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BQuat Siphoviridae Bacteriophage Genome Annotation and Analysis By: Lydia Flores, Elena Dike, Dr. Bridgette Kirkpatrick, Carole Twichell, Sophia Hines, Jonathan Lawson In 2010 , mycobacteriophage BQuat was directly isolated from soil collected on the campus of Washington University in St . Louis, Missouri . BQuat was identified to be a unique Siphoviridae that infects Mycobacterium smegmatis (mc 2 155 ) . DNA extraction was performed and the DNA was sent to the McDonnell Genome Institute at Washington University for sequencing, making it ready for annotation . BQuat’s genome is 41893 bp in length and is a member of the G 1 Cluster . Through analysis of BQuat’s genome, we will assign putative functions to BQuat proteins utilizing bioinformatic resources . BQuat’s bacterial host M . smegmatis (mc 2 155 ) is genetically similar to Mycobacterium tuberculosis . The goal of analyzing BQuat’s genome is to identify genes that likely contribute to viral fitness through interaction with described proteins of M . smegmatis (mc 2 155 ), and in turn, M . tuberculosis . Identification of these proteins will progress the aim of finding alternative treatments for humans infected with tuberculosis, as well as adding to the growing knowledge of viral and bacterial interactions and the relationships between the two pathogens . Frameshift Mutations: Frameshift mutations are deletions or insertions in a DNA sequence which shifts the sequence changing how it is read 2 . In BQuat, there is a frame shift between genes 14 and 15 . Normally, when a frameshift mutation occurs, the proteins that are coded downstream are affected and can cause them to be nonfunctional 2 . However, in Bquat’s genome after the mutation, there are still 14 functional proteins that follow . Regardless, further analysis of BQuat’s genome could help with identifying why G cluster phages infect M . smegmatis at a higher rate than M . tuberculosis and add knowledge to the understanding of infection of M. tuberculosis with G Cluster mycobacteriophages. Abstract Introduction Not only are bacteria extremely diverse, so too are the phages that infect them. In order to infect the bacteria to continue their life cycle, phages must hijack a bacterium’s DNA-replicating machinery to either replicate itself until the bacterium bursts (lytic cycle) or incorporate its viral DNA into the host genome and wait until it is ready to lyse the bacterial cells (lysogenic cycle). BQuat was found by Hillary Sigale and Sarah Jacobs at Washington University in St. Louis. BQuat’s target organism, M. smegmatis, is genetically similar to Mycobacterium tuberculosis, sharing over two thousand homologous genes as well as the unique cell wall structure found in M. tuberculosis. In analyzing BQuat’s genome, genes may be identified that specifically target structural and functional aspects of M. smegmatis and, in extension, M. tuberculosis. Discussion References Acknowledgements 1. Griffiths AJF, Gelbart WM, Miller JH, et al . Modern Genetic Analysis . New York : W . H . Freeman ; 1999 . Protein Function and Malfunction in Cells . Available from : https : //www . ncbi . nlm . nih . gov/books/NBK 21297 / 2 . Miko, L . , Ph . D (Ed . ) . (n . d . ) . Frameshift Mutation . Retrieved November 20 , 2017 , from https : //www . nature . com/scitable/definition/frameshift - mutation - frame - shift - mutation - frameshift - 203 3 . Sampson, T . , Broussard, G . W . , Marinelli, L . J . , Jacobs - Sera, D . , Ray, M . , Ko, C . - C . , … Hatfull, G . F . ( 2009 ) . Mycobacteriophages BPs, Angel and Halo : comparative genomics reveals a novel class of ultra - small mobile genetic elements . Microbiology , 155 (Pt 9 ), 2962 – 2977 . http : //doi . org/ 10 . 1099 /mic . 0 . 030486 - 0 4 . Hatfull, G . F . ( 2012 ) . The Secret Lives of Mycobacteriophages . Advances in Virus Research : Bacteriophages, Part A, 82 , 215 - 219 . Retrieved June 9 , 2017 , from https : //seaphages . org/media/docs/Hatfull_SecretLives . pdf . For educational use only 5 . SEA-PHAGES, Phamerator (2017, Oct..- Nov.). Comparative Bacteriophage Genomics Platform. Retrieved from Phamerator website http ://phamerator.org/ 6 . NCBI, National Center for Biotechnology Information (2017, Sept.- Nov.). BLAST Basic Local Alignment Search Tool. National Library of Medicine. Retrieved from https ://blast.ncbi.nlm.nih.gov/Blast.cgi 7 . Rinehart, C. A., Gaffney, B., Smith, J., & Wood, J. D. (Eds.). (n.d.). PECAAN. Retrieved November 17, 2017, from https ://pecaan.kbrinsgd.org/ 8 . Sigale, H . , & Jacobs, S . ( 2012 , January 18 ) . Mycobacterium Phage BQuat . Retrieved September, 2017 , from http : //phagesdb . org/phages/BQuat/ 9 . HATFULL, G . F . ( 2014 ) . Molecular Genetics of Mycobacteriophages . Microbiology Spectrum , 2 ( 2 ), 1 – 36 . 10 . Pellegrini - Calace, M . , & Thornton, J . M . ( 2005 ) . Detecting DNA - binding helix – turn – helix structural motifs using sequence and structure information . Nucleic Acids Research , 33 ( 7 ), 2129 – 2140 . http : //doi . org/ 10 . 