Eur. J. Biochem. 270, 3109–3121 (2003)  FEBS 2003 Gene regulation by tetracyclinesConstraints of resistance regulation in bacteria shape TetR for applicationin eukaryotes Christian Berens and Wolfgang Hillen Lehrstuhl fu¨r Mikrobiologie, Institut fu¨r Mikrobiologie, Biochemie und Genetik, Friedrich-Alexander Universita¨t Erlangen-Nu¨rnberg;Germany The Tet repressor protein (TetR) regulates transcription are fused to TetR to turn it into an efficient regulator.
of a family of tetracycline (tc) resistance determinants in Mechanistic understanding and the ability to engineer and Gram-negative bacteria. The resistance protein TetA, a screen for mutants with specific properties allow tailoring membrane-spanning H+-[tcÆM]+ antiporter, must be sen- of the DNA recognition specificity, the response to inducer sitively regulated because its expression is harmful in the tc and the dimerization specificity of TetR-based eukary- absence of tc, yet it has to be expressed before the drugs' otic regulators. This review provides an overview of the concentration reaches cytoplasmic levels inhibitory for TetR properties as they evolved in bacteria, the functional protein synthesis. Consequently, TetR shows highly speci- modifications necessary to transform it into a convenient, fic tetO binding to reduce basal expression and high affinity specific and efficient regulator for use in eukaryotes and to tc to ensure sensitive induction. Tc can cross biological how the interplay between structure ) function studies in membranes by diffusion enabling this inducer to penetrate bacteria and specific requirements of particular applica- the majority of cells. These regulatory and pharmacological tions in eukaryotes have made it a versatile and highly properties are the basis for application of TetR to selec- adaptable regulatory system.
tively control the expression of single genes in lower and Keywords: antibiotic resistance; disease models; fusion pro- higher eukaryotes. TetR can be used for that purpose in tein; inducible gene expression; ligand-binding specificity; some organisms without further modifications. In mam- mammalian cell lines; protein engineering; structure–activity mals and in a large variety of other organisms, however, relationship; Tet repressor; transgenic organism.
eukaryotic transcriptional activator or repressor domains Properties of bacterial Tet systems of transcription by the tc-responsive Tet repressor (TetR).
In the absence of inducer, TetR dimers bind to the operators Efflux-mediated tetracycline resistance is always tetO1 and tetO2, shutting down transcription of its own regulated in Gram-negative bacteria gene, tetR, and of the resistance gene, tetA. Once tc hasentered the cell, it binds TetR with high affinity as a In Gram-negative bacteria, resistance to tetracyclines (tc) [tcÆMg]+ complex [7]. This induces a conformational change is mediated by the TetA protein, a proton-[tcÆMg]+ anti- in TetR [8] resulting in dissociation from tetO [9]. The porter embedded in the cytoplasmic membrane [1,2]. Eleven following expression burst of TetA and TetR leads to a tc resistance determinants (Tet classes A–E, G, H, J, Z, 30, rapid reduction of the cytoplasmic tc concentration [10] and 33 [3–5]) share the organization of structural and which, in turn, shuts expression of both genes off again.
regulatory genes (reviewed in [6]). In enteric bacteria, the Expression of TetA is fine-tuned in the presence of tc so that efflux-encoding tetA genes are strictly regulated at the level export overcomes the slow uptake (compare below).
Regulation of Tc resistance is optimized for tightness Correspondence to W. Hillen, Lehrstuhl fu¨r Mikrobiologie, Institut fu¨r Mikrobiologie, Biochemie und Genetik, Friedrich-Alexander Regulation of tet determinants is subject to strong, opposing Universita¨t, Staudtstr. 5, D-91058 Erlangen, Germany.
selective pressures. Expression of the resistance protein Fax: +49 9131 8528082, Tel.: +49 9131 8528081,E-mail: [email protected] TetA is detrimental to the cell [11,12]. Overexpression of this Abbreviations: tc, tetracycline; dox, doxycycline; atc, anhydrotetra- integral membrane protein is lethal for Escherichia coli [13], cycline; tTA, tetracycline-dependent transactivator; rtTA, reverse probably due to the collapse of the membrane potential [14].
tetracycline-dependent transactivator; tTS, tetracycline-dependent Consequently, expression of TetA must be tightly repressed trans-silencer; CMV, cytomegalovirus; GFP, green fluorescent in the absence of the drug. However, when tc diffuses into the cell the resistance protein must be expressed before the (Received 8 April 2003, revised 14 May 2003, cytoplasmic concentration of tc reaches the micromolar accepted 15 May 2003) level necessary to inhibit translation. This requires: (a) high 3110 C. Berens and W. Hillen (Eur. J. Biochem. 270) competing nonspecific DNA to a much higher degree thanbacteria. Taken together, the evolutionary pressures ontc-dependent gene regulation have led to tight repression inthe absence of tc, without compromising sensitivity ofinduction, so that regulated tc resistance determinantsimpose no burden on the fitness of E . coli in the absence ofthe antibiotic, but still mediate high levels of resistance to tcin its presence [12].
