Status quo of tet regulation in bacteria

Abstract

The tetracycline repressor (TetR) belongs to the most popular, versatile and efficient transcriptional regulators used in bacterial genetics. In the tetracycline (Tc) resistance determinant tet(B) of transposon Tn10, tetR regulates the expression of a divergently oriented tetA gene that encodes a Tc antiporter. These components of Tn10 and of other natural or synthetic origins have been used for tetracycline-dependent gene regulation (tet regulation) in at least 40 bacterial genera. Tet regulation serves several purposes such as conditional complementation, depletion of essential genes, modulation of artificial genetic networks, protein overexpression or the control of gene expression within cell culture or animal infection models. Adaptations of the promoters employed have increased tet regulation efficiency and have made this system accessible to taxonomically distant bacteria. Variations of TetR, different effector molecules and mutated DNA binding sites have enabled new modes of gene expression control. This article provides a current overview of tet regulation in bacteria.

Fig. 1

Variables of bacterial tet systems. Key parameters and variables affecting the outcome and efficiency of tet regulation. TetR is shown in the DNA bound form. Bent arrows denote promoters, and double helical part of schematized DNA represents tetO.

Fig. 2

Different inducers and operators and suitable Tet repressors. A. Left side: Shown are selected tetracyclines, and the sequences of the Tip peptide and the 12‐1 RNA. The boxes indicate which TetR variants are (best) inducible by these compounds. TetR H64K S135L S138 is designated TetR i2. Note that inducibility by Tip in wt‐TetR is enhanced by mutations N82A F86A. Right side: TetR with the positions mutated for binding of 4‐de‐dimethylamino‐atc or enhanced interaction with Tip highlighted. B. Left side: Upper strands of the tet operator and selected variants. The grey boxes indicate which TetR variants (best) interact with these sites. Right side: TetR with the positions mutated for binding to tetO variants highlighted. C. TetR with helices α1, α4 and α6 highlighted. Mutations resulting in the reverse phenotype are mostly found in these regions.

Fig. 3

Types of Tet‐ON and Tet‐OFF control. A. upper panel: conventional Tet‐ON control with wt‐TetR; lower panel: Control by proTeOn (Volzing et al., 2011). B. 1st panel: Tet‐OFF control by revTetR; 2nd panel: Expression of AS‐RNA by wt‐TetR; 3rd panel: The TetR/Pip OFF system (Boldrin et al., 2010). A similar mode is represented by tet‐regulated expression of dCas9 (Mariscal et al., 2018) (not shown); 4th panel: Control by proTeOff. Bent arrows denote promoters. Boxes below the promoters symbolize tetO, or binding sites of Pip (TetR/Pip OFF) or LuxR (ProTeOn or ProTeOff). Effector is depicted as triangles. Light green arrows symbolize the inactive state; bright green arrows denote actively transcribed gene.

Fig. 4

Phylogenetic distribution of applied tet regulation in bacteria. The presented phylogeny is based on 16S DNA sequences of respective species, assessed from the NCBI Nucleotide database. Sequences were aligned and the phylogeny was calculated using the EMBL‐EBI web services (https://www.ebi.ac.uk/Tools/phylogeny/simple_phylogeny/) using default parameters. The visualization was done using the iTOL (version 6.3) web tool (Letunic and Bork, 2021).

Fig. 5

Selected promoters of popular bacterial tet regulation systems for Gram‐negative bacteria. A. P_A, P_R1 and P_R2 of transposon Tn10. B. P_A of Tn10 and P_bla of pBR322 in the pASK75 system. The P_A promoter is identical to that in A). bla: beta‐lactamase. C. P_LtetO‐1 and PN25 in the pZ vector system. Sequence deviations of promoter variants are given in grey dotted boxes.

Fig. 6

The P_xyl/tet promoter. P* and P_xyl/tet in pWH353 (one tetO) and pWH354 two tetO). Note that the sequence is continued from the upper to the lower part. Sequence deviations of promoter variants are given in grey dotted boxes.