We report a regulation system in Escherichia coli for independent regulation of two distinct reporter genes by application of Tet repressors with different specificities . One Tet repressor variant comprises wild-type tet operator (tetO) recognition and exclusive induction with the novel inducer 4-dedimethylamino-anhydrotetracycline . The other Tet repressor variant shows tetO-4C recognition and induction with tetracycline . We demonstrate that both variants are independently active in vivo and allow selective regulation of two genes in the same cell without any cross talk .

 
Inducible promoters are powerful tools for studying gene function in prokaryotes . The most widely used regulated expression systems in Escherichia coli have been developed from the lac, ara, and tet genes (2, 9, 14), while the tet, spac, and xyl systems have been used mostly with gram-positive bacteria (1, 6, 7, 16, 18) . The advantage of tet regulation lies in the combination of tight control and sensitive induction by compounds that diffuse passively across biological membranes and do not require the presence of uptake proteins . Since complex cellular processes are often determined by more than one gene, multiple gene regulation systems that can be distinctly addressed are of general interest . We describe here modifications of the tet regulatory system that allow differential expression control of two genes in E . coli .

Independent tet regulation of two genes requires different tet operators and TetR variants with unique recognition specificity for them . Two such pairs have been described previously (4, 5) . For differential induction, TetR variants with exclusive specificity for distinct inducers are needed . We have recently constructed a TetR variant that is inducible by 4-dedimethylamino-anhydrotetracycline (4-DDMA-ATC) (Fig. 1) but not inducible by tetracycline (TET) . This would allow independent induction of two genes . Finally, since native TetR is a homodimer, cross talk between the TetR variants needs to be avoided, which can be accomplished by using tetR alleles from different naturally occurring sequence variants, as has been described previously (8, 11) . Here, we ask whether the mutations leading to these functionally different TetR proteins can be combined to yield specifically addressable TetR variants and if their phenotypes are stringent enough for distinct regulation in E . coli . In addition to the E . coli lacZ, we used xynB from Bacillus subtilis encoding a ß-xylosidase (ß-Xyl) as a second reporter gene to quantitatively distinguish regulatory efficiencies .

 
Construction of the dual regulation system. Strains and plasmids used in this study are presented in Table 1 . Both reporter genes were expressed in the E . coli strain WH207_tet50, which carries a chromosomal tetA-lacZ transcriptional fusion (17) in which lacZ is under wild-type tetO control . This construct is regulated by the TetR(BD) mutant H64K L131I S135L, which is inducible with 4-DDMA-ATC but not with TET . The second reporter gene, xynB, is regulated by the tet operator 4C (tetO-4C) mutant, with two palindromic sequence changes from the wild type (4) . xynB was amplified by PCR from chromosomal DNA of B . subtilis strain 168, introducing a downstream BsmBI site . tetO-4C was amplified from the plasmid pWH1012-4C (13) containing an upstream BsmBI site and then fused to the xynB-carrying fragment in a subsequent PCR without primers . The resulting product was cloned in two steps into a variant of pWH1411 with the tetR mutant . The resulting plasmid was termed pWH628 (Fig . 2) . TetR-4C, the TetR(B) E37A P39Q Y42M mutant that specifically recognizes tetO-4C instead of wild-type tetO (4), was expressed from the plasmid pWH1925-tetR-4C . Due to different origins of replication (Fig . 2), pWH1925-tetR-4C and pWH628 can coexist in WH207_tet50 . This plasmid compatibility allows the regulation of xynB with TetR-4C . In addition, TetR-4C is inducible with TET but not with 4-DDMA-ATC, and the class B and BD sequence variants of TetR do not heterodimerize (11) .

 
In vivo evaluation of enzyme activities. ß-Galactosidase (ß-Gal) activity was determined as described previously (10) by using ortho-nitrophenyl-ß-D-galactopyranoside as a substrate . ß-Xyl activity was determined in the same way except with para-nitrophenyl-xylanopyranoside as a substrate . We tested for potential cross-reaction of the enzymes by adding ortho-nitrophenyl-ß-D-galactopyranoside to a culture expressing only ß-Xyl . No absorption was detected at 420 nm, and the same result was obtained with ß-Gal and para-nitrophenyl-xylanopyranoside (data not shown) . Measurements of dual regulation were performed in parallel, where one part of the culture was assayed for ß-Gal and the other for ß-Xyl activity . Both effector concentrations were adjusted to achieve optimal induction within the tolerance range of E . coli (0.2 µM for TET and 1.6 µM for 4-DDMA-ATC) (data not shown) . The regulation of ß-Gal expression is shown in Fig. 3A . Independent of the presence or absence of any inducer, TetR-4C does not repress ß-Gal expression and therefore does not recognize tetO, as is shown on the left side of Fig. 3A . The right side of Fig . 3A shows that in the absence of inducer, the 4-DDMA-ATC-specific TetR binds to tetO, as indicated by the tight repression of lacZ, while TET does not induce this TetR variant at all . In contrast, lacZ is highly expressed when 4-DDMA-ATC is added, indicating that this TetR variant is inducible only with 4-DDMA-ATC and not with TET . ß-Gal activity remains high in the presence of both inducers, suggesting that TET does not interfere with the induction of TetR H64K L131I S135L by 4-DDMA-ATC . Figure 3B shows the results of ß-Xyl activity determinations of the same cultures . TetR H64K L131I S135L does not bind to tetO-4C (Fig. 3B, left) . No XynB expression was detectable in the absence of an inducer, indicating that TetR-4C binds tightly to its cognate sequence tetO-4C . Upon addition of TET, ß-Xyl was induced to 65% activity . 4-DDMA-ATC does not induce TetR-4C, as shown by the repression of xynB, and it does not interfere with induction by TET (Fig . 3B, right) .

 
Conclusions. We present for the first time a dual regulation system that combines different TetR inducer specificities with different TetR operator recognition mutations for application in prokaryotes, allowing independent and reversible in vivo regulation of two different genes by two distinct effectors . Apparently, 4-DDMA-ATC resembles TET as a well-permeating effector showing fast and efficient induction in E . coli . In addition, this effector exhibits no antibiotic activity and may thus overcome limitations in efficiency of expression that are sometimes observed with induction by TET in sensitive strains . The use of these two independently functioning repressors should be a powerful tool for the analysis of prokaryotic phenotypes that depend on the expression of two genes . It should also have merits in target validation or inhibitor screening assays for heterooligomeric proteins .

 
This work was supported by the Deutsche Forschungsgemeinschaft through SFB 473 and Graduiertenkolleg 805 and the Fonds der Chemischen Industrie .

We thank Peter Gmeiner and Susanne Lochner for kindly providing 4-DDMA-ATC . We thank Ralph Bertram and Christian Berens for helpful discussions and for critically reading the manuscript .

 
* Corresponding author . Mailing address: Lehrstuhl für Mikrobiologie, Biochemie und Genetik, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudstrasse 5, 91058 Erlangen, Germany . Phone: 49 9131 85-28081 . Fax: 49 9131 85-28082 . E-mail: whillen@biologie.uni-erlangen.de .

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