. PCR products from the Hirosawanoike Pond samples taken on Oct 2009, Sep 2010 and Sep 2011 and Lake Shinji samples on Oct 2011 were sequenced after the construction of a clone library. Among 104 sequences obtained, 48 clones belong to type 1, three clones to type 2, eight clones to type 3, five clones to type 4, 27 clones to type 5 and others to type 6–19. PaV-LD (ADZ31529); PCC7120, Nostoc sp. [Chr, chromosome (BAB76216); PD, plasmid Delta (BAB77423)]; PCC73102, Nostoc punctiforme (ZP00345234);
PCC7942, Synechococcus sp. (ABB58157); PCC6803-1, Synechocystis sp. (NP_441275); PCC6803-2, Synechocystis sp.(NP_441274); PORPU, Porphyra purpurea (NP053976); AGLNE, Aglaothamnion neglectum (P48446); CYACA, Cyanidium caldarium (NP045072); and CYAME, Cyanidoschyzon merolae (BAC76137). The conserved motifs are shown in boxes. They contain the highly conserved motifs LRLEQ motif and two amino acid residues (Ile50 and Lys52) (Yoshida et al. 2008b; Yoshida-Takashima et al. 2012)
the Clp protease. Ile50and Lys52 (C terminus) is involved in the attachment to phycobiliproteins (Fig. 2) (Yoshida et al. 2008b; Yoshida-Takashima et al. 2012). The g04 sequences have no detectable homologs in the databases. The g03 sequences show weak sequence similarity to the DraRnl subfamily RNA ligase of Aeromonas phage (Sanmukh et al. 2012) (E value: 7 × 10−5). Except for Ma-LMM01, we could not find the genome contents on double-stranded DNA phage genomes (608 genomes completely sequenced) (data not shown).
and Lake Shinji (Table 1). The coverage value from Hirosawanoike Pond was 4.17–22.2 %. Conversely, coverage from Lake Shinji was 46.2 %. Similarly, the Shannon and Chao1 indexes from Hirosawanoike Pond (Shannon index 2.9–3.1; Chao1 index 55–162) were higher than those from Lake Shinji (Shannon index 2.1; Chao1 index 42). The genetic diversity varied by genes. The Shannon and Chao1 indexes of g03 and g04 were higher (Shannon index of g03, 1.7–2.6; Chao1 index of g03, 15–60; Shannon index of g04, 1.4–2.5; Chao1 index of g04, 10–55) than those of nblA (Shannon index, 0–1.4; Chao1 index, 2–23) (Table 1). Thus, the high divergence of the nrdA–B intergenetic region is largely caused by the diversification of g03 and g04. To determine the relationships between the nrdA–B sequences including the Ma-LMM01 sequence, we conducted a maximum parsimony network analysis with a 95 % parsimony connection limit (Fig. 3). This network showed the genotypes were largely divided into two sequence groups: group 1 (52 genotypes, 67 sequences)
Genetic diversity of Ma‑LMM01‑type phages in the two different freshwater environments The 104 nrdA–B sequences (concatenated sequence of nblA, g04, g03 and non-coding regions) were assigned to 81 genotypes (GT1–GT81) clustered at 100 % nucleotide sequence similarity. We determined the genetic diversity within the nrdA–B sequences from Hirosawanoike Pond
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Table 1 Genetic diversity of the nrdA–B sequences from the Ma-LMM01-type phages Samples
No. of sequences
Hirosawanoiek Pond (2009 Oct.) Concatenated sequence 27 nblA g04 g03 Hirosawanoike Pond (2010 Sep.) Concatenated sequence 27 nblA g04 g03 Hirosawanoike Pond (2011 Sep.) Concatenated sequence 24 nblA g04 g03 Lake Shinji (2011 Oct.) Concatenated sequence nblA
26
No. of genotypes
Coverage (%)
Shannon index
21 8
22 70
2.9 1.3
Chao1 index
55 (31–135) 23 (11–76)
11
59
1.7
47 (21–137)
18
33
2.6
53 (28–144)
25 8
7 70
3.1 1.3
162 (87–327) 23 (11–76)
16
41
2.5
38 (22–103)
22
19
3.0
60 (34–148)
23 8
4 67
3.1 1.4
127 (53–372) 13 (9–40)
16
33
2.5
55 (26–166)
16
33
2.6
38 (22–103)
14 2
46 92
2.1 0.0
42 (21–127) 2 (2–2)
g04
8
69
1.4
10 (8–20)
g03
10
62
1.7
15 (10–39)
Fig. 3 Maximum parsimony network performed using the TCS v1.21 program (Clement et al. 2000) for the sequence of nrdA–B intergenic region of Ma-LMM01 phages. One hundred and four sequences generated from the samples taken from Hirosawanoike Pond (Hirosawanoike) and Lake Shinji were used. Group 1 and 2 indicate sequences divided with 95 % parsimony connection limits. The unassigned group included sequences that did not belong to group 1 or 2. The number of nucleotide differences between the two genotypes in the sum of steps for shortest connecting path, the number beside the line, the intervening genotypes and junction nodes (small black circles)
and group 2 (14 genotypes, 26 sequences). An unassigned group formed sequences (11 genotypes, 11 sequences) that did not belong to group 1 or 2 (Fig. 3). Group 1 and
2 were dominated by GT1 (nine sequences) and by GT80 (ten sequences), respectively (Fig. 3). Group 1 and 2 consisted of sequences from Hirosawanoike Pond and Lake
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Hirosawanoike Pond and Ma-LMM01
Lake Shinji
Fig. 4 A maximum-likelihood tree of 104 nrdA–B sequences of MaLMM01-type phages and Ma-LMM01 sequence. Black boxes, black boxes included dots, and stripe box indicates group 1, group 2 and
unassigned group, respectively. Bootstrap values are indicated at the nodes with more than 50 % bootstrap support
Shinji, respectively. Comparing the sequences of representatives from each group (GT1, Group1; GT80, Group2; MaLMM01 and the unassigned group), nucleotide differences
