Isolation and characterization of the A3 bacteriophage Kady from the host Mycobacterium smegmatis John Sherwood1, Victoria Torres1, Jasmina Cunmulaj1, Amber Elinsky1, Gabrielle Ford1, Emily Gorkiewicz1, Christopher Harness1, Anthony Huffman1, Christina Jones1, Anna Julien1, Danielle Maxwell1, Elizabeth Moak1, Payal Patel1, Madeleine Reardon1, Royce Swasey1, Tamia Waller1, Katherine Yacco1, Stephanie Conant1, Joshua Thomson2, and Jacob D. Kagey1 ​ 1.Department of Biology, College of Engineering and Science, University of Detroit Mercy 2. University of Detroit Mercy School of Dentistry Abstract This fall eight novel bacteriophage were isolated as part of the HHMI SEA-Phages course at the University of Detroit Mercy. Two phages were submitted for sequencing and genomic annotation. Here we present the genomic annotation of the A3 cluster phage, Kady. Kady was isolated from an enriched soil sample from Warren, MI using Mycobacterium smegmatis as the host. After isolation, Kady was identified as a lytic phage and purified using a series of streak plates. Once purified, DNA was isolated from Kady and it was sent in for sequencing at Pittsburgh Bacteriophage Institute. In total there are 50,898 base pairs in Kady’s genome. DNA Master was used to auto-annotate the genome. In Silico methods were then used to check all of these predictions as well as look for new genes in the novel genome. The DNA Master program predicted 90 genes in Kady. In total, two protein-coding genes were predicted to be false positives and deleted, and two protein genes were not auto annotated and were added to the genome. We also supported the identification of three tRNA genes predicted by DNA Master. Of note, we also annotated a frame shift. After final predictions were made, gene functions were assigned to those genes where function predictions could be made using BLAST data as well as HHPred software. This project is intended to help understand the genetic makeup and evolution of bacteriophages, as well as add to a growing collection of isolated and annotated bacteriophages, especially those who may have the ability to infect Mycobacterium tuberculosis. tRNA In addition to 90 Genes, 3 tRNA were predicted Predicted between basepairs 3922-4221 Predicted using Aragorn and Mobyle Pasteur software Leucine, Tryptophan, and Asparagine Figure 5. This is a picture of the predicted shape of tRNA (Asn) as predicted by Aragorn software. In Silico Gene Annotation In Silico gene annotation is the prediction of genes inside of a genome done completely on the computer with different softwares shown below. Figure 3A. Example of high coding potential as predicted by GLIMMER. Figure 3B. This shows DNA masters predicted genes, ORF diagrams, and ribosomal binding scores. Gene Prediction 11 SSC: Start: 7041 Stop: 8741 CP: Yes, all coding potential is contained SD: Best possible score Final Score: -2.339 Z-Score: 3.090 SCS: Both GeneMark and GLIMMER call 7041 LO: Yes, longest open reading frame Gap: 30 base pair gap BLAST: 1:1 alignment with gp13 of Mycobacterium phage Bxz2 ST: Starterator agrees and calls for 7041 Figure 3D. This is an example of all data compiled and considered in final gene predictions as recorded in lab notebooks. Figure 3C. Examples of BLAST(left), and a Starterator (right) which were used to predict start sites. Frameshift Figure 1: Plaque Morphology Plaques are clear zones formed in a lawn of cells due to lysis by phage. The plaques are 3 mm in size and lytic. Figure 2: Transmission Electron Microscopy Image of KADY An image is formed from the interaction of electrons transmitted through the specimen. KADY was classified in the Siphoviridae family. Introduction Kady’s soil sample was from Warren, MI Kady was isolated using soil enrichment techniques and grown in Mycobacterium smegmatis Kady was then purified using multiple rounds of streak plates and titer essays A High Titer Lysate (HTL) was created and stored The HTL was then sent to the University of Pittsburgh to have Kady’s genome sequenced for the In Silico gene annotation process Figure 6A: Phamerator Image of KADY and Wooldri Figure 6B: Six Frame Translation In Figure 6A, KADY (above) and Wooldri (below) are shown in phamerator where the frameshift occurred. Gp23 of KADY aligned with the tail assembly chaperone protein of Wooldri 28. A frameshift is caused when there is a signal for a ribosome to shift from one reading frame to another resulting in an insertion or deletion. In Figure 6B, it is shown that the insertion is an extra Cytosine at the six frame translation LIDEY. Conclusion In total 90 genes were predicted in the 50,900 basepairs of Kady’s genome 2 deleted from original auto-annotation 2 called by annotator, not found by auto-annotation (ORF 2, ORF 60.5) 3 tRNA were predicted The frameshift was determined to be genes 23 and 24 The integrase protein was classified as gene 34 Kady is apart of cluster A3 Acknowledgements This work was supported by the NIH Rebuild grant and the HHMI SEA Phage program. Thank you to Wayne State University for allowing use of their electronic Microscope and the Pittsburgh Bacteriophage Institute for sequencing our DNA. Figure 4: Final Genome Prediction Along the bottom is Kady’s complete genome after all predictions were finalized. Key: Forward Gene Reverse Gene Added Genes Frameshift