The type III protein secretion system contributes to Xanthomonas citri subsp. citri biofilm formation
© Zimaro et al.; licensee BioMed Central Ltd. 2014
Received: 10 December 2013
Accepted: 9 April 2014
Published: 18 April 2014
Several bacterial plant pathogens colonize their hosts through the secretion of effector proteins by a Type III protein secretion system (T3SS). The role of T3SS in bacterial pathogenesis is well established but whether this system is involved in multicellular processes, such as bacterial biofilm formation has not been elucidated. Here, the phytopathogen Xanthomonas citri subsp. citri (X. citri) was used as a model to gain further insights about the role of the T3SS in biofilm formation.
The capacity of biofilm formation of different X. citri T3SS mutants was compared to the wild type strain and it was observed that this secretion system was necessary for this process. Moreover, the T3SS mutants adhered proficiently to leaf surfaces but were impaired in leaf-associated growth. A proteomic study of biofilm cells showed that the lack of the T3SS causes changes in the expression of proteins involved in metabolic processes, energy generation, exopolysaccharide (EPS) production and bacterial motility as well as outer membrane proteins. Furthermore, EPS production and bacterial motility were also altered in the T3SS mutants.
Our results indicate a novel role for T3SS in X. citri in the modulation of biofilm formation. Since this process increases X. citri virulence, this study reveals new functions of T3SS in pathogenesis.
The bacterial genus Xanthomonas comprises a number of Gram-negative plant pathogenic bacteria that cause a variety of severe plant diseases . Xanthomonas citri subsp. citri, the phytopathogen causing citrus canker, invades host plant tissues entering through stomata or wounds and then colonizes the apoplast of fruit, foliage and young stems, causing raised corky lesions and finally breaking the epidermis tissue due to cell hyperplasia, thus allowing bacterial dispersal to other plants .
Persistent and severe disease can lead to defoliation, dieback and fruit drop, reducing yields and causing serious economic losses . To date, no commercial citrus cultivars are resistant to citrus canker and current control methods are insufficient to manage the disease . Thus, there is a need to study the infection process in order to enable the development of new tools for disease control. Furthermore, the study of X. citri-citrus interactions has been used as a model to provide new advances in the understanding of plant-pathogen interactions .
The Type III protein secretion system (T3SS) is conserved in many Gram-negative plant and animal pathogenic bacteria . The T3SS is subdivided into (i) the non-flagellar T3SS (T3aS) involved in the assembly of the injectisome or hypersensitive response and pathogenicity (Hrp) pilus, and (ii) the flagellar T3SS (T3bS), responsible for assembly of the flagellum . The T3SS spans both bacterial membranes and is associated with an extracellular filamentous appendage, termed ‘needle’ in animal pathogens and ‘Hrp pilus’ in plant pathogens, which is predicted to function as a protein transport channel to the host-pathogen interface . Translocation of effector proteins across the host membrane requires the presence of the T3SS translocon, a predicted protein channel that consists of bacterial Type III-secreted proteins .
A number of surface appendages, such as conjugative pili, flagella, curli, and adhesins have been shown to play a role in biofilm formation [7, 8]. The role of T3SS as an effector protein delivery machine is well established, however, whether this secretion system participates in multicellular processes such as biofilm formation remains unanswered. Several studies concluded that T3SS is only necessary for pathogenicity and that expression of this secretion system is repressed in biofilm-growing bacteria. For example, Pseudomonas aeruginosa PA14 sadRS mutant strains that cannot form biofilms have enhanced expression of T3SS genes, while a P. aeruginosa PA14 T3SS mutant exhibits enhanced biofilm formation compared to wild type strain . Furthermore, in Yersinia pseudotuberculosis, it has been shown that the T3SS needle blocks biofilm formation in the model host Caenorhabditis elegans. In contrast, other studies highlighted the role of T3SS in bacterial biofilm formation. Microarray experiments performed in P. aeruginosa cystic fibrosis epidemic strain AES-2 showed expression of T3SS encoding genes up-regulated in biofilms as compared to planktonic bacteria . In the plant pathogen Erwinia chrysanthemi, it has been shown that the T3SS pilus is involved in the aggregative multicellular behavior that leads to pellicle formation . The enterohemorrhagic Escherichia coli O157 has a well-defined T3SS, termed E. coli Type III secretion system 1 (ETT1), which is involved in attachment and effacement and is critical for virulence. This strain also has a gene cluster potentially encoding an additional T3SS (ETT2) . Studies on an ETT2 deletion mutant strain showed that although ETT2 is not responsible for protein secretion, it is involved in biofilm formation and hence in virulence . Recently, it has been shown that the Salmonella enterica serovar Typhimurium T3SS secretion system SPI-1 is involved in the formation of an adherent biofilm and cell clumps in the culture media . Taken together, the evidence suggests that T3SS may play a role in bacterial biofilm formation.