The structural change of TetR associated with inductionby tetracycline is known X-ray crystal structures of free TetR [17], TetR complexedwith different tetracyclines [18–21] and with tetO [8] havebeen determined at resolutions of 1.9–2.5 A˚, revealingthe allosteric conformational change leading to induction.
These results have been reviewed in detail [22] and have beencompared to Lac repressor [23]. Thus, they are onlysummarized here (Fig. 2). The DNA reading head of TetR(magenta) is connected to the protein core (blue) by the helixa4 (green). Binding of [tcÆMg]+ (yellow) to TetR unwindsthe C-terminal residues of helix a6 (light blue), which bumpinto a4 and displace it. As the C terminus of a4 is held inplace by contacts to tc, the displacement leads to apendulum-like swing of the a4 N terminus increasing thedistance between the recognition helices by 3 A˚, so that theydo not fit into successive major grooves of DNA anymore[24]. These conformational changes are consistent withmany noninducible TetR mutants [24,25], spectroscopicanalysis of TetR in vitro [26], in vivo [27] and in vitro [28]disulfide trapping experiments. Furthermore, a movementof a9 closes the tc binding pocket after the drug has entered[17], and the loop between a8 and a9 is also important for Fig. 1. Structures of tetracyclines used in eukaryotic gene regulation.
(A) Structure of tetracycline with the pKa values of the three titratablegroups. (B) Structure of doxycycline. (C) Structure of anhydrotetra-cycline.
Tetracycline penetrates cells by diffusion Tetracyclines (Fig. 1) diffuse in their uncharged forms affinity of TetR for both tetO and tc to keep the basal through lipid bilayers without the aid of protein channels expression level of tetA low and to ensure that its [32–36]. Measuring the increase in fluorescence intensity of transcription is initiated at concentrations which are still tc observed upon binding to TetR [7] allows us to determine subinhibitory for translation; (b) low affinity of the TetR– the cytoplasmic concentration of tc and, thus, to calculate [tcÆMg]+ complex for DNA; and (c) high-level, but short- permeation coefficients for tc uptake into liposomes term expression of TetA to initially reduce the internal [(2.4 ± 0.6) · 10)9 cmÆs)1] concentration of tc. A low level of TetR is important for [(5.6 ± 1.9) · 10)9 cmÆs)1] [36]. These translate into half- sensitive induction, since E . coli strains expressing high equilibration times of 35 ± 15 min for tc to cross the levels of TetR need high concentrations of tc for full membranes and are in good agreement with the half- induction [15]. These conflicting requirements are met by equilibration time of 15 min measured for [3H]tc-uptake in the genetic organization of the resistance determinants Bacillus subtilis [37], and the slow uptake of tc observed in (reviewed in [6]) and by the ligand binding properties of Staphylococcus aureus [38]. Tetracycline diffusion through TetR. High sensitivity towards tetracyclines [see Fig. 1 for phospholipid membranes is, thus, slow and appears to be the structures of tc, doxycycline (dox) and anhydro-tc (atc)] the rate-limiting step of uptake into cells [36]. The previously is achieved by the remarkable binding constant of TetR for observed rapid uptake of tc [33,39] might rather reflect ), [doxÆMg]+ (Ka  1010 M ) or unspecific adsorption of tc to membrane surfaces [32,36]. A ) [7,9], about 103)105-fold higher detailed model explaining the transport and accumulation than the affinity of the drugs to their intracellular target, the of tc across the Gram-negative cell envelope has been ribosome [16]. Binding of two molecules of [tcÆMg]+ to a presented by Nikaido and coworkers ([40,41] and references TetR dimer diminishes repressor affinity for tetO by about cited therein). In the medium, as well as in the periplasm and nine orders of magnitude to the unusually low background cytoplasm, tc is present in one uncharged and several DNA binding level of less than 105 [9]. This high ratio charged or zwitterionic species, due to its three titratable of specific over nonspecific DNA binding enables TetR to groups (Fig. 1). The distribution between these species bind tetO efficiently, even in larger genomes containing depends on the pH of the respective compartment [40]. The Gene regulation by tetracyclines (Eur. J. Biochem. 270) 3111 Fig. 2. Structure of the TetR–[tcÆMg]+complex. Tet repressor is shown as a ribbondiagram with one monomer in gray and theother monomer color-coded as follows: TheDNA-binding region is in magenta, the helixconnecting it with the protein core is in green.
