Genomic Characterization of the Novel Aeromonas hydrophila Phage Ahp1 Suggests the Derivation of a New Subgroup from phiKMV-Like Family

Abstract

Aeromonas hydrophila is an opportunistic pathogenic bacterium causing diseases in human and fish. The emergence of multidrug-resistant A. hydrophila isolates has been increasing in recent years. In this study, we have isolated a novel virulent podophage of A. hydrophila, designated as Ahp1, from waste water. Ahp1 has a rapid adsorption (96% adsorbed in 2 min), a latent period of 15 min, and a burst size of 112 PFU per infected cell. At least eighteen Ahp1 virion proteins were visualized in SDS-polyacrylamide gel electrophoresis, with a 36-kDa protein being the predicted major capsid protein. Genome analysis of Ahp1 revealed a linear doubled-stranded DNA genome of 42,167 bp with a G + C content of 58.8%. The genome encodes 46 putative open reading frames, 5 putative phage promoters, and 3 transcriptional terminators. Based on high degrees of similarity in overall genome organization and among most of the corresponding ORFs, as well as phylogenetic relatedness among their DNAP, RNAP and major capsid proteins, we propose a new subgroup, designated Ahp1-like subgroup. This subgroup contains Ahp1 and members previously belonging to phiKMV-like subgroup, phiAS7, phi80-18, GAP227, phiR8-01, and ISAO8. Since Ahp1 has a narrow host range, for effective phage therapy, different phages are needed for preparation of cocktails that are capable of killing the heterogeneous A. hydrophila strains.

Fig 1. Transmission electron micrograph of A. hydrophila phage Ahp1.

Ahp1 was negatively stained with 2% uranyl-acetate. The bar corresponds to 100 nm.

Fig 2. Estimation of genome size of phage Ahp1 by pulsed-field gel electrophoresis.

Lanes: M, midrange I PFG markers; Ahp1, genomic DNA of Ahp1.

Fig 3. SDS-polyacrylamide gel (8–16% gradient) electrophoresis (SDS-PAGE) of Ahp1 virion proteins.

About 5 × 10¹¹ PFU of purified phage particles were boiled in sample buffer (100 mM Tris-HCl pH 6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol, 200 mM dithiothreitol) (20 μl) and loaded onto the well. Lane M, prestained middle range protein markers (Protech Technology). Estimated molecular masses are indicated to the right.

Fig 4. Genome organization of Ahp1 and similar phages.

Predicted ORFs are numbered for Ahp1 and other members. The ruler below represents the features of the genome. PhiAS7, phage of Aeromonas salmonicida; phi80-18, phage of Yersinia enterocolitica; GAP227, phage of Cronobacter sakazakii, phiKMV, phage of Pseudomonas aeruginosa. Three closely related Yersinia enterocolitica phages ISAO8, phi80-18, and phiR8-01 have been available. Shown here is only phi80-18.

Fig 5. Sequence alignment of RNA polymerase (RNAP) from T7, phiKMV, phi80-18, GAP227, phiAS7, and Ahp1 by ClustalW.

Lines superposed over the alignment show the major features obtained experimentally for T7 RNAP. Black shadowed residues indicate functionally important residues in T7 RNAP. Boldface residues are highly conserved amino acids within known RNAP. Symbols: “*”, identical residues in all sequences, “:”, highly conserved residues, “.”, weakly conserved residues.

Fig 6. Phylogenetic relatedness among DNAP (A), RNAP (B), and major capsid proteins (C) from Ahp1 and some Autographivirinae phages based on amino acid sequence.

The tree was drawn based on the neighbor joining algorithm using 1,000 bootstrap replicates, calculated from alignment results of MEGA program (version 6.0.6). Names of phages are shown on the right side.