In X. citri, biofilm formation is required for optimal virulence as revealed by several reports with different bacterial mutants. For instance, X. citri mutants that are unable to biosynthesize molecules needed for biofilm formation such as exopolysaccharide (EPS), an adhesin protein and the lipopolysaccharide show a reduced virulence [15–17]. Consistent with this, X. citri infection is reduced by foliar application of compounds that are able to inhibit X. citri biofilm formation . The role of X. citri T3SS in pathogenicity is well known since T3SS mutants are unable to grow in host plants indicating that X. citri T3SS is responsible for the secretion of effector proteins . Taking into account that biofilm formation is a requirement for X. citri to achieve full virulence, we have characterized the ability of a T3SS mutant to form biofilms and by performing a proteomic analysis we have identified differentially expressed proteins with a view to obtain a greater understanding of this process.
The T3SS contributes to X. citri in vitro biofilm formation
The T3SS is not required for attachment to host tissue but is necessary for X. citri biofilm formation on the leaf surface
T3SS is required for X. citri leaf-associated survival
Proteomic analysis of statically cultured X. citri and hrpB − strains
Differentially expressed protein spots between X. citri and hrpB − strains statically cultured in XVM2 with a change abundance of minimum 1.5 fold and p value of < 0.05 (ANOVA)
X. citri gene no.
Fold change compared to WT
01.01 Amino acid metabolism
Tryptophan synthase subunit b
Tryptophan repressor binding protein
01.02 Nitrogen, sulfur and selenium metabolism
01.03 Nucleotide/nucleoside/nucleobase metabolism
01.05 C-compounds and carbohydrate metabolism
Succinate dehydrogenase flavoprotein subunit
Phosphohexose mutases (XanA)
01.06 Lipid, fatty acid and isoprenoid metabolism
Putative esterase precursor (EstA)
Short chain dehydrogenase precursor
01.06.02 Membrane lipid metabolism
Outer membrane protein (FadL)
Outer membrane protein (FadL)
01.20 Secondary metabolism
Coproporphyrinogen III oxidase
02.01 Glycolysis and gluconeogenesis
UTP-glucose-1-phosphate uridylyltransferase (GalU)
02.07 Pentose phosphate pathway
02.11 Electron transport and membrane-associated energy conservation
Electron transfer flavoprotein a subunit
10 Cell cycle and DNA processing
10.03 Cell cycle
Cell division topological specificity factor (MinE)
10.03.03 Cytokinesis/septum formation and hydrolysis
Septum site-determining protein (MinD)
DNA-directed RNA polymerase subunit a
DNA-directed RNA polymerase subunit b
14 Protein fate (folding, modification and destination)
14.01 Protein folding and stabilization
60 kDa chaperonin (GroEL)
16 Protein with binding function or cofactor requirement
Adenine-specific methylase (Dam methylase)
OmpW family outer membrane protein precursor
30 Cellular communication/Signal transduction mechanism
Oar protein ( TonB-dependent transporter)
Oar protein ( TonB-dependent transporter)
Outer membrane protein assembly factor BamA
32 Cell rescue, defense and virulence
Regulator of pathogenicity factors (RpfN)
Alkyl hydroperoxide reductase subunit C
32.07 Cellular detoxification
34 Interaction with the environment
42 Biogenesis of cellular components
Putative membrane protein
99 Unclassified proteins
Protein of unknown function (Aminopeptidase)
Protein of unknown function (CcmA)
The lack a T3SS enhances X. citri EPS production and decreases bacterial motility
The role of T3SS in bacterial pathogenesis as a machine involved in effector protein delivery is well established, however, little is known about other functions in bacterial behavior that this system may have. Given that biofilm formation is required for X. citri to achieve full virulence, we used X. citri as a model to gain further insights into the functional role of T3SS in biofilm formation. By comparing the capacity of biofilm formation of three T3SS mutants and X. citri and also performing a proteomic assay with the hrpB − mutant, which revealed differentially expressed proteins between both strains, we demonstrated that T3SS is involved in biofilm formation in X. citri.
