Cyanophages, a group of phages that infected cyanobacteria as host cells [1], [2], play a crucial role in controlling cyanobacterial blooms [3], [4]. To lyse and kill their host cells, cyanophages commonly employ a holin-endolysin system, which consists of two adjacent genes encoding holin and endolysin proteins [5]. Holin, a small hydrophobic membrane-spanning protein (65–125 aa), facilitates the translocation of endolysin across the membrane and into the periplasmic space [6]. Endolysin, on the other hand, acts as a glycosyltransferase or peptidoglycan degrading enzyme [7], [8], responsible for lysing the peptidoglycan layer of the host cell during the late stages of phage infection [9], [10], [11]. Previous studies have revealed the presence of the holin-endolysin system in tailless cyanophage PaV-LD (Planktothrix agardhii virus from Lake Donghu), as well as the long-tailed cyanophages Mwe-Yong 1112–1 and VB_ MelS-Me-ZS1. Significantly, it was noteworthy that in the cyanophage PaV-LD, the holin and endolysin demonstrate robust synergistic bacteriolytic activity [12], [13].

Another strategy of bacteriophage-mediated bacterial lysis relies on lyases to inhibit the synthesis of peptidoglycan or hydrolyze the host cell wall [14]. Similarly, certain cyanophages lack the conventional holin-endolysin lysis system and instead possess one or two lyases. Among the known freshwater cyanophages, only three out of nineteen have been found to contain holin-endolysin systems, while the rest exhibit one or two genes associated with lysis. These genes primarily encode N-acetylmuramoyl-L-alanine amides [15], transglycosylase, endopeptidase [16], lysozyme [5], [17], GH19 lysozyme and peptidase [18], [19]. The putative GH19 and peptidase have been suggested to function as lysozymes within the Myoviridae cyanophage group [18], [19]. Two freshwater cyanophages MaMV-DH01 (Microcystis aeruginosa Myoviridae virus from Lake Donghu) and MaMV-DC (Microcystis aeruginosa Myoviridae virus from Lake Dianchi), which exhibit high lytic activity against the M. aeruginosa FACHB-524, belong to the Myoviridae family and lack the classical holin-endolysin system. Notably, MaMV-DH01 possesses two putative lysis-related enzymes: DHGH19 and DHpeptidase [20]. However, experimental confirmation regarding the functions of GH19 and peptidase in cyanophages is still lacking.

In this study, two genes (ORF91 and ORF117) encoding DHGH19 and DHpeptidase of MaMV-DH01 were first characterized with bioinformatics. The molecular experimental analyses helped us to reveal their bacteriolytic functions. Additionally, the function of homologous gene to DHGH19 in the MaMV-DC genome (ORF71) was also verified. Based on these findings, we hypothesize that GH19 and peptidase could be further developed as biological agents, holding significant potential for the development of new antibacterial treatments in the future.