between each pair were 136 (5.7 %, GT1 and GT80), 104 (4.4 %, GT1 and Ma-LMM01) and 106 (4.4 %, GT80 and Ma-LMM01). In addition, the phylogenic analysis of the
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nrdA–B sequences revealed that the group 1 and group 2 were distantly related to each other (Fig. 4). We also investigated mutation sites and frequency of mutations in the nrdA–B sequences (Fig. 5). The mutations in the nrdA–B sequences were mapped randomly and were not biased in non-coding regions. Many of the mutation sites in the sequences from Hirosawanoike Pond were different from those of Lake Shinji. Further, we detected high mutation frequency at the 12 mutation sites in the nrdA– B sequences from Hirosawanoike Pond (positions 56, 136, 424, 561, 619, 834, 881, 884, 943, 1,083, 1,178 and 1,265; mutation frequency 0.21–0.79) (Fig. 5). Of these, six sites (positions 881, 884, 943, 1,083, 1,178 and 1,265) were located in N-terminal sites and were not conserved motifs for g03 (Fig. 5). All of the high frequency mutation sites had non-synonymous substitutions. We also could not detect high frequency mutation sites in functional motifs of the genes and in the non-coding regions (Fig. 5).
Discussion Our data shown the genetic diversity of the sequences obtained was high; nevertheless, all the sequences from natural environments were conserved in Ma-LMM01 gene order and content (nblA, g04, g03 and two non-coding regions). This suggests the sequences obtained are derived
Fig. 5 Frequency and distribution of mutations in nrdA–B sequences. Each mutation is represented by a vertical black line with the height representing the observed frequency. The position of the vertical line along the horizontal axis gives its genomic location. Functional
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from Ma-LMM01-type phages and these genes may be necessary to propagation of Ma-LMM01-type phages. However, the genetic diversity varied by genes (Table 1). In all the samples, genetic diversity of nblA was lowest among the three genes. The phages often acquire metabolic or functional genes from their hosts (AMG, see introduction) (Lindell et al. 2004; Sullivan et al. 2005). Phage NblA is considered to be important to prevent photoinhibition and to maintain photosynthetic activity even after decline of the host’s photosynthesis gene expression during infection and replication (Yoshida et al. 2008b; Gao et al. 2012; YoshidaTakashima et al. 2012). Accordingly, low genetic diversity of nblA may suggest the nblA is a highly significant gene that does not allow mutation. Genetic diversity of g03 (RNA ligase) was higher than nblA (Table 1). This high genetic diversity was caused by many point mutations at the N-terminal site. Some of these mutations show high frequency and all of the high frequency of mutation sites had non-synonymous substitutions (Fig. 5). If the mutations derived from only PCR error, substitutions would occur randomly in genes. However, we observed many high frequencies of mutations in N-terminal site. Thus, these substitutions are likely to occur for co-evolution with their hosts. In general, RNA ligase participates in repair and editing pathways that reseal broken RNAs or alter their primary structure (Ho et al. 2004). Bacteriophage T4 has developed a
motifs are represented by a lattice (ClpC-binding site), stripes (phycobilisome binding site) and gray (nucleotidyltransferase motifs). HP, Hirosawanoike Pond; LS, Lake Shinji
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counter-defense mechanism using RNA ligases and polynucleotide kinase to repair breaks in the host tRNA anticodon loop (Abi system) (El Omari et al. 2006; Labrie et al. 2010). The ligation activity of DraRNA ligase subfamily varies by non-synonymous mutations at N-terminal sites (Raymond and Shuman 2007). Therefore, when variation of ligation activity by mutations (e.g., non-synonymous mutations of high frequency at the N-terminal site) is advantageous to phage populations for propagation, mutations may be fixed in Ma-LMM01-type phage populations. Logically, g04 is possibly involved in the counterphage defense system because of its mutation frequency. Previous study suggests random distribution of mutations in g91 of Ma-LMM01-type phages is driven by the host CRISPR–Cas system (Kimura et al. 2013). In addition to CRISPR, the finding in this study (i.e., high mutation frequency in g03 and g04) suggests that the phage defense system (e.g. Abi system) may also play an important role in rapid gene diversification in phage genome. Phages depend on the host cellular machinery to propagate and are exposed the host defense systems during the infection, that is, phages diversify their genes in order to escape from selective pressure created by host defense systems (Kimura et al. 2013; Labrie et al. 2010; Marston et al. 2012). Ma-LMM01-type phages which include mutation in g03 and other genes may be able to escape from selective pressure of host. Solo exceptional example has been reported; mutations of functional domain in most of the other