A Novel Freshwater Cyanophage Mae-Yong1326-1 Infecting Bloom-Forming Cyanobacterium Microcystis aeruginosa

Microcystis aeruginosa is a major harmful cyanobacterium causing water bloom worldwide. Cyanophage has been proposed as a promising tool for cyanobacterial bloom. In this study, M. aeruginosa FACHB-1326 was used as an indicator host to isolate cyanophage from Lake Taihu. The isolated Microcystis cyanophage Mae-Yong1326-1 has an elliptical head of about 47 nm in diameter and a slender flexible tail of about 340 nm in length. Mae-Yong1326-1 could lyse cyanobacterial strains across three orders (Chroococcales, Nostocales, and Oscillatoriales) in the host range experiments. Mae-Yong1326-1 was stable in stability tests, maintaining high titers at 0–40 °C and at a wide pH range of 3–12. Mae-Yong 1326-1 has a burst size of 329 PFU/cell, which is much larger than the reported Microcystis cyanophages so far. The complete genome of Mae-Yong1326-1 is a double-stranded DNA of 48, 822 bp, with a G + C content of 71.80% and long direct terminal repeats (DTR) of 366 bp, containing 57 predicted ORFs. No Mae-Yong1326-1 ORF was found to be associated with virulence factor or antibiotic resistance. PASC scanning illustrated that the highest nucleotide sequence similarity between Mae-Yong1326-1 and all known phages in databases was only 17.75%, less than 70% (the threshold to define a genus), which indicates that Mae-Yong1326-1 belongs to an unknown new genus. In the proteomic tree based on genome-wide sequence similarities, Mae-Yong1326-1 distantly clusters with three unclassified Microcystis cyanophages (MinS1, Mwe-Yong1112-1, and Mwes-Yong2). These four Microcystis cyanophages form a monophyletic clade, which separates at a node from the other clade formed by two independent families (Zierdtviridae and Orlajensenviridae) of Caudoviricetes class. We propose to establish a new family to harbor the Microcystis cyanophages Mae-Yong1326-1, MinS1, Mwe-Yong1112-1, and Mwes-Yong2. This study enriched the understanding of freshwater cyanophages.


Introduction
Cyanobacterial bloom is a disastrous ecological phenomenon in which plankton, especially cyanobacteria, proliferate abnormally and gather on the water surface, causing water discoloration [1]. Due to anthropogenic activities, global warming, and eutrophication, cyanobacteria harmful algal blooms (cyanoHABs) are becoming increasingly extensive and frequent. Cyanobacterial blooms have many negative effects. Cyanobacteria blooms reduce the water surface clarity and thus inhibit the growth of aquatic macrophytes; cyanobacterial blooms reduce the dissolved oxygen content of water, resulting in the death of aquatic organisms, including fish, crab, shrimp, etc. [2]. Furthermore, cyanobacterial blooms make water toxic, as many cyanobacteria produce highly toxic secondary metabolites known as "cyanotoxin". Cyanotoxins not only can notoriously cause liver and nervous system damage but also are immunotoxic, teratogenic, carcinogenic, and mutagenic [3][4][5][6][7][8][9]. Humans and animals can be exposed to cyanotoxins in various ways, such as through food, drink, inhalation, and dermal exposure during recreational activities. Therefore, it is urgent to solve the environmental problems caused by cyanobacterial bloom.
In this study, M. aeruginosa FACHB-1326 was used as an indicator host to isolate cyanophage from Lake Taihu. The general features (morphology, one-step growth curve, physicochemical stabilities, and host range) of the isolated Microcystis cyanophage Mae-Yong1326-1 were analyzed. The complete genome of the isolated Microcystis cyanophage Mae-Yong1326-1 was sequenced and analyzed.

