Characterization of a Novel Tectivirus Phage Toil and Its Potential as an Agent for Biolipid Extraction

The oleaginous bacterium Rhodococcus opacus PD630 is metabolically diverse and can be cultivated on various renewable resources to serve as a sustainable triacylglycerol (TAG) feedstock for biodiesel production. Current methods for TAG extraction are costly, but infection of cultures by lytic bacteriophages (phages) may be a viable approach for achieving release of intracellular lipid from oleaginous bacteria such as R. opacus. This study reports the novel tectiviral phage Toil capable of releasing intracellular contents including a fluorescent protein marker and TAGs into the supernatant after phage infection of R. opacus PD631, a domesticated derivative of strain PD630. Phage Toil is placed in the Tectiviridae by its morphology, the presence of a lipid membrane, its genome architecture and the presence of terminal covalently-linked proteins. Toil is the first tectivirus capable of infecting a member of the Actinobacteria. Microscopy shows that infected cells do not undergo sudden lysis but instead maintain their original shape for several hours, with the cellular morphology gradually deteriorating. Approximately 30% of intracellular TAGs could be recovered from the culture supernatants of Toil-infected PD631 cells. Phage Toil has potential to be used as an agent in extraction of TAGs from oleaginous bacterium R. opacus. Importance: This study reported the first tectivirus (Phage Toil) capable of infecting a member of the Actinobacteria. In this study, we showed that Phage Toil can infect oleaginous bacterium Rhodococcus opacus to release intracellular contents such as a fluorescent protein marker and TAG lipid granules, which can serve as a starting material for biodiesel production. This study demonstrates a new method to extract TAGs by using this phage. Additionally, Phage Toil can be a new model phage to advance knowledge regarding phage infection mechanisms in Rhodococcus and other mycolic acid-containing bacteria such as Mycobacterium.