noroviral sequences are induced by selective pressure, though mutations of non-functional domain in some noroviral sequences may be occurred by genetic drift (Iritani et al. 2008). Further, Genetic diversity of Ma-LMM01-type phages in Hirosawanoike Pond was higher than in Lake Shinji (Table 1). A recent study using Synechococcus and its infecting phage demonstrated antagonistic co-evolution in chemostats where the host–phage co-culture leads to increased mutation of phage gene over time (Marston et al. 2012). Microcysits blooms have occurred in Hirosawainoike Pond for several decades (Yamamoto and Nakahara 2009) while in Lake Shinji since 2010 (4 years). Therefore, genetic diversity of Ma-LMM01-type phages may be derived from periods of co-evolution. Several model studies suggest these coevolutionary interactions can readily lead to phage local adaptation (Brockhurst et al. 2007; Koskella et al. 2011; Vos et al. 2009). These findings suggest Ma-LMM01-type phages rapidly diversify their genomes through co-evolution with hosts in each location and eventually provided locality of their genomes. Acknowledgments This study was partially supported by Grant-inAid for Scientific Research (B) (No. 20310045). We thank Takahiro Miyazako for sampling help.
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409 Sullivan MB, Coleman ML, Weigele P, Rohwer F, Chisholm SW (2005) Three Prochlorococcus cyanophage genomes: signature features and ecological interpretations. PLoS Biol 3:790–806 Suttle CA (2005) Viruses in the sea. Nature 437:356–361 Suttle CA (2007) Marine viruses: major players in the global ecosystem. Nat Rev Microbiol 5:801–812 Takashima Y, Yoshida T, Yoshida M, Shirai Y, Tomaru Y, Takao Y, Hiroishi S, Nagasaki K (2007) Development and application of quantitative detection of cyanophages phylogenetically related to cyanophage Ma-LMM01 infecting Microcystis aeruginosa in fresh water. Microbes Environ 22:207–213 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739 Tucker S, Pollard P (2005) Identification of cyanophage Ma-LBP and infection of the cyanobacterium Microcystis aeruginosa from an Australian subtropical lake by the virus. Appl Environ Microbiol 71:629–635 Vos M, Birkett PJ, Birch E, Griffiths RI, Buckling A (2009) Local adaptation of bacteriophages to their bacterial hosts in soil. Science 325:833–833 Yamamoto Y, Nakahara H (2009) Seasonal variations in the morphology of bloom-forming cyanobacteria in a eutrophic pond. Limnology 10:185–193 Yoshida T, Takashima Y, Tomaru Y, Shirai Y, Takao Y, Hiroishi S, Nagasaki K (2006) Isolation and characterization of a cyanophage infecting the toxic cyanobacterium Microcystis aeruginosa. Appl Environ Microbiol 72:1239–1247 Yoshida M, Yoshida T, Kashima A, Takashima Y, Hosoda N, Nagasaki K, Hiroishi S (2008a) Ecological dynamics of the toxic bloomforming cyanobacterium Microcystis aeruginosa and its cyanophages in freshwater. Appl Environ Microbiol 74:3269–3273 Yoshida T, Nagasaki K, Takashima Y, Shirai Y, Tomaru Y, Takao Y, Sakamoto S, Hiroishi S, Ogata H (2008b) Ma-LMM01 infecting toxic Microcystis aeruginosa illuminates diverse cyanophage genome strategies. J Bacteriol 190:1762–1772 Yoshida-Takashima Y, Yoshida M, Ogata H, Nagasaki K, Hiroishi S, Yoshida T (2012) Cyanophage infection in the bloom-forming cyanobacteria Microcystis aeruginosa in surface freshwater. Microbes Environ 27:350–355
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Original Paper
Genetic diversity of Microcystis cyanophages in two different freshwater environments Ginji Nakamura · Shigeko Kimura · Yoshihiko Sako · Takashi Yoshida
Received: 28 February 2014 / Revised: 13 March 2014 / Accepted: 15 March 2014 / Published online: 27 March 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Bacteriophages rapidly diversify their genes through co-evolution with their hosts. We hypothesize that gene diversification of phages leads to locality in phages genome. To test this hypothesis, we investigated the genetic diversity and composition of Microcystis cyanophages using 104 sequences of Ma-LMM01-type cyanophages from two geographically distant sampling sites. The intergenetic region between the ribonucleotide reductase genes nrdA and nrdB was used as the genetic marker. This region contains the host-derived auxiliary metabolic genes nblA, an unknown function gene g04, and RNA ligase gene g03. The sequences obtained were conserved in the Ma-LMM01 gene order and contents. Although the genetic diversity of the sequences was high, it varied by gene. The genetic diversity of nblA was the lowest, suggesting that nblA is a highly significant gene that does not allow mutation. In contrast, g03 sequences had many point mutations. RNA ligase is involved in the counter-host’s phage defense mechanism, suggesting that phage defense also plays an important role for rapid gene diversification. The maximum parsimony network and phylogenic analysis showed the sequences from the two sampling sites were distinct. These findings suggest Ma-LMM01-type phages rapidly diversify their genomes through co-evolution with hosts in each location and eventually provided locality of their genomes.
