Stable transformation of an episomal protein-tagging shuttle vector in the piscine diplomonad Spironucleus vortens

Abstract

<p>Abstract</p> <p>Background</p> <p>Diplomonads are common free-living inhabitants of anoxic aquatic environments and are also found as intestinal commensals or parasites of a wide variety of animals. <it>Spironucleus vortens </it>is a putatively commensal diplomonad of angelfish that grows to high cell densities in axenic culture. Genomic sequencing of <it>S. vortens </it>is in progress, yet little information is available regarding molecular and cellular aspects of <it>S. vortens </it>biology beyond descriptive ultrastructural studies. To facilitate the development of <it>S. vortens </it>as an additional diplomonad experimental model, we have constructed and stably transformed an episomal plasmid containing an enhanced green fluorescent protein (GFP) tag, an AU1 epitope tag, and a tandem affinity purification (TAP) tag. This construct also contains selectable antibiotic resistance markers for both <it>S. vortens </it>and <it>E. coli</it>.</p> <p>Results</p> <p>Stable transformants of <it>S. vortens </it>grew relatively rapidly (within 7 days) after electroporation and were maintained under puromycin selection for over 6 months. We expressed the enhanced GFP variant, eGFP, under transcriptional control of the <it>S. vortens </it>histone H3 promoter, and visually confirmed diffuse GFP expression in over 50% of transformants. Next, we generated a histone H3::GFP fusion using the <it>S. vortens </it>conventional histone H3 gene and its native promoter. This construct was also highly expressed in the majority of <it>S. vortens </it>transformants, in which the H3::GFP fusion localized to the chromatin in both nuclei. Finally, we used fluorescence <it>in situ </it>hybridization (FISH) of the episomal plasmid to show that the transformed plasmid localized to only one nucleus/cell and was present at roughly 10–20 copies per nucleus. Because <it>S. vortens </it>grows to high densities in laboratory culture, it is a feasible diplomonad from which to purify native protein complexes. Thus, we also included a TAP tag in the plasmid constructs to permit future tagging and subsequent purification of protein complexes by affinity chromatography via a two-step purification procedure.</p> <p>Conclusion</p> <p>Currently, progress in protistan functional and comparative genomics is hampered by the lack of free-living or commensal protists in axenic culture, as well as a lack of molecular genetic tools with which to study protein function in these organisms. This stable transformation protocol combined with the forthcoming genome sequence allows <it>Spironucleus vortens </it>to serve as a new experimental model for cell biological studies and for comparatively assessing protein functions in related diplomonads such as the human intestinal parasite, <it>Giardia intestinalis</it>.</p