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Proc Natl Acad Sci U S A. Jun 30, 2009; 106(26): 10638–10643.
Published online Jun 22, 2009. doi:  10.1073/pnas.0901501106
PMCID: PMC2700147
Biophysics and Computational Biology

Controlling transgene expression in subcutaneous implants using a skin lotion containing the apple metabolite phloretin

Abstract

Adjustable control of therapeutic transgenes in engineered cell implants after transdermal and topical delivery of nontoxic trigger molecules would increase convenience, patient compliance, and elimination of hepatic first-pass effect in future therapies. Pseudomonas putida DOT-T1E has evolved the flavonoid-triggered TtgR operon, which controls expression of a multisubstrate-specific efflux pump (TtgABC) to resist plant-derived defense metabolites in its rhizosphere habitat. Taking advantage of the TtgR operon, we have engineered a hybrid P. putida–mammalian genetic unit responsive to phloretin. This flavonoid is contained in apples, and, as such, or as dietary supplement, regularly consumed by humans. The engineered mammalian phloretin-adjustable control element (PEACE) enabled adjustable and reversible transgene expression in different mammalian cell lines and primary cells. Due to the short half-life of phloretin in culture, PEACE could also be used to program expression of difficult-to-produce protein therapeutics during standard bioreactor operation. When formulated in skin lotions and applied to the skin of mice harboring transgenic cell implants, phloretin was able to fine-tune target genes and adjust heterologous protein levels in the bloodstream of treated mice. PEACE-controlled target gene expression could foster advances in biopharmaceutical manufacturing as well as gene- and cell-based therapies.

Keywords: synthetic biology, synthetic gene networks, transdermal gene regulation, gene therapy, biopharmaceutical manufacturing

Synthetic mammalian expression systems, which enable reversible and adjustable transgene expression, have been essential for recent advances in (i) functional genomic research (1), (ii) drug discovery (2, 3), (iii) manufacturing of difficult-to-produce protein therapeutics (4, 5), (iv) the design of synthetic gene networks replicas reaching the complexity of electronic circuits (69), and (v) gene therapy applications (1012).

To date, a multitude of heterologous transgene expression systems for use in mammalian cells and transgenic animals have been described (4). The prevailing design consists of a heterologous small molecule-responsive transactivator engineered by fusing a prokaryotic repressor to a eukaryotic transactivation domain and a transactivator-specific promoter containing the matching prokaryotic operator linked to a minimal eukaryotic promoter. Inducer-triggered modulation of the affinity of the transactivator to its cognate promoter results in adjustable and reversible transcription control of the specific target gene (1316). In recent years, a panoply of such heterologous transcription control modalities have been developed, which are responsive to various inducer molecules such as antibiotics (13, 14, 17), steroid hormones and their analogs (18, 19), quorum-sensing molecules (20, 21), immunosuppressive and antidiabetic drugs (22, 23), biotin (24), l-arginine (25), as well as volatile acetaldehyde (16). Apart from gaseous acetaldehyde, which can simply be inhaled, all other inducers need to be either taken up orally or be administered by injection in any future gene therapy application. Transdermal and topical delivery of inducer molecules, which would provide advantages over conventional injection-based or oral administration such as convenience, improved patient compliance, and elimination of hepatic first-pass effect, have not yet been developed.

Phloretin is mainly found in the root bark of apple trees and in apples where it acts as a natural antibacterial plant defense metabolite (26). Phloretin has been studied as a possible penetration enhancer for skin-based drug delivery (2731), attenuates inflammation by antagonizing prostaglandins (32), protects the skin from UV light-induced photodamage (33, 34), and is currently evaluated as a chemopreventive agent for cancer treatment (35). Because the plant rhizosphere is one of the natural habitats of Pseudomonas putida (strain DOT-T1E), this prokaryote has evolved the RND family transporter TtgABC with multidrug recognition properties, which is controlled by its cognate repressor TtgR binding to a specific operator (OTtgR) in the TtgR promoter (PTtgR). Phloretin has been shown to bind to the TtgR-operator complex at a stoichiometric ratio of 1 effector molecule per TtgR dimer, and to release TtgR from OTtgR, which results in induction of TtgABC production and effective pump-mediated efflux of the flavonoid from P. putida (26, 36).

