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Aug 19, 2004 - of 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal) per liter. α-Amylase ex- pression ..... Zhang, L., F. Fan, L. M. Palmer, M. A. Lonetto, C. Petit, L. L. Voelker, A. St. John, B. Bankosky, M. Rosenberg, and D. McDevitt. 2000.

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2005, p. 728–733 0099-2240/05/$08.00⫹0 doi:10.1128/AEM.71.2.728–733.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Vol. 71, No. 2

Tetracycline-Dependent Conditional Gene Knockout in Bacillus subtilis Annette Kamionka, Ralph Bertram, and Wolfgang Hillen* Lehrstuhl fu ¨r Mikrobiologie, Institut fu ¨r Mikrobiologie, Biochemie und Genetik, Friedrich-Alexander-Universita ¨t Erlangen-Nu ¨rnberg, Erlangen, Germany Received 19 August 2004/Accepted 20 September 2004

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 ␮M 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.

Inducible transcriptional control is an important tool for studying gene function in prokaryotes (5, 17). Tetracyclineinduced gene expression has been widely used for this purpose because tetracycline and its derivatives doxycycline and anhydrotetracycline penetrate most cells and lead to sensitive regulation in bacteria even in animal infection models (11). While TetR-mediated gene regulation requires the presence of inducer for expression (Fig. 1A), revTetR mutants characterized so far only in Escherichia coli allow the reversible silencing of genes upon addition of the drug (Fig. 1B) (14, 24). Studying the functions of bacterial genes at particular states, e.g., at a defined growth phase, presently requires the creation of a knockout mutant with the irreversible Cre/loxP or the Flp/frt system (3, 19, 30). Alternatively, this can indirectly be achieved by antisense-mediated silencing of genes (12, 13, 29). Conditional phenotypes of Salmonella enterica serovar Typhimurium have been created by transposition yielding the off state (21), where the on state can be induced but not shut down again. All limitations of these systems can be circumvented by revTetRmediated regulation. Tet regulatory elements allow a wide range of applications in enterobacteria (15, 17, 20, 26). However, their application for gram-positive bacteria is less straightforward, as the previously published tet/xyl promoters allow only a narrow regulatory window (2, 4, 27, 31). We describe the optimization of wild-type and establishment of reverse tet regulation in B. subtilis to obtain the full regulatory range in both approaches.

many). Isolation and manipulation of DNA were performed as described (23). All plasmids constructed were sequenced between the restriction sites employed for cloning. Oligonucleotides for PCR and sequencing were obtained from MWG Biotech (Ebersberg, Germany) unless stated otherwise. Sequencing was carried out according to the protocol provided by Applied Biosystems for cycle sequencing and analyzed with an ABI Prism 310 genetic analyzer (Applied Biosystems, Weiterstadt, Germany). For ␤-galactosidase assays, cells were grown in LB medium supplemented with the required antibiotics to an optical density at 600 nm of 0.4. ␤-Galactosidase activity was determined according to Miller (18). Bacterial strains and plasmids. The bacterial strains and plasmids used and constructed in this study are listed in Table 1. All B. subtilis strains are derivatives of the wild-type strain 168 and were made competent and transformed with DNA as described (16). The tet operator used in this study was tetO1 (8). Chromosomal integration into the amyE locus was verified by the loss of amylose expression (see below) and by PCR analysis of the chromosomal DNA. Culture and growth conditions. E. coli and B. subtilis were generally grown in Luria-Bertani (LB) at 37°C. Antibiotics for selection were added to the following final concentrations: ampicillin, 100 mg/liter; chloramphenicol, 5 mg/liter; and erythromycin, 2 mg/liter. When necessary, the cultures were adjusted to the final anhydrotetracycline concentrations as indicated. All anhydrotetracycline solutions were protected from light. ␤-Galactosidase indicator plates contained 40 ␮g of 5-bromo-4-chloro-3-indolyl-␤-D-galactoside (X-Gal) per liter. ␣-Amylase expression was tested by growing colonies overnight on nutrient broth plates (lab lemco powder, 2 g/liter; yeast extract, 2 g/liter; tryptone, 5 g/liter; sodium chloride, 5 g/liter; 1.2% agar) containing 10% (wt/vol) starch and staining of the plate with iodine. Design and construction of promoters. For expression of the spoVG-lacZ reporter gene from pDH32m (16), the control region containing the oligonucleotide shown in Fig. 2 was cloned via EcoRI and HindII into the linearized vector, giving rise to pWH102. For introduction of a second tetO site upstream of the spoVG-lacZ fusion, the double-stranded oligonucleotide tetO-BamHI with sticky BamHI overlaps was obtained by hybridizing the oligonucleotides 5⬘-GATC CTCTATCATTGATAGAG-3⬘ and 5⬘-GATCCTCTATCAATGATAGAG-3⬘ (purchased from TIB-Molbiol, Berlin, Germany). Based upon the Pr* promoter of pWH353 (4), a set of new promoters was synthesized for tetR expression. A degenerated oligonucleotide of 95 bp was created so that three positions each in the ⫺35 and the ⫺10 regions contained a 70% probability of the original nucleotide and 10% for each of the three remaining nucleotides. The tetO sequence contained in Pr* was mutated at four positions to disable TetR binding and one position in the Shine-Dalgarno sequence was altered to match the consensus. The sequence of the oligonucleotide designated pt_wobble (purchased from TIB-Molbiol, Berlin, Germany) was 5⬘-CTGGAATTCCCGGGAAATAAAAA ACTAGTTTGacaAATAACTCCACCAATGATAtaaTGTCAACAAAAAGG AGGTATTAATGATGTCTAGAGCAC-3⬘, with italics showing variable positions as described above. For creation of the promoter pool, the oligonucleotides were amplified with primers pt_fw, 5⬘-CTGGAATTCCCGGGAAA-3⬘. and pt_rev. 5⬘-GTGCTCTAGACATCAT-3⬘ in a thermocycle reaction with puRe-

