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.
van Poppel
Nicole F J
NF
Department of Parasitology R and D, Intervet International BV, P.O. Box 31, 5830 AA Boxmeer, The Netherlands. nicole.vanpoppel@intervet.com
Welagen
Jelle
J
Duisters
Rudy F J J
RF
Vermeulen
Arno N
AN
Schaap
Dick
D
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D013754:Tetracyclines / Q000494:pharmacology; D014122:Toxoplasma / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D013754:Tetracyclines; C034811:tetracycline resistance-encoding transposon repressor protein; C054562:yellow fluorescent protein, Bacteria