Capitalizing on the phloretin-responsive TtgR-OTtgR interaction of P. putida DOT-T1E, we have assembled a synthetic mammalian phloretin-adjustable control element (PEACE), which was able to reversibly adjust product gene expression of transgenic cells grown in culture, standard bioreactors, or implanted into mice after addition of pure phloretin or topical administration of a phloretin-containing skin lotion.

Results

Design of a Synthetic Mammalian PEACE.

With a half-life of 70 h in culture and no negative influence on viability, growth, or production of CHO-K1 cells, phloretin is a valid flavonoid candidate for trigger-inducible transcription control in mammalian cells (SI Materials and Methods and Fig. S1). Living in the plant rhizosphere, P. putida DOT-T1E has evolved resistance to various plant-derived antimicrobials (37, 38), which is triggered by phloretin-induced release of TtgR from the operator (OTtgR) of its target promoter and subsequent induction of a broadly specific TtgABC efflux pump (26, 36). By fusing TtgR (36) to the Herpes simplex-derived transactivation domain VP16 (39), we created a synthetic mammalian transactivator (TtgA1), which is able to bind and activate transcription from chimeric promoters (PTtgR1) harboring OTtgR linked to a minimal human cytomegalovirus immediate early promoter (PhCMVmin), in a phloretin-responsive manner (Fig. 1 A and B). Cotransfection of the constitutive TtgA1 expression vector pMG11 (PSV40-TtgA1-pA) and pMG10 [PTtgR1-secreted alkaline phosphatase (SEAP)-pA] encoding a TtgA1-specific PTtgR1-driven SEAP expression unit, resulted in high-level SEAP expression [23.6 ± 3.1 units (U)/L], which compares with an isogenic vector containing a constitutive PSV40-driven SEAP expression cassette (pSEAP2-Control; 21.4 ± 1.0 U/L). Addition of increasing concentrations of phloretin (0–70 μM) to a culture of pMG10- and pMG11-cotransfected CHO-K1 cells resulted in dose-dependent reduction of SEAP expression up to complete repression (Fig. 1C). These data suggest that PEACE-controlled transgene expression is adjustable and enables complete repression within a nontoxic phloretin concentration range. We have also designed PTtgR1 variants with different tandem OTtgR modules and TtgA1 variants harboring various transactivation domains, and provide a detailed combinatorial performance analysis in different cell lines and different expression configurations, including autoregulated ones that are known to be essential for the assembly of complex synthetic gene networks (SI Materials and Methods, Fig. S2, Fig. S3, and Table S1) (6, 9, 40).

Fig. 1.
Design and functionality of PEACE. The P. putida DOT-T1E-derived bacterial repressor TtgR was fused to the VP16 transactivation domain of H. simplex virus, and the resulting transactivator TtgA1 (TtgR-VP16) was cloned under control of the constitutive ...

PEACE Control by Phloretin and Other Flavonoids.

Because TtgR of P. putida was shown to bind several plant-derived flavonoids with high affinity (26), we profiled their PEACE-controlling capacities in mammalian cells. CHO-K1 were transiently (co)transfected with either pMG10 (PTtgR1-SEAP-pA) and pMG11 (PSV40-TtgA1-pA), to score regulation performance, or with pSEAP2-Control (PSV40-SEAP-pA), to assess compound-related cytotoxicity, and then cultivated for 48 h in medium containing different concentrations (0, 25, and 50 μM) of specific flavonoids (berberine, butylparaben, genistein, luteolin, β-naphthol, naringenin, phloretin, phloridzin, or quercetin) before SEAP production was profiled (Fig. 2A). Although genistein, luteolin, β-naphthol, naringenin, and quercetine were cytotoxic within the tested concentration range (genistein only at 50 μM), berberine, butylparaben, phloridzin, and phloretin did not reduce cell viability. However, berberine failed to control PEACE, and butylparaben, as well as phloridzin, were able to regulate, but not fully repress SEAP production (Fig. 2B). Therefore, phloretin, which enabled maximum expression levels, as well as full transgene repression, was chosen as the ideal PEACE inducer for all further experiments.

Fig. 2.
PEACE responsiveness to different flavonoids. (A) Toxicity of flavonoids. CHO-K1 were transiently transfected with pSEAP2-Control, cultivated in medium supplemented with different flavonoids (0, 25, and 50 μM) SEAP levels were scored after 48 ...

