Diversity of mycobacteriophages.

<p>Sequenced genomes for 471 mycobacteriophages were compared according to their shared gene contents, and the relationships are displayed using Splitstree <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003953#ppat.1003953-Huson1" target="_blank">[24]</a>. The genomes are clustered according to overall nucleotide sequence similarity, and the clusters (A, B, C…) correlate closely with their gene content. Colored circles encompass Clusters A–T as indicated, and grey circles represent singleton genomes that have no close relatives. Ten of the clusters are divided into subclusters (e.g., A1, A2, A3….) and are shown as circles within each cluster. Micrographs show the two morphotypes observed, typified by the myoviral Cluster C phages and the siphoviruses (all others) that primarily differ in tail length (scale bars, 100 nm). With the exception of one singleton (DS6A), all of the phages infect <i>M. smegmatis</i> mc²155. DS6A, the Cluster K phages and a subset of Cluster A phages also infect <i>M. tuberculosis</i>.</p

Network representation of mycobacteriophage Lysin A relationships.

<p>A matrix of the presence/absence of each of the 15 individual domains in the 224 genomes was analyzed using Splitstree and its NeighborNet function. Genomes within each of the 25 organizations (Org-A – Org-Y) are circled and individual genome names are colored according to cluster/subcluster. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034052#pone-0034052-g002" target="_blank">Figure 2</a> for specific cluster/subcluster designations.</p

Comparison of the left parts of Cluster A2 genomes.

<p>Maps of the Subcluster A2 genomes were generated using the program Phamerator <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034052#pone.0034052-Cresawn1" target="_blank">[58]</a> and the left parts (approximately 20–25 kbp) are shown. Each of the predicted protein-coding genes is shown as a box with its color corresponding to its phamily designation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034052#pone.0034052-Cresawn1" target="_blank">[58]</a>; the number of the phamily is shown above the gene with the number of phamily members in parentheses. Coloring of the regions between the genomes reflects the strength of pairwise nucleotide similarity, with the strength reflected according to the color spectrum (violet being the most similar, and the red the least similar). Note that there is higher divergence among the lysis genes and the flanking parts of the genome. An example of intragenic mosaicism reflected at the nucleotide sequence level is provided in the comparison of the L5, Trixie, and Turbido endolysins (Lysin As), reflecting domain organizations Org-C, Org-E, and Org-F respectively.</p

Domain organization of mycobacteriophage endolysins.

<p>Each of the mycobacteriophage endolysins is designated according to the presence (+) or absence (−) of each of three domain types: an N-terminal domain (N1, N2, N3, N4, N5, M23), a central catalytic domain (Ami-2A, Ami-2B, GH19, GH25, or TG), and a C-terminal domain (C1, C2, C3, or LGFP). Each is designated with an organizational type (Org) according to the domain content as listed in the rightmost column.</p

Schematic representation of mycobacterial peptidoglycan and generalized target bonds of peptidoglycan hydrolases.

<p>The type A1γ peptidoglycan of <i>M. tuberculosis</i> is proposed to contain the typical 4→3 interpeptide bridges between m-DAP and D-Ala but also 3→3 m-DAP to m-DAP bonds <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034052#pone.0034052-Gupta1" target="_blank">[30]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034052#pone.0034052-Lavollay1" target="_blank">[31]</a>. There are seven positions within peptidoglycan where phage endolysins are known or are proposed to cleave (red arrows): 1, N-acetyl-β-D-muramidase (lysozyme, lytic transglycosylase); 2, N-acetyl-β-D-glucosaminidase; 3, N-acetylmuramoyl-L-alanine amidase; 4, L-alanoyl-D-glutamate (LD) endopeptidase; 5, γ-D-glutamyl-meso-diaminopimelic acid (DL) peptidase; 6, D-Ala-m-DAP (DD) endopeptidase; 7, m-DAP-m-DAP (LD) endopeptidase. Mycobacteriophage endolysins contain domains that are predicted to cleave all of these sites with the exception of position #2. The GH19, GH25, and TG domains cleave at position #1, Ami-2A and Ami-2B cleave at position #3, the N1 domain is predicted to cleave at position #4, the N5 position is predicted to cleave at #5, and the M23, N2 and N3 domains are predicted to cleave interpeptide bridges such as #6 and possibly #7. Peptide linkages to the central MurNAc residues, glycolylated muramic acid residues, and the amidation of D-Glu and m-DAP are not shown. GlcNAc, N-acetyl glucosamine; MurNAc, N-acetyl muramic acid; m-DAP, meso-diaminopimelic acid.</p

Features of bioinformatically-defined Mycobacteriophage endolysin domains.

<p>Features of bioinformatically-defined Mycobacteriophage endolysin domains.</p

Identification of Corndog gp69 fragment with increased lytic activity.

<p><b>A.</b> Left, SDS PAGE with increasing amounts of Corndog gp69 sample illustrating the minute amount of protein (red asterisk) corresponding to one of the bands of high activity seen in the zymogram on the right (red box). <b>B.</b> Highly concentrated sample of Corndog gp69 from which a sample was taken (red asterisk) and submitted to Edman degradation. The control sequencing of the large band matched the N-terminal sequence of Corndog gp69 (blue brackets), while the smaller fragment corresponded to a sequence 153 aa C-terminal to the start of Corndog gp69 (orange brackets).</p

ATP release upon induction of lysin expression.

<p>Cultures of <i>M. smegmatis</i> mc2155 carrying pLAM12-based plasmids with different lysins were split and half were induced with 0.2% acetamide. <b>A.</b> ATP release was measured for 7 hours and the fold-difference between induced and uninduced ATP release calculated for each lysin and plotted versus the time. <b>B.</b> After more than 16 hours of induction several of the cultures had lysed completely.</p

Recombination of domains between LysinAs.

<p>ClustalW alignments of four LysA proteins were used to construct a phylogenetic tree for the N-terminal and amidase domains. <b>A</b>. Proteins are identified by phage name, gp#, and Organizations. <b>B.</b> and <b>C.</b> Tree illustrating the most parsimonious phylogeny for the (<b>B</b>) N-terminal and (<b>C</b>) amidase domains. Bootstrap values are 100 for the division of recombination between the domains, and an SH test rejected alternate topologies (<i>P-</i>value <0.001). Branch lengths do not represent evolutionary distance.</p

Modular organizations of mycobacteriophage endolysins.

<p>Through combinations of 15 domains and sequence elements, 26 different organizations are observed in the 240 mycobacteriophage Lysin As. Representative Lysin A structures shown for each organization. N1–N5 (red and pink shades) and C1–C3 (purple shades) are N-terminal and C-terminal regions, respectively. Predicted catalytic functions include Ami-2A and Ami-2B (amidases, green and aquamarine), GH19 (glycoside hydrolase family 19, yellow), GH25 (glycoside hydrolase family 25, orange), M23 (M23 family peptidase, blue), and TG (lytic transglycosylase, cyan). Also identified are the predicted binding motifs PGBD (peptidoglycan binding domain, gray) and LGFP repeats (rose).</p