3.1 Phage morphology
Phage Kayfunavirus TM1 was isolated from pig farm sewage in Guangxi, China. Phage Kayfunavirus TM1 formed small, clear, and round plaques approximately 2 mm in diameter after incubating on soft agar plates with host bacteria for 5 h at 37 ◦C(Fig. 1A). Electron microscopy showed that the phage tubular tail was approximately 36 nm in length, and the head was 54 nm in diameter (Fig. 1B). In accordance with the International Classification Committee of Viruses, the phage belonged to the order Caudovirales, family Autographiviridae, subfamily Studiervirinae, genus Kayfunavirus according to its morphology, named as Kayfunavirus TM1.
3.2 Host range of phage Kayfunavirus TM1
As shown in Table 1, twenty-two strains were tested. Host range analysis revealed that phage Kayfunavirus TM1 had a very high specificity and only cleaved GXEC-TM1, and showed no activity against any of the other strains examined in this study.
Table 1. Host strains information of phage Kayfunavirus TM1.
Strains
|
Genus
|
Serotype
|
Place
|
Source
|
Sensitivity
|
GXEC-34-2
|
Escherichia coli
|
Undetected
|
Guangxi, China
|
Pig
|
-
|
GXEC-2-3
|
Escherichia coli
|
O127a:K63(B8)
|
Guangxi, China
|
Pig
|
-
|
GXEC-2-8
|
Escherichia coli
|
Undetected
|
Guangxi, China
|
Pig
|
-
|
GXEC-2-11
|
Escherichia coli
|
Undetected
|
Guangxi, China
|
Pig
|
-
|
GXEC-2-2
|
Escherichia coli
|
O114:K90(B90)
|
Guangxi, China
|
Pig
|
-
|
GXEC-K5
|
Escherichia coli
|
O126:K71(B16)
|
Guangxi, China
|
Pig
|
-
|
GXEC-36
|
Escherichia coli
|
Undetected
|
Guangxi, China
|
Pig
|
-
|
GXEC-H9
|
Escherichia coli
|
O157:H7
|
Guangxi, China
|
Pig
|
-
|
CVCC-4050
|
Escherichia coli
|
O157:H7
|
Guangxi, China
|
Pig
|
-
|
GXEC-TM1
|
Escherichia coli
|
Undetected
|
Guangxi, China
|
Pig
|
+
|
GXEC-H5
|
Escherichia coli
|
Undetected
|
Guangxi, China
|
Pig
|
-
|
GXEC-3J
|
Escherichia coli
|
Undetected
|
Guangxi, China
|
Pig
|
-
|
GXEC-15-2-1
|
Escherichia coli
|
Undetected
|
Guangdong, China
|
Pig
|
-
|
GXEC-15-3
|
Escherichia coli
|
Undetected
|
Guangdong, China
|
Pig
|
-
|
GXEC-16-1
|
Escherichia coli
|
Undetected
|
Guangdong, China
|
Pig
|
-
|
GXEC-16-3
|
Escherichia coli
|
Undetected
|
Guangdong, China
|
Pig
|
-
|
GXEC-17-4
|
Escherichia coli
|
Undetected
|
Guangdong, China
|
Pig
|
-
|
GXEC-18-1
|
Escherichia coli
|
Undetected
|
Guangdong, China
|
Pig
|
-
|
GXEC-18-2
|
Escherichia coli
|
Undetected
|
Guangdong, China
|
Pig
|
-
|
GXEC-19-2
|
Escherichia coli
|
Undetected
|
Guangdong, China
|
Pig
|
-
|
GXEC-19-3
|
Escherichia coli
|
Undetected
|
Guangdong, China
|
Pig
|
-
|
GXEC-20-4
|
Escherichia coli
|
Undetected
|
Guangdong, China
|
Pig
|
-
|
+, clear lysis zone; -, no lysis zone;
|
3.3 Optimal MOI and one-step growth assays
Phage Kayfunavirus TM1 was mixed with the host bacteria in a certain ratio, When the MOI was 0.01, the phage Kayfunavirus TM1 produced the maximum progeny virus (Fig. 2A). which indicated that 0.01 is the optimal MOI of phage Kayfunavirus TM1. The one-step growth experiment was conducted to determine the latent time period and burst size of phage Kayfunavirus TM1 (Fig. 2B). The results showed that the latent period was 5 min and the outbreak period was 40 min. The burst size was calculated as the ratio of the final number of free phage particles to the number of infected bacterial cells during the latent period, and the burst size of phage Kayfunavirus TM1 was determined to be 20 plaque-forming units PFU/ cell.
