Abstract 1865 Isolation, Bioinformatic Characterization, and Functional Genomic Analyses of the Bacteriophage MossRose against Actinomycete Gordonia rubripertincta

Bacteriophages are a type of virus that uses bacterial cells as their host. The number of antibiotic-resistant bacterial strains is ever increasing and the choices for effective treatments using traditional chemical antibiotic compounds limited. Since bacteriophages infect only prokaryotic cells (bacteria) and not eukaryotic cells, studies into whether using bacteriophages as a tool towards treating these infections may be extremely promising. Equally as important as identifying viruses that infect and lyse bacterial cells is an understanding of the mechanisms associated with host/virus interactions. The purpose of our project was to isolate and purify novel bacteriophages and then annotate their genome. We attempted to isolate phages from a variety of environmental samples focusing on the Actinobacterial host, Gordonia rubripertincta NRRL B-16540. We used both direct and enriched protocols to isolate bacteriophage. Serial dilutions, 'pick-n-jiggle/pick-a-plaque,' and spot-plating techniques were employed resulting in pure viral colonies from which genomic DNA could be isolated. Genomic DNA was mailed to the Pittsburgh Bacteriophage Institute (University of Pittsburgh, PA) for Illumina sequencing. Completed bioinformatic analysis will be completed later this spring, 2024; however, preliminary annotation of both viruses has been performed using several programs including PECAAN, DNA Master, Phamerator, Starterator, NCBI BLAST, and HHPred. Initial phylogenetic analysis positions virus MossRose (∼61,000 bp) within the DR cluster of Actinomycete bacteriophages. Studies surrounding host-virus range specificity and viral titers have also been initiated amongst several Gordonia species (G. terrae, G. rubripertincta, G. westfalica, G. lacuna). Functional genetics experiments were conducted using a mutated strain of G. rubripertincta. An insertional element containing kanamycin resistance was engineered and introduced into G. rubripertincta, which was subsequently used for large-scale screening for alternative host-virus phenotypes. The genomes of G. rubripertincta mutants exhibiting altered phenotypes were analyzed at the molecular level using bioinformatic/proteomic tools. It is anticipated that these findings will help contribute toward the further understanding of the mechanisms associated with bacteriophages and their host specifications. We thank the Northern State University and the colleagues in the Department of Sciences and Mathematics for the resources. Additionally, we would like to express our sincere appreciation to the Hatfull Lab, University of Pittsburgh Sequencing Facility, and the entire HHMI SEA community for supporting this work and sharing their experiences and advice.