Isolation and Purification of Cyanophage
Cyanophage isolation was carried out according to the reported method [15]. The surface water samples were collected from Lake Taihu (North latitude, 31.246,376; East longitude, 120.371,044), Suzhou, China on 1 July 2021. The water samples were centrifuged at 10,000× g for 20 min at 4 • C. The supernatant was successively filtered through 0.45 µm and 0.22 µm pore size nitrocellulose membrane. Each 80 mL filtrate was mixed with 20 mL of 5 × BG11 liquid medium and 20 mL logarithmic-phase M. aeruginosa FACHB-1326 (OD680 ≈ 0.738, 2.34 × 10 7 CFU/mL). In the control group, sterile water was substitute for the filtrate of water sample. The mixtures were cultured in a light incubator under a light/dark cycle of 12 h:12 h with a constant illumination of 30-40 µmol-photons/(m 2 × s) at 25 • C until yellowing (about seven days). Lysates were centrifuged at 10,000× g for 10 min, and the supernatant was cultured again with fresh FACHB-1326 (about 2 × 10 7 CFU/mL) until yellowing. Lysates were centrifuged at 10,000× g for 10 min. The supernatants were successively filtered through 0.45 µm and 0.22 µm pore size nitrocellulose filters. The filtrates were diluted (10 −1 -10 −9 ) with BG11. Each 100 µL of dilution was mixed with 900 µL of logarithmic-phase FACHB-1326 cultures and incubated at 25 • C for 30 min, then mixed quickly with 8 mL of molten BG11 agar medium (0.7% agar, pre-incubated at 42 • C), and poured into a BG11 agar plate (1.5% agar). Clear plaques emerged in 7-10 days. Unique plaque was suspended in 3 mL of logarithmic-phase FACHB-1326 cultures and subsequently used for a new round of plaque isolation. Five rounds were carried out until plaques show uniform shape and size.

Transmission Electron Microscopy (TEM)
The cyanophage lysates were centrifuged at 10,000× g for 10 min. The supernatants were centrifuged at 35,000× g for 60 min. The precipitates were cleaned twice with 0.01 M PBS, suspended in PBS, and then deposited on a carbon-coated copper grid for 5 min, negatively stained with 3% uranyl acetate for 25 s, and observed under TEM (Hitachi-7650, Japan) as described [21].

One-Step Growth Curve Experiment
Fresh logarithmic-phase FACHB-1326 cultures (2.35 × 10 7 CFU/mL) were mixed with Mae-Yong1326-1 suspension at optimal MOI of 0.1 in triplicates. After incubation for 30 min at 25 • C, the mixtures were centrifuged at 10,000× g for 10 min at 4 • C. The sediments were washed twice with BG11 and resuspended in an equal volume of BG11 medium. Samples were taken at 0, 30, 60, 120, 180, 360, 540, 720, 1440, 2160, and 2880 min, respectively. The titers in the samples were immediately determined using the double-layer plate method. The burst size of the Microcystis cyanophage Mae-Yong1326-1 was calculated as the ratio of the final number of released virions to the initial count of infected bacterial cells at the beginning of the latent period.

Host Range Experiments of Cyanophage
Thirty-nine freshwater cyanobacteria strains (Table 1) obtained from the Freshwater Algal Culture Bank of Institute of Hydrology (Wuhan, China), Academy of Sciences were used to determine the host range of cyanophage. In the experimental groups, each 300 µL of Mae-Yong1326-1 suspension (2.8 × 10 5 PFU/mL) and 600 µL of cyanobacterial cultures in logarithmic growth phase were added to48-well plates in triplicates and incubated in the light incubator (25 • C, 2000 Lux, with a 12 h:12 h light-dark cycle). In the negative control group, the cyanophage suspension was replaced with BG11 medium. Three parallel experiments were performed. The lysis of the culture was observed daily, and OD 680 measurements were also performed daily.

Isolation and Morphology of Cyanophage Mae-Yong1326-1
The experimental group turned yellow in seven days ( Figure 1A). The quantity of the cyanobacterial cells in the yellowing experimental groups ( Figure 1B) was much less than that in the control group ( Figure 1C) under microscopic observation. Mae-Yong1326-1 developed clear and circular plaques with diameter up to 5 mm in five days ( Figure 1D). Cyanophage Mae-Yong1326-1 has an elliptical head of about 47 nm in diameter and a slender flexible tail of about 340 nm in length ( Figure 1E).

One-Step Growth Curve
The one-step growth curve (Figure 2) of the cyanophage Mae-Yong1326-1 (at MOI = 0.1) showed that the titer of Mae-Yong1326-1 did not change significantly within 180 min post infection, increased slowly from 180 to 540 min, increased sharply from 540 to 1440 min, and remained relatively stable after 2160 min. Results indicated a latent period of 180 min and a burst period of 1980 min with the burst size of 329 PFU/cell [32].

