Hypochlorous acid and hydrogen peroxide-induced negative regulation of Salmonella enterica serovar Typhimurium ompW by the response regulator ArcA
- Eduardo H Morales†1,
- Iván L Calderón†1,
- Bernardo Collao1,
- Fernando Gil1,
- Steffen Porwollik2,
- Michael McClelland2, 3 and
- Claudia P Saavedra1Email author
© Morales et al.; licensee BioMed Central Ltd. 2012
Received: 2 February 2012
Accepted: 30 April 2012
Published: 22 May 2012
Hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) are reactive oxygen species that are part of the oxidative burst encountered by Salmonella enterica serovar Typhimurium (S. Typhimurium) upon internalization by phagocytic cells. In order to survive, bacteria must sense these signals and modulate gene expression. Growing evidence indicates that the ArcAB two component system plays a role in the resistance to reactive oxygen species. We investigated the influx of H2O2 and HOCl through OmpW and the role of ArcAB in modulating its expression after exposure to both toxic compounds in S. Typhimurium.
H2O2 and HOCl influx was determined both in vitro and in vivo. A S. Typhimurium ompW mutant strain (∆ompW) exposed to sub-lethal levels of H2O2 and HOCl showed a decreased influx of both compounds as compared to a wild type strain. Further evidence of H2O2 and HOCl diffusion through OmpW was obtained by using reconstituted proteoliposomes. We hypothesized that ompW expression should be negatively regulated upon exposure to H2O2 and HOCl to better exclude these compounds from the cell. As expected, qRT-PCR showed a negative regulation in a wild type strain treated with sub-lethal concentrations of these compounds. A bioinformatic analysis in search for potential negative regulators predicted the presence of three ArcA binding sites at the ompW promoter region. By electrophoretic mobility shift assay (EMSA) and using transcriptional fusions we demonstrated an interaction between ArcA and one site at the ompW promoter region. Moreover, qRT-PCR showed that the negative regulation observed in the wild type strain was lost in an arcA and in arcB mutant strains.
OmpW allows the influx of H2O2 and HOCl and is negatively regulated by ArcA by direct interaction with the ompW promoter region upon exposure to both toxic compounds.
Hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) are reactive oxygen species that are part of the oxidative burst encountered by S. Typhimurium upon internalization by phagocytic cells. Under acidic conditions, such as those found inside the phagosome, H2O2 is generated spontaneously by the reaction of two superoxide anion (O2−) molecules . Moreover, S. Typhimurium encodes both periplasmic and cytoplasmic superoxide dismutases that catalyze O2− dismutation to generate H2O2 and molecular oxygen [2–4]. HOCl is produced by the action of myeloperoxidase (MPO) in a reaction that depends on H2O2, Cl−and acidic conditions [5, 6]. Taken together, H2O2 and HOCl react with thiol and heme groups, copper and iron salts generating the reactive hydroxyl radical (OH.). As a consequence, they produce lipid peroxidation, chlorination of tyrosine residues, oxidation of iron centers, protein cross linking and DNA damage [5–8].
In order to enter Gram negative bacteria, H2O2 and HOCl must be able to cross the outer membrane (OM) and even though several biological membranes are permeable to H2O2, studies in E. coli and Saccharomyces cerevisiae showed that this compound cannot diffuse freely [9, 10]. For HOCl, diffusion through the OM is reported to be limited . One possibility for H2O2 and HOCl influx through the OM is diffusion through porins. In this context, we recently reported that OmpD, S. Typhimurium most abundant OM porin, allows H2O2 diffusion . OM porins are organized as homo-trimers (classic porins) or monomers (small porins) forming aqueous channels that allow the influx of hydrophilic solutes with a molecular weight ≤ 600 Da . Classic porins, including OmpC and OmpF, form β-barrels with 12–22 transmembrane segments while small porins (OmpW) are composed of 8–10 [14, 15]. The crystal structure of OmpW from E. coli revealed that it forms an 8-stranded β-barrel and functions as an ion channel in lipid bilayers [16, 17]. In Vibrio cholerae, OmpW was described as an immunogenic 22 KDa protein  and its expression is altered by factors such as temperature, salinity, nutrient availability and oxygen levels . Additionally, several studies show that porins are regulated by ROS. Due its oxidant nature and diffusion through the OM, regulation of porin expression must be tightly regulated as a mechanism of controlling OM permeability. Accordingly, S. Typhimurium ompD and ompW expression is regulated in response to H2O2 and paraquat [12, 20], respectively, and S. Enteritidis and Typhimurium exposure to HOCl results in lower levels of ompD ompC and ompF transcripts .