The protein core is dark blue, with the helix a6in light blue. Tetracycline is displayed asspace-filling CPK model in yellow. For clarity,the helices a1–a10 of one monomer are num-bered and the N and C termini of both sub-units are indicated. The coordinates weretaken from the PDB entry 2TRT [18].
uncharged form of tc can penetrate the outer membrane repression by unmodified TetR [53]. Here, TetR most directly. But the major fraction of tc equilibrates as a likely acts by interfering sterically with binding of RNA [tcÆM]+-complex rapidly through the outer membrane via polymerase or auxiliary transcription factors [42,54]. To porins, with the Donnan potential across the outer mem- achieve this, one or more tetO elements are placed in brane leading to a two- to threefold accumulation of this proximity of either the TATA box or the transcriptional charged complex in the periplasm. Tc then diffuses passively start site of the respective target gene and TetR is expressed in its uncharged form through the cytoplasmic membrane.
concomitantly by a strong, constitutive promoter. Promot- Due to the pH gradient across the cytoplasmic membrane, ers of all three eukaryotic RNA polymerases have been a larger fraction of the uncharged tc dissociates in the targeted in the manner described. Unfortunately, as will cytoplasm than in the periplasm. Since equilibrium is become evident in the following paragraph, the published, reached when the concentration of uncharged tc is identical successful approaches do not yet allow formation of a in both compartments, this results in a higher intracellular simple strategy for establishing a TetR-repressed system, concentration of [tcÆM]+, the biologically active compound.
although they clearly point out that the positioning of the Again, accumulation of tc is the product of this passive tetO boxes is crucial for efficient regulation.
equilibration across the inner membrane [40,41].
In Leishmania donovani, an RNA polymerase I promo- ter was brought under tc-control by placing a single tetO site Tc-based gene regulation functions in 2–24 bp upstream of the transcriptional start site [55], different setups in many eukaryotic systems whereas in Trypanosoma brucei at least one tetO elementhad to be inserted at a position +2 or )2 relativ e to the The evolved properties of TetR described above combined transcription start site of an RNA polymerase I-like with the favorable pharmacokinetics of tetracyclines and promoter [56]. For RNA polymerase III-mediated tran- their long record of safe use in clinical practice make the Tet scription of suppressor tRNA genes, induction factors system a good candidate to fulfill the criteria that are required between two- to fivefold were observed in Saccharomyces for an ideal transcriptional regulator in eukaryotic cells as cerevisiae, Dictyostelium discoideum and carrot protoplasts given by Saez and others [42,43]. Consequently, the past when tetO was introduced within 10 bp upstream of the 15 years have seen the broad application of tc-dependent transcriptional start site [57–59]. A regulated version of the regulatory systems, mainly in mammalian cell culture, but to human U6 snRNA promoter, also transcribed by RNA an increasing degree in transgenic organisms like plants, polymerase III, was developed by replacing sequences yeasts, protozoan parasites, slime molds, flies, and rodents.
between the proximal sequence element and the transcrip- These topics have been extensively reviewed [42–52]. The tional start site with tetO [60]. Flanking the TATA-box with following section presents an overview of the basic Tet two operators completely abolished transcriptional activity.
systems used to regulate gene expression in eukaryotes.
In contrast, introduction of a single tetO element affectedtranscription only slightly, but led to up to 25-foldrepression in the presence of TetR. A regulated U6 snRNA Gene regulation by TetR in eukaryotes promoter with a defined expression window [61,62] would The most basic and first published application of be a very powerful tool as this promoter is used to express tc-mediated gene regulation in eukaryotes is transcriptional the small interfering RNA [63] needed for silencing gene

3112 C. Berens and W. Hillen (Eur. J. Biochem. 270) expression by RNA interference [64]. Repression of RNA magnitude can be reached with sensitive reporter genes like polymerase II promoters exerted by TetR is strongest in firefly luciferase [75]. Luciferase activity is expressed within plants [65,66], mammalian cells [67] and fungi like Schizo- 4 h of removal of tc and about 20% of the steady-state level saccharomyces pombe [68,69] when multiple tet operators is reached after 12 h. While the use of a strong, constitutive are positioned within a region from 5 bp upstream to 35 bp promoter (CMV IE, EF-1a, Ubiquitin C) is common in cell downstream of the TATA-element. In contrast, placement culture applications, the use of tissue-specific promoters in of one to four tet operators immediately downstream of the transgenic animals provides spatial control to the Tet transcription initiation site has been shown to be most system, restricting expression of the Tet transregulator and, effective in the parasitic protozoa Entamoeba histolytica subsequently, the transgene to the desired tissue [84,85]. In [70,71], Toxoplasma gondii [72] and Giardia lamblia [73].
