The LIKE system, a novel protein expression toolbox for Bacillus subtilis based on the liaI promoter
© Toymentseva et al.; licensee BioMed Central Ltd. 2012
Received: 31 May 2012
Accepted: 15 October 2012
Published: 30 October 2012
Bacillus subtilis is a very important Gram-positive model organism of high biotechnological relevance, which is widely used as a host for the production of both secreted and cytoplasmic proteins. We developed a novel and efficient expression system, based on the liaI promoter (P liaI ) from B. subtilis, which is under control of the LiaRS antibiotic-inducible two-component system. In the absence of a stimulus, this promoter is kept tightly inactive. Upon induction by cell wall antibiotics, it shows an over 100-fold increase in activity within 10 min.
Based on these traits of P liaI , we developed a novel LiaRS-controlled gene expression system for B. subtilis (the “LIKE" system). Two expression vectors, the integrative pLIKE-int and the replicative pLIKE-rep, were constructed. To enhance the performance of the P liaI -derived system, site-directed mutagenesis was employed to optimize the ribosome binding site and alter its spacing to the initiation codon used for the translational fusion. The impact of these genetic modifications on protein production yield was measured using GFP as a model protein. Moreover, a number of tailored B. subtilis expression strains containing different markerless chromosomal deletions of the liaIH region were constructed to circumvent undesired protein production, enhance the positive autoregulation of the LiaRS system and thereby increase target gene expression strength from the P liaI promoter.
The LIKE protein expression system is a novel protein expression system, which offers a number of advantages over existing systems. Its major advantages are (i) a tightly switched-off promoter during exponential growth in the absence of a stimulus, (ii) a concentration-dependent activation of P liaI in the presence of suitable inducers, (iii) a very fast but transient response with a very high dynamic range of over 100-fold (up to 1,000-fold) induction, (iv) a choice from a range of well-defined, commercially available, and affordable inducers and (v) the convenient conversion of LIKE-derived inducible expression strains into strong constitutive protein production factories.
Bacillus subtilis is a widely exploited bacterium for basic research, but also industrial and biotechnological applications  owing to the ease of genetic manipulation, a systems level understanding of its genome and physiology [2–4], its efficient protein secretion systems , non-pathogenic GRAS-status  and well-characterized mechanisms for gene expression . Over the years, numerous genetic devices and expression systems have been developed for this organism to facilitate the production of homologous or heterologous proteins [7–14], usually based on strong inducible promoters. Such systems can either be integrated into the chromosome or located on replicative plasmids to increase the gene copy number under the control of the inducible promoter.
A number of new expression systems based on induction by peptide antibiotics were described for Gram-positive bacteria [9, 15, 16]. The nisin-controlled gene expression (NICE) system was developed for different species of Lactococcus and Lactobacillus and allows the production of the desired proteins in high amounts (comparable to other expression systems), reaching a maximum 3 h after nisin induction [15, 16]. A very similar subtilin-regulated expression system (SURE) was recently constructed for B. subtilis. Both systems enable the controlled overexpression of a variety of homologous and heterologous proteins and enzymes and show a number of advantages to other control elements, such as the strict control of gene expression, no leakage of the promoter regulation under non-inducing conditions, high levels of expression upon induction and almost no limitations in the choice of sugar-containing media [9, 15]. For the use in B. subtilis, the SURE system has several advantages over the NICE system: (i) The SURE system only requires a single plasmid, thereby ensuring a stable expression platform; (ii) the expression levels achieved by the SURE system are significantly higher; and (iii) it also requires lower concentrations of the inducer molecule [9, 17].
Despite significant progress in the field, no exisiting system works equally well for all proteins and none of the existing expression systems for B. subtilis is without pitfalls or limitations. While the SURE system represents a major improvement, its inducer, the lantibiotic subtilin, is not commercially available. Instead, culture supernatant of the lantibiotic producer must be used, which introduces a source of variation and requires testing the potency each time a new supernatant is used for induction. Therefore, novel tightly controllable gene expression systems are still in demand to expand and complement the existing repertoire in order to find the optimal solution for a given protein to be produced in B. subtilis.
