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

Abstract

To date, Certrevirus is one of two genera of bacteriophage (phage), with phages infecting Pectobacterium atrosepticum, an economically important phytopathogen that causes potato blackleg and soft rot disease. This study provides a detailed description of Pectobacterium phage CB7 (vB_PatM_CB7), which specifically infects P. atrosepticum. 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 Certrevirus, sharing sequence similarity with other members, highlighting conservation within the genus. Conserved elements include a putative early promoter like that of the Escherichia coli 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 Vequintavirus and Seunavirus. The results of this study provide greater insights into the phages of the Certrevirus genus as well as the subfamily Vequintavirinae.

The component parts of bacteriophage virions accurately defined by a machine-learning approach built on evolutionary features

“…The mass spectrometry data is crucial as it enables a discrimination between false positive (FP; predicted but not present by mass spectrometry of the virion) and true positive (TP; predicted and found present by mass spectrometry of the virion). Phage vB_EfaS_271 infects Enterococcus faecalis (26), phage vB_PatM_CB7 infects Pectobacterium atrosepticum (27), and phage vB_Eco4M-7 infects enteropathogenic Escherichia coli (28). STEP 3 was benchmarked against equivalent predictors: PVPred, PVP-SVM, PVPred-SCM and Pred-BVP-Unb (Fig.…”

“…(c) For phage vB_PatM_CB7, the bar chart counts the virion proteins correctly retrieved as TP and non-virion proteins mistakenly predicted as FP. (d) Detailed predictions from STEP 3 and other tools for vB_PatM_CB7 virion proteins defined by mass spectrometry (27). (e) For phage vB_Eco4M-7, the bar chart counts the virion proteins correctly retrieved as TP and non-virion proteins mistakenly predicted as FP.…”

Component Parts of Bacteriophage Virions Accurately Defined by a Machine-Learning Approach Built on Evolutionary Features

“…The mass spectrometry data are crucial, as it enables discrimination between false-positive (FP; predicted but not present by mass spectrometry of the virion) and true-positive (TP; predicted and found present by mass spectrometry of the virion) results. Phage vB_EfaS_271 infects Enterococcus faecalis ( 26 ), phage vB_PatM_CB7 infects Pectobacterium atrosepticum ( 27 ), and phage vB_Eco4M-7 infects enteropathogenic Escherichia coli ( 28 ). STEP 3 was benchmarked against equivalent predictors: PVPred, PVP-SVM, Pred-BVP-Unb, and PVPred-SCM ( Fig.…”

“…(c) For phage vB_PatM_CB7, the bar chart shows the numbers of virion proteins correctly retrieved as TP and nonvirion proteins mistakenly predicted as FP. (d) Detailed predictions from STEP 3 and other tools for vB_PatM_CB7 virion proteins defined by mass spectrometry ( 27 ). (e) For phage vB_Eco4M-7, the bar chart shows the numbers of virion proteins correctly retrieved as TP and nonvirion proteins mistakenly predicted as FP.…”

“…Finally, a training data set (243 positive samples and 694 negative samples) and an independent data set (96 positive samples and 96 negative samples) were obtained, where each had less than 40% sequence similarity against each other. Three very recently reported phage genomes vB_EfaS_271 ( 26 ), vB_PatM_CB7 ( 27 ), and vB_Eco4M-7 ( 28 ), as well as two newly sequenced phage genomes MMNM and MMBB in this study, were used to validate the prediction capability of STEP 3 in practical scenarios.…”