The cellular response to oxidative stress is regulated at the transcriptional level by activating the SoxRS and OxyR regulons in response to O2− and H2O2, respectively [22, 23], however, several studies have provided evidence for a role of the ArcAB two component system in the resistance to ROS induced damage [12, 24–26]. ArcA is essential for S. Enteritidis, Typhimurium and E. coli resistance to ROS [24, 26, 27]. ArcB is a sensor member of the histidine kinase family that is anchored to the inner membrane . In response to oxygen availability, ArcB autophosphorylates in an ATP dependant intramolecular reaction at position His-292 [29, 30] and transfers the phosphate group to the cytoplasmic response regulator ArcA [31–33], which binds to promoter regions regulating gene expression [34, 35]. ArcB activity is regulated in response to oxygen conditions by the redox state of both the ubiquinone and menaquinone pools [29, 36–38]. However, recent studies in E. coli show that the system is regulated by the degree of aerobiosis but not by the redox state of the ubiquinone pool, challenging the idea that the system is inhibited by oxidized quinones .
In this work we provide further evidence of the role of the ArcAB two component system in the response to ROS under aerobic conditions and show that this system mediates regulation of ompW expression in response to a novel signal, HOCl. First we demonstrate, both in vivo and in vitro, that OmpW mediates diffusion of H2O2 and HOCl and that exposure of S. Typhimurium to these compounds results in a negative regulation of ompW. By EMSA and using transcriptional fusions, we demonstrate that the global regulator ArcA binds to the ompW promoter region. Furthermore, we show that ompW negative regulation observed in wild type cells treated with H2O2 and HOCl was not retained in an arcA or arcB mutant strain, indicating that the ArcAB two component system mediates ompW negative regulation in response to H2O2 and HOCl. These results further expand our knowledge in both the mechanisms of ROS resistance and the role of ArcAB in this process.
Results and discussion
The OmpW porin facilitates H2O2 and HOCl diffusion through the OM and reconstituted proteoliposomes
Hydrogen peroxide and hypochlorous acid are ROS generated by phagocytic cells and in order to enter Gram-negative bacteria they must be able to cross the OM. Even though several biological membranes are permeable to H2O2, studies in E. coli and S. cerevisiae demonstrate that this compound cannot diffuse freely [9, 10]. Additionally, the dielectric properties of H2O2 are comparable to those of water and this compound has a slighter larger dipolar moment, further limiting its diffusion through the OM lipid bilayer. For HOCl, diffusion through the OM is also reported to be limited . Therefore, H2O2 and HOCl must be channeled through the lipid bilayer and one possibility is the influx through porins. We recently demonstrated that the most abundant OM protein in S. Typhimurium, OmpD, allows H2O2 diffusion and is regulated by ArcAB . Little is known about the diffusion of HOCl, but genetic evidence has suggested that in E. coli porins might be used as entry channels for hypothiocyanate ions (OSCN−), a molecule with a similar chemical structure generated by lactoperoxidase using thiocyanate and H2O2 as an oxidant . In one study, ompC and ompF knockout mutants showed an increased resistance to OSCN−, however, a direct role of porins in mediating HOCl diffusion was not evaluated.
To establish a direct contribution of OmpW in H2O2 and HOCl transport, we used reconstituted proteoliposomes. OmpW-proteoliposomes showed a decrease in H2O2 and HOCl extra/intraliposomal ratios (3.5 and 5-fold respectively) when compared to free liposomes (Figure 1B and D). Proteoliposomes with S. Typhimurium OmpA porin were used as a negative control as previously described . As expected, OmpA-proteoliposomes showed similar levels to those of free liposomes, indicating that OmpW facilitates H2O2 and HOCl uptake.