Here, such an addition to the existing bioengineering toolbox for B. subtilis will be described. The LIKE (from the German “LIa-Kontrollierte Expression”) system is based on the cell envelope stress-responsive liaI promoter. This promoter was initially identified in the course of studies on the response of B. subtilis to the presence of harmful concentrations of various cell wall antibiotics . The underlying regulatory network of the cell envelope stress response in this organism is rather complex and consists of at least four extracytoplasmic function (ECF) σ factors and a similar number of two-component systems (TCS) and has been extensively studied [19, 20]. One such TCS, LiaRS, is a central player in the envelope stress response network of B. subtilis. It strongly responds to antibiotics that interfere with the lipid II cycle, such as bacitracin. Activation of the LiaRS system of B. subtilis specifically leads to the strong induction of a single target promoter, P liaI , which drives the expression of the liaIH operon. This promoter is basically shut off in the absence of inducing condition during logarithmic growth and shows an impressive dynamic range of over 100- up to 1,000-fold in the presence of suitable stimuli [21–23].
Because of its specificity and sensitivity, P liaI has already been developed as a powerful screening tool for mechanism-of-action studies of novel peptide antibiotics interfering with envelope integrity [22, 24, 25]. But its tightly regulated, concentration-dependent and highly dynamic behavior also makes this promoter a very promising candidate for the development of a novel gene expression system. This prospect is further supported by transcriptome studies of mutants that are constitutively switched Lia-ON or Lia-OFF, which revealed a very specific response with only very few genes being indirectly affected . Moreover, B. subtilis is highly resistant to bacitracin, a commercially available compound, which can be used as an ideal inducer to activate P liaI -driven gene expression in growing cultures of B. subtilis. Moreover, a simple gene deletion can convert the inducible into a high-level constitutive promoter activity. Based on these traits of P liaI , we developed vectors and strains to apply this promoter as a powerful protein expression system in B. subtilis.
Results and discussion
Features of the native liaI promoter (P liaI )
Design and construction of P liaI -based expression vectors and B. subtilis protein production strains for the LIKE system
For the construction of new P liaI -derived bacitracin-inducible gene expression systems, we chose two vectors as backbones: the E. coli/B. subtilis shuttle vector pGP380, and pDG1662 for ectopic integration at the amyE locus of B. subtilis[28, 29], thus enabling both expression from a multi-copy replicative vector, as well as the stable chromosomal integration at single copy. The optimized regulatory element PliaI(opt) was amplified by PCR and cloned into the two vectors (see Materials and Methods for details) resulting in the expression vectors pLIKE-rep and pLIKE-int, respectively (Figure 2A/B).
Previous work has demonstrated that the liaIH operon is the only relevant target of LiaFSR-dependent gene expression, and that activation of P liaI results in a strong accumulation of LiaH in the cytosol (Figure 1B) . Based on the organization and expression of genes in the liaIH-liaGFSR locus, activation of P liaI also leads to an increased expression of liaGFSR, due to read-through transcription . Such positive autoregulatory feedback loops often have beneficial effects on the activity of their target genes . Hence, it might be desirable to maintain this feedback loop. On the other hand, the observed very strong production of the native LiaFSR-target proteins LiaIH is not desired in a protein production host, since it depletes the cells of energy, amino acids and ribosomes required for heterologous protein production.
To account for these two opposing goals, we constructed a number of clean deletion mutants as potential hosts of the LIKE-system. The features of the resulting strains are summarized in Figure 2C. Strain TMB604 lacks both the liaIH operon including the native liaI promoter. Hence, no autoregulation can occur under inducing conditions. In contrast, strains TMB1151/TMB1152, which are also deleted for the liaIH operon, still maintain P liaI and therefore autoregulation. They differ in the presence or absence of the weak terminator located downstream of liaH (Figure 2C).
As a measure for PliaI(opt)-dependent protein production in the two expression plasmids, gfpmut1 gene was used as a reporter gene . Translational fusions of PliaI(opt) with gfpmut1 were constructed in both pLIKE-int and pLIKE-rep and subsequently introduced into the aforementioned B. subtilis strains.
