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

Abstract

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.

Keywords: Microcystis aeruginosa; cyanophage; genome; phylogenetic analysis.

Figure 1

Micro- and macrographs of M. aeruginosa FACHB-1326 cultures, plaques, and negatively stained Mae-Yong1326-1. (A) Macrograph of a normal culture (left picture) and a M. aeruginosa FACHB-1326 culture infected with Mae-Yong1326-1 (right picture); (B) micrograph of a M. aeruginosa FACHB-1326 culture infected with cyanophage Mae-Yong1326-1. Scale bar =  100 µm; (C) micrograph of a normal culture of M. aeruginosa FACHB-1326. Scale bar =  100 µm; (D) plaques developed by Mae-Yong1326-1 on M. aeruginosa FACHB-1326 lawn; (E) a transmission electron micrograph of cyanophage Mae-Yong1326-1. Scale bar represents 200 nm.

Figure 2

One-step growth curve of Mae-Yong1326-1 developed under the MOI of 0.1. Each dot represents the average titer at each time from the three parallel experiments. Error bars indicate standard deviations.

Figure 3

Physical and chemical tolerance test. (A) pH stability; (B) UV stability; (C) thermostability; (D) chloroform stability. All tests are performed in triplicate. Error bars indicate standard deviations.

Figure 4

Genome map of Microcystis cyanophage Mae-Yong1326-1. The outermost circle represents 57 ORFs encoded in the genome, with different colors representing different functions (clockwise arrow indicates the forward reading frame; counterclockwise arrow indicates the reverse reading frame); the dark circles in the middle represent the GC content (Black indicates greater than the average GC content compared with the whole genome, and gray indicates the opposite); the innermost circle represents the GC skew (G − C/G + C: Outwards indicates > 0, and inwards indicates < 0).

Figure 5

Phylogenetic proteomic tree of Mae-Yong1326-1, 9 reported Microcystis cyanophages, and 91 classified phages of the 33 families.

Figure 6

Genome comparison of the Microcystis cyanophage Mae-Yong1326-1, MinS1, and Mwe-Yong1112-1.