Date Thesis Awarded

5-2022

Access Type

Honors Thesis -- Access Restricted On-Campus Only

Degree Name

Bachelors of Science (BS)

Department

Biology

Advisor

Margaret S. Saha

Committee Members

Shantá D. Hinton

Mark H. Forsyth

Randolph A. Coleman

Abstract

The genus Mycobacterium contains over 190 species and is responsible for significant morbidity and mortality in humans and other animals. In addition to well-studied human pathogens such as Mycobacterium tuberculosis, leprae, and abscessus, the genus includes many non-tuberculosis environmental species that dwell in the soil and water supply, species that may serve as useful models for the pathogenic species. With the recently reported successes in phage therapy, there has been an increased interest in expanding the diversity of phages that infect hosts within the Mycobacterium genus. This project aimed to identify phages that infected Mycobacterium aichiense, a host for which there are currently no reported bacteriophages. Using standard methods to isolate phages from soil samples, we isolated approximately 50 phages infecting this host from local soil samples. All of the phages had consistently low titers and required optimization of DNA isolation techniques. Ten isolates were sequenced. In each case, sequencing resulted in a ~50 kb phage representing a new subcluster within the A cluster of mycobacterial phages; this sequence corresponds to a prophage in the M. aichiense genome that we called Herbert WM. Surprisingly, there was also an 11 kb “phagelet” in each sample that shared little identity with the 50 kb HerbertWM prophage. The phagelet encodes 20 genes, including a terminase, a serine-threonine kinase, a head-to-tail adaptor, a head-to-tail stopper, and 16 hypothetical proteins of unknown function. While all the isolates contained both the prophage and the phagelet, the stoichiometry of the two phages differed among the various samples. In order to investigate this novel “phagelet” further, we performed RNA-Seq analysis at different time points following infection of the host, including a time point before phage was added to the sample; and then at 0, 5, 15, 30, 60, and 120 minutes following infection. This analysis revealed, somewhat surprisingly, that in addition to expression of the repressor, many prophage genes were expressed at low levels even in the absence of infection with the phagelet. Phagelet genes were expressed as early as t=5 minutes post-infection and continued to increase throughout the time periods analyzed, with the few structural genes being expressed late in infection. Prophage (HerbertWM) genes did not significantly upregulate until t=60 min. This represents the first instance of satellite-like phage in the mycobacterial genus, one that may provide valuable tools for manipulating and understanding mycobacterial genomes.

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