Phloretin-Controlled Transgene Expression Is Functional in Different Mammalian Cell Lines and Human Primary Cells.

To assess its versatility, we tested PEACE in several immortalized mammalian cell lines, as well as in human primary cells. Therefore, pMG10 (PTtgR1-SEAP-pA) and pMG11 (PSV40-TtgA1-pA) were cotransfected into BHK-21, COS-7, HaCaT, HEK-293, HT-1080, and NIH/3T3 cell lines, as well as into primary human fibroblasts and keratinocytes, and cultivated for 48 h in the presence (50 μM) and absence of phloretin, followed by scoring of SEAP levels (Table 1). PEACE-controlled transgene expression was functional in all tested cell lines, suggesting that this technology will be broadly applicable.

Table 1.
PEACE-controlled transgene expression in various mammalian cells

Expression Kinetics, Adjustability, and Reversibility of PEACE-Controlled Transgene Expression in a Stable Transgenic CHO-K1 Cell Line.

We have generated 5 double-transgenic cell lines (CHO-PEACE) by sequential transfection and clonal selection of pMG11 and pMG10 into CHO-K1. All of these PEACE-transgenic cell lines showed phloretin-regulated SEAP expression, but differed in their overall regulation performance (maximum and leaky expression levels) as a result of differences in transgene copy number and integration sites that remains beyond control using standard transfection technology (Fig. S4) (41). CHO-PEACE8, which was considered the best cell line and was, therefore, used in all follow-up experiments, showed (i) unchanged maximum SEAP expression levels and unaffected regulation performance in long-term cultures over 60 days (day 0, ON: 70.9 ± 3.1 U/L, OFF: 1.8 ± 0.1 U/L; day 60, ON: 67.6 ± 2.7 U/L, OFF: 2.1 ± 0.2 U/L; OFF at 50 μM phloretin), (ii) excellent adjustability (Fig. 3A), (iii) exponential SEAP expression kinetics (Fig. 3B), (iv) full reversibility of transgene expression (Fig. 3C), and (v) optimal compatibility with other transgene regulation systems (SI Materials and Methods and Tables S2 and S3).

Fig. 3.
Design and characterization of the stable CHO-PEACE8 cell line transgenic for phloretin-responsive SEAP expression. (A) Dose-response profile of CHO-PEACE8. (B) SEAP expression kinetics of CHO-PEACE8 cultivated for 72 h in the presence and absence of ...

Time-Delayed Induction of Product Gene Expression in a Prototype Biopharmaceutical Manufacturing Scenario.

Because phloretin is a nontoxic fruit component, it could be an ideal product gene inducer for biopharmaceutical manufacturing scenarios that require precise timing or dosing of difficult-to-express protein pharmaceuticals (4, 4244). Also, because phloretin has a determined half-live of 70 h in mammalian cell culture systems (SI Results), any production culture can be programmed to start product gene expression at a predefined point in time as phloretin concentrations drop below a repressing threshold level. We have inoculated a 1-L BioWave bioreactor with 2 × 103 CHO-PEACE8 and different transgene-repressing phloretin doses of 60, 80, or 100 μM. Although CHO-PEACE8 grew exponentially from the start of the bioprocess, SEAP production gradually increased once phloretin was degraded to nonrepressive levels (40 μM; Fig. 4). Using PEACE, mammalian production cultures could indeed be programmed for timely induction of product gene expression without any process intervention.

Fig. 4.
Automatically programmed product gene expression in bioreactors using well-defined phloretin degradation profiles. The 2 × 103 cells/mL CHO-PEACE8 were cultivated in a bioreactor containing 1-L culture medium supplemented with either 60, 80, or ...

Phloretin-Mediated Transdermal Gene Expression in s.c. Implants in Mice.

Because phloretin has been suggested as a penetration enhancer for transdermal drug delivery (2731), and was shown to propagate systemically in rodents after local skin-based administration (28), we have evaluated phloretin as a potential transdermal therapeutic transgene expression inducer. Therefore, we have microencapsulated CHO-PEACE8 in coherent alginate-PLL-alginate capsules and implanted them s.c. into mice. The back of treated mice was partially shaved, and petroleum jelly-based skin lotions (200 μL) containing different amounts of phloretin (0–42 mg) were put on every day. The SEAP levels detected in treated mice 72 h after implantation showed phloretin-dependent dose-response characteristics akin to the ones observed with the same microencapsulated implant batch cultivated and exposed to phloretin in vitro (Fig. 5 A and B). Control mice receiving CHO-K1 cells transgenic for constitutive SEAP expression were insensitive to any treatment with phloretin-containing skin lotion (0 mg phloretin: 6.2 ± 0.43 U/L; 42 mg phloretin: 6.02 ± 0.58 U/L).