1093 /nar/gki 349 Special thanks to our professor, Dr. Bridgette Kirkpatrick, and supporting staff at Collin College, notably Professor Carole Twichell, Professor Sophia Hines, Dr. Jonathan Lawson for their direction, time, and immense effort in supporting this project and providing the materials and assets necessary to perform this research. Additional gratitude for Austin Community College and the SEA-PHAGES program for their cooperation with Collin College and allowing us to present this research. Partial funding provided by the Community College Undergraduate Research Initiative. Materials and Methods After 454 Pyrosequencing was done by the McDonnell Genome Institute at Washington University, annotations were performed. Sequencing brought BQuat’s membership in G Cluster into light. For initial annotations, DNA master, Genemark, and the National Center for Biotechnology Information (NCBI) database were used. After the first round of annotations were performed, PECAAN was used to verify previous notes taken for BQuat’s genome. Then, the genome was compared to other members of G Cluster, including Aroostook, Chance64, Gideon, Jane, and Zombie. After comparison, genes were chosen by the uniqueness against these other G Cluster phages and further analyzed. Results BQuat Genes 14 and 15: Between gene 14 and gene 15 there is a -1 frameshift with a 9bp overlap. Both gene 14 and gene 15 share the common function of a tail assembly chaperone (source from PhagesDB). Frame shifts in mycobacteriophages of G cluster are common in these tail assembly chaperones and can possibly explain why there is a frame shift between gene 14 and gene 15 of BQuat’s genome 4 . Since frameshifts are technically a framing error mutation, it is not understood why this mutation is so common in G cluster phages while still functioning properly. Frameshifts are caused by a deletion or insertion of a single nucleotide, so this should mean that all other genes downstream of the frameshift should also be off by one, allowing nonfunctional proteins to occur 1 . However, there are 14 genes that have called putative functions downstream of said frameshift within BQuat’s genome. Figure 1: A) BQuat’s Plaque morphology on M. smegmatis lawn B) TEM Image of BQuat, courtesy of the University of North Texas. 1A Genes 45 and 57: Members of only six clusters, including G cluster, are known for possessing unique proteins called ultra-small mycobacteriophage mobile elements (MPMEs) with two subtypes MPME1 and MPME2 3,9 . After annotations and anaylsis were performed, BQuat’s Gene 45, which is 252bp in length, is a member of Pham 31918 and has 100% identity, 100% alignment, and 88% coverage (per NCBI via PECAAN) with the MPME1 protein 6,7 . Since identified MPME1 proteins are usually approximately 370bp-440bp long and Gene 45 is 252bp long, this could explain why the PECAAN hit for BQuat’s Gene 45 is only for 88% coverage. BQuat’s gene 57, 183bp in length, does not have a known function, however, it is also a member of Pham 31918. None of the genes that are members Gene 51: Out of 21 annotated phages, 8 were very similar in length and members of the same phamily to gene 51. This postulates as to why there is movement of gene 51 on the pham map. During annotation of Gene 51, it was found to be 135 bp in length and a member of Pham 3075 8 . Per PhagesDB and NCBI the gene has has no known function 6,8 . However, when the Pham was analyzed, BQuat’s annotated fellow member of G Cluster phage and fellow member of Pham 3075, phage Avocado, its Gene 57 is given the function of a Helix-turn-Helix DNA binding domain protein. When BQuat’s Gene 51 was compared to phage Avocado’s Gene 57, there was 57% identity and 68% coverage between the two genes 6 . These results are not high enough to confirm that BQuat’s Gene 51 and Avocado’s Gene 57 are of the same function, however, there is a possibility that BQuat’s Gene 51 is a partial code of Avocado’s Helix-turn-Helix DNA binding domain protein. MPME Proteins: The primary cause of differences in genome length among G Cluster mycobacteriophages is the presence or absence of MPME proteins 4 .These proteins can provide further knowledge for the advancement of knowledge with viral and bacterial interactions. Helix-Turn-Helix Proteins and the Relation to Gene 51: Helix- turn-helix proteins are motifs made up of two alpha helices which interact by binding DNA 10 .In BQuat, Gene 51 had no known function however, there is a possibility that Gene 51 is a partial code of Avocado’s helix-turn-helix binding domain protein. Since Gene 51 was found to move around the pham map, this could possibly be due to a mutation or discrepancy with the helix-turn- helix binding protein that it received as a partial code from the phage Avocado. of this Pham that are less than 300bp have been given a function. It is possible for BQuat’s Gene 57 to be a partial coding for the MPME protein, since the members of this Pham do code for MPME proteins. Aroostook BQuat Chance64 Gideon Jane Zombie Aroostook BQuat Chance64 Gideon Jane Zombie Figure 3: A) Framshift Mutation between genes 14 and 15 within BQuat’s genome provided by DNA Master B) Phamerator Map comparison between BQuat and its closest G1 Cluster relatives displaying all of their frameshift mutation 5 . Gene 14 Gene 15 3A Figure 2: A) Phamerator Map comparison between BQuat and its most closely related G1 Cluster members 5 . Image features BQuat’s genes 45 and 57, in comparison to Aroostook, Chance64, Gideon, Jane and Zombie’s MPME proteins. B) Details of Pham 31918, which BQuat’s genes 45 and 57 are members of 8 . C) PECAAN hit for BQuat’s Gene 45. Image displays Identity, Alignment, and Coverage of protein function from NCBI. Bubble → 2A 2B 2C 3B 4A 4B Figure 4: A) NCBI Blastp hit when comparing BQuat’s Gene 51 to Avocado’s Gene 57 that is assigned a Helix- Turn-Helix DNA Binding Domain protein function. B) Example of a Helix-Turn- Helix DNA Binding Domain protein and described characteristics of this protein. 5 1B Figure 5: Above is an example of a frameshift mutation, showing a shift in the nucleotides, thus changing the coding of the amino acids. Courtesy of the U.S. National Library of Medicine.
