1. Cluster frequency for Phams found in Tortellini Genes 30-73
Genomic Analysis of Mycobacteriophage Tortellini Reveals Significant Genetic Differences from Current P Cluster Phages, Establishing this Phage as the First Member of the P2 Subcluster
Kyla Foster, Nathaniel Moore, Paul Bisesi, Tyler Yates, Christy Fillman, Nancy Guild
Figure 1: Comparative Genome Map of Phages Purky, Tortellini, and Phayonce. Phayonce was chosen as a representative member of P1, and Purky was selected as the P1 phage most related to Tortellini. This Phamerator map shows that considerable genetic differences exist between Tortellini and the P1 cluster, especially from genes 30 to 73. The most notable feature of this region is the bacterial integrase, located at gene 33, found in a region of the genome where 10 genes (30-34, 36-39, 42) have higher sequence similarities to bacterial genes than phage genes suggesting the occurrence of lateral gene transfer between Tortellini and its bacterial hosts.
Abstract
Mycobacteriophage are classified and subdivided into clusters and subclusters based on genetic similarities. Preliminary results indicated Mycobacteriophage Tortellini contained considerable genome sequence similarity to phages in the P cluster. However, annotation and analysis of the Tortellini genome revealed the presence of substantial genetic differences when compared to other members of the P cluster. These differences supported the creation of a new P subcluster (P2) with one member: Tortellini. We describe the genome differences in detail, focusing on the second half of the genome and its sequence similarity to phages in other clusters. This second half of Tortellini’s genome also contains several genes whose sequences are similar to bacterial genes from other Actinobacterial hosts.
Cluster frequency for Phams found in Tortellini Genes 30-73
Frequency
Figure 7A: ClustalX Comparative Amino Acid Analysis between Tortellini and Mycobacterium canariasense BLASTp analysis of gene 32 of Tortellini (33 in Figure 1) showed the highest similarity with integrase of Mycobacterium canariasense as supported in this ClustalX analysis of the two integrases. Tortellini’s integrase is a member of pham consisting of 7 other phages, all of which were isolated on other Actinobacterial hosts. Tortellini may be able to infect other mycobacterial species as well as other Actinobacterial hosts.
Figure 7B: ClustalX Comparative Amino Acid Analysis between Tortellini and Purky. The amino acid sequence of the integrases from Tortellini (P2) and Purky (P1) are compared. There is substantially less amino acid similarity between Tortellini and Purky’s integrases when compared to the integrases from Tortellini and Mycobacteria canariansense, supporting Tortellini’s distinction into a new P subcluster and its potential to acquire bacterial genes through lateral gene transfer.
MW Uncut BamHI
EcoRI EcoRV
Background
Mycobacteriophage Tortellini was identified as a P cluster phage from a pool of unspecified phage genomes through PCR and restriction digestion analysis (Figure 2). Temperate phages, such as Tortellini, can integrate their DNA into the host genome where they will be replicated along with the host chromosome until unfavorable environmental conditions cause the phage to enter the lytic cycle. During the induction of lysis phage can pick up host genes, resulting in lateral gene transfer of bacterial genes. Using the bioinformatics programs Phamerator and DNA Master mycobacteriophage have been classified into a total of 26 clusters containing phage with over 50% similarity, and 55 subclusters, which have an average threshold of 75% similarity (3, 5).
A C E F G I J K M N P T Y
Cluster
Figure 4: Cluster Frequency for Phams Found in Tortellini Genes Clusters present in each pham for genes were counted and the total frequency for each cluster is displayed. Figure 5B compares the nucleotide sequences of Tortellini (P2) and Awesomesauce (F1) . The similarities between the two phages in the later half of their genomes is consistent with the high frequency of the F cluster phams shown in this bar graph.
Figure 5A: Dotplot comparison of nucleotide sequences of Tortellini and Purky. Nucleotide sequences of Purky, a P1 phage, and Tortellini, a P2 phage, are compared. The beginning of the genome (upper right) and the end of the genome (bottom left) display strong similarity. However, very little nucleotide similarity is seen in the second half of the genome, indicating enough nucleotide differences to justify Tortellini's classification into a separate subcluster.
Figure 5B: Dotplot comparison of nucleotide sequences of Tortellini and Awesomesauce. Nucleotide sequences of Awesomesauce, a F1 phage, and Tortellini, a P2 phage, are compared. During comparative genomic analysis, Awesomesauce showed several regions of genome similarities with the second half of Tortellini’s genome; the region which diverges most from other P1 phages. A B
Conclusions
The first half of Tortellini’s genome shows strong sequence similarity to other P cluster phages, while the second half of the genome does not share the sequence similarity when comparing the phamerator maps (Figure 1) and the nucleotide sequences (Figure 5A). When the phams of genes of Tortellini were analyzed, F cluster phages were the most abundant members in these phams (Figure 4), further supporting the classification of Tortellini as the first member of the P2 subcluster. This also suggests a possible common ancestry between Tortellini and F cluster phages. Interestingly, Tortellini has 10 genes of bacterial origin (Figure 1) near the integrase gene which not only implies recombination between this region of Tortellini’s genome and its bacterial hosts, but also supports Tortellini having a wider host range.
Figure 2: Restriction Digest of Tortellini Compared to a Virtual Digest of a P phage. Based on preliminary PCR experiments with primers specific for the P-cluster contig (identified through the DNA sequencing of a pool of phage genomic DNA) Tortellini was a strong candidate for the P cluster classification. Tortellini DNA was digested with BamHI, EcoRI, and EcoRV and the similarities between this digest and a virtual digest of the P-cluster contig provided sufficient data to support the hypothesis that Tortellini was the P cluster phage.
Acknowledgments
This research would not have been possible without the support of HHMI, SEA-PHAGES, the University of Colorado at Boulder, and the mentorship of Dr. Christy Fillman and Dr. Nancy Guild. Thank you.
References
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Figure 3A: Electron Microscopy Image of Mycobacteriophage Tortellini shows a siphoviridae morphotype. Tortellini’s tail length was 203.5nm and its head diameter was 62.58nm. Phage lysates were stained with uranyl acetate and visualized using electron microscopy.
Figure 6: Comparative Genome Map of Phages Tortellini and Awesomesauce. Tortellini is a P2 cluster phage and Awesomesauce is F1. The Phamerator map above only displays the second half of the two genomes indicating considerable similarity between these two phages. The first half of Tortellini’s genome is very similar to P1 cluster phage (Figure 1). However, within this first half of the genome there is another similarity to Awesomesauce: gene 17 of Tortellini and gene 16 of Awesomesauce (not shown) are members of the same phamily and encode a minor tail protein.
Figure 3B: Plaque morphology of Tortellini indicates classification as a temperate phage. The
10-3 dilution of a titer assay of Tortellini revealed a plaque size of approximately 1.5mm in diameter. These plaques were turbid with halos suggesting that Tortellini was temperate. Almost all of the other P cluster phage produce plaques that are turbid in appearance with defined halos.