Fig. 5.
PEACE-controlled transgene expression in mice. (A) Microencapsulated CHO-PEACE8 were implanted s.c. into female OF1 mice (2 × 106 cells per mouse); 200 μL of a cream containing different amounts of phloretin (0, 5.25, 10.5, 21, and 42 ...

Discussion

Flavonoids such as phloretin are polyphenols widely distributed in the plant kingdom, and they are present in fruits and vegetables regularly consumed by humans. The recent findings that phloretin and some of its derivatives have potent antiinflammatory (32), antioxidative (33, 45), and even some anticancer (35, 46) activities form the scientific basis for the common saying “an apple a day keeps the doctor away.” Phloretin has also been successfully evaluated as a penetration enhancer for transdermal drug delivery (2731) and as skin protectant reducing oxidative stress resulting from external insults, such as UV irradiation, which triggers skin cancer and photo aging (33, 34, 47). Recent studies in rodents have confirmed that dermal administration of phloretin will systemically spread in the animal (28), and that phloretin has a rather short half-life in vivo (48). All of the aforementioned characteristics corroborate phloretin to be a nontoxic natural compound with high metabolic turnover that could be ideal for reversible transdermal induction of therapeutic transgenes. Transdermal and topical delivery of drugs and regulating molecules provide advantages over conventional oral or injection-based administrations, such as convenience, improved patient compliance and elimination of hepatic first-pass effect. However, most molecules are not applicable to dermal administration due to the excellent barrier properties of the skin, which requires penetrating molecules to pass the stratum corneum with its compact keratinized cell layers and the viable epidermis before reaching the papillary dermis and crossing the capillary walls into systemic circulation. Phloretin-containing skin lotions put on the skin of mice containing cell implants harboring a synthetic phloretin-responsive expression system were able to precisely fine-tune target gene expression in the animal. This pioneering transdermal transcription control system may enable precise patient-controlled dosing of protein pharmaceuticals that are produced in situ by cell implants contained in clinically licensed devices (4952). There should be no risk of nutrition-based interference of PEACE-controlled therapeutic transgene expression in transgenic cell implants, because a patient (70 kg) would need to eat >2,000 apples to reach PEACE-modulating phloretin levels in his bloodstream (48, 53). Besides this gene therapy-focused in vivo scope of phloretin-responsive transgene expression, the system showed excellent regulation performance, including adjustability and reversibility in vitro. Owing to its short half-life in culture, phloretin-responsive production cultures grown in bioreactors could be preprogrammed for timely production initiation by inoculation with excessive phloretin concentrations. While the production cell cultures grow and phloretin levels decrease after precise kinetics, production will be initiated at a defined point in time, which is a function of the inoculum and the initial phloretin concentration. Such a time-delayed production concept would be particularly valuable for difficult-to-express protein therapeutics like those that impair growth or are cytotoxic (4, 4244). The fact that phloretin is a natural fruit component regularly consumed by humans and licensed for use in skin lotions (34) may facilitate approval of such biopharmaceutical manufacturing protocols by governmental agencies. Also, because the PEACE system has been assembled after a standard binary transactivator/promoter design, it is conceivable that its performance can easily be adapted to specific control requirements using an established refinement program (54, 55).

Considering all facts, the pioneering phloretin-based transgene control technology is unique, and may foster advances in the production of difficult-to-express protein pharmaceuticals, as well as in cell implant-based therapeutic applications.

Materials and Methods

Expression Vector Design.

The pMG10 (PTtgR1-SEAP-pA) harbors a phloretin-responsive SEAP expression unit, and pMG11 (PSV40-TtgA1-pA) encodes constitutive expression of the phloretin-dependent transactivator. Detailed information on expression vector design and plasmids used in this study is provided in Table S4.

Cell Culture and Transfection.