Drosophila, usage of the Gal4-UAS system to control Tettransregulator expression allows the generation of spatiallydelimited expression patterns by simple crossing with one of Gene regulation by TetR-based transregulators the many Gal4 driver lines available in the Drosophila While unmodified TetR acts as a transcriptional repressor in research community [86].
plants and lower eukaryotes, it can be, but not always is One concern has been the expression levels of Tet efficient in mammalian cells [67,74]. A consistently func- transregulators as influenced by a potentially low mRNA tional version for yeasts, flies and mammalian cell lines is stability or efficiency of translation. This was recently TetR fused to an eukaryotic regulatory domain, such as an addressed by generating a synthetic coding sequence for acidic activation domain (Fig. 3A; tTA or Tet-Off) [75–80] tetR. Potential splice donor and acceptor sites identified by or a repression domain (Fig. 3B; tTS) [81–83]. The trans- sequence analysis, several potential endonuclease cleavage activator tTA directs expression from a tc-dependent sites, and potential stable hairpin structures in the mRNA promoter that contains seven repeats of a tetO2 element were eliminated and human codon usage was used [87–90].
from the transposon Tn10. The palindromic centers of two The consequence of this optimization protocol is a higher adjacent operators are separated by 41 bp. This element is protein level in Drosophila, HeLa and HEK293 cells.
fused to a minimal promoter, typically derived from the Another concern voiced was that the CMV-derived human cytomegalovirus (CMV) immediate early promoter minimal promoter was not transcriptionally silent under all [75]. When both components are stably integrated into experimental conditions [91–93]. This promoter leakiness proper chromosomal loci of mammalian cell lines, tran- can be caused by promoter-dependent or integration site- scription from the hybrid promoter is silent in the presence dependent effects and has been discussed in detail [94].
of more than 10 ngÆmL)1 dox. Removal of dox leads to Promoter-dependent leakiness has been addressed by the binding of tTA to tetO and subsequent activation of use of alternative minimal promoters [75,95,96]. In transient transcription. Regulatory factors of up to five orders of transfection experiments, these show lower basal activities Fig. 3. Regulation of gene expression by Tet transregulators. The promoter proximal tetO boxes are represented by black boxes. The transregulatorsare shown as follows: the DNA reading heads are in light gray, the inducer-binding and dimerization domain is in dark gray, activation domains areblack boxes, and the silencing domain is stippled. The conformational change leading to the loss of DNA-binding activity is pictured as a light graybox. High-level activated transcription is displayed by a bold arrow, low-level basal transcription by a dotted arrow. (A) tTA. (B) tTS. (C) rtTA.
Gene regulation by tetracyclines (Eur. J. Biochem. 270) 3113 than Ptet-1, but also do not reach its maximal activation tion can be used to find solutions to some of the problems level. Thus, the regulatory window for target gene expres- and limitations that arise for Tet system applications in sion is shifted and expanded due to the stronger reduction of the basal activity. Integration site-dependent leakiness hasbeen attributed to enhancers located close to the integration Alterations of the activator domain of tTA site of the target gene construct. Besides screening additionalclones until one harboring the desired properties is found, Especially for gene therapy, concern about a viral protein is the problem has been approached by insulating Ptet-1 from often voiced, as humoral as well as cellular immune external activating signals through insertion of a chicken response against the VP16 protein has been found in herpes lysozyme matrix attachment region just upstream of Ptet-1 simplex infected humans [106–108]. Thus, immune res- [87] or by flanking the target gene expression unit with either ponses against transactivators containing the VP16 domain chicken b-globin insulators [90] or SCS and SCS' boundary cannot be rigorously excluded, although they have not been elements from Drosophila [86].
observed so far in a mouse model using reverse tTA (rtTA; A different strategy was adopted by engineering a Tet-On) [109]. Two solutions circumventing this concern tc-controlled trans-silencer protein [81]. Fusion of the have been developed: (a) the VP16 domain has been KRAB domain of Kox1 [97] to TetR yielded a hybrid replaced by three repeats of a minimal activation domain protein called tTS, that not only substantially repressed derived from a 12-amino acid activating patch of the VP16 basal transcription from Ptet-1 even if the tet operators were protein (tTA2 [76]); and (b) a variety of human activator located 3 kbp distant from the minimal promoter, but also domains from the acidic, glutamine-rich, serine/threonine- efficiently down-regulated gene expression from a CMV rich and proline-rich functional groups were tested for their enhancer-driven Ptet-1 [83]. This strategy therefore appears ability to replace the VP16 domain. When fused to TetR, to be more versatile in coping with unwanted target gene only acidic activation domains were highly active [78–80].
expression than the promoter adaptation proposed above.