MATERIALS AND METHODS General materials and methods. Anhydrotetracycline was purchased from Acros (Geel, Belgium). All other chemicals were from Merck (Darmstadt, Germany), Roth (Karlsruhe, Germany), or Sigma (Munich, Germany) at the highest purity available. Enzymes for DNA restriction and modification were from New England Biolabs (Frankfurt am Main, Germany) or Roche (Mannheim, Ger-

* Corresponding author. Mailing address: Lehrstuhl fu ¨r Mikrobiologie, Institut fu ¨r Mikrobiologie, Biochemie und Genetik, FriedrichAlexander-Universita¨t Erlangen-Nu ¨rnberg, Staudtstrasse 5, 91058 Erlangen, Germany. Phone: 49 9131 85-28081. Fax: 49 9131 85-28082. E-mail: [email protected] 728


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dilution of rabbit polyclonal antibodies raised against TetR(BD) (Seqlab, Go ¨ttingen, Germany). Antibodies were detected with anti-rabbit peroxidase conjugate (ECL; Amersham Biosciences, Buckinghamshire, England) as described by the manufacturer and exposed on a Hyperfilm (Amersham Biosciences, Buckinghamshire, England) film for 0.5 to 5 min.

RESULTS Construction of chromosomal lacZ fusions. The goal of this study was to accomplish full range tet regulation with TetR and revTetR in B. subtilis, and we constructed all the components needed for that purpose. For chromosomal integration of the reporter gene and its regulatory region, we used the spoVGlacZ translational fusion preceded by three successive stop codons in all three reading frames present on plasmid pDH32m, which is nonreplicative in B. subtilis (16). The spoVG-lacZ fusion contains 12 codons from spoVG of B. subtilis followed by the 18th codon of lacZ from Escherichia coli (9, 16). pDH32m and the oligonucleotide containing the improved control region (Fig. 2) were restricted with EcoRI and HindIII. The 9,891-bp vector was ligated with the 68-bp insert, and the resulting plasmid was termed pWH102. Since one tetO might not be sufficient to yield the full regulatory range (4), we introduced a second tetO site. Therefore, pWH102 was linearized with BamHI and ligated with the tetOcontaining oligonucleotide (see Materials and Methods section), giving rise to pWH105. Unlike the plasmid-based systems (4), we aimed to integrate the regulatory region together with a chloramphenicol resistance cassette for selection into the chromosome to obtain single-copy reporters. Therefore, the strain Bacillus subtilis 168 ⌬RA was transformed with either pWH102 or pWH105, resulting in strains designated WH476 and WH478, respectively. In order to verify spoVG-lacZ expression in the strains generated, colonies of both strains were tested on X-Gal plates (data not shown).

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.

TaqReady-To-Go PCR beads (Amersham Biosciences, Buckinghamshire, England). For amplification of the B. subtilis spoVG and xylA promoters, we used the oligonucleotides SpoVG_vorne (5⬘-AAGCTTTATGACCGAATTCTGTAACT ATATCC-3⬘), SpoVG_hinten (5⬘-ATCTAATCTAGACATCATAGTAGTTCA CCACC-3⬘), and XylA_vorne (5⬘-CGAATCTTCCCTTTATGAATTCTAATG TGTTC-3⬘) and XylA_hinten (5⬘-ATCTAATCTAGACATCATGTGATTTCC CCC-3⬘). Western blot analysis. Detection of TetR was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 60 ␮g of B. subtilis cell extract on a 10% polyacrylamide gel. Proteins were transferred by electroblotting onto a PhotoGene nylon membrane (Gibco-BRL, Karlsruhe, Germany). After blocking with 0.3% I-Block solution (Tropix), the membrane was flushed with a 1:20,000

