Genomic Analysis of a New Freshwater Cyanophage Lbo240-yong1 Suggests a New Taxonomic Family of Bacteriophages

Abstract

A worldwide ecological issue, cyanobacterial blooms in marine and freshwater have caused enormous losses in both the economy and the environment. Virulent cyanophages-specifically, infecting and lysing cyanobacteria-are key ecological factors involved in limiting the overall extent of the population development of cyanobacteria. Over the past three decades, reports have mainly focused on marine Prochlorococcus and Synechococcus cyanophages, while information on freshwater cyanophages remained largely unknown. In this study, a novel freshwater cyanophage, named Lbo240-yong1, was isolated via the double-layer agar plate method using Leptolyngbya boryana FACHB-240 as a host. Transmission electron microscopy observation illustrated the icosahedral head (50 ± 5 nm in diameter) and short tail (20 ± 5 nm in length) of Lbo240-yong1. Experimental infection against 37 cyanobacterial strains revealed that host-strain-specific Lbo240-yong1 could only lyse FACHB-240. The complete genome of Lbo240-yong1 is a double-stranded DNA of 39,740 bp with a G+C content of 51.99%, and it harbors 44 predicted open reading frames (ORFs). A Lbo240-yong1 ORF shared the highest identity with a gene of a filamentous cyanobacterium, hinting at a gene exchange between the cyanophage and cyanobacteria. A BLASTn search illustrated that Lbo240-yong1 had the highest sequence similarity with the Phormidium cyanophage Pf-WMP4 (89.67% identity, 84% query coverage). In the proteomic tree based on genome-wide sequence similarities, Lbo240-yong1, three Phormidium cyanophages (Pf-WMP4, Pf-WMP3, and PP), one Anabaena phage (A-4L), and one unclassified Arthronema cyanophage (Aa-TR020) formed a monophyletic group that was more deeply diverging than several other families. Pf-WMP4 is the only member of the independent genus Wumpquatrovirus that belongs to the Caudovircetes class. Pf-WMP3 and PP formed the independent genus Wumptrevirus. Anabaena phage A-4L is the only member of the independent Kozyakovvirus genus. The six cyanopodoviruses share similar gene arrangements. Eight core genes were found in them. We propose, here, to set up a new taxonomic family comprising the six freshwater cyanopodoviruses infecting filamentous cyanobacteria. This study enriched the field's knowledge of freshwater cyanophages.

Keywords: Leptolyngbya boryana; cyanophage; genome; isolation; phylogenetic tree.

Figure 1

Normal and microscopic pictures of L. boryana FACHB-240 cultures: (A) plaques developed by Lbo240-yong1 on the FACHB-240 lawn; (B) picture of a FACHB-240 culture (left) and of a FACHB-240 culture infected with Lbo240-yong1 (right); (C) microscopic picture of a normal FACHB-240 culture; (D) microscopic picture of a FACHB-240 culture infected with Lbo240-yong1.

Figure 2

Transmission electron microscopy images of negatively stained free Lbo240-yong1 (A,B) and L. boryana FACHB-240 cells absorbed by Lbo240-yong1 (C,D). Lbo240-yong1 had an icosahedral head of 50 ± 5 nm in diameter and a short tail of 20 ± 5 nm in length. The white arrow indicates the negatively stained Lbo240-yong1. Black Arrows indicate a large number of cyanophage particles, adsorbed on the surfaces of host cells. White triangles indicate particles are intact and full of DNA.

Figure 3

Genome map of cyanophage Lbo240-yong1. The outermost circle represents the 44 ORFs encoded in the genome, with different colors representing different functions (the clockwise arrow indicates the forward reading frame, and the counterclockwise arrow indicates the reverse reading frame); the dark circles in the middle represent the GC content (outwards indicates greater than the average GC content compared with the whole genome, and inwards indicates the opposite); the innermost circle represents the GC skew (G-C/G+C; outwards indicates >0, and inwards indicates <0).

Figure 4

Proteomic tree based on the complete genome sequences of L. boryana cyanophage Lbo240-yong1 (red star), all six remaining Podovirus-like freshwater cyanophages with reported genomes, one Siphoviridae-like freshwater cyanophage (black star) capable of infecting L. boryana, five Podovirus-like marine cyanophages (black hollow star), and 59 representative bacteriophages of 33 families of the Caudovirales class. The proteomic tree was generated using ViPTree online, based on the genome-wide similarities determined by tBLASTx. Cyanophage family assignments, according to the official ICTV classification (2022), are provided with different color bars.

Figure 5

Genome comparison of the Lbo240-yong1 (A) and Phormidium cyanophage Pf-WMP4 (B). The color of each arrow refers to the functional groups. The orientation of the arrows indicates the direction of gene transcription. The homologous regions are represented by gray bars, with their depth reflecting the degree of sequence similarity.

Figure 6

(A) Concatenated phylogenetic tree byViPTree (left) and by Easyfig of the six freshwater cyanopodoviruses. The homologous regions are represented by green bars. Light green to dark green represent low to high similarity. (B) Percent intergenomic similarities between the six freshwater cyanopodoviruses, calculated by the VIRIDIC server. (C) Integrated visualization of the viral clustering output of VirClust. 1. Hierarchical tree calculated using the protein cluster-based intergenomic distances. 2. Heatmap representation of the protein cluster distribution in the viral genomes. Rows represent the individual viral genomes. Columns represent the individual protein clusters. 3. Viral genome-specific statistics: genome length, the proportion of proteins shared (dark grey) from all proteins (light grey bar), and the proportion of proteins shared in the own viral genome cluster.