| Bacterial diseases are one of the important threats to the freshwater aquaculture industry,among which Aeromonas veronii is a representative pathogen causing aquatic diseases.In recent years,green and efficient microbial preparations have attracted our attention due to the promulgation of aquaculture drug regulations in China.Lytic phage is a ubiquitous bacterial killer in nature.As an ideal medicine for controlling pathogens,it has the advantages of targeted sterilization,high efficiency,and convenience due to the characteristics of specific lysis of host proliferation.In this study,the gram-negative bacteria Aeromonas veronii AV1212 was used as a host to isolated the siphoviridae family double-stranded DNA phage from sewage.A strain of phage-resistant bacteria AV0110 was isolated in the process of continuous transfer and culture.The genome-wide comparison analysis of the two strains based on reads and SNPs as well as the secondary sequencing verification of the mutation region revealed that the man B gene encoding(Phomannomutase,PMM)had a frameshift mutation.PMM is closely related to the synthesis of O-antigen of lipopolysaccharide(LPS).Specific adsorption is a necessary step for phage to cause infection,and the O-antigen of the lipopolysaccharide of gram-negative bacteria is a common phage adsorption receptor.Therefore,the mutation of the PMM of AV0110 may cause the phage Avs-1 to fail to adsorb.By expressing the man B gene in the resistant mutant bacteria AV0110,it was found that the resistance of the engineered bacteria was lost,which means that the mutation of man B is the reason why AV0110 gained resistance.In this study,the potential endolysin Lys40 in the genome of the phage Avs-1 was heterologously expressed in E.coli.It was found that Lys40 could significantly inhibit the growth of E.coli,but could not lyse E.coli.The amino acid sequences with perforin characteristics in the 60 ORFs in the Avs-1 phage genome were analyzed using bioinformatics methods.It was found that ORF42 and ORF44 have transmembrane domains and the C-terminus has hydrophilic and highly charged.ORF42 is also Contains signal peptides,and both belong to hypothetical proteins.Therefore,we speculated that they might be involved in the lysis of the host by the phage Avs-1,and constructed their individual expression and co-expression vectors.In this study,the resistance of Aeromonas veronii AV0110 to phage Avs-1 and lytic host-related enzyme of phage Avs-1 were studied.Experiments showed that phage resistance-mutations are genetically stable.Genomic analysis showed that phage resistance has nothing to do with the CRISPR-Cas system.The whole-genome comparison results showed that it is related to man B gene mutations.Genome analysis and experiments showed that there were no lysogenic factors and virulence factors in the Avs-1 genome,and there might be perforin that is quite different from the known protein sequence or a new lysis mechanism.The research on Aeromonas veronii and the new phage Avs-1 expands the phage gene library and provides a theoretical basis for further research on the interaction between pathogens and phages and the prevention and treatment of Aeromonas veronii. |