Communicated by Erko Stackebrandt. G. Nakamura · S. Kimura · Y. Sako · T. Yoshida (*) Laboratory of Marine Microbiology, Graduate School of Agriculture, Kyoto University, Kitashirakawa‑Oiwake, Sakyo‑ku, Kyoto 606‑8502, Japan e-mail: [email protected]‑u.ac.jp
Keywords Cyanobacteria · Microcystis aeruginosa · Ma-LMM01 · Cyanophage · Co-evolution
Introduction Bacteriophages (phages) are abundant in marine and freshwater ecosystems (Suttle 2005). They infect bacterial hosts and eventually contribute to biogeochemical cycles (Suttle 2005, 2007) and may be directly involved in the genetic diversity of bacteria mediating horizontal gene transfer (Lindell et al. 2004). In addition, phages also affect genetic diversity of hosts via two ways: (1) frequency-dependent selection; host-selective phage infection influences and maintains bacterial diversity through a mechanism where phage infection checks bacterial populations that become dominant in populations (e.g., constant-diversity dynamics) and thereby enables coexistence of multiple bacterial host and phage populations (Rodriguez-Valera et al. 2009); (2) co-evolution (the continual evolution of host defense system and phages counter-defense); a hosts have various phage defense systems, such as the clustered regularly interspaced short palindromic repeats (CRISPR)–Cas (CRISPR-associated genes) systems, the toxin–antitoxin (TA) systems, the classical restriction–modification (RM) systems and the abortive infection (Abi) (Labrie et al. 2010; Marston et al. 2012). These defense systems are thought to promote the high evolution rate of phage genes through overcoming of defense systems by phages (Labrie et al. 2010). Among defense systems, the CRISPR–Cas system provides adaptive immunity in prokaryotes against phages and plasmids via incorporation of short sequences called spacers (Kuno et al. 2012; Labrie et al. 2010). A single mutation the origin of the spacers (protospacers) can abolish CRISPR-mediated host immunity against phages (Kuno
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et al. 2012; Labrie et al. 2010). Thus, phages rapidly diversify their genes for the continual host-phage co-evolution in the presence of CRISPR–Cas system (Iranzo et al. 2013). Microcystis aeruginosa is a bloom-forming cyanobacterium from freshwater environments worldwide. Some of this species produce potent hepatotoxins called microcystins that are involved in many cases of animal and human poisonings (Jochimsen et al. 1998; Codd et al. 2005). Many studies show each M. aeruginosa bloom consist of genetically distinct populations that undergo temporal changes in genetic composition (Yoshida et al. 2008a; Moisander et al. 2009; Sabart et al. 2009; Gaevsky et al. 2011). Further, the M. aeruginosa NIES-843 genome contains a large number of phage defense systems, the largest among the bacteria and archaea (Makarova et al. 2011). Our recent study shows CRISPR spacers in M. aeruginosa are diversified (Kuno et al. 2012). This suggests diverse viral communities infect to M. aeruginosa and co-evolve with their hosts. Several studies suggest M. aeruginosa is attacked by morphologically and genetically diverse cyanophages (Tucker and Pollard 2005; Honjo et al. 2006; Hargreaves et al. 2013; Ou et al. 2013). However, detailed genomic information of Microcystis cyanophages has not been reported except Ma-LMM01. Ma-LMM01 is assigned as a member of a new lineage in the Myoviridae family and is a lytic cyanophage that specifically infects a toxic strain of M. aeruginosa (Yoshida et al. 2006, 2008b; Lavigne et al. 2009; Carstens 2010). Recently, clone analysis of a phage g91 gene shows that high frequency of clone sequences almost always co-occurred with their variants (one to two nucleotides mutants) (Kimura et al. 2013). This suggests that increased contact frequency with a host–phage population promotes rapid co-evolution and generates phage diversity (Kimura et al. 2013). Several studies show coevolutionary interactions can readily lead to local adaptation in phages, where the phages often have high infectivity to hosts from the same location rather than hosts from different locations (Brockhurst et al. 2007; Vos et al. 2009; Koskella et al. 2011). We hypothesized the genetic composition and gene diversity of Ma-LMM01-type phages differs in each location. Ma-LMM01 genome harbors nrdA and nrdB coding for the alpha- and beta-subunits of ribonucleotide reductase, an essential enzyme that generates precursors of DNA by converting ribonucleotides to deoxyribonucleotides (Yoshida et al. 2006, 2008b). The nrdA and nrdB genes are often found in double-stranded DNA phages and thus are recognized as core genes (Sullivan et al. 2005; Ignacio-Espinoza and Sullivan 2012; Dwivedi et al. 2013). The intergenetic region between nrdA and nrdB in Ma-LMM01 contains nblA that is involved in degradation of the phycobilisome and is an example of host-derived auxiliary metabolic genes (AMGs) (Yoshida et al. 2006, 2008b; Sanmukh et al.
