Characterization of Actinobacteriophage LittleTokyo Reveals Variable Diversity Within Cluster AS ●Phages possess incredible genetic diversity; however, there is a very small number of sequenced phages (Pope et al., 2017) ○Many phages are poorly characterized due to a lack of information ○There is limited information on Arthrobacter phages, as only 270 are currently sequenced ●One of the ways in which phages are classified is by placing them into clusters ○Clusters are groups of phages that have high nucleotide sequence similarities, shared life styles, and shared unique genes ●This project sequenced and classified a novel Arthrobacter phage, LittleTokyo ○LittleTokyo belongs to cluster AS ■Has three subclusters: AS1, AS2, and AS3 ■Formed in 2018, when phage Galaxy was found to be sufficiently similar to Abidatro (Klyczek et al., 2018) ●LittleTokyo is a temperate Arthrobacter globiformis phage ●TEM revealed LittleTokyo is a member of the Siphoviridae family through its icosahedral head diameter and tail length ●39.73% of genes were not assigned a function ●ANI heatmap and SplitsTree analyses show LittleTokyo is distinct from the AS2 subcluster ●Full genome Phamerator comparison reveals similarity between LittleTokyo and Kuleana that sets them apart from the AS2 subcluster ●Lysin A and Tape Measure Protein revealed inconsistent evolutionary origins ○In Lysin A, AS1 phages formed the outgroup from AS2 ○In TMP, AS1 phages were more closely related to the rest of the AS2 phages than LittleTokyo or Kuleana ●Evidence of horizontal gene transfer within the genome due to the presence of unique genes ○PAPS reductase-like domain ●Ackermann, HW. Bacteriophages: Methods in Molecular Biology, vol. 501, 2009, pp. 127-140. ●Dereeper, A. et al. Nucleic Acids Res, vol. 36, no. 2, 2008, pp. 465-469. ●Huson, DH. Bioinformatics, vol. 14, no. 1, 1998, pp. 68-73. ●Klyczek, KK. et al. Genome Announc, vol. 6, no. 5, 2018, pp. 1474-1517. ●Krumsiek, J. et al. Bioinformatics, vol. 23, no. 8, 2007, pp. 1026-1028. ●Papadopoulos, JS. and Agarwala, R. COBALT Bioinformatics, vol. 23, no. 9, 2007, pp. 1073-1079. ●Pope, WH. et al. mBio, vol. 8, no. 4, 2017, pp. 1069-1117. ●Savage, H. et al. Cell, 1997, vol. 5, pp. 865-906. ●Yoon, SH. et al. Antonie van Leeuwenhoek, vol. 110, 2017, pp. 1281-1286. Figure 1. LittleTokyo Genome Map A gene map of LittleTokyo was made using Inkscape and LittleTokyo’s genome from Phamerator. Highlighted genes represent genes of interest that were examined later in this project. The genome is 38969 base pairs long with 73 genes, 44 of which were assigned functions and 29 of which could not be assigned a function. Gene product functions were determined through Phamerator, PhagesDB, HHpred, the NCBI Basic Local Alignment Search Tool (BLAST), and the NCBI Conserved Domains Database (CDD). Annotated Genome of LittleTokyo Figure 2. TEM of LittleTokyo Transmission electron microscopy of LittleTokyo. Tail morphology was indicative of the Siphoviridae family. The tail was approximated to be 130 ± 2 nm and the head diameter was estimated to be 50 ± 2 nm. LittleTokyo Morphology Figure 3. Phamerator Genome Comparison Alignment of LittleTokyo’s genome with AS2 phages created through Phamerator and Inkscape. Comparison of LittleTokyo’s whole genome sequence to AS2 phages reveals higher synteny between LittleTokyo with Kuleana and slightly lower synteny compared to the other phages in the AS2 subcluster. (A) (B) Genome Comparison of Cluster AS2 Phages LittleTokyo is Distinct From Other Phages in Subcluster AS2 Figure 4. Phylogenetic Relationships of Cluster AS A Nexus file containing the shared gene content of the cluster AS and AZ phages was created using PhamNexus, which was used to create a SplitsTree diagram (Huson & Bryant, 2006). The AZ phages were included as an intercluster reference. In the tree, LittleTokyo and Kuleana are located on separate branches from the rest of AS2. The tree shows that LittleTokyo and Kuleana branch separately from o