Cluster J Mycobacteriophages: Intron Splicing in Capsid and Tail Genes

Bacteriophages isolated on Mycobacterium smegmatis mc2155 represent many distinct genomes sharing little or no DNA sequence similarity. The genomes are architecturally mosaic and are replete with genes of unknown function. A new group of genomes sharing substantial nucleotide sequences constitute Cluster J. The six mycobacteriophages forming Cluster J are morphologically members of the Siphoviridae, but have unusually long genomes ranging from 106.3 to 117 kbp. Reconstruction of the capsid by cryo-electron microscopy of mycobacteriophage BAKA reveals an icosahedral structure with a triangulation number of 13. All six phages are temperate and homoimmune, and prophage establishment involves integration into a tRNA-Leu gene not previously identified as a mycobacterial attB site for phage integration. The Cluster J genomes provide two examples of intron splicing within the virion structural genes, one in a major capsid subunit gene, and one in a tail gene. These genomes also contain numerous freestanding HNH homing endonuclease, and comparative analysis reveals how these could contribute to genome mosaicism. The unusual Cluster J genomes provide new insights into phage genome architecture, gene function, capsid structure, gene mobility, intron splicing, and evolution

Cluster J mycobacteriophages: intron splicing in capsid and tail genes

Bacteriophages isolated on Mycobacterium smegmatis mc2155 represent many distinct genomes sharing little or no DNA sequence similarity. The genomes are architecturally mosaic and are replete with genes of unknown function. A new group of genomes sharing substantial nucleotide sequences constitute Cluster J. The six mycobacteriophages forming Cluster J are morphologically members of the Siphoviridae, but have unusually long genomes ranging from 106.3 to 117 kbp. Reconstruction of the capsid by cryo-electron microscopy of mycobacteriophage BAKA reveals an icosahedral structure with a triangulation number of 13. All six phages are temperate and homoimmune, and prophage establishment involves integration into a tRNA-Leu gene not previously identified as a mycobacterial attB site for phage integration. The Cluster J genomes provide two examples of intron splicing within the virion structural genes, one in a major capsid subunit gene, and one in a tail gene. These genomes also contain numerous free-standing HNH homing endonuclease, and comparative analysis reveals how these could contribute to genome mosaicism. The unusual Cluster J genomes provide new insights into phage genome architecture, gene function, capsid structure, gene mobility, intron splicing, and evolution

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

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

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

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

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

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

Genome maps of Mycobacteriophage LittleE.

<p>The annotated map of LittleE 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