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Current Genetics

, Volume 64, Issue 1, pp 303–316 | Cite as

Improved Tet-On and Tet-Off systems for tetracycline-regulated expression of genes in Candida

  • Swati Bijlani
  • Anubhav S. Nahar
  • K. GanesanEmail author
Technical Notes

Abstract

Tetracycline-regulated expression of genes is often used for functional analysis of Candida albicans genes. However, the widely used Tet-On system has certain limitations such as prolonged lag time (up to 8 h) for induction and non-uniform expression among the cells. We speculated that poor expression of tetracycline-controlled transactivator (Tet-transactivator) from CaADH1 promoter could be responsible for this, and thus compared the effect of expressing this protein under the control of CaADH1, CaTDH3 and CaRP10 promoters on the expression of GFP from the TET promoter. Only CaRP10 promoter facilitated a more uniform and rapid induction of GFP. However, a high concentration of doxycycline was needed for induction, which is not desirable for assessing certain phenotypes. Tet-Off systems are known to require a low concentration of doxycycline, but a limitation of the widely used Tet-Off system for C. albicans is the use of CaENO1 promoter, which is known to be repressed in the presence of gluconeogenic carbon source, for expression of transactivator. Thus, we have converted the above-mentioned Tet-On systems to Tet-Off systems by site-directed mutagenesis of the Tet-transactivator. Compared to the Tet-On systems, the Tet-Off systems required about 200-fold less concentration of doxycycline for modulation of gene expression. Only the Tet-Off system with CaRP10 promoter driving the expression of transactivator allowed rapid and high level expression of GFP compared to those with CaADH1 or CaTDH3 promoters. The utility of CaRP10 based Tet-On and Tet-Off systems was further validated by the conditional expression of the CaTUP1 gene. We have also adapted these systems for use with Candida tropicalis and find that the Tet-Off system is functional in this species. The Tet systems reported here will be useful for conditional expression of genes in C. albicans as well as C. tropicalis.

Keywords

Functional genomics Conditional expression of genes Tetracycline transactivator Reverse tetracycline transactivator 

Notes

Acknowledgements

We wish to thank Joachim Morschhäuser and Christophe d’Enfert for providing us with the plasmids pNIM1 and pNIMX, respectively, and Surjeet Singh for technical assistance. S.B. and A.S.N. acknowledge University Grants Commission, New Delhi, for fellowships.

Compliance with ethical standards

Funding

This work was supported by Council of Scientific & Industrial Research (CSIR) projects on “Infectious Diseases” (No. BSC0210) and “Bugs to Drugs” (BSC0211).

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

294_2017_720_MOESM1_ESM.pdf (927 kb)
Supplementary material 1 (PDF 927 kb)

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Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.CSIR-Institute of Microbial TechnologyChandigarhIndia

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