Complete Genome Sequence of Aeromonas hydrophila Phage CC2

Abstract

Aeromonas hydrophila is one of the major pathogenic bacteria for fish and people. To develop an effective antimicrobial agent, we isolated a bacteriophage from sewage, named CC2, and sequenced its genome. Comparative genome analysis of phage CC2 with its relatives revealed that phage CC2 has higher sequence homology to A. salmonicida phage 65 than to A. hydrophila phage Aeh1. Here, we announce the complete genome sequence of CC2 and report major findings from the genomic analysis.

GENOME ANNOUNCEMENT

Aeromonas hydrophila, a Gram-negative bacterium widely distributed in aquatic environments, is a pathogen of aquatic and terrestrial animals, and its pathogenicity in humans has been recognized for decades (3, 7). Bacteriophage therapy is considered to be an alternative method for the treatment of bacterial infection (6). To date, however, only one phage from A. hydrophila, named Aeh1, has been sequenced and annotated. Here, a new member of the Aeromonas phages, named CC2, infecting Chinese vaccine strain A. hydrophila J-1 was isolated from local sewage in China; it belongs to the Myoviridae family of T4-like viruses, efficiently inhibiting several virulent strains of A. hydrophila from diseased fish (data not shown).

The phage DNA was extracted using the alkaline lysis method (9). The DNA was sequenced, using a 454 GS-FLX Titanium sequencing system (Roche), by Encode Genomics, Shanghai, China. A total of 12,106 reads with an average length of 346 bp were assembled into one contiguous sequence (contig) using a 454 Newbler assembler. The putative opening reading frames (ORFs) were predicted using Glimmer 3.0 (4) and Prodigal (5). Annotation of predicted ORFs was conducted using BLASTP searches (1) and RPSBLAST searches (2) at the NCBI Conserved Domain database (CDD) and Clusters of Orthologous Groups of proteins (COG) database. Prediction of tRNA genes was carried out using tRNAscan-SE 1.23 software (8).

The phage CC2 genome has a full length of 231,743 bp, with GC content of 38.82%, 427 ORFs, and 9 tRNAs. While the gene coding percentage is 94.2% in the genome, 74.71% of the ORFs were annotated as hypothetical or lacking any reliable database matches, probably due to insufficient database information about the functional genes of Aeromonas phage genomes. The genome of phage CC2 encodes structural and packaging proteins, such as terminase, head outer capsid protein, head vertex assembly chaperone, prohead core scaffold protein, prohead protease, neck protein, fibritin neck whiskers, tail tube protein, tail sheath protein, tail fibers, tail pin, baseplate hub, and baseplate tail tube cap. In addition, this genome contains functional genes related to DNA metabolism and replication (endonuclease, topoisomerase, exonuclease, primase, helicase, recombination protein, polymerase, RNaseH RNase, thymidylate synthetase, reductase, kinase, dCMP deaminase, translation repressor protein, RNA polymerase sigma factor, RNA ligase, and anti-sigma70 protein). Furthermore, one lysozyme-encoding gene is found in this genome. Expression of the lysozyme gene using an Escherichia coli gene expression system could lyse the host strain (data not shown). Interestingly, comparative genome analysis revealed that phage CC2 has the highest degree of similarity (64%) to A. salmonicida phage 65 (GenBank accession number NC015251) but shows only 12% similarity to A. hydrophila phage Aeh1 (GenBank accession number NC005260). The number of tRNAs in phage CC2 is the lowest among all reported T4-like Aeromonas phages and is notably 50% less than the number seen with phage 65, the phage with which it shares the greatest homology. Studies investigating the complete genome of phage CC2 would provide novel information about A. hydrophila-targeting phage.

Nucleotide sequence accession number.

The complete genome sequence of Aeromonas phage CC2 is available in GenBank under accession number JX123262.