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2012). Here, to determine whether the gene diversification of phages leads to locality in phage genome, we investigated the genetic diversity of Ma-LMM01-type phages by sequencing the phage intergenetic region between nrdA and nrdB from two geographically distant natural freshwater environments.
Materials and methods Sampling sites and sample collection Surface water samples were collected from Hirosawanoike Pond (Kyoto, Japan) (35°26′N, 135°690′E) on Oct 21, 2009, Sep 13, 2010 and Sep 15, 2011. Also, a water sample was collected from Lake Shinji (Shimane, Japan) (35°28′N, 133°1′E) on Oct 31, 2011. Microcysits blooms have occurred in the lake since 2010. To concentrate the phage particles, 10 mL of samples water was filtered through a 0.2-μm polycarbonate filter and ultracentrifuged at 111,000 × g for 1.5 h at 4 °C as previously described (Takashima et al. 2007). The pellets was re-suspended in 200 μL sterilized milliQ water and stored at −80 °C. DNA extraction DNA was extracted from the phage pellets using the xanthogenate-SDS method as previously described (Takashima et al. 2007). To avoid contamination with dissolved DNA, the stored pellets were treated with mixture contained 10 U of Turbo DNase (Ambion, Austin, TX) and 1 × DNase buffer (final concentration) at 37 °C for 1 h before DNA extraction. Purified DNA was suspended in 20 μL sterilized milliQ water. Each DNA extract was used as a PCR template. Primer design, PCR, cloning and sequencing We designed a degenerate primer set (Ma-nrdA3/nrdB3) for this study (Fig. 1). As no strain closely related to MaLMM01 has been isolated, the degenerated primer set was designed based on Ma-LMM01 and 24 sequences (18 sequences from Hirosawanoike Pond and six sequences from Lake Shinji) obtained using PCR products with the primer sets Ma-nrdA1 (5′-CAGGCCAGTGTAGAACTAGCAGAG-3′) and Ma-nrdB1 (5′-GTCTACGCTTCTGTGGGCTATC-3′) designed based on corresponding environmental sequences of Ma-LMM01. This strategy is detailed in our previous paper (Kimura et al. 2013). PCR amplification with the degenerate primer sets ManrdA3 (5′-GGTCAGMRATAATAGCRCCGAC-3′) and Ma-nrdB3 (5′-GTARCGGTA ACACGCGGGTCT-3′) was performed using an i Cycler (Bio-Rad, Hercules, CA).
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403
4.9 kbp Ma-nrdA1
2.6 kbp
Ma-nrdA3
2.4 kbp
Ma-nrdB1 Ma-nrdB3
5’
3’ nrdA (2228)
nblA g04 (560) (239)
g03 (794)
nrdB (1046)
Fig. 1 Design of primer sets Ma-nrdA3 and Ma-nrdB3 for clonal analysis of the nrdA–nrdB intergenetic region of Ma-LMM01-type phages. Based on sequences obtained using a combination of Ma-
nrdA1 and Ma-nrdB1 PCR products from environmental samples. Gray shades represent non-coding regions. The numbers in parentheses indicate the number of base pairs
The reaction conditions were 2 min of initial denaturing at 94 °C followed by 30 cycles: 94 °C for 30 s, 60 °C for 30 s and 72 °C for 2.5 min. and a final extension at 72 °C for 7 min. The 25 μL reaction mixture contained 10 × Ex Taq Buffer (TaKaRa Bio Inc., Shiga, Japan), 200 μM each dNTP mix, 10 μM primers, 2.5 U of Ex Taq polymerase (TaKaRa Bio Inc.) and 1 μL of each DNA template (q.s. to 25 μl). The PCR products (25 μl) were electrophoresed on a 1.0 % (wt/vol) agarose gel in 1 × TAE buffer and stained with GelRed (Biotium, Hayward, CA). The gel image was captured and analyzed with the Gel Doc XR system (BioRad Laboratories). Visually confirmed bands were purified with a Wizard Miniprep Purification Kit (Promega, Madison, WI) according to the manufacturer’s instructions. The purified PCR products were cloned into the pTAC-1 vector (BioDynamics Laboratory Inc., Tokyo, Japan) and then transformed into E. coli DH5α-competent cells according to the manufacturer’s instructions. Positive clones containing an insert of the correct size from each clone library were verified using colony PCR. The plasmids were extracted using a Mini Plus™ Plasmid DNA Extraction System (VIOGENE, Taipei, Taiwan) from positive clones. Sequence templates were amplified by PCR using the commercial primers M13 BDFw and M13 BDRev for the pTAC-1 vector. The sequencing was performed using a 3130 Genetic Analyzer (Applied Biosystems) with a BigDye Terminator v3.1 Cycle Sequencing Kit according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). The sequences obtained were aligned using MEGA5 (Tamura et al. 2011) where the primer sequences were removed from all sequences.