To date the involvement of X. citri T3SS in bacterial attachment and survival on leaf tissue has not been studied. In this work we observed that the adherence of different T3SS mutants to host cell tissue was not altered. Studies in several pathogenic bacteria, such as Salmonella typhimurium, E. coli[36, 37] and the plant pathogen P. syringae revealed that mutants unable to produce T3SS appendages become affected in their interactions with host cells. However, in the phytopathogen Ralstonia solanacearum, it has been shown that the lack of a T3SS pilus does not affect attachment to plant cells , and this is consistent with our observation that adherence of X. citri to the host tissue was not affected by the absence of a functional T3SS. In addition, we determined that T3SS is required for X. citri survival on citrus leaves and that T3SS genes are expressed while bacteria reside on the plant surface. Expression of T3SS genes on the leaf surface was also detected in Xanthomonas euvesicatoria cells suggesting a role for T3SS in epiphytic survival of the bacteria . In a recent report, it was revealed that the survival of Pseudomonas syringae T3SS-deficient strains on leaf surfaces is reduced, supporting a role of T3SS and effector proteins in the promotion of epiphytic bacterial survival . Our results suggest that T3SS plays a role in X. citri leaf-associated survival on the leaf surface by enabling biofilm formation.
The proteomic study revealed differentially expressed proteins between X. citri and the hrpB − mutant strain and GO analysis detected enrichment of up-regulated proteins in different metabolic processes and generation of energy in the hrpB − mutant. Similarly, in a previous proteomic study, these categories were also enriched with up-regulated proteins in X. citri planktonic cells compared to biofilm, suggesting a slower metabolism and reduction in aerobic respiration in the X. citri biofilm . Therefore, the higher expression of proteins involved in these processes in the hrpB − mutant compared to X. citri may be caused by the lack of biofilm formation of the mutant.
It is remarkable that among the differentially expressed proteins between the mutant and the wild type strain, some have been previously characterized as involved in biofilm formation in X. citri or in other pathogenic bacteria. Such is the case of DNA-directed RNA polymerase subunit β , tryptophan synthase , GroEL [44, 45], FadL [32, 42, 46] and several TBDTs [42, 47]. Interestingly, high intracellular L-tryptophan concentration prevents biofilm formation and triggers degradation of mature biofilm in E. coli. The proteomic assay showed that tryptophan synthase (XAC2717) was up-regulated, while the tryptophan repressor binding protein (XAC3709) was down-regulated in hrpB − strain suggesting a link also between tryptophan metabolism and biofilm formation in X. citri. Another example is the outer membrane protein XAC0019 that displays high homology to the fatty acid transport porin FadL. Pseudomonas fluorescens mutants in the fadL gene showed defects in their ability to develop a biofilm on a abiotic surfaces leading to the suggestion that long chain fatty acids bind to FadL thereby altering surface hydrophobicity, and adhesion characteristics . Consistent with this, a recent work showed that a X. citri mutant in XAC0019 displays reduced capacity to form a biofilm  and its expression is increased during X. citri biofilm formation . In the present study, XAC0019 protein was down-regulated in the hrpB − mutant impaired in biofilm formation, reinforcing the role of this protein in this process.
