Molecular Characterization of Podoviral Bacteriophages Virulent for Clostridium perfringens and Their Comparison with Members of the Picovirinae

Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium responsible for human food-borne disease as well as non-food-borne human, animal and poultry diseases. Because bacteriophages or their gene products could be applied to control bacterial diseases in a species-specific manner, they are potential important alternatives to antibiotics. Consequently, poultry intestinal material, soil, sewage and poultry processing drainage water were screened for virulent bacteriophages that lysed C. perfringens. Two bacteriophages, designated ΦCPV4 and ΦZP2, were isolated in the Moscow Region of the Russian Federation while another closely related virus, named ΦCP7R, was isolated in the southeastern USA. The viruses were identified as members of the order Caudovirales in the family Podoviridae with short, non-contractile tails of the C1 morphotype. The genomes of the three bacteriophages were 17.972, 18.078 and 18.397 kbp respectively; encoding twenty-six to twenty-eight ORF's with inverted terminal repeats and an average GC content of 34.6%. Structural proteins identified by mass spectrometry in the purified ΦCP7R virion included a pre-neck/appendage with putative lyase activity, major head, tail, connector/upper collar, lower collar and a structural protein with putative lysozyme-peptidase activity. All three podoviral bacteriophage genomes encoded a predicted N-acetylmuramoyl-L-alanine amidase and a putative stage V sporulation protein. Each putative amidase contained a predicted bacterial SH3 domain at the C-terminal end of the protein, presumably involved with binding the C. perfringens cell wall. The predicted DNA polymerase type B protein sequences were closely related to other members of the Podoviridae including Bacillus phage Φ29. Whole-genome comparisons supported this relationship, but also indicated that the Russian and USA viruses may be unique members of the sub-family Picovirinae

Comparative Genome Maps for Podoviral Bacteriophages Isolated from the Russian Federation and the USA.

<p>The %GC plot displays regions (500-bp window) above and below the average GC content. Open reading frames (ORFs) are depicted as arrows in the predicted direction of transcription and identified putative protein domains are listed in the legend with their respective ORF designation.</p

Shared and Unique Genes present in Bacillus phage Φ29 versus Bacteriophages ΦCPV4, ΦZP2 and ΦCP7R.

<p>Gene clustering was determined by uclust and blastp as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038283#s3" target="_blank">methods</a> section.</p

Whole-genome Tetra-nucleotide Frequency Comparisons Relative to Bacteriophages ΦCPV4, ΦZP2 and ΦCP7R.

<p>Lower panel shows normalized frequencies of 256 tetranucleotides for each genome, upper panel shows Pearson correlation coefficients. In lower panel, the most closely-related genomes are shown in blue and red. Genome comparisons with correlation coefficients >0.5 are shown with blue points, r-squared values from simple linear regression >0.5 are shown with red lines. Genome names are shown on diagonal axis.</p

Phylogenetic Relationships of ΦCPV4, ΦZP2 and ΦCP7R among other related Bacteriophages based on the DNA Polymerase Protein.

<p>Phylogenetic analysis of polymerase amino acid sequences were completed in MEGA5 utilizing MUSCLE for alignments with two thousand bootstrap replications and the <i>Clostridium</i> phage ΦCTP1 polymerase outgroup.</p

Electron Micrograph of Bacteriophage ΦCP7R.

<p>Scale bar represents 100 nm.</p

Whole-genome Similarities as Determined by Tetranucleotide Frequencies.

<p>Dendrogram is based on a dissimilarity matrix for whole bacteriophage genomes constructed in R as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038283#s3" target="_blank">methods</a> section.</p

Inverted Terminal Repeat Genomic Nucleotide Sequences for Bacteriophages ΦCPV4, ΦZP2 and ΦCP7R.

<p>Inverted terminal repeat (ITR) sequences were identified by whole-genome analysis utilizing the EMBOSS 6.3.1: palindrome program.</p