Comparative genomics of Cluster O mycobacteriophages

Mycobacteriophages - viruses of mycobacterial hosts - are genetically diverse but morphologically are all classified in the Caudovirales with double-stranded DNA and tails. We describe here a group of five closely related mycobacteriophages - Corndog, Catdawg, Dylan, Firecracker, and YungJamal - designated as Cluster O with long flexible tails but with unusual prolate capsids. Proteomic analysis of phage Corndog particles, Catdawg particles, and Corndog-infected cells confirms expression of half of the predicted gene products and indicates a non-canonical mechanism for translation of the Corndog tape measure protein. Bioinformatic analysis identifies 8-9 strongly predicted SigA promoters and all five Cluster O genomes contain more than 30 copies of a 17 bp repeat sequence with dyad symmetry located throughout the genomes. Comparison of the Cluster O phages provides insights into phage genome evolution including the processes of gene flux by horizontal genetic exchange

Comparative Genomics of Cluster O Mycobacteriophages

Mycobacteriophages – viruses of mycobacterial hosts – are genetically diverse but morphologically are all classified in the Caudovirales with double-stranded DNA and tails. We describe here a group of five closely related mycobacteriophages – Corndog, Catdawg, Dylan, Firecracker, and YungJamal – designated as Cluster O with long flexible tails but with unusual prolate capsids. Proteomic analysis of phage Corndog particles, Catdawg particles, and Corndog-infected cells confirms expression of half of the predicted gene products and indicates a non-canonical mechanism for translation of the Corndog tape measure protein. Bioinformatic analysis identifies 8–9 strongly predicted SigA promoters and all five Cluster O genomes contain more than 30 copies of a 17 bp repeat sequence with dyad symmetry located throughout the genomes. Comparison of the Cluster O phages provides insights into phage genome evolution including the processes of gene flux by horizontal genetic exchange

Comparative genomics of Cluster O mycobacteriophages.

Mycobacteriophages--viruses of mycobacterial hosts--are genetically diverse but morphologically are all classified in the Caudovirales with double-stranded DNA and tails. We describe here a group of five closely related mycobacteriophages--Corndog, Catdawg, Dylan, Firecracker, and YungJamal--designated as Cluster O with long flexible tails but with unusual prolate capsids. Proteomic analysis of phage Corndog particles, Catdawg particles, and Corndog-infected cells confirms expression of half of the predicted gene products and indicates a non-canonical mechanism for translation of the Corndog tape measure protein. Bioinformatic analysis identifies 8-9 strongly predicted SigA promoters and all five Cluster O genomes contain more than 30 copies of a 17 bp repeat sequence with dyad symmetry located throughout the genomes. Comparison of the Cluster O phages provides insights into phage genome evolution including the processes of gene flux by horizontal genetic exchange

Comparative Genomics of Cluster O Mycobacteriophages

<div><p>Mycobacteriophages – viruses of mycobacterial hosts – are genetically diverse but morphologically are all classified in the Caudovirales with double-stranded DNA and tails. We describe here a group of five closely related mycobacteriophages – Corndog, Catdawg, Dylan, Firecracker, and YungJamal – designated as Cluster O with long flexible tails but with unusual prolate capsids. Proteomic analysis of phage Corndog particles, Catdawg particles, and Corndog-infected cells confirms expression of half of the predicted gene products and indicates a non-canonical mechanism for translation of the Corndog tape measure protein. Bioinformatic analysis identifies 8–9 strongly predicted SigA promoters and all five Cluster O genomes contain more than 30 copies of a 17 bp repeat sequence with dyad symmetry located throughout the genomes. Comparison of the Cluster O phages provides insights into phage genome evolution including the processes of gene flux by horizontal genetic exchange.</p></div

Dylan MPME element.

<p>Phage Dylan contains a Mycobacteriophage Mobile Element (MPME) inserted between genes <i>46</i> and <i>48</i>. The Dylan MPME contains an open reading frame (<i>47</i>) that is transcribed leftwards, such that the MPME left inverted repeat (IR-R) is 48-proximal. Alignment of the Dylan MMPE sequence with MPME1 and MPMP2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118725#pone.0118725.ref027" target="_blank">27</a>] shows that one half (green box) is identical to MPME1 and the other half (yellow box) is identical to MPME2. The Dylan MPME is thus a hybrid of MPME1 and MPME2, presumably generated by homologous recombination with the intervening sequence (grey box).</p

Unusual translation initiation of the Corndog tape measure protein gene.

<p><b>A</b>. Two organizations of the tape measure genes are present in the Cluster O phages. In Dylan and Catdawg the <i>tmp</i> gene is predicted to start translation immediately downstream of the tail assembly chaperone genes that are translated via a programmed translational frameshift. In contrast, Corndog, Firecracker, and YungJamal have a non-coding gap prior to the <i>tmp</i> start site. However, LC-MS/MS identified Corndog peptides corresponding to this gap and the sequence of the most N-terminal peptides are shown in bold type. Translation presumably either initiates at the ACG threonine codon or starts further upstream and involves a ribosome bypass event. <b>B</b>. RT-PCR of Corndog transcripts. PCR products were generated using a Corndog lysate (lane 2) or RNA isolated from uninfected cells (lanes 3 and 4), or at different times after infected by Corndog: 30 min (lanes 5 and 6), 2.5 h (lanes 7 and 8), 3.5 h (lanes 9 and 10), and 4.5 h (lanes 11 and 12. Lanes 3, 5, 7, 9, and 11 are controls lacking reverse transcriptase. A DNA ladder marker (M) is shown with sizes in base pairs. Genomic DNA and unspliced RNAs generate an expected product of ~1.7 kbp. No smaller spliced products are observed.</p

Genome map of Mycobacteriophage Firecracker.

<p>The genome of phage Firecracker is represented as a scale bar (major intervals: 1 kbp) with predicted genes shown as boxes either above (rightwards transcribed) or below (leftwards transcribed). Gene number is shown within each box and the phamily designation is shown either above or below with the number of phamily members shown in parentheses. Putative gene functions are indicated. The positions of putative SigA-like promoters (P_L1—P_L6 and P_R1—P_R3) are shown as large arrows. Small vertical arrows show the locations of the palindromic repeat 5′-TGTTCGGNNNCCGAACA.</p

Genome map of Mycobacteriophage Dylan.

<p>The genome of phage Dylan is represented as a scale bar (major intervals: 1 kbp) with predicted genes shown as boxes either above (rightwards transcribed) or below (leftwards transcribed). Gene number is shown within each box and the phamily designation is shown either above or below with the number of phamily members shown in parentheses. Putative gene functions are indicated. The positions of putative SigA-like promoters (P_L1—P_L6 and P_R1—P_R3) are shown as large arrows. Small vertical arrows show the locations of the palindromic repeat 5′-TGTTCGGNNNCCGAACA.</p

Cluster O mycobacteriophage virion morphologies.

<p><b>A</b>. Electron micrographs of Cluster O phages. Scale bar corresponds to 100 nm. <b>B</b>. SDS-PAGE analysis of Corndog virions.</p