One-Step Growth Curve
The one-step growth curve (Figure 2) of the cyanophage Mae-Yong1326-1 (at MO 0.1) showed that the titer of Mae-Yong1326-1 did not change significantly within 180 m post infection, increased slowly from 180 to 540 min, increased sharply from 540 to 14 min, and remained relatively stable after 2160 min. Results indicated a latent period of 1 min and a burst period of 1980 min with the burst size of 329 PFU/cell [32].
The literature review revealed that the burst size of the previously reported Micr cystis cyanophagesranged from 28-127 PFU/cell [17,18,[33][34][35]. The burst size of Mae-Yon 1326-1 is much larger than them. That is, among all the Microcystis cyanophages studi so far, Mae-Yong 1326-1 has the largest burst.

Temperature, pH, UV and Chloroform Stability
The physicochemical stabilities (pH, UV, temperature, and chloroform) of c anophages are important factors affecting the application potential. Mae-Yong1326-1 h a wide pH tolerance range. Itsactivity was relatively stable at pH3 to 12 although almo inactivated at pH 2 ( Figure 3A). UV irradiation reduced the activity of Mae-Yong1326 and caused complete inactive in 50 min( Figure 3B). The activity of Mae-Yong1326-1 stay at high levelsat temperatures ranging from 0 °C to 40 °C, yet decreased to 0 within 20 m at the temperatures over 60 °C ( Figure 3C). The best storage and transportation temper ture for Mae-Yong1326-1 is room temperature (RT, 25 °C), as the activity of which w most stable at RT. Mae-Yong1326-1 maintained infectivity under chloroform treatmen but the activity of it decreased. The literature review revealed that the burst size of the previously reported Microcystis cyanophages ranged from 28-127 PFU/cell [17,18,[33][34][35]. The burst size of Mae-Yong 1326-1 is much larger than them. That is, among all the Microcystis cyanophages studied so far, Mae-Yong 1326-1 has the largest burst.

Temperature, pH, UV and Chloroform Stability
The physicochemical stabilities (pH, UV, temperature, and chloroform) of cyanophages are important factors affecting the application potential. Mae-Yong1326-1 has a wide pH tolerance range. Its activity was relatively stable at pH3 to 12 although almost inactivated at pH 2 ( Figure 3A). UV irradiation reduced the activity of Mae-Yong1326-1 and caused complete inactive in 50 min ( Figure 3B). The activity of Mae-Yong1326-1 stayed at high levels at temperatures ranging from 0 • C to 40 • C, yet decreased to 0 within 20 min at the temperatures over 60 • C ( Figure 3C). The best storage and transportation temperature for Mae-Yong1326-1 is room temperature (RT, 25 • C), as the activity of which was most stable at RT. Mae-Yong1326-1 maintained infectivity under chloroform treatment, but the activity of it decreased.
Although most isolated cyanophages have a narrow host range,Mae-Yong1326-1 and four cyanophages, reported recently, have broad host range [15,16,18,20]. A wide host rangemay be advantageous for the application because cyanobacterial blooms are usually caused by multiple cyanobacteria [16].
Although most isolated cyanophages have a narrow host range, Mae-Yong1326-1 and four cyanophages, reported recently, have broad host range [15,16,18,20]. A wide host range may be advantageous for the application because cyanobacterial blooms are usually caused by multiple cyanobacteria [16]. antibiotic-tolerance gene and virulence gene was found in Mae-Yong1326-1 genome, which proposes the security of the application potential of Mae-Yong1326-1 as a candidate for controlling Microcystis bloom. The genome was deposited in GenBank under the accession number OP028995.   By utilizing RAST, Blastp, HHpred, and HMMER, 20 ORFs in Mae-Yong1326-1 genome were predicted as known functional genes, accounting for about 35% of the total 57 ORFs. The remaining 37 ORFs, accounting for 65% of the total ORF, were unannotated. The annotated ORFs could be classified into four functional categories: DNA replication/regulation, structure, packaging, and lysis ( Figure 4).
DNA replication and regulation genes: ORF 3 and ORF 4 of Mae-Yong1326-1 were predicted to encode CobS and CobT subunit of cobaltochelatase. CobST gene cluster is found to be widely encoded in tailed viruses that infect members of eight bacterial or archaeal orders [39]. In T4-like cyanophages, cobST gene cluster is part of the core genome [39][40][41], i.e., cobS and cobT genes are reported to be core genes in T4-likecyanophages, although cobT is usually mistakenly annotated as a peptidase [39,40].CobS and CobT were reported to play the role in the biosynthesis of cobalamin (vitamin B12), which is an important cofactor in various metabolic pathways, including DNA biosynthesis and replication of the virus [39,41]. ORF 52 encoded Zinc finger proteins that may be involved in transcriptional regulation or mediate protein-protein interactions [42]. ORF 44 encoded H-N-H endonuclease. HNH endonucleases were suggested to play an important role in the phage life cycle, fitness, and DNA packaging as well as in the response to environmental stress conditions [43].
Lysis, DNA packing, and structure genes: ORF 38 of Mae-Yong1326-1 was predicted to encode a putative peptidoglycan transglycosylase, which can crack the peptidoglycan cell wall of host cell [44]. ORF 50 encodes a putative terminase large subunit, which mediates DNA packaging and performs nuclease activity, thus generating the terminal of the phage chromosome [45]. ORF 43 was predicted to encode a packaged DNA stabilization protein, the function of which is involved with stabilizing the condensed DNA within the capsid [46]. ORF 45,47, and 49 encoded putative tail tubular protein, major capsid protein, and portal protein, respectively.