Minor activation was observed with the serine/threonine- In addition and in contrast to tTA, the tc-dependent rich domains from the transcription factors ITF-1, ITF-2, silencing of complex promoters offers the unique possibility and MTF-1. Transactivators with activation potentials of reversibly down-regulating the expression of cellular spanning more than three orders of magnitude have been genes on top of their normal regulation. The KRAB generated by combination of various minimal activation domain is inactive in S. cerevisiae and Drosophila where it domains (see above; [76]). They are attractive for combined was replaced with repression domains from the proteins knock-in/knock-out strategies to convey tissue-specific SSN6 [82], knirps, giant or dCtBP [83].
expression of the transactivator, while at the same time The expression of transfected genes can be rapidly inactivating expression of the genomic copy of the target repressed in mammalian cells by epigenetic mechanisms gene. Expression of the regulatory protein is then an [98]. Although this transgene silencing is not specific for invariant function of the genomic locus and, if too high, can the Tet system, it is often observed for genes under tc lead to squelching [110]. This can be addressed by control due to its frequent usage as conditional expression employing a transactivator with reduced activation poten- system. Approaches to achieve stable gene expression have tial as these are tolerated in the cell at higher concentrations been to: (a) screen many transfected clones; (b) the use of lentiviral vectors [99]; (c) replace the viral promoters thatdirect expression of the transregulators with promoters of Conversion of TetR to reverse TetR human origin [100]; (d) use chromatin insulator sequencesto protect transgene expression [98]; or (e) couple transgene Eukaryotic gene regulation by tTA shows a high dynamic expression to a selectable marker via an IRES element range and works consistently well, but has several practical [101] or by fusion of the transregulator with green drawbacks. Tc has to be continually present to keep fluorescent protein (GFP) [102]. Note that in this fusion expression of the gene of interest downregulated. Although protein GFP is connected to the DNA-binding domain of tc is not toxic at the levels utilized in gene regulation, TetR which can interfere with nonspecific DNA-binding prolonged exposure to the antibiotic is not always desirable activity of TetR at low levels of dox (see Fig. 2A in [102] in transgenic animals nor is it possible in gene therapy.
and [78]). The few published examples make it impossible Furthermore, induction of target genes is mostly slow as it to recommend one of the strategies for use in establishing requires removal of the drug from the culture or organism.
homogenous expression of transgenes, but silencing of To be able to control the time point of induction more transregulator expression is not completely suppressed by precisely, and since organisms are more easily saturated the use of lentiviral vectors [103] or insulator sequences with an effector than depleted of it [111,112], reverse TetR variants which bind tetO only in the presence of tc weresearched for and found (Fig. 3C). Screening in E . coli [113] Modifications of the Tet transregulators and in S. cerevisiae [88] revealed that a small number ofmutations in TetR can lead to that phenotype [113]. Once The TetR–VP16 fusion works very well in many cases, but this was discovered, intensive screening led to rtTA alleles in may not be optimal for all applications. Structure–function which the initial disadvantages of occasional background studies based on powerful selection and screening systems in expression and low sensitivity for dox were eliminated [88].
E . coli [104,105] and in S. cerevisiae [88] have lead to a The rtTA-S2 allele was obtained by screening for reduced profound understanding of how DNA binding, inducer background expression and rtTA-M2 was the result of binding and dimerization function in TetR. This informa- screening for higher sensitivity towards dox starting from 3114 C. Berens and W. Hillen (Eur. J. Biochem. 270) the alterations in rtTA-S2 that are responsible for the DNA binding of the modified transactivators is efficient; reverse phenotype [88]. None of the exchanges found in in transient transfections in HeLa cells, they specifically these new alleles were present in the original rtTA. The achieve induction factors between 2000 and 8000 and are, mutations leading to the reverse phenotype are located at thus, as active as wild-type tTA2. Moreover, they are also the interface between the DNA reading head and the highly specific, as they induce the converse operator less protein core or in the last turn of helix a6 that undergoes a than twofold [114]. Modulation of the DNA-binding conformational change upon inducer binding. Structural specificity is not confined to tTA. Alleles specific for the analysis of the DNA-bound form of TetR has led to the 4C- [120] and 6C-tet operators [114] have been constructed proposal that the mutations present in rtTA [113] restrict with rtTA and also regulate tc-dependent expression units the repressor to a noninducible conformation and lock the efficiently. This now leaves us with different tTA- and rtTA- DNA-binding domains in the position necessary for oper- operator combinations capable of controlling gene expres- ator binding [8]. Taken together, the phenotype of rtTA can sion tightly over a wide range of inducer concentrations.
be improved and designed by using appropriate screens.