3.4 The Stability Analysis of phage Kayfunavirus TM1
The stability of the phage Kayfunavirus TM1 is shown in Fig. 2C/D. For pH stability, the results showed optimal stability of Kayfunavirus TM1 at pH 10, the phage activity stayed at high levels after 2h of incubation at pH 3–11. No viable phage was detected at pH 2 and 12 (Fig. 2C). For thermostability, temperatures ranging from 4 ℃ to 60 ℃ won't affect phage vitality, however, temperatures higher than 60 ℃ will cause vitality decrease. Almost all phage was inactivated at temperatures higher than 80 ℃ (Fig. 2D).
3.5 Antibacterial curve in vitro of phage Kayfunavirus TM1
The bacteriolytic effect of phage Kayfunavirus TM1 was tested on early-phase cultures of GXEC-TM1 at the best MOI (Fig. 3). The OD450 value continued to increase when the GXEC-TM1 was not infected by phage Kayfunavirus TM1. When the GXEC-TM1 was infected, its growth was inhibited and after 7h the OD450 value started to increase.
3.6 Genomic analysis of Kayfunavirus TM1
According to the genome sequencing results, the linear double-stranded DNA of phage Kayfunavirus TM1 is 39,948 bp in length, with a GC content of 50.03%. Based on RAST and BLASTn analysis, the results revealed that the Kayfunavirus phage DY1 (MT808983.1) had the highest query coverage (89%) containing 94.82% identity. The RAST annotation results indicated that the genome contained only 53 coding domain sequences (CDS), and ATG was the start codon for all CDS. However, only 32 CDS were determined to be functional based on gene predictions and annotation of the genome, the rest of CDS’ functions remain unknown (Table 2). The annotated genes mainly involved 4 functional modules, including phage lysis-related genes, phage morphogenesis genes, DNA packaging genes, replication and regulation related genes. As shown in Table 2 and Fig. 4, the morphogenesis proteins CDS24 and CDS25 were the major capsid proteins, with the length of 349 and 294 bp respectively, while CDS15 (666bp), CDS21 (785bp), CDS23 (188bp) and CDS26 (522bp) appeared to be phage head-tail connector proteins. In addition, CDS27 (82bp) and CDS34 (97bp) had a supporting and protective function for the overall structure of phage as assembly protein and binding protein. Specifically, nineteen proteins were involved in the replication and regulation of phage DNA. Meanwhile, CDS17 (149bp), CDS36 (89bp), and CDS43 (150bp) had the functional characteristics of endonuclease,and played a vital role in Phage DNA replication regulation. CDS1 (893bp) and CDS53 (893bp) showed the greatest identity to phage DNA-dependent RNA polymeras proteins. CDS41 (152bp), CDS12 (150bp) and CDS14 (64bp) was associated with phage cleavage.
As shown in Table 2, phage Kayfunavirus TM1 has 12 CDS, which shared a high identity with Escherichia phage LM33_P1(LT594300.1). However, only 3 CDS have explicit functionality, including DNA packing, phage morphogenesis, and phage DNA replication and regulation. Moreover, phage Kayfunavirus TM1 contains 2 CDS (Rz-like lysis protein, endolysin) required for phage lysis, which are similar to those in Escherichia phage P762, and 1 CDS (Phage holin) that are similar to Cronobacter phage Dev2, indicating that phage Kayfunavirus TM1 may have similar lysis system to that of Escherichia phage P762(MW876471.1) and Cronobacter phage Dev2 (HG813241.1). The phage Kayfunavirus TM1 also has 4 CDS (CDS15, CDS21, CDS23, CDS26) which showed the greatest identity to phage tail-associated protein. And these proteins play a significant role in phage infection and the adsorption of host bacteria(11).
In adition, there were no antibiotic resistance and virulence genes were detected in the whole genome of the phage Kayfunavirus TM1.
3.7 Phylogeny analysis of phage Kayfunavirus TM1
To analyze the relationships between phage Kayfunavirus TM1 and other phages, evolutionary trees were constructed based on the Phage terminase large subunit Gp19 DNA packaging protein (Fig. 6). The tree showed that phage Kayfunavirus TM1 is closely related to the Escherichia phage Mt1B1_P3 genome, and was closely related to the other phages that grouped into a clade of Kayfunavirus genus, Autographviridae family in the analyses. And the result was consistent with the results of the whole genome similarity analysis.