# Isolation and Characterization of a Novel Cyanophage Encoding Multiple Auxiliary Metabolic Genes
## 3 Results and Discussion
### Cyanophages Structural Proteome  
The LC-MS based proteomics analysis has led to the identification of 30 proteins from cyanophage S-SZBM1 (Mascot score > 50), including 24 structural proteins (a total of 32 structural proteins were detected in cyanophage S-SZBM1 genome), one enzyme (ctidylyltransferase), and five proteins with unknown function (Table 1). As a T4-like phage, cyanophage S-SZBM1 assembly consists of three independent stages, including head (proteins of S-SZBM1_72, S-SZBM1_176-178, S-SZBM1_180, S-SZBM1_185, S-SZBM1_196, S-SZBM1_198, and S-SZBM1_199 were detected), tail (proteins of S-SZBM1_61, S-SZBM1_69, S-SZBM1_74, S-SZBM1_174, S-SZBM1_181, S-SZBM1_182, S-SZBM1_197, S-SZBM1_204, S-SZBM1_206, S-SZBM1_209, and S-SZBM1_210 were detected), and long tail fiber (no protein was detected) assembly pathways [82]. Portal vertex protein (S-SZBM1_180) is involved in the formation of a membrane-spanning initiation complex; scaffolding proteins (S-SZBM1_177, S-SZBM1_178) are responsible for the formation of a scaffolding core of prohead on this initiation complex [83]. Around this scaffolding core, the major capsid protein (S-SZBM1_176) builds an outer shell [84; 85]. Then, enzyme zymogen (S-SZBM1_178) activates itself to be a T4 FPase and cleaves related proteins to free the space for DNA genome [86; 87; 88]. When the genome is loaded into the head, terminates will initiate the DNA packing, which consists of multiple copies of small subunit (S-SZBM1_196) and large subunit (S-SZBM1_185). Head completion protein (S-SZBM1_72) and neck protein (S-SZBM1_198, S-SZBM1_199) promote maturation of the head, bind with portal vertex protein, and set up a site for the attachment of phage tail [88; 89]. The tail assembly is divided into several modules. A wedge is formed by multicopy baseline wedge subunit protein (S-SZBM1_74, S-SZBM1_204, S-SZBM1_206, S-SZBM1_209, S-SZBM1_210); the central hub consists of hub subunit protein and tail lysozyme (S-SZBM1_61, S-SZBM1_69); then, six identical wedges surround a central hub to make up a baseplate [82]; multicopy tube protein (S-SZBM1_181) and sheath protein (S-SZBM1_182) separately build a tail tube and its outer retractable sheath [90]; this stage of assembly is completed by tail completion protein (S-SZBM1_174) and sheath stabilizer (S-SZBM1_197) [82]. According to the result of TEM images, long tail fiber is absent in S-SZBM1, which explains the missing of corresponding genes or translated products from its genome and proteome. The lack of long tail fibers or related gene/protein has also been reported in several other closely related cyanophages, such as cyanophages S-WAM1, P-RSM6, and P-TIM40, which differs from typical T4-like phages. The LC-MS analysis also revealed two proteins, namely YadA domain-containing structural protein and DUF680 domain-containing protein, that have been commonly found in a number of cyanophages but not in classic T4-like assembly. They were thought of as the targets of positive selection to adapt sub-optimal hosts and to promote genetic diversity of phage populations [91]. The _YadA_ gene was first identified in a Gram-negative bacillus-shaped bacterium _Yersinia enterocolitica_, and was reported to encode an agglutinating adhesin (YadA, _Yersinia_ adhesion), a homotrimeric outer membrane protein belonging to the trimeric autotransporter adhesin (TAA) family [92]. This protein forms a fibrillar matrix at bacterial surface and acts as an adhesin and an invasin, mediating _Yersinia_ adherence and enhancing its entry to epithelial cells [93; 94]. In phages, its homolog, _YadA_ domain-containing structural protein, could be found in various phages, including _Enterobacteria_ phage \(\Phi\)Eco32, _Pectobacterium_ phage My1, _Synechococcus_ phages Syn19 and Syn30, and _Prochlorococcus_ phages P-HM1 and P-HM2 [95].