Mastering subunit recognition of TetR Tet transregulators vary in their sensitivity towards Comparison of the TetR primary structures reveals 38–90% tetracyclinic inducers identical amino acids overall, but only 18% in the four-helix The tTA and rtTA variants presently employed in eukary- bundle involved in dimerization. Detailed structural infor- otic gene regulation display differential sensitivity towards mation [19] of the dimerization interface [121] suggested that tc and its derivatives. While tTA can be induced by tc, TetR proteins from individual classes would not readily dox and atc [114], reverse transactivators respond only to form heterodimers. The modular architecture of TetR dox and atc [113] and tTSG is about twofold less sensitive to allows the combination of a class B DNA-binding domain dox than rtTA [115]. The response range of tTA to dox (0.1– with the inducer-binding and/or dimerization domains of 10 ngÆmL)1) is clearly lower and, more importantly, non- Tet repressors from other classes [121]. Fusion to the overlapping with that of rtTA to dox (100–3000 ngÆmL)1) reading head from TetR(B) increases activity of Tet [114], but slightly overlapping with that of the more sensitive repressors from several other classes [121] and ensures tight rtTA2s-M2 allele (2–200 ngÆmL)1) [88]. The molecular binding to the tetO boxes from Tn10 [122]. Class B TetR mechanisms responsible for these different sensitivities are does not form heterodimers with Tet repressors from classes presently unknown. The isolation of a tc-like antagonist for D [121], E [93,114,120], or G [115]. The fusion points can be TetR [116] and the demonstration of its activity in chosen with some flexibility; functional chimeras have been transgenic plants [117] make it seem likely that alternative obtained either by connecting the entire protein core from inducers for TetR can be identified by screening.
TetR(D) or TetR(E) to a TetR(B) DNA-reading head[114,120,121] or by replacing the four-helix bundle formedby the helices a8 and a10 from both subunits (see Fig. 2), The DNA binding specificity of Tet-transregulators with the respective region from TetR(G) [115]. The resulting transrepressors or transactivators regulate gene expression Structure–function analyses of TetR–tetO interactions had efficiently and do not form heterodimers as demonstrated in shown that only few changes (shown in Fig. 4) in the DNA DNA-retardation assays [114], immunoprecipitation and binding helix–turn–helix motif of TetR suffice to switch the FACS analysis [115] opening up the possibility to introduce recognition specificity from the 19-base pair wild-type tetO two or more TetR-based regulatory proteins into the same to variants containing symmetric exchanges of bases at cell without having to cope with the disadvantages of position 4 (tetO-4C [118]) and position 6 (tetO-6C [119]).
heterodimer formation [114,115].
The TetR mutants were converted into the transactivatorstTA24C or tTA26C and minimal promoters Ptet4 and Ptet6 Combinatorial Tet regulation solves special were constructed with the respective tetO variants [114].
problems and allows sophisticatedapplications The previous section has shown that DNA-binding speci-ficity, subunit recognition and response to the inducer canbe altered in TetR. Fig. 5 gives an overview of the presentstate of the Tet modules that are available for use and thefollowing section presents a few principles of how themodular nature of the transregulators can be exploited toaddress specific experimental requirements and open up newapplications for conditional regulation.
Fig. 4. Operator specificity combinations for the Tet system. The pri-mary structure of the TetR(B) recognition helix a3 and the flanking Expression can be switched between two alleles loops is given in standard one-letter abbreviations. The entire sequence of tetO2 is shown with the palindromic center marked by an asteriskand the base numbering shown above one operator half-side. The The expression of two genes or of two alleles of one gene can exchanges in TetR and tetO are highlighted in inverse print for each be controlled in a mutually exclusive manner by combining matching pair (wt, 4C, 6C).
different dimerization domains, different operator-binding

Gene regulation by tetracyclines (Eur. J. Biochem. 270) 3115 therapeutic goal has been reached or, if necessary, in caseof emergency.
One gene can be regulated stringently by converselyacting transrepressor and transactivator Detectable levels of transgene expression in animals or cellsin which the transactivator is not active can limit theusefulness of any conditional expression system for mode-ling complex biological processes or evaluating the effects ofa gene product. For the Tet system, this transgene leakagehas been attributed either to basal activity of the respectivetetO-based minimal promoter used (see above; [115]); or, insystems with rtTA, to residual binding of the reversetransactivator to tetO in the absence of dox [123,124]. Astringently controlled regulatory system can now be Fig. 5. The Tet toolbox. TetR modules and regulatory domains are accomplished by combining a trans-silencer with a reverse displayed with the possible combinations. The different binding func- transactivator, since heterodimer formation and concomit- tions of TetR were coded in different shades of gray and placed at theirapproximate position in the protein, but not drawn to scale. The TetR ant phenotype blurring will be prevented if the trans-silencer variants characterized were classified in the corresponding module.
is equipped with a dimerization domain from the TetR The regulatory domains that can be fused to TetR are coded in dif- classes E or G. Thus, both transregulators bind in a ferent shades of gray according to their viral, human, insect or fungal mutually exclusive manner. Gene expression is actively origin. Note that not every possible combination of modules need repressed in the absence of dox by the binding of tTSE/tTSG result in a transregulator with acceptable regulatory properties.
