Tight control of transcription in Toxoplasma gondii using an alternative tet repressor

Fusion of yellow fluorescent protein (YFP) to the N-terminus of the Escherichia coli Tn10 tet repressor (TetR) created a functional YFP–TetR repressor with the capacity of 88-fold repression of transcription when expressed in Toxoplasma gondii. As a test promoter we used the T. gondii ribosomal protein RPS13 promoter for which we provide experimental evidence of having a single major transcriptional start site, a condition favourable to the design of inducible expression systems. Integration of four tet operator (tetO) elements, 23–43 bp upstream of the RPS13 transcriptional start site, resulted in maximal repression of transcription (88-fold). Moreover, integration of these four tetO elements reduced the promoter activity only 20% in comparison with the wildtype promoter. Regulation was six-fold higher compared with an inducible expression system employing wildtype TetR. Importantly, only 0.1 μg/ml tetracycline was required for maximal induction demonstrating a higher affinity of tetracycline for YFP–TetR than for wildtype TetR which required 1 μg/ml tetracycline for maximal induction. The use of 0.1 μg/ml tetracycline allows prolonged continuous culturing of T. gondii for which levels of 1 μg/ml tetracycline are toxic. Our results show that YFP–TetR is superior to TetR for transcriptional regulation in T. gondii and we expect that its improved characteristics will be exploitable in other parasites or higher eukaryotes.