by: lydia flores, elena dike, dr. bridgette kirkpatrick, carole … · 2018-03-07 · bquat...
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BQuat Siphoviridae Bacteriophage Genome Annotation and AnalysisBy: Lydia Flores, Elena Dike, Dr. Bridgette Kirkpatrick, Carole Twichell, Sophia Hines, Jonathan Lawson
In 2010, mycobacteriophage BQuat was directly isolated from soilcollected on the campus of Washington University in St. Louis,Missouri. BQuat was identified to be a unique Siphoviridae thatinfects Mycobacterium smegmatis (mc2155). DNA extraction wasperformed and the DNA was sent to the McDonnell GenomeInstitute at Washington University for sequencing, making it readyfor annotation. BQuat’s genome is 41893 bp in length and is amember of the G1 Cluster. Through analysis of BQuat’s genome,we will assign putative functions to BQuat proteins utilizingbioinformatic resources. BQuat’s bacterial host M. smegmatis(mc2155) is genetically similar to Mycobacterium tuberculosis. Thegoal of analyzing BQuat’s genome is to identify genes that likelycontribute to viral fitness through interaction with describedproteins of M. smegmatis (mc2155), and in turn, M. tuberculosis.Identification of these proteins will progress the aim of findingalternative treatments for humans infected with tuberculosis, aswell as adding to the growing knowledge of viral and bacterialinteractions 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 read2. 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
nonfunctional2. 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
ofM. tuberculosiswith G Cluster mycobacteriophages.
Abstract
IntroductionNot only are bacteria extremely diverse, so too are the phages thatinfect them. In order to infect the bacteria to continue their lifecycle, phages must hijack a bacterium’s DNA-replicatingmachinery to either replicate itself until the bacterium bursts (lyticcycle) or incorporate its viral DNA into the host genome and waituntil it is ready to lyse the bacterial cells (lysogenic cycle). BQuatwas found by Hillary Sigale and Sarah Jacobs at WashingtonUniversity in St. Louis. BQuat’s target organism, M. smegmatis, isgenetically similar to Mycobacterium tuberculosis, sharing overtwo thousand homologous genes as well as the unique cell wallstructure found in M. tuberculosis. In analyzing BQuat’s genome,genes may be identified that specifically target structural andfunctional 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/NBK21297/
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/gki349
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 GenomeInstitute at Washington University, annotations wereperformed. Sequencing brought BQuat’s membership in GCluster into light. For initial annotations, DNA master,Genemark, and the National Center for BiotechnologyInformation (NCBI) database were used. After the first round ofannotations were performed, PECAAN was used to verifyprevious notes taken for BQuat’s genome. Then, the genomewas compared to other members of G Cluster, includingAroostook, Chance64, Gideon, Jane, and Zombie. Aftercomparison, genes were chosen by the uniqueness against theseother 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 genome4. 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 occur1. However, there are 14 genes that have called putative
functions downstream of said frameshift within BQuat’s genome.
Figure 1: A)BQuat’sPlaquemorphologyon M.smegmatislawnB) TEMImage ofBQuat,courtesy oftheUniversityof NorthTexas.
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 MPME23,9. 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 protein6,7. 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 30758. Per
PhagesDB and NCBI the gene has has no known function6,8. 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 genes6. 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: Theprimary cause ofdifferences in genomelength among G Clustermycobacteriophages isthe presence or absenceof MPME proteins 4.Theseproteins can providefurther knowledge for theadvancement ofknowledge with viral andbacterial 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 DNA10.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) FramshiftMutation between genes 14and 15 within BQuat’sgenome provided by DNAMasterB) Phamerator Mapcomparison between BQuatand its closest G1 Clusterrelatives displaying all oftheir frameshift mutation5.
Gene 14
Gene 15
3A
Figure 2: A) Phamerator Map comparison between BQuat and its most closely related G1Cluster members5. 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’sgenes 45 and 57 are members of8. 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
4BFigure 4: A) NCBI Blastp hitwhen comparing BQuat’sGene 51 to Avocado’s Gene57 that is assigned a Helix-Turn-Helix DNA BindingDomain protein function.B) Example of a Helix-Turn-Helix DNA Binding Domainprotein and describedcharacteristics of thisprotein.
5
1B
Figure 5: Above is an example of a frameshift mutation,showing a shift in the nucleotides, thus changing the codingof the amino acids. Courtesy of the U.S. National Library ofMedicine.