Genome maps of Mycobacteriophage BAKA.

<p>The annotated map of BAKA is represented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069273#pone-0069273-g003" target="_blank">Figure 3</a>.</p

Dotplot of Cluster J and two Subcluster F1 phages.

<p>Whole genome nucleotide sequences of the Cluster J phages and phages Wee and DeadP from Cluster F1 were compared to themselves and to each other using the program Gepard [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069273#B48" target="_blank">48</a>]. Dotted lines are used to indicate the correspondence of the common segment in Wee and DeadP with the Thibault genome.</p

HNH endonuclease insertion regions.

<p><b>A</b>. Regions including <i>lysA</i> genes showing insertion of a free-standing HNH endonuclease into Courthouse upstream of <i>lysA</i>. <b>B</b>. LysA region of Optimus and Omega, showing two different predicted HNH endonucleases, Optimus gp51 and Omega gp51. <b>C</b>. Sequence alignment of Courthouse and Thibault shows a precise insertion of predicted HNH endonuclease. <b>D</b>. Sequence alignment of Optimus and Omega, showing the HNH insertion site.</p

HNH endonuclease phamilies in Cluster J phages.

<p><b>A</b>, <b>B</b>. Pham circles for phams 3687 (A) and 6944 (B). Phage names are organized by cluster/subcluster sequentially in a clockwise direction around the edge of the circle. Phages containing a gene within each pham are connected by an arc in blue (BLASTP) or red (ClustalW). Cirlces were drawn using Phamerator and thresholds of 32% identity and an E value of 10^-50 for ClustalW and BlastP respectively. <b>C</b>, <b>D</b>. Phylogenetic trees of pham 3687 genes (C) and pham 6944 genes (D). Trees were generated using ClustalW multisequence alignments and neighbor-joining. Trees were drawn using NJPlot. Phages are color coded to designate cluster assignment, as shown in the key.</p

Morphologies of Cluster J phages.

<p><b>A</b>. Electron micrographs of Cluster J phages. From left to right, top row: BAKA, Courthouse, LittleE; Bottom row: Omega, Optimus, Thibault. Phages were negatively stained with 1% uranyl acetate. Scale bar = 100nm. <b>B</b>. Phage BAKA Cryo-EM. The DNA-filled capsids are icosahedral with planar sides, and the long striated tail ends in a small two-layered base structure and tail tip. Capsids appear regular in the cryo-micrograph with small features evident at the outer surface and the tightly packaged dsDNA revealing the typical “fingerprints” of swirls and punctate patterns. Bar = 50nm. <b>C</b>, <b>D</b>. The capsid structure calculated from 250 particles to 2.2nm resolution is represented by surface shading colored by radius (C), or as a central thin section where dark regions correspond to the capsid (outer layer) and DNA (inner layers) spaced ~2.3nm apart (D). Capsomers on the vertices in panel C are at higher radii and appear dark blue, with adjacent hexavalent capsomers light blue and the remaining capsomers around the icosahedral 3-fold axes in green. The capsid diameter varies from 72nm across the 2-fold axis to 85nm across the 5-fold vertices. Organization of capsomers on the icosahedral lattice is described by a Triangulation number [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069273#B49" target="_blank">49</a>], T=13, in which the facet of the icosahedron is tiled by 13 smaller 3-subunit triangles (see inset). This organization is similar to that observed for coliphage T5 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069273#B31" target="_blank">31</a>]. Bar = 10nm.</p

Genome maps of Mycobacteriophage Courthouse.

<p>The annotated map of Courthouse is represented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069273#pone-0069273-g003" target="_blank">Figure 3</a>.</p

Genome maps of Mycobacteriophage Thibault.

<p>The annotated map of Thibault is represented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069273#pone-0069273-g003" target="_blank">Figure 3</a>.</p

Genome maps of Mycobacteriophage Optimus.

<p>The annotated map of Optimus is represented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069273#pone-0069273-g003" target="_blank">Figure 3</a>.</p

Intron splicing in LittleE and BAKA.

<p><b>A</b>. 12% polyacrylamide-SDS gels of CsCl-purified Omega particles (left and center) and LittleE particles (right). In the left lane, Omega particles were heavily loaded to view less-abundant proteins. The major dark band corresponds to the major capsid as well as the major tail subunit protein that co-migrate. The lightly loaded sample in the right show the masses of the dominant protein species. <b>B</b>. Organizations of the capsid genes in Omega and LittleE. The major capsid genes are shown in blue, with the two exons of LittleE’s capsid gene connected by a black line. The HNH endonuclease (14) encoded within the LittleE intron is transcribed in the leftwards direction. Shading between the two genomes reflects nucleotide sequence similarity as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069273#pone-0069273-g001" target="_blank">Figure 1</a>. <b>C</b>. Nucleotide sequence alignment of the intron-splicing regions of LittleE and Omega. Amino acids translations are shown above the nucleotide sequence, with those determined by N-terminal sequencing in red. <b>D</b>. Agarose gel electrophoresis of RT-PCR amplification of the spliced LittleE intron. Lane M is a molecular weight ladder, with markers shown in kbp. cDNA amplification using LittleE primers used RNA from uninfected cells (lane 1), RNA from LittleE-infected cells (lane 2), LittleE phage genomic DNA (lane 3), and using RNA from infected cells but without reverse-transcriptase (lane 4). <b>E</b>. Map of minor tail protein regions of BAKA and Thibault, with the two exons of BAKA’s minor tail gene connected by a black line. <b>F</b>. Agarose gel electrophoresis of RT-PCR-amplification of BAKA intron region. Lane M is a molecular weight ladder, with markers shown in kbp. cDNA amplification using BAKA primers used RNA from BAKA-infected cells (lane 1), BAKA phage genomic DNA (lane 2), using RNA from infected cells but without reverse-transcriptase (lane 3), and RNA from uninfected cells (lane 4). <b>G</b>. Nucleotide sequence alignment of the spliced region in the minor tail gene in BAKA. Amino acid translations are shown above the nucleotide sequence, with exon-encoded residues in bold type.</p

Integration functions of Cluster J phages.

<p><b>A</b>. Architecture of the integration cassette in Cluster J phages. The <i>attP</i> site is located downstream of the integrase gene with the 44 bp common core – which is shared by the <i>attB</i> site – located approximately 100bp from the 3’ end of <i>int</i>. The common core is flanked by pairs of putative arm-type integrase binding sites, P1 and P2 to its left, and P3 and P4 to its right. At the start of the <i>int</i> genes of Omega and BAKA there two arm-type-like binding sites in inverted orientation overlapping the putative integrase start codons. <b>B</b>. Sequences of the putative arm-type binding sites in BAKA and Omega, with the consensus sequences (con). <b>C</b>, <b>D</b>. Schematic representations of the tRNA-Leu genes overlapping the common core at <i>attB</i> in <i>M. smegmatis</i> (C) and <i>M. tuberculosis</i> (D). Sequence differences between the two tRNAs are circled. The left end of <i>attB</i> site is indicated by a horizontal line in the anticodon loop of the Msmeg_3245 tRNA. <b>E</b>. Agarose gel of PCR products demonstrating integration of plasmid pKR03. Lane 1 is a control using pKR03 DNA and primers deigned to amplify <i>attR</i>. Lane 2 uses the same primers and DNA from a pKR03 transformant.</p