Isolation and Characterization of Pectobacterium Phage vB_PatM_CB7: New Insights into the Genus Certrevirus

Abstract

To date, <i>Certrevirus</i> is one of two genera of bacteriophage (phage), with phages infecting <i>Pectobacterium atrosepticum</i>, an economically important phytopathogen that causes potato blackleg and soft rot disease. This study provides a detailed description of <i>Pectobacterium</i> phage CB7 (vB_PatM_CB7), which specifically infects <i>P. atrosepticum</i>. Host range, morphology, latent period, burst size and stability at different conditions of temperature and pH were examined. Analysis of its genome (142.8 kbp) shows that the phage forms a new species of <i>Certrevirus,</i> sharing sequence similarity with other members, highlighting conservation within the genus. Conserved elements include a putative early promoter like that of the <i>Escherichia coli</i> sigma70 promoter, which was found to be shared with other genus members. A number of dissimilarities were observed, relating to DNA methylation and nucleotide metabolism. Some members do not have homologues of a cytosine methylase and anaerobic nucleotide reductase subunits NrdD and NrdG, respectively. Furthermore, the genome of CB7 contains one of the largest numbers of homing endonucleases described in a single phage genome in the literature to date, with a total of 23 belonging to the HNH and LAGLIDADG families. Analysis by RT-PCR of the HNH homing endonuclease residing within introns of genes for the large terminase, DNA polymerase, ribonucleotide reductase subunits NrdA and NrdB show that they are splicing competent. Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) was also performed on the virion of CB7, allowing the identification of 26 structural proteins-20 of which were found to be shared with the type phages of the genera of <i>Vequintavirus</i> and <i>Seunavirus.</i> The results of this study provide greater insights into the phages of the <i>Certrevirus</i> genus as well as the subfamily <i>Vequintavirinae</i>.