Allan J. Somers, Elizabeth Rutledge PhD., Salish Kootenai College Investigation of immunity class and repressor gene sequence in novel mycobacteriophages. Allan J. Somers, Elizabeth Rutledge PhD., Salish Kootenai College B. A. C. Abstract Methods Mycobacteriophage phages are found in many environments including land and marine environments. Our results show the M. smegmatis lysogen of phage Flapjack has immunity to WishMePhage infection. Flapjack lysogen phage resists infection because all of the repressor genes are located in the same cluster. This allows the Flapjack repressor protein to silence the WishMePhage infection. If a repressor protein keeps the phage silent it will not allow invading phages to infect it. The goal of this study was to investigate why Flapjack phage, in a lysogenic state, was immune to the infection of WishMePhage, and to compare Tikka phages repressor gene sequence to six other phage lysogens. Immunity tests were conducted. Crude, filtered lysates of the phages were used for PCR of the repressor gene. Bioinformatics analysis was performed after PCR sequencing. We analyzed two phages using this method and compared their repressor genes. Tikkas repressor gene sequence was nearly identical to that of phage SKCA1, suggesting they are in the same cluster group. It’s also possible that Tikka is a sub-cluster of SKCA1. Using this method, we can screen for more phages to understand their genetic exchanges and could use this method as a tool to understand horizontal genetic transfer as well as to better understand repressor genes. This technique could be used to monitor viruses and their evolution and as a tool to understand genetic inhibition, and gene expression. D. E. F. G. Figure 4. After all 6 lysogens were tested the only lysogen that showed immunity to WishMe phage was FlapJack phage figure D. Phage names with a protein identity match of 100% with TIKKA except BxB1 Plant 7149-Draft_73 Ichabod-Draft_77 Dynamix_Draft_Draft_74 Dlane_38, Repressor BpBiebs31_80, repressor Bobi_BoBI_40 Batiatus_Draft_41 Astraea_48 Fruitloop_37 Alice_41 BxB1 97 % Figure 2. Lytic and Lysogenic pathway after a phage attaches itself to a host (3). Samples. Soil samples were collected from various locations around Montana, including Salish Kootenai campus. WishMe phage was captured by using a .22um filter and processed for DNA extraction. DNA extraction procedure. Wish me Phage was processed using Qiagen DNA easy kit protocol. PCR preparation. Samples were carried out in 25uL volume reaction using Qiagen taq polymerase. The primers used were Repressor HTH_F1- aggctctggtaccttgaacg and RepressorHTH_R1- acaaggaggaccgatgagaausing. PCR products were verified on a 0.8% agarose gel. PCR product purification. A Mini Elute PCR purification kit protocol was used and sent to Murdock lab for DNA sequencing. Introduction Figure 5. This graph represents 11 phages amino acid sequence that were matched With other phages using blastp on the phage website. Research on phage is imperative to better understand repressor genes and their evolution as a tool to understand genetic inhibition, and gene expression. A repressor protein has the ability to inhibit expression of certain genes by binding to regions in DNA and silencing it. An immunity test works by infecting lysogens with a virus, if the lysogen contains a repressor gene, a silence protein will turn off gene expression from the invader resulting in a plaque not developing which means the lysogen is immune to the phage. Clusters of phage are determined by looked at their amino acid sequence classified by similarity into a phylogeny. The method of collection were sampled locally around the flathead reservation in Montana. The samples were then tested for DNA and analyzed looking at the phage repressor genes. *Flap Jack phage is the only phage out of 6 other lysogens that showed resistance to Wish Me phage. All other lysogens that were tested showed plaques. Results Conclusion A repressor gene was identified and amplified in Wish Me phage, which was compared to other sequences based on their amino acid matches. Using the results from this screening, the sequences were analyzed for differences in their nucleotide composition because if they are in the same cluster the invading phage will not be able to infect the lysogen. By identifying resulting amino acid matches within different phage genome, a phylogeny can be developed with further investigation. A. Score- Method Identities- 94.0 bits(232) Compositional matrix adjust 45/45 100% SKCA1 TREQLPRLSLEVIEALKATGETEADIARMYGVTPQAVSWHVHTYG 45 TREQLPRLSLEVIEALKATGETEADIARMYGVTPQAVSWHVHTYG BxB1 TREQLPRLSLEVIEALKATGETEADIARMYGVTPQAVSWHVHTYG 92.4 bits(228) Compositional matrix adjust 44/47 94% SKCA1 TREQLPRLSLEVIEALKATGETEADIARMYGVTPQAVSWHVHTYG TIKKA TREQLPRLSLEVIEALKAAGETEADIARMYGVTPQXVSWHVHTYGTR 47 90.1 bits(222) Compositional matrix adjust 43/47% 91% Wish Me TREQLPRLSLEVIEALKAAGETEADIARMYGVTPQAVSWHVHTYG 52 TREQLPRLSLEVIEALKAAGETEADIARMYGVTPQ VSWHVHTYG TIKKA TREQLPRLSLEVIEALKAAGETEADIARMYGVTPQXVSWHVHTYG 49 90.5 bits(223) Compositional matrix adjust 43/45 96% TREQLPRLSLEVIEALKA GETEADIARMYGVTPQ VSWHVHTYG BxB1 TREQLPRLSLEVIEALKATGETEADIARMYGVTPQAVSWHVHTYG 52 A. B. B. References November 10, 2014. Mycobacterium Phage. Retrieved from: http://dc359.4shared.com/doc/LgwdpskZ/preview.html 2. November 11, 2014. Photograph of Phag mycobacterium phage. Retrieved from : http://phagehuntnz.wordpress.com. 3. November 09, 2014 Photo retrieved from: https://www.google.com/search? q=lytic+and+lysogenic +diagram&client. 4. Carlton, M. R., ( 1999) “ Phage Therapy: Past History and future prospects.” 5. Payne D Phil., H. J. R., Jansen, A.A. V., ( 2000) “ Phage therapy: The peculiar kinetics of self-replicating pharmaceuticals.” Clinical Pharmacology & Therapeutics: 68, 225-230. C. D. Acknowledgments Figure 3. Once the amino acids were obtained using Blastp data base, the amino acids were Compared to each other looking at their amino acid similarities. (3a), SKCA1 compared with BxB1. (3b), SKCA1 compared to WishMe Phage. (3c), SKCA1 to TIKI. (3d), WishMe phage to TIKKA to BxB1 sequence. Identities were based on number of amino acid matches. Figure 1a. Representative diagram of a mycobacterium phage (1). Each phage has a characteristic based on its size, and specificity to attach to protein receptors on its host. Figure 1b. A photograph of a phage taken using microscopy(2).