Evaluation of the LIKE-system, based on the bacitracin-induced GFP production
The range of inducers for the envelope-stress responsive LiaFSR three-component system is well-defined and includes, amongst others, the cell wall antibiotic bacitracin [22, 25]. As an inducer for protein production in B. subtilis, this compound has a number of advantages: (i) It is one of the strongest inducers for the Lia-system and is easily commercially available. (ii) B. subtilis is highly resistant against bacitracin, and even above inhibitory antibiotic concentrations, cellular damage occurs only very slowly [18, 32]. (iii) The maximum P liaI activity occurs well below the inhibitory concentration, thereby avoiding any damage to the producing cultures. (iv) In addition to its major inhibitory activity on cell wall biosynthesis, bacitracin also acts as a weak protease inhibitor , which can be viewed as a beneficial side effect of using this inducer. For all of these reasons, bacitracin will be used as the model inducer for the subsequently described evaluation of the LIKE expression system.
Nevertheless, it should be pointed out that a number of other compounds and conditions can also be considered as suitable alternative inducers, including antibiotics such as vancomycin or nisin, as well as non-antibiotic conditions such as alkaline shock , making the LIKE-system highly variable even in cases where bacitracin is not suitable for a given application (for example for heterologous protease production).
Effect of mutations in the liaIH operon on the expression of translational P liaI (opt) - gfp fusions
Promoter activity (fluorescence)b
amyE:: pSJ5101 (P liaI -gfp)
(WT168) P liaI liaIHTerm+
(WT168) P liaI liaIHTerm+
(TMB604) ∆P liaI liaIH
(WT168) P liaI liaIHTerm+
(TMB604) ∆P liaI liaIH
Determination of the P liaI activity revealed that the window of promoter activity was narrower in case of the integrated promoter, both for activation and shut-off, relative to the replicative derivatives (Figure 3B). For the pLIKE-int derivatives, maximum promoter activity was reached already 20–30 min after addition of bacitracin and the total window of activity was less than 60 min. In contrast, pLIKE-rep derivatives required almost 60 min to reach maximum promoter activity and the total window of activity was about 120 min. But in light of the overall 10-times higher promoter activity in case of the latter, this result is maybe not too surprising.
All major conclusions drawn above were verified at the protein level by Western analysis, using antibodies against GFP and LiaH. Both proteins were not detectable in uninduced cultures, supporting the previously demonstrated tight control of P liaI and the absence of any significant promoter activity under non-inducing conditions. Upon addition of bacitracin, both proteins accumulated to different level, depending on strain background. These studies demonstrate both the positive effect of improving the ribosome binding site and the negative effect of deleting the autoregulatory feedback loop at the level of protein production (Figure 3C).
Taken together, both pLIKE-int and pLIKE-rep were successfully established as vectors for bacitracin-dependent protein production in strains that maintain the positive autoregulatory feedback loop. While expression based on the replicative vector yields higher protein amounts, the integrative system has the advantage of genetic stability and does not require any selection.
Effect of the inducer (bacitracin) concentration on the activity of PliaI(opt)
Overproduction of YdfG using the LIKE system
To demonstrate the suitability of the LIKE system for the overexpression of a heterologous protein, we performed an expression experiment using the protein YdfG of Bacillus licheniformis. This protein is a putative carboxymuconolactone decarboxylase. We could recently demonstrate that its gene represents the only target of the extracytoplasmic function σ factor ECF41Bli. It consists of 148 amino acids and an estimated molecular weight of 16,6 kDa.
We have developed a novel and efficient LiaFSR-dependent gene expression system, which places target proteins under the control of an optimized bacitracin-responsive P liaI promoter. The LIKE system offers first a single-copy, integrative option (pLIKE-int in strain Bsu-LIKE2), which is genetically stable without selective pressure, but reaches lower protein production levels. The second option consists of the replicative vector pLIKE-rep in combination with strain Bsu-LIKE1 to obtain a maximum gene expression. The LIKE-system has a number of important features: (i) There is no detectable background expression in the non-induced state. (ii) Using bacitracin as inducer, the promoter has an impressive dynamic range of up to 1,000-fold above background level that can be titrated as a function of inducer concentration. (iii) The described range of inducers is wide, including cell wall antibiotics that function as strong (bacitracin, nisin, daptomycin) or intermediately strong (vancomycin) inducers, as well as non-antibiotic conditions that act as intermediate to weak inducers of the Lia-system, including pH-upshift, organic solvents, some detergents, or ethanol [22, 25, 36–38]. All of these inducers are well-defined and readily available at low prices. Moreover, a recent study demonstrated that the Lia-system can also be induced by the overexpression of certain heterologous and secreted proteins, especially the universal shock protein USP45 from Lactococcus lactis and the TEM-1 β-lactamase from E. coli. (iv) Lastly, an antibiotic-inducible LIKE-expression strain can easily be converted into a strong constitutive expression platform by the simple deletion of liaF, encoding the LiaRS-specific inhibitor protein [21–23]. The effect of such a deletion is shown in Figure 1, which demonstrates that a liaF deletion results in a protein production that even surpasses that of the fully induced strains, even in the absence of an inducer.