Our data supports the proposed model where OmpW allows the influx of small polar molecules, like H2O2 and HOCl. The crystal structure of OmpW from E. coli revealed that the cross-section of the barrel has approximate dimensions of 17 × 12 Å along the length of the barrel and although the interior of the channel has a hydrophobic character, the observed single channel activities shows that polar molecules traverse the barrel . Taken together, these results provide biochemical and genetic evidence indicating that both toxic compounds are channeled through OmpW. From our knowledge, this is the first direct evidence of HOCl diffusion through porins. Furthermore, preliminary analyses indicate that H2O2 and HOCl channeling is common for S. Typhimurium OmpD, OmpC and OmpF porins (unpublished data).
Hydrogen peroxide and hypochlorous acid exposure results in ompW negative regulation
ArcA binds the ompW promoter region
Evaluating ArcA binding site 1 (ABS-1) functionality
The activity of the constructions was compared to the untreated 14028s strain with the wild type fusion. Treatment of this strain with H2O2 and HOCl resulted in lower activity levels (0.58 ± 0.008 and 0.53 ± 0.095, respectively), in agreement with qRT-PCR experiments. However, a 5 nucleotide substitution of the most conserved residues at ABS-1 site (pompW/ABS1-lacZ) resulted in no regulation after exposure to either of the toxic compounds (1,09 ± 0.104 and 0,93 ± 0.061), indicating that they are relevant for the transcriptional activity of ompW in response to H2O2 and HOCl (Figure 5B). Furthermore, these results are in agreement with EMSAs which indicate that ArcA only binds to fragments containing ABS-1.
The ArcAB two component system mediates ompW negative regulation
To determine whether the negative regulation by ArcA was dependant on its cognate sensor ArcB, ompW mRNA levels were evaluated in a ∆arcB strain. In contrast to the negative regulation observed in wild type cells, ompW mRNA levels were further increased in a ∆arcB strain after exposure to HOCl (3.37 ± 1.09). Transcript levels after treatment with H2O2 were similar as those observed in untreated cells (Figure 6B). One possibility for this result is that in the absence of ArcA, ArcB might phosphorylate (i.e ArcB-OmpR, ) one or more response regulators, either unspecifically or due to cross-talk, which could bind to the promoter region and therefore prevent binding of positive regulators like SoxS, which has been demonstrated to regulate ompW and is up-regulated in response to HOCl [20, 44]. This could result in constant ompW transcript levels as shown in Figure 6A. On the other hand, in the absence of ArcB no phosphorylation occurs and SoxS or other positive regulator(s) might have free accessibility to the ompW promoter and therefore increase its expression (Figure 6B), although this possibility has not been evaluated in this study. Genetic complementation of ∆arcB restored the negative regulation observed in wild type cells exposed to H2O2 and HOCl (0.19 ± 0.04 and 0.24 ± 0.11, respectively, Figure 6C). The ompD and ompC transcripts levels remained down-regulated after exposure to H2O2 and HOCl in the ∆arcB strain, while the negative control arcA remained unaltered (Figure 6B).
The ArcA regulon in anaerobically grown S. Typhimurium was recently determined . Interestingly, neither ompD nor ompW expression was down-regulated in an ArcA dependant manner, suggesting that the ArcA regulon under anaerobic and aerobic ROS conditions could be different. Even in E. coli ompW expression is suggested to be regulated by FNR in response to oxygen availability . The difference between the ArcA regulons under aerobic and ROS conditions might be explained by studies suggesting that the mechanism of ArcA activation under aerobic conditions is different from those classically described. E. coli mutant strains in residue H-717 of ArcB are able to phosphorylate and activate ArcA through the transfer of the phosphate group from residue His-292 under aerobic conditions  and Loui et al. (2009) suggested that H2O2 resistance is independent of ArcA phosphorylation at residue Asp-54. To the date, the detailed molecular mechanism of ArcAB activation in response to ROS remains unsolved. Therefore, further experiments to unveil the molecular mechanism by which the S. Typhimurium ArcAB two component system is activated are needed and under way in our laboratory.
We provide both genetic and biochemical evidence indicating that the OM porin OmpW mediates the influx of H2O2 and HOCl. The results revealed that the S. Typhimurium ompW gene is negatively regulated upon exposure to both toxic compounds. Furthermore, we demonstrate that the response regulator ArcA mediates ompW negative regulation in response to H2O2 and HOCl via a direct interaction with the upstream region of ompW. Taken together, with our previous observation that OmpD mediates influx of H2O2 and is negatively regulated by ArcA in response to H2O2, these results further expand our knowledge regarding the coordinated regulatory mechanisms of ROS resistance and the role of ArcAB in this process.
