Tetracycline-dependent conditional gene knockout in Bacillus subtilis

Abstract

Reversible tetracycline-dependent gene regulation allows induction of expression with the tetracycline repressor (TetR) or gene silencing with the newly developed reverse mutant revTetR. We report here the implementation of both approaches with full regulatory range in gram-positive bacteria as exemplified in Bacillus subtilis. A chromosomally located gene is controlled by one or two tet operators. The precise adjustment of regulatory windows is accomplished by adjusting tetR or revtetR expression via different promoters. The most efficient induction was 300-fold in the presence of 0.4 microM anhydrotetracycline obtained with a Pr-xylA-tetR fusion. Reversible 500-fold gene knockouts were obtained in B. subtilis after adjusting expression of revTetR by synthetically designed promoters. We anticipate that these tools will also be useful in many other gram-positive bacteria.

FIG. 1.

Gene regulation by TetR and revTetR. (A) Regulation by TetR. In the absence of anhydrotetracycline (white triangles), TetR (depicted by gray ovals) binds to tetO, thereby repressing transcription of spoVG-lacZ. Anhydrotetracycline binds to TetR, causes dissociation from tetO and induction of transcription (black arrow). (B) Regulation by revTetR. revTetR cannot bind to tetO unless the corepressor anhydrotetracycline is present, which results in switching transcription off.

FIG. 2.

spoVG-lacZ control region of the integration plasmid pWH102. The sequence of the promoter for transcription of the chromosomal reporter gene is shown. The sequence was adapted for B. subtilis based on the synthetic tet/xyl construct described before (4). Restriction sites are underlined; −35 and −10 region and the Shine-Dalgarno sequence (SD) are indicated by boxes. tetO is indicated in large letters and underlined; the central nucleotide is marked with an asterisk. The start codon of spoVG-lacZ is underlined twice. pWH105 carries a second tetO inserted at the BamHI site (see Results section).

FIG. 3.

Regulation of spoVG-lacZ by TetR. Panel A shows a schematic representation of the different promoters driving tetR expression. The organization of poly(A) tracts, −35 (horizontally dashed), −10 (vertically dashed), and Shine-Dalgarno boxes is depicted. tetO and xylO denote the operator position. Panel B shows the β-galactosidase activities of B. subtilis WH476 transformed either with a plasmid bearing no tetR (pHT304) or with plasmids expressing tetR from different promoters (pWH116 to pWH119) in the absence and presence of anhydrotetracycline (black, gray, and white columns show no anhydrotetracycline, 0.4 μM anhydrotetracycline, and 0.8 μM anhydrotetracycline, respectively). β-Galactosidase activity in the absence of tetR was about 2,700 Miller units and was set to 100%. The right panel shows a comparison of WH476 (one tetO) and WH478 (two tetO) transformed with pWH119. The bottom panel shows the Western blots of 60 μg of soluble protein from the strains indicated above; 60 ng of purified TetR is shown as a control in the left lane.

FIG. 4.

Establishing revTetR regulation in B. subtilis. Panel A displays the degenerate promoter for revtetR expression. Variable positions of the −35 and −10 sequences are given in small letters. Panel B shows the β-galactosidase activities of B. subtilis WH478 transformed with a plasmid bearing either no tetR (pHT304), tetR (pWH119), or revtetR expressed from different promoters (pWH123 to pWH127, see panel C) in the absence (black columns) and presence (white columns) of 0.4 μM anhydrotetracycline. The bottom panel shows the corresponding Western blots of 60 μg of soluble protein of the corresponding strains; 60 ng of purified TetR is shown as a control in the left lane. Panel C shows the promoter sequences present in the indicated plasmids in comparison with the consensus promoter sequence.

FIG. 4.

Establishing revTetR regulation in B. subtilis. Panel A displays the degenerate promoter for revtetR expression. Variable positions of the −35 and −10 sequences are given in small letters. Panel B shows the β-galactosidase activities of B. subtilis WH478 transformed with a plasmid bearing either no tetR (pHT304), tetR (pWH119), or revtetR expressed from different promoters (pWH123 to pWH127, see panel C) in the absence (black columns) and presence (white columns) of 0.4 μM anhydrotetracycline. The bottom panel shows the corresponding Western blots of 60 μg of soluble protein of the corresponding strains; 60 ng of purified TetR is shown as a control in the left lane. Panel C shows the promoter sequences present in the indicated plasmids in comparison with the consensus promoter sequence.

FIG. 4.

Establishing revTetR regulation in B. subtilis. Panel A displays the degenerate promoter for revtetR expression. Variable positions of the −35 and −10 sequences are given in small letters. Panel B shows the β-galactosidase activities of B. subtilis WH478 transformed with a plasmid bearing either no tetR (pHT304), tetR (pWH119), or revtetR expressed from different promoters (pWH123 to pWH127, see panel C) in the absence (black columns) and presence (white columns) of 0.4 μM anhydrotetracycline. The bottom panel shows the corresponding Western blots of 60 μg of soluble protein of the corresponding strains; 60 ng of purified TetR is shown as a control in the left lane. Panel C shows the promoter sequences present in the indicated plasmids in comparison with the consensus promoter sequence.