Genomics of Three New Bacteriophages Useful in the Biocontrol of Salmonella

Abstract

Non-typhoid Salmonella is the principal pathogen related to food-borne diseases throughout the world. Widespread antibiotic resistance has adversely affected human health and has encouraged the search for alternative antimicrobial agents. The advances in bacteriophage therapy highlight their use in controlling a broad spectrum of food-borne pathogens. One requirement for the use of bacteriophages as antibacterials is the characterization of their genomes. In this work, complete genome sequencing and molecular analyses were carried out for three new virulent Salmonella-specific bacteriophages (UAB_Phi20, UAB_Phi78, and UAB_Phi87) able to infect a broad range of Salmonella strains. Sequence analysis of the genomes of UAB_Phi20, UAB_Phi78, and UAB_Phi87 bacteriophages did not evidence the presence of known virulence-associated and antibiotic resistance genes, and potential immunoreactive food allergens. The UAB_Phi20 genome comprised 41,809 base pairs with 80 open reading frames (ORFs); 24 of them with assigned function. Genome sequence showed a high homology of UAB_Phi20 with Salmonella bacteriophage P22 and other P22likeviruses genus of the Podoviridae family, including ST64T and ST104. The DNA of UAB_Phi78 contained 44,110 bp including direct terminal repeats (DTR) of 179 bp and 58 putative ORFs were predicted and 20 were assigned function. This bacteriophage was assigned to the SP6likeviruses genus of the Podoviridae family based on its high similarity not only with SP6 but also with the K1-5, K1E, and K1F bacteriophages, all of which infect Escherichia coli. The UAB_Phi87 genome sequence consisted of 87,669 bp with terminal direct repeats of 608 bp; although 148 ORFs were identified, putative functions could be assigned to only 29 of them. Sequence comparisons revealed the mosaic structure of UAB_Phi87 and its high similarity with bacteriophages Felix O1 and wV8 of E. coli with respect to genetic content and functional organization. Phylogenetic analysis of large terminase subunits confirms their packaging strategies and grouping to the different phage genus type. All these studies are necessary for the development and the use of an efficient cocktail with commercial applications in bacteriophage therapy against Salmonella.

Keywords: Myoviridae; Podoviridae; Salmonella; bacteriophage; chromosomal ends; genomics.

Figure 1

Genomic structure of bacteriophage UAB_Phi20 including the Rho-indepedent terminators. Arrows represent genes, and the different colors identify the functional category into which the homologous genes were classified. Gene functions are indicated where they are known. The color code for gene function is provided at the bottom of the figure. ORFs are numbered consecutively from left to right as described in Table 1, and are indicated by arrows pointing to the direction of transcription.

Figure 2

Alignment of the UAB_Phi20 (A), UAB_Phi78 (B), and UAB_Phi87 (C) genomes and their counterparts belonging to the Podoviridae and Myoviridae families using Progressive MAUVE. The names of the different bacteriophages are indicated under their maps. Colored blocks correspond to regions of nucleotide similarity which is indicated by its height and regions with a lack of homology are outside these blocks or indicated in white inside the blocks. The tRNA genes are indicated by the filled gene blocks.

Figure 3

Genomic structure of UAB_Phi78 including the Rho-indepedent terminators. Arrows represent genes, and the different colors identify the functional category into which the homologous genes were classified. Gene functions are indicated where they are known. The color code for gene function is provided at the bottom of the figure. ORFs are numbered consecutively from left to right as described in Table 2, and are indicated by arrows pointing to the direction of transcription.

Figure 4

Genomic structure of UAB_Phi87, including the Rho-independent terminators and tRNAs. Arrows represent genes, and the different colors identify the functional category into which the homologous genes were classified. Gene functions are indicated where they are known. The color code for gene function is provided at the bottom of the figure. ORFs are numbered consecutively from left to right as described in Table 3, and are indicated by arrows pointing to the direction of transcription.

Figure 5

Determination of genome ends of UAB_Phi20 phage after digestion with EcoRI enzyme. Genome of bacteriophage P22 digested with EcoRI was used as control. Arrows indicate the 4007-bp fragment containing the pac sequence. Lambda DNA digested with HindIII (M1) or BstEII (M2) were used as molecular markers. Sizes (bp) are indicated on both sides of the image.

Figure 6

Time-limited digestion with Bal31 exonuclease of UAB_Phi78 and UAB_Phi87 DNA followed by digestion with HindIII and SpeI, respectively. Arrows indicate the sequentially degraded DNA bands of 2200 and 2080 bp for UAB_Phi78 (A) and of 4322 and 2819 bp for UAB_Phi87 (B). M: marker lanes containing a mixture of λ DNA digested with BstEII and φX174 digested with HinfI (M1), λ-DNA-digested HindIII (M2), and λ-DNA-digested BstEII (M3). Sizes (bp) are indicated on the left side of the images.

Figure 7

Neighbor-joining phylogenetic tree of large terminase subunit sequences of bacteriophages UAB_Phi20, UAB_Phi78, and UAB_Phi87 (indicated by arrows) and comparison to other phages with known packaging mechanisms. Bootstrap analysis was performed with 1000 repetitions. The node of phylogenetic tree shows the bootstrap confidence values above 70%.