The First Pseudomonas Phage vB_PseuGesM_254 Active against Proteolytic Pseudomonas gessardii Strains

Abstract

Bacteria of the Pseudomonas genus, including the Pseudomonas gessardii subgroup, play an important role in the environmental microbial communities. Psychrotolerant isolates of P. gessardii can produce thermostable proteases and lipases. When contaminating refrigerated raw milk, these bacteria spoil it by producing enzymes resistant to pasteurization. One possible way to prevent spoilage of raw milk is to use Pseudomonas lytic phages specific to undesirable P. gessardii isolates. The first phage, Pseudomonas vB_PseuGesM_254, was isolated and characterized, which is active against several proteolytic P. gessardii strains. This lytic myophage can infect and lyse its host strain at 24 °C and at low temperature (8 °C); so, it has the potential to prevent contamination of raw milk. The vB_PseuGesM_254 genome, 95,072 bp, shows a low level of intergenomic similarity with the genomes of known phages. Comparative proteomic ViPTree analysis indicated that vB_PseuGesM_254 is associated with a large group of Pseudomonas phages that are members of the Skurskavirinae and Gorskivirinae subfamilies and the Nankokuvirus genus. The alignment constructed using ViPTree shows that the vB_PseuGesM_254 genome has a large inversion between ~53,100 and ~70,700 bp, which is possibly a distinctive feature of a new taxonomic unit within this large group of Pseudomonas phages.

Keywords: Pseudomonas bacteriophage; Pseudomonas gessardii; environmental P. fluorescens complex; genome inversion; myovirus; proteolytic activity.

Figure 1

Phage PseuGes_254 characteristics. (A) Electron micrograph of phage PseuGes_254. (B) Phage adsorption assay. (C) One-step growth experiments. (D) Multistep bacterial lytic curves for the host bacterium P. gessardii CEMTC 4637, infected with phages, are shown. The growth at 25 °C is indicated by boxes (red boxes for the phage-infected culture and black ones for the control culture), and the growth at 10 °C is shown by triangles (blue triangles for the phage-infected culture and black ones for the control culture). The bars show standard deviations for each point.

Figure 2

Phylogenetic analysis of P. gessardii subgroup isolates. Concatenated gene sequences of rpoB, rpoD, and gyrB were used. Sequences were aligned using the ClustalW algorithm. Phylogenetic trees were constructed using the maximum likelihood (ML) method based on the JTT matrix-based LG model in MEGA 11.0 with 1000 bootstrap replicates. Bacterial strains sensitive to the PseuGes_254 were marked with red circles, other investigated Pseudomonas isolates are marked with black circles.

Figure 3

Pseudomonas phage vB_PseuGes_254 genome map. Genes encoding structural proteins are marked with blue arrows; genes corresponding to nucleic acids metabolism are marked with red; terminase subunits are yellow; genes encoding proteins of lysis cassette are rose; tRNA genes are green; other genes encoding hypothetical proteins are brown; DTRs and origin of replication are marked with grey and black boxes.

Figure 4

Phage origin prediction in the PseuGes_254 genome Ori-Finder software [52]. (A) The scheme of the putative origin of replication: CtrA binding motif is marked with green circles, GATC-region is marked with red hexagon, DnaA boxes are marked with brown arrows. (B) 70,536–70,927 nt sequence with highlighted elements of a putative origin: CtrA binding motif is marked with green, AT-containing region is underlined, AT-repeats are marked with bold, GATC-region is marked with red, DnaA boxes are marked with brown italics.

Figure 5

Alignment of the nucleotide sequences of the PseuGes_254 genome and two contigs from BioProject ID:PRJEB4728, namely NODE_144_length_46990_cov_333.200810 (CALTWU010000030) and NODE_148_length_46611_cov_317.059283 (CALTWU010000032). Several ORFs encoding signature proteins are indicated: TLS—terminase large subunit, MCP—major capsid protein, TMP—tape measure protein, DNA-pol A—DNA polymerase A.

Figure 6

ViPTree analysis for phage PseuGes_254 and related phages. PseuGes_254 phage and putative phage Wonh are marked with red lines, PseuGes_254 is marked with red circle.

Figure 7

A pairwise comparison of the PseuGes_254 genome and a number of the most similar Pseudomonas phages was performed using the ViPTree tool.

Figure 8

VIRIDIC heatmap indicating intergenomic similarity between the genomes of PseuGes_254 and related Pseudomonas phages.