Exploration of Putative Riboswitches Involved in Bacteriophage Temperate Life Cycle

Background: Bacteriophages are viruses that survive by infecting and then replicating using a bacterial host’s genetic machinery, leading to the destruction of the host cell. Bacteriophages and their hosts are locked in an evolutionary struggle that has led to the development of new mechanisms of defense and infection. Bacteria have regulatory structures known as a riboswitch, an ancient regulation mechanism composed of a single piece of RNA that alters its self-annealed structure in the presence or absence of cellular metabolites. The cellular metabolite interacts with the portion of the RNA structure forming a hairpin-loop and making the Shine-Delgarno sequence unavailable to ribosomes. Methods: We used an enrichment method to isolate phage that infect the host Mycobacterium smegmatis. Phage were purified and classification was assisted by TEM imaging. The quality and quantity of DNA harvested from ‘Chupacabra’ were measured through restriction enzyme digest. After genomic sequencing, bioinformatic analyses of the isolated phage genome were used to classify the phage and annotate its genome. Putative riboswitches were located in our isolated phage using the Denison Riboswitch Detector (DRD). In addition, FASTA files available from GenBank for four bacteria and 94 bacteriophage were analyzed using the DRD. Results: During this project, the novel bacteriophage ‘Chupacabra’ was isolated. This phage belongs to the cluster A and subcluster A10 of bacteriophages. In culture, ‘Chupacabra’ exhibits a temperate life cycle and forms plaques approximately 3 mm in diameter. Its capsid and tail were 60nm in diameter and 140nm long, respectively, and its genome was 50,286 base pairs in length. Annotation of the ‘Chupacabra’ genome revealed genes that were atypical when compared to related lytic phages. Among the four bacterial species included in our study, we identified 322 total putative riboswitches. We also located 110 putative riboswitches across the bacteriophage genomes. Lysogenic and lytic phages appear to favor different types of riboswitches. Conclusions: To our knowledge, this is the first time that putative riboswitches have been located in an actinobacteriophage genome. These findings suggest that riboswitches may have functioned as metabolite sensors in primitive organisms, and actinobacteriophage and modern cells still retain some of the ancient regulatory control systems. We postulate that riboswitches are able to regulate phage gene expression and are, therefore, able to control the transition from a lysogenic to a lytic lifestyle.