Enzymes involved in EPS production XanA and GalU, [30, 31] were up-regulated in the hrpB − mutant. Consistently, all the hrp mutant analyzed in this work produced larger amounts of EPS in comparison with X. citri and also had higher expression levels of gumD. Recent reports have shown that X. citri galU mutant strain is not pathogenic and also loses its capacity to form a biofilm due to a reduction in EPS production [30, 32], and that a X. citri xanA mutant has an altered capacity for biofilm formation . Although, the hrp mutants are impaired in biofilm formation, these mutants produce more EPS than X. citri. This interesting result open new hypotheses about the link between T3SS and EPS production, thus further studies are needed to unravel this issue. In other pathogens, such as P. aeruginosa, T3SS gene expression is coordinated with many other cellular activities including motility, mucoidy, polysaccharide production, and also biofilm formation .
Bacterial motility was impaired in the hrp mutants and consistently, proteins known as involved in these processes such as the outer membrane protein XAC0019  and the bactofilin CcmA [33, 34] were down-regulated in the hrpB − mutant. Besides, swarming motility was less affected than swimming in the hrp mutants compared with X. citri. This may be due to the fact that in X. citri swarming motility depends on flagella and also on the amount of EPS secreted , and since these mutants over-produced EPS swarming was less affected than swimming.
This work demonstrated that in X. citri T3SS is involved in multicellular processes such as motility and biofilm formation. Furthermore, our results suggest that T3SS may also have an important role in modulating adaptive changes in the cell, and this is supported by the altered protein expression when this secretion system is not present. It was previously shown that an E. coli O157 strain mutant in the additional T3SS named ETT2 is impaired in biofilm formation . It was also suggested that deletion of ETT2 might cause structural alterations of the membrane modifying bacterial surface properties, thus affecting bacteria-bacteria interactions or the interaction with host cells . Further, it was proposed that these structural alterations could trigger a signal that activates differential gene expression and/or protein secretion . In line with this, we propose that in X. citri the ‘Hrp pilus’ structure per se, or its interaction with a solid surface, stabilizes the outer membrane structure, hence the lack of T3SS may trigger membrane remodeling itself. These membrane modifications in turn may change the pattern of protein expression, leading to the impairment of cellular processes directly related to bacterial virulence including biofilm formation. Another possibility is that the ‘Hrp pilus’ may function like an attachment device or flagellum. Future studies are likely to add further insights into the exact role and modes of operation of X. citri ‘Hrp pilus’ in biofilm formation and motility.
This work demonstrates that the presence of T3SS in X. citri, besides its participation in the secretion of effector proteins is also required for biofilm formation, motility and survival on leaf tissue revealing novel functions for this secretion system in X. citri. In biofilm formation, T3SS may have an important role in modulating adaptive changes that lead to this process. Some of these changes are revealed by variations in proteins involved in metabolic processes, energy generation, EPS production and bacterial motility as well as in outer membrane proteins between the wild type strain and the T3SS mutant. In summary, the present study reveals novel contributions of this protein secretion system to bacterial virulence.
Bacterial strains, culture conditions and media
X. citri strain Xac99-1330 was isolated from C. sinensis and kindly provided by Blanca I. Canteros (INTA Bella Vista, Argentina). The hrpB − mutant was constructed in previous work . Here, hrpB −c complemented strain was constructed by cloning the region from hrpB5 to hrcT in the replicative plasmid pBBR1MCS-5  under the control of the lacZ promoter. This region was amplified from X. citri genomic DNA with the oligonucleotides: HrpB5F-Hind (5′ ATAGAAGCTTCATGCGTCTCTGGTTGAGGTC 3′) and HrcTR-Bam (5′ ATCAGGATCCTCAGTGCGACGCGGCTCTCT 3′) and cloned into pBBR1MCS-5 previously digested with the restriction enzymes Hind III and Bam HI. The resulting construction was electroporated into the hrpB − strain and the complemented mutant strain was selected by for gentamicin antibiotic resistance. For confocal laser scanning microscopy analyses, a GFP-expressing hrpB − strain was obtained. To this end, the coding sequence for EGFP from the broad-host-range vector pBBR1MCS-2EGFP  was digested with BamH I and Xba I and ligated in frame with the LacZ-α-peptide of the pBBR1MCS-5 vector  previously digested with the same enzymes, rendering the plasmid pBBR1MCS-5EGFP. E. coli S17-1 cells transformed with this plasmid were conjugated with the hrpB − strain and the cells carrying the plasmid pBBR1MCS-5EGFP were selected for Gm resistance. All strains were grown at 28°C in Silva Buddenhagen (SB) medium  or in XVM2 medium . Antibiotics were used at the following final concentrations: 25 μg/ml ampicillin (Amp), 5 μg/ml gentamicin (Gm) and 40 μg/ml kanamycin (Km).
