Two E. amylovora bacteriophages were isolated from soil samples near apple trees in different districts of Minsk, Belarus on June (Loshitsa2) and July (Micant). Loshitsa2 and Micant produced 3-3.5 mm diameter plaques with translucent halo on double agar overlay plates (Fig. 1). Their plaques were turbid that was the hint of incomplete lysis. It may indicate the presence of lysogens or phage-resistant variants in bacterial culture.
The morphology of Loshitsa2 and Micant virion was revealed using TEM of the negatively stained samples. The bacteriophages had icosahedral heads measuring 59.80 ± 2.60 (Loshitsa2) and 56.39 ± 2.69 (Micant) and short non-contractile tails (Fig. 2). According to Bradley’s morphological groups of bacteriophages [33] two E. amylovora bacteriophages were classified as belonging to C type. It should be added that among the isolated E. amylovora bacteriophages representatives of three morphotypes are described in the literature: myoviruses, siphoviruses and podoviruses, with a capsid size of 53–143.2 nm [34, 14, 35]. Recently, there the first filamentous E. amylovora bacteriophages isolation was reported, the PEar viruses carrying a single-strand DNA genome between 6608 and 6801 nucleotides [36].
The host range analysis revealed that Loshitsa2 and Micant bacteriophages were polyvalent and able to infect strains of different genera (Table 1). Among sensitive bacteria there were strains of E. amylovora, P. agglomerans and P. ananatis. It should be noted that antagonistic relationships of E. amylovora and Pantoea species were described [37, 38]. P. agglomerans has a potential as biocontrol agent and can be used as a phage carrier [39, 40].
Table 1
The host range of two E. amylovora bacteriophages
Phages
|
Bacterial strains
|
1/79Sm
|
E2
|
D4
|
L-3-1
|
L-3-2
|
L-3-5
|
L-3-6
|
L-3-8
|
E3
|
E4
|
E5
|
133/95
|
DH5α
|
B
|
14a
|
36A
|
194
|
197
|
198
|
208
|
216
|
219
|
220
|
245
|
246
|
Loshitsa2
|
+
|
-
|
-
|
-
|
-
|
-
|
-
|
+
|
+
|
+
|
-
|
+
|
-
|
-
|
-
|
-
|
-
|
+
|
-
|
+
|
-
|
-
|
-
|
+
|
-
|
Micant
|
+
|
-
|
-
|
-
|
-
|
-
|
-
|
+
|
+
|
+
|
-
|
+
|
-
|
-
|
-
|
-
|
-
|
+
|
-
|
-
|
-
|
-
|
-
|
+
|
-
|
The double-stranded DNA genomes of Loshitsa2 and Micant bacteriophages consists of 43092 bp and 43028 bp, respectively sharing 98.18% of nucleotide identity between them (coverage 99%). The GC% content is 54% for both bacteriophages. In total, 53 putative genes were predicted in each of two genomes, including 51 protein coding genes and 2 tRNA genes (Fig. 3). All Loshitsa2 and Micant genes have the same orientation. Among the genes with predicted function there were 8 putative structural genes: tail tubular protein A, tail tubular protein B, internal virion proteins, tail fiber protein with EPS depolymerase, head-tail connector and major capsid protein (MCP). Gene of viral scaffolding protein which is essential for phage capsid assembly is located upstream of the MCP gene. Putative genes of DNA maturase A and B crucial for DNA packaging follow the structural genes cluster. Two genes identified to be involved in host lysis by bacteriophages (holin and endolysin genes). There are 9 genes playing a vital role in DNA/RNA metabolism, replication and repair: ATP-dependent DNA-ligase, DNA primase, DNA helicase, DNA polymerase A, DNA polymerase II small subunit, DNA exonuclease, DNA endonuclease, nucleotide kinase, DNA-dependent RNA-polymerase.
Using PhageTerm analysis multiple preferred termini on the forward strand and unique termini on the reverse strand were predicted. Phage termini determined at 15277 (+ strand), 15534 (- strand) positions for Loshitsa2 and at 16880 (+ strand), 17138 (- strand) positions for Micant (Li's method data [28]). The headful mode of packaging PAC is concluded for both bacteriophages, when the terminase initiates packaging at a specific pac site of the phage concatemer, as described for P1 and P22 phages [28].
Bacteriophage genome sequences were queried against the viruses (taxid:10239) nucleotide collection (nr/nt) using blastn (carried out on 22/02/2022). Sequence similarity searches revealed that Loshitsa2 and Micant present 76.13% (coverage 38%) and 76.29% (coverage 40%) nucleotide sequence identity with Pantoea phage LIMElight [41], respectively. Bacteriophage LIMElight has also relatively small dsDNA genome of 44,546 bp, encoding 55 open reading frames. Nowadays bacteriophage LIMElight is classified in the family Autographiviridae, within the genus of the “Limelightvirus” (NCBI:txid881915). Interestingly, bacteriophage formed a small clear plaque of 1 mm in diameter on its host P. agglomerans strain GBBC 2043 and no plaques on E. amylovora strain GBBC 403. Primer-walking revealed that LIMElight genome has direct terminal repeats (DTRs) of 277 bp, suggesting other packaging mechanisms.
The novel E. amylovora bacteriophages also have limited similarity with phages of different host specificity. Bacteriophage Loshitsa2 has sequence similarity with Erwinia phage vB_EamP-L1 (coverage 2%, identity 81.19%, HQ728265.1), Klebsiella phages KMI6 (coverage 3%, identity 72.04%, MN101220.1), KMI5 (coverage 5%, identity 72.04%, MN101219.1) and KMI3 (coverage 5%, identity 72.04%, MN101217.1). Bacteriophage Micant has sequence similarity with Erwinia phage vB_EamP-L1 (coverage 2%, identity 81.28%, HQ728265.1), Shigella phage HRP29 (coverage 3%, identity 73.44%, NC_048174.1), Enterobacter phage ENC16 (coverage 3%, identity 71.61%, OL355133.1). Erwinia phage vB_EamP-L1 (NCBI:txid1051673), Klebsiella phages KMI6 (NCBI:txid2601617), KMI5 (NCBI:txid2601616), KMI3 (NCBI:txid2601614), Shigella phage HRP29 (NCBI:txid2530183) are also classified in the family Autographiviridae. Enterobacter phage ENC16 (NCBI:txid2906747) at the moment is of unclassified Caudovirales. Gene cluster comparison of listed bacteriophages is presented in Fig. 4.
Generated phylogenetic trees of the single phage proteins using “one click” at Phylogeny.fr (Fig. 5) and the VICTOR whole-genome sequence analysis placed bacteriophages Loshitsa2 and Micant in separate cluster closely related to LIMElight bacteriophage. Loshitsa2 and Micant bacteriophages should be classified in the family Autographiviridae and could be proposed to share novel subfamily with LIMElight bacteriophage.
Summing up, whole-genome sequence analysis revealed two novel bacteriophages - Loshitsa2 and Micant, isolated in Belarus using E. amylovora for its propagation. Bacteriophages have rather small genomes and share sequence similarity with Pantoea phage LIMElight. According to protein-based phylogeny and whole-bacteriophage genome sequence phylogeny Loshitsa2, Micant and LIMElight are proposed to form a new subfamily within the family Autographiviridae.