TABLE 1. Bacterial strains and plasmids used in this study Strain or plasmid

Relevant characteristics ⫺

Reference or source

Escherichia coli DH5␣ Bacillus subtilis 168 Bacillus subtilis 168 ⌬RA WH476 WH478

recA1 endA1 gyrA96 thi relA1 hsdR17 (rk mk ) supE44 ⌽80dlacZ⌬m15 ⌬lacU169 trpC2 trpC2 ⌬xylRA trpC2 ⌬xylRA amyE (control region and spoVG-lacZ from pWH102, Cmr) trpC2 ⌬xylRA amyE (control region and spoVG-lacZ from pWH105, Cmr)

6 Laboratory stock Laboratory stock This study This study

pWH1411-BD pWH1520 pWH353 pDH32M pHT304 pWH102

Cmr, tetR(BD) Apr Tcr, xylR xylA⬘ Kmr Apr, improved Prⴱ promoter upstream of tetR(B) Apr Cmr, spoVG-lacZ Apr Err, pUC19 polylinker Apr Cmr, 68-bp control region with tetO bearing EcoRI-HindIII fragment upstream of spoVG-lacZ pWH102, second tetO cloned in BamHI site pWH1520 with a 668-bp tetR(BD)-containing HincII fragment from pWH1411-BD cloned in SmaI site pHT304 with a xylR⬘-xylA⬘-tetR(BD)-bearing 1,453-bp EcoRI-SphI fragment from pWH1520-BD pHT304 with Prⴱ from pWH353 cloned via EcoRI-ApaI pHT304 with spoVG promoter from B. subtilis 168 cloned via PCR and EcoRI-XbaI pHT304 with xylA promoter from B. subtilis 168 cloned via PCR and EcoRI-XbaI pHT304 with synthetic promoter cloned via EcoRI-XbaI (see Fig. 4C)

25 22 4 9 1 This study

pWH105 pWH1520-BD pWH116-BD pWH117-BD pWH118-BD pWH119-BD pWH123-127

This study This study This study This This This This

study study study study




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).

Introduction of different promoters in tetR expression plasmids. To obtain different levels of tetR expression, we placed the xylA promoter from Bacillus megaterium (Prmeg-xylA), P* from pWH353, and the spoVG and xylA promoters from B. subtilis (Pr-spoVG and Pr-xylA, respectively) upstream of tetR. In order to create the Prmeg-xylA-tetR fusion, pW1411-BD was restricted with HincII, generating a blunt end fragment bearing tetR with its natural Shine-Dalgarno sequence. This fragment was cloned into pWH1520 linearized with SmaI, and the resulting plasmid was termed pWH1520-BD. Since it harbors a tetracycline resistance cassette which would interfere with induction of TetR, Prmeg-xylA-tetR was excised with EcoRI and SphI. This fragment was cloned into pHT304 restricted with the same enzymes, and the resulting plasmid was named pWH116. pWH353 and pWH116 were both restricted with EcoRI and ApaI and ligated to yield pWH117 bearing the P*-tetR fusion. In order to exchange this promoter for PrspoVG and Pr-xylA, we isolated chromosomal DNA and amplified the fragments with the specific primers (see Materials and Methods section). The PCR products and pWH117 were restricted with EcoRI and XbaI and ligated to yield pWH118 (Pr-spoVG) and pWH119 (Pr-xylA), respectively. A schematic overview of the promoter regions is given in Fig. 3A. Efficiency of regulation with different expression levels of TetR. The regulatory properties of the promoter-tetR fusions were determined in strain WH476 (Fig. 3B). No repression of spoVG-lacZ was detected in the absence of tetR (pHT304). All tetR-containing constructs mediated repression of spoVG-lacZ, albeit to different extents, namely 38% (pWH118), 22% (pWH116), 10% (pWH117), and 1.7% ␤-galactosidase activity (pWH119). We initially used 0.8 and 0.4 ␮M anhydrotetracycline for induction but detected no difference in ␤-galactosidase activity. Therefore, all subsequent measurements were done with 0.4 ␮M anhydrotetracycline. Since the differences in spoVG-lacZ expression vary with different promoters, we asked if this might be correlated to different amounts of TetR. As shown in the Western blot (Fig. 3B), the repression levels correspond to the TetR amounts expressed from the promoters. The most efficient construct was used to examine the effect of a tandem tetO arrangement in WH478. As also shown in the right part of Fig. 3B, an approximately fivefold-stronger repressed ␤-galactosidase activity (0.3% compared to 1.6%) was obtained, and yet induction was still complete in the presence of 0.4 ␮M anhydrotet-

