Genomic characterization of three novel Pseudomonas phages within the subfamily of Autographivirinae isolated from organic waste

Three phages targeting Pseudomonas syringae GAW0113 have been isolated from organic waste samples: Pseudomonas phage Bertil, Misse and Strit. The phages have double-stranded DNA genomes ranging from 41342 to 41374 bp in size comprising 50 to 51 open reading frames. The three phages genomes are highly similar and genomic comparison analyses shows that they all belong to the Autographivirinae subfamily of the family Podoviridae. The phages are however only distantly related to other members of this family, and have limited gene synteny with type-phages of other genera within Autographivirinae, suggesting that the newly isolated phages could represent a new genus.


Introduction
Pseudomonas syringae is a gram-negative, rod-shaped bacterium. Strains of P. syringae are ubiquitous epiphytic plant pathogens and different strains can infect a wide range of important agricultural plant species, causing diseases such as bacterial canker of wild cherries [1] and kiwifruit [2], bacterial speck of tomato [3] and leaf blight in wheat [4]. The widespread problem with P. syringae in agricultural plants calls for an inexpensive, sustainable method for preventing or treating bacterial infections.
Bacteriophage therapy is a promising approach to disease management [5], but requires a extensive library of relevant phages and understanding of phage-host interaction between phages and the P.

Materials and methods
Samples of organic waste (OW) was collected, aliquoted and filtered with 0.45 μm syrin m syringe filters. The filtrate were tested for phage activity against P. syringae GAW0113 using a double agar overlay assay [7]. Plaques with distinct appearance or size were purified thrice. The phage DNA was extracted from lysate, as previously described [7] Phage DNA libraries were prepared using NEBNext Ultra II FS library Prep and Kit for Illumina (New England Biolabs, Ipswich, USA). The sequencing was performed on the Illumina iSeq platform as part of a flowcell (2 x 151, 300 cycles; Illumina). The genomes were trimmed and assembled in CLC Genomics Workbench version 12.0.3. Blastn [8] were used to search for sequence similarity against the non-redundant NCBI databases. Three of the phages are presented here because of low similarity with other known phages, but high similarity with each other.
The position of the terminal repeats were identified by the presence of two distinct spikes in read-start coverage at two nearby sites in the genome, encompassing areas with approximately double readcoverage. Open reading frames (ORFs) were identified automatically with GeneMark [9] and Glimmer [10] and revised manually in DNA Master [11]. The genomes were searched for tRNA sequences using Aragorn[12] and tRNAscan-SE [13], but none were found. The ORFs were manually annotated using both HHPred [14] against three downloaded databases the pFam 32.0, SCOP70 1.75 and PDB70 databases and blastp against the NCBI databases [8]. The nucleotide similarity heatmap   2   20   25   30   35   40 between Autographivirinae phages was made with Gegenees version 3.1.0 (500 nt fragment size and 500 nt step size ) [15]. A genome comparison between the isolated phages was made with Easyfig version 2.2.2. (default blastn settings with 2.9.0+ NCBI blast) [16].

Results and Discussion
The 2), gene synteny and gene size seems to be conserved for some of the structural genes ( fig. 1). The similarity in gene synteny is however limited and is notably not the case for ex. the DNA polymerase, terminase and the internal virion protein A genes ( fig. 1). Altogether, this suggests that the three phages could represent a new genus within the Autographivirinae subfamily.
The phages also share little identity with other known phages targeting P. syringae. Increased diversity within the known phages targeting P. syringae strains allow for development of more complex phage cocktails, which could decrease the possibility for the target bacteria to develop resistance to the treatment without losing fitness or virulence [17].