Abstract
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.
Highlights
Among many oleaginous microorganisms, the bacterium Rhodococcus opacus PD630 has attracted increasing interest due to its ability to accumulate TAGs up to 76% of its cell dry weight under nitrogen-limited conditions[5]
Phage Toil was isolated on R. opacus strain PD631 from a soil sample collected near an open-air aerobic digestion basin of a local wastewater treatment plant in College Station, TX
Strain PD631 is a domesticated derivative of strain PD630 that was obtained by repeated subculture of strain PD630 on Reasoner’s 2 A (R2A) medium
Summary
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. 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. R. opacus is a mycolic acid- producing member of the order Corynebacteriales, is metabolically diverse, and has been showed to produce TAGs from a broad range of organic compounds including lignocellulosic biomass[6,7,8,9,10,11,12,13] Using this model oleaginous bacterial strain to produce TAGs offers a unique opportunity for us to develop a new means for TAG extraction –using bacteriophages to lyse cells for TAG release. We report isolation and characterization of a novel R. opacus phage and demonstrate its ability to be used as bioextraction agent in an environmentally friendly, safe and relatively low-cost approach for lipid extraction
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