Orange (Citrus sinensis cv. Valencia) was used as the host plant for X. citri. All plants were grown in a growth chamber with incandescent light at 28°C with a photoperiod of 16 h.
For biofilms development, bacteria were grown in SB with shaking until exponential growth phase and then diluted 1:10 in fresh XVM2 medium containing appropriate antibiotics. A 2 ml aliquot of diluted bacterial suspension was placed in borosilicate glass tubes or in 24-well PVC plates and incubated statically for seven days at 28°C. The quantification of biofilm formation by CV staining was carried out as previously described . Briefly, the culture medium was decanted and the absorbance of planktonic cells was measured at 600 nm using a UV-visible spectrophotometer (Synergy 2 Reader, BioTek). After washing the tubes three times with distilled water (dH2O) during 10 min with gentle agitation, the remaining attached cells were incubated for 10 min at 60°C and stained with 0.1% (w/v) CV for 30 min at room temperature. Excess CV stain was removed by washing under running tap water. The CV stain was solubilized by the addition of 1.5 ml ethanol:acetone (80:20, v/v) to each tube and quantified by measuring the absorbance at 600 nm. The relative absorbance (Relative abs.) was calculated as: CV Abs. 600 nm/Planktonic cells Abs. 600 nm. Values represent the mean from seven tubes for each strain, data were statistically analyzed using one-way analysis of variance (ANOVA) (p < 0.05).
Confocal analysis of biofilm architecture
In vitro biofilm of the GFP-expressing hrpB − mutant and X. citri previously constructed  grown in 24-well PVC plates in XVM2 medium were analyzed after seven days by confocal laser scanning microscopy (Nikon Eclipse TE-2000-E2). For biofilms assays on leaf surfaces, overnight cultures of both GFP-expressing strains grown in XVM2 medium were centrifuged, washed and resuspended in phosphate buffer (pH 7.0) to the same OD600 and 20 μl of each bacterial suspension were applied on abaxial leaf surfaces. These biofilms were also analyzed after seven days by confocal laser scanning microscopy (Nikon Eclipse TE-2000-E2).
The adhesion capacity to leaf surfaces was measured as described previously . Overnight cultures of the different strains in XVM2 medium were centrifuged to recover cell pellets, washed and resuspended in phosphate buffer (pH 7.0) to the same optic density measured at 600 nm (OD600). Then, 20 μl of each bacteria suspension were place on abaxial leaf surfaces and incubated for 6 h at 28°C in a humidified chamber. After washing the non-adhered cells, bacteria were stained with CV, the CV stain was extracted from the bacterial drops with 95% (v/v) ethanol by pipetting up and down with a 20 μl micropipette. Quantification of the extracted CV stain was carried out by measuring the absorbance at 590 nm as described above. For each strain 20 stained drops were quantified and data were statistically analyzed using one-way ANOVA (p < 0.05).
Quantification of leaf-associated survival
Leaf-associated fitness was evaluated as previously described . Briefly, overnight cultures in SB medium were centrifuged to recover bacteria cell pellets, washed and resuspended in 10 mM phosphate buffer (pH 7.0) at a concentration of 109 CFU/ml. These bacterial suspensions were sprayed onto leaves until each leaf surfaces were uniformly covered. Old citrus leaves were used since the greater thickness of the cuticles of these leaves naturally render the leaves resistant to bacterial entry (unpublished results). Four different leaves were inoculated with each strain, leaves were photographed and the surfaces were quantified using the software Image-Pro (Media Cybernetics). Leaves were collected on different days post-inoculation and transferred to borosilicate glass flasks containing 10 mM potassium phosphate buffer (pH 7.0). Flasks were submerged in a sonicator (Branson model #5510) for 10 min. Subsequently, each flask was vortexed for 5 sec, bacteria were recovered by centrifugation and serial dilutions were plated on SB plates containing Ap to count bacterial colonies. Results were expressed in CFU/cm2 of inoculated leaves. Values represent an average of four leaves assayed for each strain, the data were statistically analyzed using one-way ANOVA (p < 0.05).