Bacterial strains and growth conditions
Bacterial strains used in this study
wild type strain
14028s transformed with a derivative of plasmid pLacZ-Basic carrying the ompW promoter (nt −600 to +1)
14028s transformed with a derivative of plasmid pLacZ-Basic carrying the ompW promoter (nt −600 to +1) with substitution GTTAA to TCCGG into position −70 to −66
ΔompW strain complemented with pBAD vector carrying the S. Typhimurium ompW gene
ΔarcA strain complemented with pBAD vector carrying the S. Typhimurium arcA gene
ΔarcB strain complemented with pBAD vector carrying the S. Typhimurium arcB gene
F- mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacΧ74 recA1 araD139 Δ(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG
Top10 transformed with the pBAD vector carrying the S. Typhimurium ompW gene
Top10 transformed with the pBAD vector carrying the S. Typhimurium ompA gene
Top10 transformed with the pBAD vector carrying the S. Typhimurium arcB gene
BL21(DE3) transformed with the pET-TOPO101ArcA vector carrying the S. Typhimurium arcA gene
Strain construction and genetic complementation
S. Typhimurium arcB gene was interrupted by gene disruption as previously described . Strain 14028s (wild type) harboring plasmid pKD46 was grown in the presence of arabinose (10 mM) and ampicillin (100 μg ml−1) to OD600 ~ 0.4, made electrocompetent and transformed with a PCR product generated with plasmid pKD3 as template and primers 5′ ATTGGGTATTATGTGCGAAGTTGTGGTGAAGGAATCCTCTTGTAGGCTGGAGCTGCTTCG 3′ (WarcBF) and 5′ GGTGTTGGCGCAGTATTCGCGCACCCCGGTCAAACCGGGGCATATGAATATCCTCCTTAG 3′ (WarcBR). Transformants were selected on LB plates supplemented with chloramphenicol (20 μg ml−1) and confirmed by PCR using primers 5′ GCTACGCATATTTCGCACAA 3′ (arcBF) and 5′ GCGCCTTTGACATCATCATA 3′ (arcBR).
Genetic complementation of the ∆arcB strain was performed using plasmid pBAD-arcB. To generate this plasmid, S. Typhimurium arcB gene was amplified by PCR using primers 5′ ATGAAGCAAATTCGTATGCTG 3′ (pBADarcBF) and 5′ TCATTTTTTTTCCGCGTTTGCCACCC 3′ (pBADarcBR) and cloned into plasmid pBAD-TOPO TA® (Invitrogen) according to manufacturer’s instructions. Insertion was verified by DNA sequencing.
Bacterial survival after exposure to oxidative stress
Bacteria were cultured in 5 ml of LB medium at 37°C overnight with shaking. Antibiotics were added as appropriate. 1:1000 dilutions of the overnight cultures were grown in 25 ml to OD ~ 0.4 and H2O2 4 mM or NaOCl 5 mM (final concentration) were added. In all the assays the cultures were grown aerobically at 250 rpm. Aliquots of cultures were withdrawn at the different time points, diluted and plated in triplicate. Bacterial cultures were enumerated by counting the number of CFU after overnight incubation to determine the bacterial concentrations.