This flexibility distinguishes the LIKE system from other available expression systems. Taken together, the expression vectors and strains described in this report expand the genetic toolbox already available for protein production, based on the tight and highly dynamic bacitracin-inducible promoter P liaI . We hope and believe that the vectors and strains described in this report will provide valuable tools for protein expression in B. subtilis. The LIKE system, consisting of both expression vectors as well as the host strains Bsu-LIKE1 and Bsu-LIKE2, is available for the scientific community through the Bacillus Genetic Stock Center (http://www.bgsc.org; accession numbers ECE255, ECE256 for the two vectors and 1A1070, 1A1071 for the two B. subtilis expression strains).
Bacterial strains used in this study
Source and/or reference
E. coli DH5α
recA 1 endA 1 gyrA 96 thi hsdR 17(rK- mK+) relA 1 supE 44 ϕ80ΔlacZ ΔM15 Δ(lacZYA-argF)U169
Wild type, trpC 2
W168 att SPβ2∆2 trpC 2, liaR::kan
W168 amy E::(cat P liaI -lacZ)
HB0933 amyE::(cat P liaI -lacZ)
W168 ∆liaF (clean deletion)
TMB329 amyE::(cat P liaI -lacZ)
W168 amyE::pSJ5101 (P liaI -gfp)
W168 ΔP liaI -liaIH (clean deletion)
W168 ∆liaIH (clean deletion)
W168 ∆liaIH-terminator (clean deletion)
W168 amyE::pAT6203 (pLIKE-int PliaI(opt)-gfp)
W168 pAT3803 (pLIKE-rep PliaI(opt)-gfp)
TMB604 amyE::pAT6203 (pLIKE-int PliaI(opt)-gfp)
TMB604 pAT3803 (pLIKE-rep PliaI(opt)-gfp)
TMB1151 amyE::pAT6203 (pLIKE-int PliaI(opt)-gfp)
TMB1151 pAT3803 (pLIKE-rep PliaI(opt)-gfp)
TMB1152 amyE::pAT6203 (pLIKE-int PliaI(opt)-gfp)
TMB1152 pAT3803 (pLIKE-rep PliaI(opt)-gfp)
TMB1151 pKSLIKEr01 (pLIKE-rep PliaI(opt)-His6-ydfG)
TMB1152 pKSLIKEi01 (pLIKE-int PliaI(opt)-His6-ydfG)
DNA manipulations, transformation and PCR
Oligonucleotides used in this study
Sequence (5' to 3')a
BsaI; 5' end of P liaI
CTTGTTGGATCCATCGAT GATCCTCCT TACGTTTTCCTTGTCTTC
Strong SD region; BamHI, ClaI; 3' end of P liaI
EcoRI; 5' end of P liaI
ATTTTCTCTAGA ATCCTCCT TACGTTTTCCTTGTCTTC
Strong SD region; XbaI; 3' end of P liaI
TCCTATCG ATG AGTAAAGGAGAAGAACTTTTCACTGG
ATG start codon; ClaI; 5' end of gfpmut1
HindIII; 3' end of gfpmut1
TTCCTCTAG ATG AGTAAAGGAGAAGAACTTTTC
ATG start codon; XbaI; 5' end of gfpmut1
SalI; 3' end of gfpmut1
CCATATCG ATG CAT CATCATCATCATCAC GAAACGAGATTTCTAATGGAAAAAG
ATG start codon; ClaI; His6-tag; 5’ end of ydfG
HindIII; 3’ end of ydfG
CCATTCTAG ATG CAT CATCATCATCATCAC GAAACGAGATTTCTAATGGAAAAAG
ATG start codon; XbaI; His6-tag; 5’ end of ydfG
BamHI; upstream of P liaI
3' end of P liaI ; 3' end of liaH
3' end of liaH
EcoRI; inside the liaG gene
upstream of liaI gene; upstream of liaG
upstream of liaG
EcoRI; center of liaG gene
Construction of markerless deletion mutant strains
Vectors and plasmids used in this study
Primer pair(s) used for cloning
cat, spc, bla, amyE' … 'amyE integrative vector
erm, bla, Strep-Tag, PdegQ36, replicative vector
erm, ori(pE194-Ts), MCS-P clpB -bgaB, ori(pBR322), bla
bla, cat, gfpmut1
pDG1662 derivative; spc gene deleted
pAT6200 derivative; PliaI(opt); integrative protein expression vector
pGP380 derivative; PliaI(opt); replicative protein expression vector
pLIKE-int, PliaI(opt) translationally fused to gfp
pLIKE-rep, PliaI(opt) translationally fused to gfp
pMAD ΔliaIH up/down overlap
TM2130/ TM2131, TM1055/ TM1058
pMAD ΔliaIHTerminator up/down overlap
TM2130/ TM2132, TM2133/ TM2134
pLIKE-rep, PliaI(opt) translationally fused to His6-ydfG
pLIKE-int, PliaI(opt) translationally fused to His6-ydfG