the number of genotypes appearing only once). Maximum parsimony network analysis was performed using the statistical parsimony program TCS v1.21 (Clement et al. 2000). According to the estimated Ex Taq error rate (a single error in 5.0 × 103 nucleotides), approximately one in the 3.3 amplified nrdA–B sequences is expected to contain a single PCR error. However, phages can abolish CRISPRmediated immunity for a single mutation in the protospacers (Labrie et al. 2010). Thus, we evaluated the genetic diversity using 100 % nucleotide identity due to detection of a single mutation. Other studies also use same identity for genetic diversity analysis of phage or virus (Bellec et al. 2010; Kimura et al. 2013).
Sequence analysis The Shannon index and Chao1 index were estimated for the obtained sequences using EstimateS v8.2.0 (Colwell 2004). Coverage index (C) was calculated as C = (1 − n/N) × 100 (where N is the number of sequences in each sample, n is
Nucleotide sequence The nucleotide sequences determined in this study are deposited in the DDBJ/EMBL/GenBank database. The accession numbers are AB812896-AB812999 and AB818405-AB818452.
Results Characterization of nrdA–B intergenetic region sequences from natural freshwater environments In all, 104 nrdA–B intergenetic region sequences (approximately 2.4 kbp, 24–27 sequences from each sample) were obtained. The sequences obtained showed significant similarities to the corresponding regions of Ma-LMM01 when searched against the NCBI non-redundant protein sequence database using BLASTx (data not shown). The gene order and contents of Ma-LMM01 (Fig. 1: nblA, g04, g03 and two non-coding regions) were conserved in all sequences. The nblA from those sequences possessed highly conserved motifs. LRLEQ (N terminus) is involved in the attachment to ClpC which is an HSP100 chaperone partner of
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Fig. 2 Alignment of deduced amino acid sequences on the gene encoding the nblA. PCR products from the Hirosawanoike Pond samples taken on Oct 2009, Sep 2010 and Sep 2011 and Lake Shinji samples on Oct 2011 were sequenced after the construction of a clone library. Among 104 sequences obtained, 48 clones belong to type 1, three clones to type 2, eight clones to type 3, five clones to type 4, 27 clones to type 5 and others to type 6–19. PaV-LD (ADZ31529); PCC7120, Nostoc sp. [Chr, chromosome (BAB76216); PD, plasmid Delta (BAB77423)]; PCC73102, Nostoc punctiforme (ZP00345234);
PCC7942, Synechococcus sp. (ABB58157); PCC6803-1, Synechocystis sp. (NP_441275); PCC6803-2, Synechocystis sp.(NP_441274); PORPU, Porphyra purpurea (NP053976); AGLNE, Aglaothamnion neglectum (P48446); CYACA, Cyanidium caldarium (NP045072); and CYAME, Cyanidoschyzon merolae (BAC76137). The conserved motifs are shown in boxes. They contain the highly conserved motifs LRLEQ motif and two amino acid residues (Ile50 and Lys52) (Yoshida et al. 2008b; Yoshida-Takashima et al. 2012)
the Clp protease. Ile50and Lys52 (C terminus) is involved in the attachment to phycobiliproteins (Fig. 2) (Yoshida et al. 2008b; Yoshida-Takashima et al. 2012). The g04 sequences have no detectable homologs in the databases. The g03 sequences show weak sequence similarity to the DraRnl subfamily RNA ligase of Aeromonas phage (Sanmukh et al. 2012) (E value: 7 × 10−5). Except for Ma-LMM01, we could not find the genome contents on double-stranded DNA phage genomes (608 genomes completely sequenced) (data not shown).