Construction of transcriptional fusions with reporter gene lacZ
The native ompW promoter region from positions +1 to −600 (with respect to the translation start) site was amplified by PCR with primers ompW_pLacZ_-600F_ATG 5′ CGGGGTACCCCCGATATCGAAAATTCGCG 3′ and ompW_pLacZ_-1R_ATG 5′ CCCAAGCTTACCCGCTCCATCGTTATGGT 3′ using genomic DNA from S. Typhimurium (strain 14028s). The restriction sites (KpnI and HindIII, respectively) at the ends of the DNA fragment were introduced by the PCR primers (underlined sequences) and digested with the corresponding enzymes. The digested PCR product was cloned into the multiple cloning site (MCS) of the β-galactosidase reporter vector pLacZ-Basic (GenBank accession no. U13184), Clontech, generating plasmid pompW-lacZ. To generate plasmid pompW/ABS1-lacZ, primers ompW_pLacZ_-600F_ATG with Mut_sit_arcAR 5′ TGTTCTTATAATGCGGAATTTATTGATCCAG 3′ and ompW_pLacZ_-1R_ATG with Mut_sit_arcAF 5′ CTGGATCAATAAATTCCGGAATTATAAGAACA 3′ were used to generate overlapping PCR products spanning the whole length of the ompW promoter. Mutation of ABS-1 was generated by incorporating substitutions in primers Mut_sit_arcAF and Mut_sit_arcAR (underlined sequences). The resulting PCR products were used as templates in a second reaction with primers ompW_pLacZ_-600F_ATG and ompW_pLacZ_-1R_ATG to generate the mutated ompW promoter, which was digested and cloned into the MCS of plasmid pLacZ-Basic. Constructions were confirmed by DNA sequencing. The generated constructs were transformed into wild type strain 14028s. To evaluate activity, cells at OD600 ~ 0.4 were grown for 20 min in the presence of H2O2 (1.5 mM) or NaOCl (530 μM). Control cells received no treatment. β-galactosidase activity was determined as previously described .
His-tagged ArcA used in EMSAs was purified as previously described . Briefly E. coli BL21 cells harboring plasmid pET-TOPO-arcA were grown in 500 ml of LB medium supplemented with amplicillin (100 μg ml−1) to OD600 ~ 0.4 and protein overexpression was carried out by adding 1 mM IPTG and further growth for 6 h. Protein was purified by affinity chromatography as described by Georgellis et al., (1997).
Outer membrane proteins used in proteoliposomes were purified as described by Calderón et al. (2011). E. coli Top10 cells carrying pBAD-ompA or pBAD-ompW were grown in 500 ml to OD600 ~ 0.6 at 37°C and overexpression was performed for 5 h in the presence of 1 mM arabinose. His-tagged porins were purified by affinity chromatography using HisTrap HP columns (Amersham) according to the manufacturer’s instructions.
Plasmid pBAD-ompW was generated amplifying the coding region of S. Typhimurium ompW by PCR using primers 5′ ATGAAAAAATTTACAGTGGC 3′ (pBAD-ompWF) and 5′ GAAACGATAGCCTGCCGAG 3′ (pBAD-ompWR) and cloned into plasmid pBAD-TOPO TA® (Invitrogen) according to the manufacturer’s instructions. Insertion was verified by DNA sequencing.
RNA isolation and ompW mRNA detection
Overnight cultures were diluted (1:100) and cells were grown to OD600 ~ 0.4. Genetically complemented cells (∆arcA/pBAD-arcA and ∆arcB/pBAD-arcB) were grown in the presence of arabinose (1 mM) and ampicillin (100 μg ml-1). At this point, H2O2 (1.5 mM) or NaOCl (530 μM) was added and cells were grown for 20 min. Control cells received no treatment. After exposure to the toxic compounds, 4 ml were withdrawn from the culture and used to extract total RNA using GenElute Total RNA purification Kit® (Sigma). Total RNA treatment with DNase I and cDNA synthesis was performed as previously described .
Relative quantification of ompW mRNA was performed using Brilliant II SYBR Green QPCR Master Reagent Kit and the Mx3000P detection system (Stratagene). 16S rRNA was used for normalization. Specific primers were 5′ ATGAAAAAATTTACAGTGG 3′ (RTompWF) and 5′ GAAACGATAGCCTGCCGA 3′ (RTompWR) for the ompW gene; 5′ GTAGAATTCCAGGTGTAGCG 3′ (16SF) and 5′ TTATCACTGGCAGTCTCCTT 3′ (16SR) for 16S rRNA gene (16S). The reaction mixture was carried out in a final volume of 20 μl containing 1 μl of diluted cDNA (1:1000), 0.24 μl of each primer (120 nM), 10 μl of 10 x Master Mix, 0.14 μl of diluted ROX (1:200) and 8.38 μl of H2O. The reaction was performed under the following conditions: 10 min at 95°C followed by 40 cycles of 30 s at 95°C, 30 s at 53°C and 45 s at 72°C. Finally a melting cycle from 53 to 95°C was performed to check for amplification specificity. Amplification efficiency was calculated from a standard curve constructed by amplifying serial dilutions of RT-PCR products for each gene. These values were used to obtain the fold change in expression for the gene of interest normalized with 16S levels according to . Experiments were performed in three biological and technical replicates.