racycline. Therefore, spoVG-lacZ can be efficiently regulated in B. subtilis with 300-fold induction. revTetR regulation in B. subtilis. More than 100 TetR mutants which require anhydrotetracycline as a corepressor have recently been constructed and analyzed (14, 24). These socalled revTetR variants allow the switching off of gene expression by addition of the corepressor anhydrotetracycline (see Fig. 1B). We chose a revTetR mutant carrying the mutations E15V, L17G, and L25V since it shows the most efficient regulation in E. coli (24). The allele was cloned via XbaI and StuI into pWH119, replacing tetR. The resulting plasmid was named pWH119-r1.7. revTetR exhibited poor regulation properties, showing only 12% corepression with anhydrotetracycline. Western blot analysis indicated that this might be due to the low amounts of revTetR in the cytoplasm (see below). We asked if increasing the revTetR level by replacing PrxylA with a stronger promoter would lead to more efficient regulation. To establish a relationship between the amount of revTetR and regulation efficiency, a promoter pool was constructed with the pt_wobble oligonucleotide (see Materials and Methods section). An overview of the promoter pool is given in Fig. 4A. The variable positions of the ⫺35 and ⫺10 regions were chosen based on a hidden Markov model, which incorporates information about conserved ␴A binding sites (7, 10, 28). The promoter pool was cloned via EcoRI and StuI into pWH119-r1.7. The ligation products were transformed into B. subtilis WH478, and the resulting candidates were streaked out on X-Gal plates with and without 0.4 ␮M anhydrotetracycline. Colonies which grew white on anhydrotetracycline containing plates and blue without anhydrotetracycline were chosen for ␤-galactosidase measurements. The results are shown in Fig. 4B. Plasmids were prepared from overnight cultures of the strains and sequenced. Four of them contained alterations in the ⫺35 and ⫺10 regions (Fig. 4C). The repressed levels of spoVG-lacZ expression dropped below 0.5% of the maximal ␤-galactosidase activity for several candidates. The most efficient repression of 0.2% combined with an induction factor of 500 was detected for pWH125, representing a 60-fold improvement over pWH119-r1.7. As shown in the Western blot in the lower panel of Fig. 4B, this efficient repression corresponds to increased revTetR levels. Thus, the use of optimized promoters for revTetR expression leads to very tight repression of spoVG-lacZ in combination with complete induction in B. subtilis.

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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 ␤-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 ␮M anhydrotetracycline, and 0.8 ␮M anhydrotetracycline, respectively). ␤-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 ␮g 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 ␤-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 ␮M anhydrotetracycline. The bottom panel shows the corresponding Western blots of 60 ␮g 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.

DISCUSSION TetR-mediated control of transcription in B. subtilis as the archetype of gram-positive bacteria was first described in 1990 (4). Since then, the tet system has been employed in Streptococcus and Staphylococcus spp. as well (2, 27, 31). Most of these systems are based on plasmids pWH353 and pWH354, which, however, have limitations concerning the regulatory windows. While the first construct allows full induction but incomplete repression, the latter has been described to entirely shut down the reporter gene at the expense of full induction. For improvement of regulation, we separated tetR and the gene under tet control: New vectors for tetR expression in B. subtilis were constructed and assayed with a chromosomally integrated transcriptional spoVG-lacZ fusion downstream of a tet/xyl promoter equipped with one or two tet operators. The second tetO improves repression 5.3-fold. A corresponding relationship between the TetR amount and repression efficiency was observed when TetR was expressed from different promoters. Despite the presence of high TetR expression levels, induction with 0.4 ␮M anhydrotetracycline was complete. RevTetR mutants bind to tetO only upon addition of anhydrotetracycline and therefore allow the rapid turn-off of gene expression (24). It would be desirable to accomplish such conditional knockout phenotypes in gram-positive bacteria. However,

the revTetR variant that is most efficient in E. coli failed to display efficient regulation when expressed from Pr-xylA in B. subtilis. RevTetR may exhibit lower stability in B. subtilis than in E. coli, where a larger amount of the protein is present (E. Henssler, personal communication). This problem was solved by using degenerate promoters from which a phenotype with tight corepression and complete derepression was obtained. The different ⫺35 and ⫺10 sequences found in pWH123-127 (see Fig. 4C) had no apparent influence on revTetR expression, which was highly similar for these five constructs and exceeded that of wild-type TetR expressed from Pr-xylA. We demonstrate here that proper adjustment of the TetR or revTetR expression levels leads to efficient regulation in both cases. Thus, it is now possible to switch expression on or off at any given time by addition of anhydrotetracycline in B. subtilis, which could provide a valuable tools for basic and applied research. It is quite likely that these regulatory elements can be used for constructing conditional knockouts in other grampositive bacteria with a low GC content. ACKNOWLEDGMENTS This work was supported by the Deutsche Forschungsgemeinschaft through SFB 473, the Volkswagenstiftung, and the Fonds der Chemischen Industrie.


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