and Lake Shinji (Table 1). The coverage value from Hirosawanoike Pond was 4.17–22.2 %. Conversely, coverage from Lake Shinji was 46.2 %. Similarly, the Shannon and Chao1 indexes from Hirosawanoike Pond (Shannon index 2.9–3.1; Chao1 index 55–162) were higher than those from Lake Shinji (Shannon index 2.1; Chao1 index 42). The genetic diversity varied by genes. The Shannon and Chao1 indexes of g03 and g04 were higher (Shannon index of g03, 1.7–2.6; Chao1 index of g03, 15–60; Shannon index of g04, 1.4–2.5; Chao1 index of g04, 10–55) than those of nblA (Shannon index, 0–1.4; Chao1 index, 2–23) (Table 1). Thus, the high divergence of the nrdA–B intergenetic region is largely caused by the diversification of g03 and g04. To determine the relationships between the nrdA–B sequences including the Ma-LMM01 sequence, we conducted a maximum parsimony network analysis with a 95 % parsimony connection limit (Fig. 3). This network showed the genotypes were largely divided into two sequence groups: group 1 (52 genotypes, 67 sequences)
Genetic diversity of Ma‑LMM01‑type phages in the two different freshwater environments The 104 nrdA–B sequences (concatenated sequence of nblA, g04, g03 and non-coding regions) were assigned to 81 genotypes (GT1–GT81) clustered at 100 % nucleotide sequence similarity. We determined the genetic diversity within the nrdA–B sequences from Hirosawanoike Pond
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Table 1 Genetic diversity of the nrdA–B sequences from the Ma-LMM01-type phages Samples
No. of sequences
Hirosawanoiek Pond (2009 Oct.) Concatenated sequence 27 nblA g04 g03 Hirosawanoike Pond (2010 Sep.) Concatenated sequence 27 nblA g04 g03 Hirosawanoike Pond (2011 Sep.) Concatenated sequence 24 nblA g04 g03 Lake Shinji (2011 Oct.) Concatenated sequence nblA
26
No. of genotypes
Coverage (%)
Shannon index
21 8
22 70
2.9 1.3
Chao1 index
55 (31–135) 23 (11–76)
11
59
1.7
47 (21–137)
18
33
2.6
53 (28–144)
25 8
7 70
3.1 1.3
162 (87–327) 23 (11–76)
16
41
2.5
38 (22–103)
22
19
3.0
60 (34–148)
23 8
4 67
3.1 1.4
127 (53–372) 13 (9–40)
16
33
2.5
55 (26–166)
16
33
2.6
38 (22–103)
14 2
46 92
2.1 0.0
42 (21–127) 2 (2–2)
g04
8
69
1.4
10 (8–20)
g03
10
62
1.7
15 (10–39)
Fig. 3 Maximum parsimony network performed using the TCS v1.21 program (Clement et al. 2000) for the sequence of nrdA–B intergenic region of Ma-LMM01 phages. One hundred and four sequences generated from the samples taken from Hirosawanoike Pond (Hirosawanoike) and Lake Shinji were used. Group 1 and 2 indicate sequences divided with 95 % parsimony connection limits. The unassigned group included sequences that did not belong to group 1 or 2. The number of nucleotide differences between the two genotypes in the sum of steps for shortest connecting path, the number beside the line, the intervening genotypes and junction nodes (small black circles)
and group 2 (14 genotypes, 26 sequences). An unassigned group formed sequences (11 genotypes, 11 sequences) that did not belong to group 1 or 2 (Fig. 3). Group 1 and
2 were dominated by GT1 (nine sequences) and by GT80 (ten sequences), respectively (Fig. 3). Group 1 and 2 consisted of sequences from Hirosawanoike Pond and Lake
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Hirosawanoike Pond and Ma-LMM01
Lake Shinji
Fig. 4 A maximum-likelihood tree of 104 nrdA–B sequences of MaLMM01-type phages and Ma-LMM01 sequence. Black boxes, black boxes included dots, and stripe box indicates group 1, group 2 and
unassigned group, respectively. Bootstrap values are indicated at the nodes with more than 50 % bootstrap support
Shinji, respectively. Comparing the sequences of representatives from each group (GT1, Group1; GT80, Group2; MaLMM01 and the unassigned group), nucleotide differences
between each pair were 136 (5.7 %, GT1 and GT80), 104 (4.4 %, GT1 and Ma-LMM01) and 106 (4.4 %, GT80 and Ma-LMM01). In addition, the phylogenic analysis of the
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nrdA–B sequences revealed that the group 1 and group 2 were distantly related to each other (Fig. 4). We also investigated mutation sites and frequency of mutations in the nrdA–B sequences (Fig. 5). The mutations in the nrdA–B sequences were mapped randomly and were not biased in non-coding regions. Many of the mutation sites in the sequences from Hirosawanoike Pond were different from those of Lake Shinji. Further, we detected high mutation frequency at the 12 mutation sites in the nrdA– B sequences from Hirosawanoike Pond (positions 56, 136, 424, 561, 619, 834, 881, 884, 943, 1,083, 1,178 and 1,265; mutation frequency 0.21–0.79) (Fig. 5). Of these, six sites (positions 881, 884, 943, 1,083, 1,178 and 1,265) were located in N-terminal sites and were not conserved motifs for g03 (Fig. 5). All of the high frequency mutation sites had non-synonymous substitutions. We also could not detect high frequency mutation sites in functional motifs of the genes and in the non-coding regions (Fig. 5).
