Promoter strength driving TetR determines the regulatory properties of Tet-controlled expression systems.

Christiane Georgi,Julia Buerger,Christian Berens,Wolfgang Hillen,Axel Cloeckaert

doi:10.1371/journal.pone.0041620

Abstract

Bacteria frequently rely on transcription repressors and activators to alter gene expression patterns in response to changes in the surrounding environment. Tet repressor (TetR) is a paradigm transcription factor that senses the environmental state by binding small molecule effectors, the tetracyclines. However, recently isolated peptides that act as inducers of TetR after having been fused to the C-terminus of a carrier protein, suggest that TetR can also regulate gene expression in a signal-transduction pathway. For this shift in regulatory mechanism to be successful, induction of TetR must be sensitive enough to respond to an inducing protein expressed at its endogenous level. To determine this regulatory parameter, a synthetic Tet-regulated system was introduced into the human pathogen Salmonella enterica serovar Typhimurium and tested for inducibility by a peptide. Reporter gene expression was detected if the peptide-containing carrier protein Thioredoxin 1 was strongly overproduced, but not if it was expressed at a level similar to the physiological level of Thioredoxin 1. This was attributed to high steady-state amounts of TetR which was expressed by the promoter of the chloramphenicol acetyl transferase gene (Pcat). Reducing Pcat strength either by directed or by random mutagenesis of its -10 element concomitantly reduced the intracellular amounts of TetR. Sensitive and quantitative induction of TetR by an inducing peptide, when it was fused to Thioredoxin 1 at its native locus in the genome, was only obtained with weak Pcat promoter variants containing GC-rich -10 elements. A second important observation was that reducing the TetR steady-state level did not impair repression. This permits flexible adjustment of an inducible system’s sensitivity simply by altering the expression level of the transcription factor. These two new layers of expression control will improve the quality and, thus, the applicability of the Tet and other regulatory systems.

Highlights

Survival and proliferation of bacteria depend on their expressing the right amounts of the right genes at the right time
The Tet-controlled expression system, which was studied here and is displayed schematically in Fig. 1A, is an example of such a synthetic regulatory system. (I) The cat promoter (Pcat) from Tn9 [34,35] constitutively expresses the Tet repressor protein (TetR). (II) The reporter gene mRNA, which is transcribed by the Tet-regulated promoter of the resistance gene tetA (PtetA), contains a modified 59 untranslated region. (III) Each expression cassette was inserted in single copy at a different attachment site in the Salmonella genome. (IV) TetR is induced either by tetracycline derivatives, or by an artificially selected TetR-inducing peptide (TIP), fused to a plasmid- or chromosomally-encoded carrier protein
Because the regulatory properties of the strain carrying this new and artificial genetic circuit were unknown, we analyzed the inducibility of TetR using both types of effector 2 anhydrotetracycline representing a potent natural inducer [36], and the peptide TIP2 [21] fused to the C-terminus of Thioredoxin 1 (Trx1, trxA) as alternative inducer representing a signal transduction pathway

Summary

Introduction

Survival and proliferation of bacteria depend on their expressing the right amounts of the right genes at the right time. What is ‘‘right’’ at any given time-point will vary with the environmental conditions and the specific growth phase. Bacteria often respond to these changing environmental stimuli by switching the expression of specific genes ‘‘on’’ or ‘‘off’’. To ensure that target gene expression is optimal, will require finetuning of the regulatory parameters that control the switch, and this fine-tuning can affect each individual step of gene expression. Gene expression is frequently controlled by proteins that activate or repress transcription by binding to specific DNA sequences close to a promoter [1]. The DNA binding activity of these transcription factors is triggered by small molecules or, less often, by protein-protein interactions

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Conclusion