Wild-type Chinese hamster ovary cells (CHO-K1, ATCC CCL-61) and their derivatives were cultivated in standard medium: ChoMaster HTS (Cell Culture Technologies) supplemented with 5% (vol/vol) FCS (cat. no. 3302, lot no. P251110; PAN Biotech) and 1% (vol/vol) penicillin/streptomycin solution (cat. no. P4458; Sigma). Human embryonic kidney cells (HEK-293) (56), African green monkey kidney cells (COS-7, ATCC: CRL-1651), baby hamster kidney cells (BHK-21, ATCC: CCL10), human fibrosarcoma cells (HT-1080, ATCC: CCL-121), the human keratinocyte cell line HaCaT (57), and mouse fibroblasts (NIH/3T3, ATCC CRL-1658) were cultured in DMEM (cat. no. 52100-39; Invitrogen) supplemented with 10% (vol/vol) FCS and 1% (vol/vol) penicillin/streptomycin solution. Primary human foreskin fibroblasts were cultivated in DMEM supplemented with 20% FCS (vol/vol) and 1% (vol/vol) penicillin/streptomycin solution, and primary human foreskin keratinocytes were cultured in chemically defined serum-free keratinocyte medium (cat. no. 10744019; Invitrogen), all kindly provided by Sabine Werner. All cell types were cultivated at 37 °C in a 5% CO2-containing humidified atmosphere. For transient transfection of CHO-K1, 1 μg of total plasmid DNA (for cotransfection an equal amount of each plasmid) was transfected into 50,000 cells per well of a 24-well plate according to an optimized calcium phosphate protocol, which resulted in typcal transfection efficiencies of 35 ± 5% (58). Plasmid DNA was diluted to a total volume of 25 μL of 0.5M CaCl2 solution, which was mixed with 25 μL 2× HBS solution (50 mM Hepes/280 mM NaCl/1.5 mM Na2HPO4, pH 7.1). After incubation for 15 min at room temperature, the precipitates were immediately added to the well and centrifuged onto the cells (5 min at 1,200 × g) to increase transfection efficiency. After 3 h, the cells were treated with 0.5 mL glycerol solution (ChoMaster HTS medium containing 15% glycerol) for 60 s. After washing once with PBS (cat. no. 21600-0069; Invitrogen), cells were cultivated in 0.5 mL of standard ChoMaster HTS medium in the presence or absence of different concentrations of phloretin. For transfection of BHK-21, COS-7, and HEK-293, plasmid DNA-Ca3(PO4)2 precipitate was prepared and applied to the cells as described above. HEK-293 and COS-7 cells were washed once with PBS after 3-h incubation with the DNA-Ca3(PO4)2 precipitate and subsequently cultivated in standard DMEM, whereas BHK-21 cells were incubated overnight with the precipitates and then cultivated in DMEM after being washed once with PBS. HaCaT, HT-1080, NIH/3T3, as well as primary human fibroblasts and keratinocytes, were transfected with Fugene 6 (cat. no. 11814443001; Roche Diagnostics AG) according to the manufacturer's protocol, and cultivated in the cell culture medium specified above. After transfection, all cells were cultivated in DMEM supplemented with various concentrations of phloretin, and reporter protein levels were profiled 48 h after transfection, unless otherwise indicated.

Construction of Stable Cell Lines.

The stable CHO-PEACE8 cell line, transgenic for phloretin-controlled SEAP expression, was constructed in 2 steps: (i) CHO-K1 cells were cotransfected with pMG11 (PSV40-TtgA1-pA) and pSV2neo (cat. no. 6172-1; Clontech) at a ratio of 20:1, and clonal selection resulted in the cell line CHO-TtgA; and (ii) CHO-TtgA was cotransfected with pMG10 (PTtgR1-SEAP-pA) and pPur (cat. no. 6156-1; Clontech) at ratio of 20:1, and the phloretin-responsive SEAP-producing double-transgenic cell line CHO-PEACE8 was chosen after clonal selection. Phloretin-dependent dose-response characteristics of CHO-PEACE8 were analyzed by culturing 100,000 cells per mL for 48 h in standard ChoMaster HTS medium at various phloretin concentrations ranging from 0 to 70 μM. Reversibility of phloretin-mediated SEAP production was assessed by cultivating CHO-PEACE8 (100,000 cells pr mL) for 144 h while alternating phloretin concentrations from 0 to 50 μM every 48 h.

Quantification of Reporter Protein Production.

Production of the human placental SEAP was quantified using a p-nitrophenylphosphate-based light absorbance time course (59). 1 SEAP unit corresponds to the conversion of 1 μmol pNPP per minute.