to the minimal promoter. Upon addition of dox, tTSE/tTSGdissociates from tetO, allowing the reverse transactivator tobind and activate transcription. This setup efficiently specificities and by exploiting the differential sensitivity of reduces background expression in yeast [82], in mammalian Tet transregulators towards tetracyclines [114]. Interference cell lines [93,115,120] and in transgenic animals [125–127], between the two expression units is excluded by using a tTA while affecting the maximal expression level only slightly allele with an alternative class E or G dimerization domain [128] or not at all [93].
and by furnishing rtTA with a modified DNA-bindingdomain that contacts the tetO-4C operator in Ptet4 speci- Transgenes can be expressed in a graded fically. Expression of the wild-type allele, for example, is or in a binary manner placed under tTA control and represents the normal state ofthe cell. A knockout situation can be generated by adding Transcriptional control has generally been assumed to either tc or, alternatively, atc or dox at concentrations operate as a binary switch with on/off characteristics between 10 and 100 ngÆmL)1 which dissociates tTA from [129,130], but several examples displaying graded changes the promoter but does not lead to DNA binding by rtTA in gene expression have recently been published [131,132].
[114]. Maintaining the intermediate concentration of dox The manner of gene expression might well be a key factor in needed to shut down expression of both alleles will be programs of cell differentiation or stimulus response.
feasible in cell culture applications. In transgenic animals, Different regulatory setups of the Tet system allow a however, the necessary fine-tuning of a dox or atc concen- transgene to be expressed in one or the other manner [133– tration may prove impossible suggesting instead the use of tc 135], enabling not only an analysis of a gene's function, but to shut down tTA-dependent gene expression without also of its mode of expression. When tTA and rtTA are interfering with regulation by rtTA. To switch to the expressed constitutively in mammalian cells and also in expression of the mutant allele requires atc or dox concen- S. cerevisiae, they drive transgene expression in a dose- trations of 1 lgÆmL)1 or more.
dependent, graded manner [133,135]. However, when rtTA Such a dual control system can provide valuable was expressed in S. cerevisiae under conditions of positive insights into developmental and pathogenic processes.
feedback using an autoregulatory circuit, the cell population One can imagine shutting down expression of a tumor was clearly divided into regulator-expressing and nonex- suppressor while inducing expression of an oncogene to pressing cell pools [135]. In mammalian cells, the combined study cancerogenesis. Switching off expression of the usage of tTSG and rtTA also led to bimodal expression of oncogene after tumor formation can establish whether the the GFP reporter (see Fig. 3 in [134]). Although not respective protein is a valid target for therapeutic inter- formally proven, we assume that a bimodal expression vention. One could also switch from a wild-type to a pattern will not be observed for all repressor/activator mutant allele at a defined developmental state of the combinations, but only for those in which the sensitivity of organism and then return to wild-type expression at a later tTS for the inducer is lower than that of the rtTA allele used, stage. This type of regulatory circuit can also deliver an as is the case for tTSG (compare the dose–response curve of additional degree of freedom to gene therapeutic strat- tTSE and rtTA of Fig. 4 in [93] with the one for tTSG and egies ) one regulatory circuit may be used to control a rtTA of Fig. 2 of [134]). This will ensure that rtTA is therapeutic gene, while the other may be exploited to serve preloaded with inducer and ready to activate transcription as a suicide switch to terminate the treatment once the the moment the dox concentrations needed for binding to 3116 C. Berens and W. Hillen (Eur. J. Biochem. 270) tTSG are reached and tetO is subsequently released. In fragment in the brain demonstrated that its continuous principle, only two regulatory states are observed: either the supply was needed to maintain the characteristic neuro- tetO sites are fully occupied with tTSG and gene expression pathology and behavioral phenotype, raising the possibility is shut off, or they are saturated with the rtTA variant, that the disease may be reversible by targeting the causative resulting in full transcriptional activation. The consequence agent [151].
is a binary expression pattern of the target gene. While this Regulation by the Tet system has also had a significant setup already works with rtTA, the effect should be even impact on behavioral studies. Expression of constitutively more pronounced with rtTA2s-M2, as its inducer response active mutant forms of the calcium/calmodulin dependent range overlaps completely with that of tTSG.
kinase II or calcineurin in the brain of adult mice resulted inaltered synaptic plasticity and impairments in spatial Highlighting the regulatory potential memory storage and retrieval, but these deficits were fully and looking into the future reversed when transgene expression was suppressed[84,152]. Because expression of the transgene was limited The properties and the adaptability of Tet regulation as to the hippocampus, this structure was additionally proven presented in the previous sections allow its use in many to be the site responsible for the behavioral effects. In a different applications. We would like to demonstrate this different example, knockout mice lacking the serotonin 1A enormous variability by referring to a few key studies that, receptor show increased anxiety-like behavior which could in our opinion, highlight the potential of Tet regulation.
be rescued by conditional expression, but only if the Regulation by tetracyclines is sensitive and efficient receptor was synthesized during the early postnatal period enough to control target gene expression in pathogenic in the hippocampus and cortex [153].