Plasmid and strain construction
Bacterial strains used in this study are derivates of the laboratory wild type strain B. subtilis W168 and are listed in Table 2. Plasmids used in this study are listed in Table 4. The promoter of the liaIH operon for integrative and replicative vectors was obtained from strain B. subtilis W168 by PCR, using primers TM2064/TM1980 and TM1991/TM1992 (Table 3), respectively. During the amplification, bases in the ribosome-binding site (RBS) were mutated to a strong B. subtilis Shine-Dalgarno (SD) sequence (TAAGGAGG)  to yield the optimized liaI promoter PliaI(opt).
The integrative expression vector pLIKE-int, containing P liaI with an optimized SD site (PliaI(opt)) was generated in two steps. First, the B. subtilis integrative vector pDG1662 was treated with BstBI to remove the spectinomycin resistance gene. The truncated (6141 bp) fragment was self-ligated, yielding vector pAT6200. During this step, the multiple cloning site (MCS), containing unique BamHI, HindIII, and EcoRI sites was expanded by an additional unique ClaI restriction site, which is required for introducing genes at the ATG start codon (see Figure 2 for details): a PCR product encompassing PliaI(opt) was digested with BsaI and BamHI and cloned into pAT6200 digested with BamHI, resulting in pLIKE-int. The ClaI restriction site is recommended to use for reconstruction of the ATG start codon, but it is not strictly necessary. The use of BamHI, HindIII, or EcoRI has the disadvantage of fusing additional amino acids to the N-terminus of the target protein which can cause undesired disabilities.
To construct the replicative expression vector pLIKE-rep, again harboring PliaI(opt), the promoter fragment was amplified by PCR using primers TM1991/TM1992 (Table 3). After digest of the PCR product with EcoRI and XbaI, the promoter region was ligated into the corresponding sites of pGP380, resulting in vector pLIKE-rep. For cloning of a gene into pLIKE-rep, XbaI must be used as restriction enzyme to generate the ATG start codon (see Figure 2).
For the determination of the properties of the two expression vectors, the genes gfpmut1 and ydfG were used. The gfpmut1 gene was amplified using primers TM1981/TM1982 and TM1993/TM1994, respectively (Table 3), using plasmid pSG1151 as the template. The 720-bp amplicon obtained was cloned into ClaI/HindIII-digested pLIKE-int or XbaI/SalI-digested pLIKE-rep, resulting in translational fusions with PliaI(opt) in pAT6203 and pAT3803, respectively (Table 4). Next, the B. subtilis strains W168, TMB604, TMB1151, and TMB1152 (Table 2) were transformed with the pAT6203 integrative plasmid. The resulting strains were designated TMB1172, TMB1174, TMB1153, TMB1318, respectively (Table 2). Strains bearing the replicative pAT3803 GFP-expression plasmid were constructed by transformation of the above strains with plasmid DNA and selection for MLS resistance, resulting in strains TMB1176, TMB1178, TMB1342, and TMB1343, respectively. The ydfG gene was amplified from Bacillus licheniformis genomic DNA using primers TM2545/TM2536 and TM2535/TM2536, respectively (Table 3). The PCR product was cloned into ClaI/HindIII digested pLIKE-int or XbaI/HindIII digested pLIKE-rep, resulting in plasmids pKSLIKEi01 and pKSLIKEr01, respectively (Table 4). Next, the B. subtilis strain TMB1151 was transformed with pKSLIKEr01 replicative plasmid and TMB1152 was transformed with the linearized pKSLIKEi01 integrative plasmid, resulting in strains TMB1566 and TMB1570 (Table 2).
