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

Genome map of Mycobacteriophage YungJamal.

<p>The genome of phage YungJamal 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 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 Mycobacteriophages.

<p>Cluster O Mycobacteriophages.</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

Sequence features of Cluster O genomes.

<p><b>A</b>. The AT rich element between Corndog genes <i>12</i> and <i>13</i> is highlighted in cyan, and two sets of flanking sequence repeats are shown in red and green. A similar arrangement of these sequences is observed in the other Cluster O phages. Residues in these sequence elements that differ across the phages (in the case of the AT rich element), or from the repeat consensus sequences are shown in lower case. <b>B</b>. A portion of Corndog genes <i>120</i> (underlined in black) and <i>121</i> (underlined in gray). The conserved T₅CCT₆GT₆GT₅ sequence is shown in cyan and flanking sequence repeats are shown in green and red. Residues in these sequence elements that differ across the phages (in the case of the T rich element), or from the repeat consensus sequences are shown in lower case.</p

ANI values for cluster O phages.

<p>ANI values for cluster O phages.</p

Genome map of Mycobacteriophage Catdawg.

<p>The genome of phage Catdawg 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. Catdawg proteins identified in a phage lysate using LC-MS/MS with at least two high confidence peptides per product are indicated (L).</p

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