Discipline: Biological Sciences
Malcolm Bass - Norfolk State University
Co-Author(s): Dr. Nazir Barekzi, Norfolk State University, Norfolk, Virginia
If we use a non-pathogenic Mycobacterium to isolate novel phage, then we can use newly discovered and isolated phage as an alternative to antibiotics against Mycobacterium tuberculosis. M. tuberculosis is a biosafety level 2 organism. Our lab works with a model organism (Mycobacterium smegmatis) that is biosafety level 1 and much safer for undergraduate research. The significance of this research is threefold: (1) the novel phage discovered and isolated could be used as an alternative treatment to pathogenic bacteria and multi-drug resistant Mycobacterium; (2) provide a better understanding of phage genomics and horizontal gene transfer; and (3) provide a better understanding of phage-bacteria interaction.
Methods and Controls:
The control host being used was Mycobacterium smegmatis. The control phage for the project were phage that had been previously isolated and annotated. The project utilized common phage biology techniques. Briefly, soil samples were collected (composition, temperature and depth were recorded), phage buffer was added to soil and the supernatant was filtered using a 0.2 um filter to remove any bacteria. The flow through was collected and the titer of the phage was determined. The presence of phage plaques was used to determine if a single or multiple type of phage was present. Phage were purified using three distinct procedures: a Spot Test procedure, serial dilution procedure, and pick-a-plaque procedure. When the phage is isolated and confirmed to be of a single morphology, the DNA of the phage was extracted using a Promega Wizard DNA Clean-up System. The extracted DNA was used in a Restriction Enzyme Digest in order to further characterize the phage providing a unique restriction enzyme digest profile. To further characterize the phage based on genome architecture, the extracted DNA was sequenced using an Illumina MiSeq Next Generation Sequencer (Old Dominion University). The whole-genome sequencing and de novo assembly was used to annotate, characterize and study the similarity of the phage discovered with published phage genomes. The controls for annotation were phage genomes that have already been submitted and accepted into the Actinobacteriophage database phagesdb.org.
A mycobacteriophage named Bassalto was isolated. The size of the plaques of Bassalto were ~1.0 mm and the morphology of the plaque resembled lytic phage. The lytic phage have complete clearing of the plaques and this was the characteristic observed for Bassalto plaques. A high concentration of Bassalto phage was used (~10e7 plaques/ml) to isolate DNA. Multiple rounds of DNA extraction was performed to obtain enough DNA for restriction digests and DNA sequencing. The Bassalto DNA concentration was 42.7 ug/ml and that was sufficient to produce sequence data. After the DNA of Bassalto was sequenced, the genome of Bassalto was calculated to be 69,044 base pairs in length. A comparison of genomes using other closely related bacteriophage from phagesdb.org revealed that Bassalto was similar to phage belonging to the B3 cluster. Bassalto shares 99% nucleotide similarity with phages RomaT, Chandler, and RagingRooster. In addition, there is a significant difference between these genomes, confirming that Bassalto is a novel bacteriophage. The difference was that Bassalto contained an additional ~500-1000 bases in the far right region approximately between nucleotides 65,000 to 67,000.
Conclusions and Future research questions:
The project was successful in isolating a novel mycobacteriophage. The results indicate that a common soil bacterium can be used to isolate different types of mycobacteriophage (lytic and lysogenic). This project lends itself for authentic research experiences by undergraduate researchers. This project was also successful in combining wet-lab experiments and bioinformatics to discover and characterize new viruses that infect bacteria. Some additional research questions need to be studied. First, it is clear that the bacteriophage isolated in this study can kill Mycobacterium smegmatis. However, investigations that can determine if this phage can infect other mycobacterium species must be conducted in the future. Furthermore, now that we have the genome sequence of Bassalto, it will be important to investigate the genes and gene products that make this phage lytic. To answer this last question further comparison of the Bassalto genome with other B3 cluster mycobacteriophages such as RomaT, Chandler, and RagingRooster, will be investigated. By further studying phage, the goal is to advance and contribute to the general knowledge of genetics, biodiversity, and microbial ecology. Beyond basic scientific research, the applications of phage therapy should be investigated to control microbial populations, including different Mycobacterium species. Ultimately, this will fill the gap of knowledge in phage genomics and phage therapy.
Funder Acknowledgement(s): NSF HBCU-UP, LSAMP, College of Science, Engineering and Technology, STARS program, and the Biology Department of Norfolk State University.
Faculty Advisor: Dr. Nazir Barekzi, firstname.lastname@example.org
Role: I began working on this project in the Fall of 2017. I was trained on the different procedures for phage isolation during the first 3 weeks. After that, I was experienced and completed all the steps to isolate the phage Bassalto, including soil sample collection, lysate collection, phage purification, high titer lysate production, phage DNA extraction, and restriction endonuclease digests. I also completed the annotation and comparison of Bassalto's genome under my adviser's supervision. The one thing that was not done by me was the DNA sequencing, which was sent out to Old Dominion University and our collaborators there.