Activation of P liaI by bacitracin and analysis of gfp gene expression
For bacitracin-mediated induction of gene expression, the appropriate B. subtilis strains were inoculated from overnight LB cultures into a final volume of 150 μL LB medium in a 96-well plate with optical bottom (Sarstedt) and were incubated in a Synergy™ 2 multimode microplate reader (Biotek) at 37°C with constant medium shaking. When the culture reached an OD600 of 0.45, bacitracin (30 μg mL-1 final concentration) was added to one half of the wells (induced sample), and the other half was left untreated (uninduced control). Plates were covered with lids to prevent evaporation and incubated for 4 h. Growth was monitored by measuring absorbance at 600 nm. Fluorescence readings were taken from the bottom by using a GFP-specific filter pair (excitation 485/20 nm, emission 528/20 nm). Measurements were taken in 10 min intervals. To calculate expression levels, the natural fluorescence of three cultures of wild type B. subtilis strain 168 (containing no reporter gene) were averaged and subtracted from the raw fluorescence value of each reporter strain at the same OD600 value . Determination of P liaI activity was calculated as described in  as the derivative of the fluorescence divided by the OD600 (dGFP/dt/OD600) for each time point. Expression values were averaged from three independent samples of the same time points ((P1+P2+P3)/3). Polynomial and exponential functions were used to fit the experimental GFP dataset; promoter activities (dGFP/dt/OD600) were calculated using these functions .
Total cytoplasmic proteins were prepared from 15 mL culture per time point by sonication. Proteins (20 μg per lane) were separated by SDS-PAGE, according to standard procedure . After electrophoresis and equilibration of the gels in transfer buffer [15.2 g Tris; 72.1 g glycine; 750 mL methanol (100%) in a final volume of 5 L with deionized water] the proteins were blotted to a PVDF membrane using a mini-trans blot apparatus (Bio-Rad) according to standard procedure . The LiaH antibody (polyclonal rabbit antisera raised against purified His10–LiaH ), GFP antibody (rabbit monoclonal antibody against the green fluorescent protein, Epitomics), and the secondary antibody (anti-rabbit IgG HRP conjugate, Promega) were diluted 1:20,000, 1:3,000, and 1:100,000, respectively. For LiaH/GFP detection, AceGlowTM (PeqLab) was used according to the manufacturer’s instructions. Blots were documented on a QUANTUM-ST4-3026 chemiluminescence documentation system (PeqLab).
Overproduction of YdfG
For the overexpression of ydfG, strains TMB1566 and TMB1570 were grown in LB medium at 37°C until they reached an OD600 of ~0.4-0.5. Cultures were split and one half was induced with 30 μg ml-1 bacitracin for 30 min. The other half was left untreated. 20 ml of each culture was harvested by centrifugation and cell pellets were kept at −80°C until further use. For total protein preparation, the cell pellets were resuspended in 1 ml of cold disruption buffer (50 mM Tris–HCl, 100 mM NaCl, pH 7.5) and cells were disrupted by sonication on ice. Proteins (10 μg per lane) were separated by 14% tricine SDS-PAGE, according to standard procedure  and gels were subsequently stained by colloidale Coomassie staining solution .
The authors are indebted to S. Hübner for invaluable assistance in establishing the BioTek microplate reader set-up in our laboratory, as well as with the corresponding data analysis. Moreover, we would like to thank D. Wolf, and S. Jordan for Bacillus strains used in this study and F. Kalamorz for the SDS-PAGE picture shown in Figure 1B. This work was supported by grant MA2837/1-3 from the Deutsche Forschungsgemeinschaft (to TM) and a Federal grant "Science and teaching program of innovative Russia" for 2009–2013 (to MRS). AAT was supported by a DAAD research fellowship “Forschungsstipendien für Doktoranden und Nachwuchswissenschaftler” (325-A/09/84065).
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