Phylogenetic Analysis of Cyanophage Mae-Yong1326-1
BLASTn search resulted that Mae-Yong1326-1 had the highest sequence similaritywith Leisingera sp. NJS201 (accession number CP038234.1), but the query cover was close to 0; i.e., in practical terms,there is no homologous genome in the database. PASC is a web tool for the analysis of pairwise identity distribution within viruses [29]. PASC scanningwith Mae-Yong1326-1 genome resulted that the maximum nucleotide sequence similarity between Mae-Yong1326-1 and the closest relative (Microcystis cyanophage MinS1) was only 17.75%, which was much lower than the threshold value of 70% to discriminate viral genus according to the International Committee on Taxonomy of Viruses (ICTV). In the VIRIDIC scanning with Mae-Yong1326-1 genome, the highest intergenomic similarities between Mae-Yong1326-1 and the closest relative (Microcystis cyanophage MinS1) was as low as 2%, which was far below the≥70% boundary to define a genus. Results demonstrate that cyanophage Mae-Yong1326-1 reveals an unknown new genus. The ANI and isDDH values for Microcystis cyanophage Mae-Yong1326-1 and the closest relative, Microcystis phage MinS1, were only −1 and 12.5%, respectively. Terminase genes are considered to be a relatively conservative genes in Caudoviricetes class. In Blastp analysis, the large terminase subunit of Mae-Yong1326-1 shared only 55% of identity with the top hitand 33% with MinS1.The genome of a total of 91 classified phages of the class Caudoviricetes and the 9 reported freshwater Microcystis cyanophages were used as reference sequences to develop a proteomic tree, applying the online software ViPTree. In the proteomic tree ( Figure 5) based on genome-wide sequence similarities, Mae-Yong1326-1 distantly clustered with three unclassified Microcystis cyanophages (MinS1, Mwe-Yong1112-1, and Mwes-Yong2). Like Mae-Yong1326-1, the highest similarities between MinS1, Mwe-Yong1112-1, Mwes-Yong2, and their closest relatives in PASC and VIRIDIC scanning were

