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 microg/ml tetracycline was required for maximal induction demonstrating a higher affinity of tetracycline for YFP-TetR than for wildtype TetR which required 1 microg/ml tetracycline for maximal induction. The use of 0.1 microg/ml tetracycline allows prolonged continuous culturing of T. gondii for which levels of 1 microg/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.