In Vivo Methods.

CHO-PEACE8 and CHO-SEAP18 (60) were encapsulated in alginate-poly(l-lysine)-alginate beads (400 μm; 200 cells per capsule) using an Inotech Encapsulator Research IE-50R (Recipharm) according to the manufacturer's instructions and the following parameters: 0.2-mm nozzle, 20-mL syringe at a flow rate of 405 units, nozzle vibration frequency of 1,024 Hz, and 900 V for bead dispersion. The backs of female OF1 mice (oncins France souche 1; Charles River Laboratories) were shaved, and 300 μL of ChoMaster HTS containing 2 × 106 encapsulated CHO-PEACE8 were injected s.c. Control mice were injected with microencapsulated CHO-K1. Shaving ensured direct contact of the phloretin-containing cream with the skin of the animal; 1 h after implantation, 200 μL of the phloretin-containing cream was applied to the skin around the injection site. The phloretin amounts in creams ranged from 0 to 42 mg. The cream was applied once a day for up to 3 days. Thereafter, the mice were killed, blood samples were collected, and SEAP levels were quantified in the serum, which was isolated by using microtainer SST tubes according to the manufacturer's instructions (Beckton Dickinson). All of the experiments involving mice were performed according to the directives of the European Community Council (86/609/EEC), approved by the French Republic (no. 69266310), and performed by Marie Daoud El-Baba at the Université de Lyon.

Bioreactor Operation.

CHO-PEACE8 (inoculum of 2 × 103 cells per mL) were cultivated in a BioWave 20SPS-F bioreactor (Wave Biotech) equipped with a 2-L Wave Bag optimized for optical pH and dissolved oxygen concentration control of the 1-L culture. The bioreactor was operated at a rocking rate of 15 min−1, a rocking angle of 6°, and an aeration rate of 100 mL/min with inlet gas humidification (HumiCare 200; Gruendler Medical) to prevent evaporation of the medium. The medium (ChoMaster HTS, 5% FCS, 1% penicillin/streptomycin) was supplemented with 60, 80, or 100 μM phloretin.

Inducer Compounds and Formulation of the Skin Lotion.

Berberine (cat. no. 20425-0100; Acros) and luteolin (cat. no. L14186; Alfa Aesar) were prepared as 10 mM stock solutions in 1:5 (vol/vol) DMSO/H2O. Butylparaben (cat. no. AV14043; ABCR), genistein (cat. no. A2202.0050; Axonlab), β-naphthol (cat. no. 185507; Sigma), naringenin (cat. no. N5893; Sigma), phloretin (cat. no. P7912; Sigma), phloridzin (cat. no. P3449; Sigma), and quercetin (cat. no. Q0125; Sigma) were prepared as 50 mM stock solution in DMSO, and were used at a final concentration of 50 μM unless indicated otherwise. The petroleum jelly-based phloretin-containing creams were professionally formulated (Pharmacy Hoengg) and contained 25, 12.5, 6.25, and 3.125% (wt/wt) phloretin; 200 μL of skin lotion was topically applied per mouse and treatment, which corresponds to a respective total phloretin amount per dose of 42, 21, 10.5, and 5.25 mg.

To quantify phloretin in cell culture medium, the samples were added to 5 × 104 CHO-PEACE8 and incubated for 48 h before SEAP quantification. Phloretin levels were calculated by comparing SEAP production with a calibration curve (Fig. 3A), established using the same parameters, and defined phloretin concentrations. Similarly, the half-life of phloretin was estimated based on the degradation dynamics observed in cell culture (61, 62).

Supplementary Material

Supporting Information:

Acknowledgments.

We thank Martine Gilet for skilled technical assistance; Sabine Werner (Eidgenössische Technische Hochschule Zürich, Zurich, Switzerland) for providing primary human fibroblasts and keratinocytes; Beat Kramer and Cornelia Fux (Eidgenössische Technische Hochschule Zürich, Zurich, Switzerland) for providing pBP99 and pCF59, respectively; and Marcia Schoenberg, Marcel Tigges, and William Bacchus for critical comments on the manuscript. This work was supported by the Swiss National Science Foundation Grant 3100A0-112549, and in part by the European Commission Framework Program 7 PERSIST.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0901501106/DCSupplemental.

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