Biological Characteristics And Genomic Analysis Of Aeromonas Hydrophila Bacteriophages

Aeromonas hydrophila is widespread in the environment, which is the causative agent of hemorrhagic septicemia in aquatic and terrestrial animals. Also, this bacterial species can cause human gastroenteritis. Nowadays, with the abuse of antibiotics, many strains acquired multidrug-resistance, which challenged prevention and control of the bacterial infection. As bacteria virus, the phages have the ability to lyse bacteria effectively. Thus, phages play an important role in controlling the A. hydrophila infection.In the study we characterized two bacteriophages from local sewage and polluted river, named CC2and9-1, and investigated their morphological, physiological and molecular characteristics. Both two phages belong to family Myoviridae with an elongate hexagonal head and a collapsible tail. Phage CC2could lyse6of25tested A. hydrophila strains. It might have an application value in controlling A. hydrophila infection. The host range of phage9-1included at least four species of Aeromonas. CC2had latent period of20min, growing period of140min and average burst size of303PFU/cell.9-1had latent period of25min, growing period of105min and average burst size of177PFU/cell. Both two phages were sensitive to high temperature and low pH.To get thorough knowledge of phage CC2’s genetic background, we purified phage CC2by glycerol gradient centrifugation and extracted its DNA. And then, we sequenced its genome using a454GS-FLX Titanium sequencing system. The length of the genome is231,743bp. The base constituents are A=30.05%[69645], G=17.82%[41297], C=21.00%[48668], T=31.13%[72133], A+T=61.18%and G+C=38.82%respectively. We predicted427open reading frames(ORFs) using Glimmer3.0and Prodigal, of which only108ORFs have predicted functions searched by BLASTP and RPSBLAST tools. These ORFs can be divided into three functional modules:DNA metabolism and replication, DNA packing and structure formation and cell lysis. Comparative genome analysis revealed that phage CC2has the highest degree of similarity (64%) to A. salmonicida phage65but shows only12%similarity to A. hydrophila phage Aehl. Phylogenetic tree of three conserved proteins indicated that terminase, tail tube protein and DNA polymerase of phage CC2were mostly close to those of Aeromonas phages.To detect the bacteriolytic rate of lysozyme of A. hydrophila phage CC2, LysC, we obtained the purified LysC using the prokaryotic expression system. First, a pair of primers was designed according to the lysozyme gene. The lysC gene was amplified by PCR and inserted into pET-28a(+). The recombinant plasmid was transformed into E. coli BL21cells and induced to express. SDS-PAGE analysis showed the expected band with the molecular weight of18.4kD. After purification, the recombinant LysC was demonstrated to have bacteriolytic activity by slip method. And turbidimetry showed a50%turbidity decrease of sensitive bacteria using LysC with final concentration200μg/mL.