Discussion Our data shown the genetic diversity of the sequences obtained was high; nevertheless, all the sequences from natural environments were conserved in Ma-LMM01 gene order and content (nblA, g04, g03 and two non-coding regions). This suggests the sequences obtained are derived
Fig. 5 Frequency and distribution of mutations in nrdA–B sequences. Each mutation is represented by a vertical black line with the height representing the observed frequency. The position of the vertical line along the horizontal axis gives its genomic location. Functional
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from Ma-LMM01-type phages and these genes may be necessary to propagation of Ma-LMM01-type phages. However, the genetic diversity varied by genes (Table 1). In all the samples, genetic diversity of nblA was lowest among the three genes. The phages often acquire metabolic or functional genes from their hosts (AMG, see introduction) (Lindell et al. 2004; Sullivan et al. 2005). Phage NblA is considered to be important to prevent photoinhibition and to maintain photosynthetic activity even after decline of the host’s photosynthesis gene expression during infection and replication (Yoshida et al. 2008b; Gao et al. 2012; YoshidaTakashima et al. 2012). Accordingly, low genetic diversity of nblA may suggest the nblA is a highly significant gene that does not allow mutation. Genetic diversity of g03 (RNA ligase) was higher than nblA (Table 1). This high genetic diversity was caused by many point mutations at the N-terminal site. Some of these mutations show high frequency and all of the high frequency of mutation sites had non-synonymous substitutions (Fig. 5). If the mutations derived from only PCR error, substitutions would occur randomly in genes. However, we observed many high frequencies of mutations in N-terminal site. Thus, these substitutions are likely to occur for co-evolution with their hosts. In general, RNA ligase participates in repair and editing pathways that reseal broken RNAs or alter their primary structure (Ho et al. 2004). Bacteriophage T4 has developed a
motifs are represented by a lattice (ClpC-binding site), stripes (phycobilisome binding site) and gray (nucleotidyltransferase motifs). HP, Hirosawanoike Pond; LS, Lake Shinji
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counter-defense mechanism using RNA ligases and polynucleotide kinase to repair breaks in the host tRNA anticodon loop (Abi system) (El Omari et al. 2006; Labrie et al. 2010). The ligation activity of DraRNA ligase subfamily varies by non-synonymous mutations at N-terminal sites (Raymond and Shuman 2007). Therefore, when variation of ligation activity by mutations (e.g., non-synonymous mutations of high frequency at the N-terminal site) is advantageous to phage populations for propagation, mutations may be fixed in Ma-LMM01-type phage populations. Logically, g04 is possibly involved in the counterphage defense system because of its mutation frequency. Previous study suggests random distribution of mutations in g91 of Ma-LMM01-type phages is driven by the host CRISPR–Cas system (Kimura et al. 2013). In addition to CRISPR, the finding in this study (i.e., high mutation frequency in g03 and g04) suggests that the phage defense system (e.g. Abi system) may also play an important role in rapid gene diversification in phage genome. Phages depend on the host cellular machinery to propagate and are exposed the host defense systems during the infection, that is, phages diversify their genes in order to escape from selective pressure created by host defense systems (Kimura et al. 2013; Labrie et al. 2010; Marston et al. 2012). Ma-LMM01-type phages which include mutation in g03 and other genes may be able to escape from selective pressure of host. Solo exceptional example has been reported; mutations of functional domain in most of the other noroviral sequences are induced by selective pressure, though mutations of non-functional domain in some noroviral sequences may be occurred by genetic drift (Iritani et al. 2008). Further, Genetic diversity of Ma-LMM01-type phages in Hirosawanoike Pond was higher than in Lake Shinji (Table 1). A recent study using Synechococcus and its infecting phage demonstrated antagonistic co-evolution in chemostats where the host–phage co-culture leads to increased mutation of phage gene over time (Marston et al. 2012). Microcysits blooms have occurred in Hirosawainoike Pond for several decades (Yamamoto and Nakahara 2009) while in Lake Shinji since 2010 (4 years). Therefore, genetic diversity of Ma-LMM01-type phages may be derived from periods of co-evolution. Several model studies suggest these coevolutionary interactions can readily lead to phage local adaptation (Brockhurst et al. 2007; Koskella et al. 2011; Vos et al. 2009). These findings suggest Ma-LMM01-type phages rapidly diversify their genomes through co-evolution with hosts in each location and eventually provided locality of their genomes. Acknowledgments This study was partially supported by Grant-inAid for Scientific Research (B) (No. 20310045). We thank Takahiro Miyazako for sampling help.
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