organisms even when they have been injected into a Nevertheless, improvement and additions to the Tet mammalian host. The role of individual genes in infection system, among them the regulatory components, are still and pathogenesis can, thus, be probed and their validity as possible and necessary. Promoter development has not targets for therapeutic intervention determined in an in vivo received the same degree of attention as the transregulators.
disease model [136]. This has not only been demonstrated The number of tetO elements and their spacing [154], as well for trypanosomes [137,138], but also for common human as the linker sequence separating the operators [155] have pathogens like Staphylococcus aureus [139] and Candida not been optimized yet. It remains to be seen if an ideal glabrata [140]. In the fungus, squalene synthase [136] and minimal promoter with no intrinsic leakiness supporting sterol 14a-demethylase [141] were, thus, shown not to be very high-level activation can be identified or designed.
ideal targets for antifungal development.
Fortunately, screens for regulators with improved prop- The successful expression of the diphtheria toxin A erties can now be performed in eukaryotic systems [88] subunit by tTA/Ptet-1 in transgenic mice has demonstrated and, as an example, the isolation of novel Tet regulators the stringency of regulation that can be reached with the Tet which recognize nonantibiotically active tetracyclines or system [142]. Although mouse lines that carried the target even nontetracyclinic inducers, would be of great benefit.
transgene were obtained at an approximately 10-fold lower They would not only facilitate gene therapy applications frequency than normal, those that were established regula- which, at the moment, can be impaired by the use of ted the transgene efficiently. Induction of toxin expression tetracyclines in anti-infective therapy or their misuse as led to cell death and development of cardiomyopathies.
growth promoting additives to animal food. If these novel Stringent control of transgene expression using rtTA has inducers are not only ecologically safe, but also easy and also been achieved in HeLa cells for the Shiga toxin B nonexpensive to manufacture, the inducer–regulator pair- subunit [143], for the proapoptotic gene PUMA in SAOS-2 ing could also be useful in insect population control using and H1299 cell lines [144] and, using rtTA2s-S2 in transgenic dominant, repressible, lethal genetic systems [156,157] and mice, for Cre-recombinase [145].
might even introduce regulation by the Tet system to crop The strength of a true conditional system ) the possibi- plants. They would add to the repertoire of transregulators lity to switch gene expression on and off at leisure and and finally, since multiple dimerization and DNA-binding repeatedly ) represents a powerful method with which to specificities are already present, allow fully independent explore the relationship between mutant protein expression expression control of more than one gene by the Tet and disease progression. This has become evident upon studying transgenic mouse models for cancer and neuro- A major experimental challenge will be to express a target logical disorders. Here, the use of tTA and rtTA to control gene within its physiological window, which might depend expression of an oncogene revealed for solid tumors on environmental stimuli and even change during develop- [146,147] and for leukemias [148,149] that the oncogene is ment, since over- or underexpression often results in altered not only necessary for tumor formation but also for tumor phenotypes [131] or pathologies. While tc-controlled expres- maintenance, suggesting pharmacological inactivation of sion can mimic the natural level [146], this must not always oncogenes as a possible therapeutic strategy for cancer. This be the case. A solution might be precise promoter targeting assumption has been substantiated by the unexpected by tetO elements, to minimally interfere with gene expres- observation that, after having gone through one cycle of sion. This will be difficult and will require extensive MYC-gene expression and silencing, reactivation of the knowledge about the influence of chromatin structure on oncogene does not lead to tumor regrowth, but rather to gene expression and its sensitivity to perturbation, partic- apoptosis [150]. Similar effects have also been found for ularly when regulatory regions are modified [158]. But, if neurological disorders. In a conditional model of Hunting- successful, this approach will provide an additional degree ton's disease, mice expressing a mutated huntingtin of freedom to manipulate gene expression, as the existing Gene regulation by tetracyclines (Eur. J. Biochem. 270) 3117 transregulators can be used to activate or silence gene 6. Hillen, W. & Berens, C. (1994) Mechanisms underlying expres- expression, in addition to and independent of the promo- sion of Tn10 encoded tetracycline resistance. Annu. Rev. Micro- ter's natural expression pattern.
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Original Investigation ASSOCIATION OF VA SURGEONS Effect of a Preoperative Decontamination Protocol onSurgical Site Infections in Patients Undergoing ElectiveOrthopedic Surgery With Hardware Implantation Serge P. Bebko, MD; David M. Green, MD; Samir S. Awad, MD, MPH IMPORTANCE Surgical site infections (SSIs), commonly caused by methicillin-resistantStaphylococcus aureus (MRSA), are associated with significant morbidity and mortality,specifically when hardware is implanted in the patient. Previously, we have demonstratedthat a preoperative decontamination protocol using chlorhexidine gluconate washcloths andintranasal antiseptic ointment is effective in eradicating MRSA in the nose and on the skin ofpatients.

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