Phylogenetic Analysis of Cyanophage Mae-Yong1326-1
BLASTn search resulted that Mae-Yong1326-1 had the highest sequence similarity with Leisingera sp. NJS201 (accession number CP038234.1), but the query cover was close to 0; i.e., in practical terms, there is no homologous genome in the database. PASC is a web tool for the analysis of pairwise identity distribution within viruses [29]. PASC scanning with Mae-Yong1326-1 genome resulted that the maximum nucleotide sequence similarity between Mae-Yong1326-1 and the closest relative (Microcystis cyanophage MinS1) was only 17.75%, which was much lower than the threshold value of 70% to discriminate viral genus according to the International Committee on Taxonomy of Viruses (ICTV). In the VIRIDIC scanning with Mae-Yong1326-1 genome, the highest intergenomic similarities between Mae-Yong1326-1 and the closest relative (Microcystis cyanophage MinS1) was as low as 2%, which was far below the ≥70% boundary to define a genus. Results demonstrate that cyanophage Mae-Yong1326-1 reveals an unknown new genus. The ANI and isDDH values for Microcystis cyanophage Mae-Yong1326-1 and the closest relative, Microcystis phage MinS1, were only −1 and 12.5%, respectively. Terminase genes are considered to be a relatively conservative genes in Caudoviricetes class. In Blastp analysis, the large terminase subunit of Mae-Yong1326-1 shared only 55% of identity with the top hit and 33% with MinS1.The genome of a total of 91 classified phages of the class Caudoviricetes and the 9 reported freshwater Microcystis cyanophages were used as reference sequences to develop a proteomic tree, applying the online software ViPTree. In the proteomic tree ( Figure 5) based on genome-wide sequence similarities, Mae-Yong1326-1 distantly clustered with three unclassified Microcystis cyanophages (MinS1, Mwe-Yong1112-1, and Mwes-Yong2). Like Mae-Yong1326-1, the highest similarities between MinS1, Mwe-Yong1112-1, Mwes-Yong2, and their closest relatives in PASC and VIRIDIC scanning were far below the ≥70% threshold to define a genus. Results indicated that Microcystis cyanophages Mae-Yong1326-1, MinS1, Mwe-Yong1112-1, and Mwes-Yong2 each reveal a new genus. These four Microcystis cyanophages form a monophyletic clade, separating at a node from the other clade formed by two families, Zierdtviridae and Orlajensenviridae, which are independent families of the Caudoviricetes class. Compared to Mwes-Yong2 and Mwe-Yong1112-1, the two Microcystis cyanophages, MinS1 and Mae-Yong1326-1, are more related. Genome comparison between Mae-Yong1326-1, MinS1, and Mwe-Yong1112-1 showed very low homology among them ( Figure 6). Core Genes 5.0 (https://coregenes.ngrok.io/ (accessed on 10 July 2022)) analysis revealed no homologs shared by Mae-Yong1326-1, MinS1, Mwe-Yong1112-1, and Mwes-Yong2. Manual analysis revealed that all or some of them shared the homologs, including terminase, integrase, DNA polymerase, HNH endonuclease, and portal protein ( Table 3). As mentioned earlier, cobT and cobS genes were reported to be core genes in cyanophages [39][40][41]. Yet, except Mae-Yong1326-1, no cobT or cobS genes were found in the genomes of Mwes-Yong2, MinS1, and Mwe-Yong1112-1 in bioinformatics analysis. In addition, unlike Mae-Yong1326-1, no fixed phage terminus and direct terminal repeat was found in Mwes-Yong2, MinS1, and Mwe-Yong1112-1 genomes. This corresponded with the very low nucleotide sequence similarity and intergenomic similarities among them. All the above results suggest more diverse characteristics of freshwater Microcystis cyanophages than have been previously known. We propose to establish a new family and four subfamilies to harbor the four Microcystis cyanophages including Mae-Yong1326-1, MinS1, Mwe-Yong1112-1, and Mwes-Yong2.

Conclusions
The newly isolated freshwater M. aeruginosa cyanophage Mae-Yong1326-1 is a novel virus species delegating a novel and genetically distinct evolutionary lineage of phages. This study enriches our understanding of freshwater cyanophage.
Mae-Yong1326-1 owns optimal characters beneficial to application. It is stable, maintaining high titers at 0-40 °C andat a wide pH range of 3-12.It has a big burst size of 329 PFU/cell, which is much larger than the reported Microcystis cyanophages. It has broad host range, capable oflysing toxic cyanobacterial strains across orders (Chroococcales, Nostocales,and Oscillatoriales). No Mae-Yong1326-1 ORF was found to be associated with virulence factor or antibiotic resistance.