DNA binding assays
Non-radioactive EMSAs were performed as described . Briefly, increasing amounts of purified ArcA (phosphorylated and unphosphorylated) were incubated with 20 or 60 ng of PCR product(s) in binding buffer (100 mM Tris-Cl [pH 7.4], 100 mM KCl, 10 mM MgCl2, 10% glycerol, and 2 mM dithiothreitol) for 20 min at 30°C. Reaction mixtures were immediately loaded on prerun 4% native polyacrylamide gels. The DNA-protein complexes were visualized by ethidium bromide staining. PCR fragments used in EMSAs were generated by PCR using reverse primer 5′ ACCCGCTCCATCGTTATGGT 3′ (ompWR) in combination with 5′ GAGCAGACAAATATTTGCAT 3′ (300WF) or 5′ TATTAGATCACTTATTACTT 3′ (170WF) to generate fragments W1 and W2, respectively. Fragment W3 was generated using primers 300WF and 5′ GATCCAGATTAATTTAGAAC 3′. Fragments W4 and W5 were generated by using reverse primer 5′ AATTTTTTCATACCCGCTCC 3′ in combination with primers 5′ CCTATAACCAGGATTTTCAA 3′ and 170WF, respectively. ArcA phosphorylation was carried out as described by Linch and Lin (1996). Briefly purified ArcA was incubated with 50 mM disodium carbamoyl phosphate (Sigma) in a buffer containing 100 mM Tris-Cl (pH 7.4), 10 mM MgCl2, 125 mM KCl, for 1 h at 30°C and used immediately in EMSA assays.
In vivo and in vitro determination of hydrogen peroxide and hypochlorous acid diffusion
In vivo diffusion of H2O2 was assessed as previously described . For HOCl detection, overnight cultures were diluted and cells were grown to OD600 ~ 0.5. Two ml of bacterial culture were centrifuged for 5 min at 4500 x g and resuspended in 1 ml of 100 mM phosphate buffer (pH 7.2). A 200 μl aliquot was incubated for 5 min with 530 μM NaOCl and constant agitation. Following, cells were vacuum filtered using polycarbonate filters of 0.025 μm (Millipore) and pass through was collected (extracellular fraction). Bacteria retained in the filter were recovered with 1 ml of 50 mM phosphate buffer (pH 7.2) and disrupted by sonication (intracellular fraction). Both fractions (190 μl) were incubated separately with dihydrorhodamine-123 to a final concentration of 5 μM as previously described . The fluorescent product, rhodamine-123, was measured by fluorescence detection with excitation and emission wavelengths of 500 and 536 nm, respectively. HOCl and H2O2 uptake was determined as the extracellular/intracellular fluorescence ratio. The background fluorescence from a bacterial suspension not exposed to either of the toxic compounds was subtracted and results were normalized by protein concentration.
Proteoliposomes were prepared as described  with modifications . For in vitro diffusion, proteoliposomes were incubated with 1.5 mM H2O2 or 530 μM NaOCl for 5 min, vacuum filtered and pass through was recovered (extraliposomal fraction). Proteoliposomes were recovered from the filters with 2 ml of 50 mM phosphate buffer (pH 7.2) and disrupted by sonication (intraliposomal fraction). Fluorescence was measured in both fractions as described above and H2O2 or HOCl uptake was determined as the extraliposomal/intraliposomal fluorescence ratio.
Eduardo H Morales and Iván L Calderón contributed equally to this work
This work was supported by grants from FONDECYT #1085131 and #1120384 (to CPS), Universidad Andres Bello DI-34-11/R (to CPS), POSTDOC FONDECYT 3095013 (to ILC) and Universidad Andres Bello DI-50-09/R (to ILC). EHM and BC received doctoral fellowships by CONICYT and MECESUP UAB0802 additionally to EHM. We would like to thank Nicolás Pacheco for his assistance in the UFC experiments. The authors have declared that no competing interests exist. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Publication fees were covered by FONDECYT grant # 1120384 and from Universidad Andres Bello DI-34-11/R (to CPS).
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