RNA preparation and RT-qPCR
Total RNA from bacterial cultures grown at the indicated conditions and from bacteria recovered from leaves at the indicated times were isolated using TRIzol® reagent (Invitrogen), according to the manufacturer’s instructions. The RT-qPCRs were performed as previously described  with the specific oligonucleotides detailed in Additional file 3: Table S1. As a reference gene, a fragment of 16S rRNA (XAC3896) was amplified using the same RT-qPCR conditions. To control that no bacterial DNA contamination was present in the samples, the same PCR reactions were carried out without retrotranscription and non amplification was observed. To ascertain the absence of plant RNA in bacterial samples controls with plant actin primers were carried out (data not shown). Values were normalized by the internal reference (Ctr) according to the equation ΔCt = Ct – Ctr, and quantified as 2–ΔCt. A second normalization using a control (time = 0 days) (Ctc), ΔΔCt = Ct – Ctc, producing a relative quantification: 2–ΔΔCt. Values are the means of four biological replicates with three technical replicates each. Results were analyzed by Student t-test (p < 0.05) and one-way ANOVA (p < 0.05).
Protein extraction and resolubilization for the proteomic analysis
Biofilms of statically grown bacterial cultures were obtained as previously described . After seven days of static growth, the XVM2 medium was carefully removed and biofilms were collected by pipetting, transferred to a new tube and pelleted by centrifugation prior to protein extraction. Biofilm proteins were extracted and resuspended in urea buffer (9 M urea, 2 M thiourea and 4% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)) with vigorous vortexing at room temperature. Concentration of total protein extracts was estimated using a modified Bradford assay  and using bovine serum albumin as standard. Protein extracts were prepared from three biological replicates for each strain.
Total proteins from biofilm cells were extracted and labeled using the fluorescent cyanine three-dye strategy (CyDyes; GE Healthcare), as described in . X. citri and hrpB − protein samples were labeled with Cy3 and Cy5, respectively, according to manufacturer’s instructions. Protein extractions were performed from three independent biological samples, and two technical replicate gels for each experiment were run. Protein separation, quantification by two-dimensional-difference in-gel electrophoresis (2D-DIGE), comparative analysis and protein identification were also carried out as previously described . Normalized expression profile data were used to statistically assess changes in protein spot expression. Differentially expressed protein spots between the two groups were calculated using the Student t-test with a critical p-value ≤ 0.05 and the permutation-based method to avoid biased results that may arise within replicate gels if spot quantities are not normally distributed. The adjusted Bonferroni correction was applied for false discovery rate (FDR) to control the proportion of false positives in the result set. Principal component analysis was performed to determine samples and spots that contributed most to the variance and their relatedness. Protein spots with a minimum of 1.5 fold change and p values < 0.05 only were considered as significantly differentially expressed between the two strains.
Quantification of EPS production
Quantification of EPS production was performed as previously described . Briefly, bacterial strains were cultured to the stationary growth phase in 50 ml of SB liquid medium supplemented with 1% (w/v) glucose in 250 ml flasks, using an orbital rotating shaker at 200 rpm at 28°C. Cells were removed by centrifugation at 2,500 × g for 30 min at room temperature, and the supernatant fluids were separately supplemented with KCl at 1% (w/v) and 2 volumes of 96% (v/v) ethanol and then incubated for 30 min at -20°C to promote EPS precipitation. Precipitated crude EPS were collected, dried and weighed. Results were expressed in grams per culture liter. Quadruplicate measurements were made for each strain and an average of all measurements was obtained, data were statistically analyzed using one-way ANOVA (p < 0.05).
Swimming and swarming assays
Swimming and swarming motility were measured as previously described . The SB plates fortified with 0.3% (w/v) or 0.7% (w/v) agar respectively were centrally inoculated with 5 μl of 1 × 107 CFU/ml cultures in exponential growth phase. Inoculated Petri dishes were then incubated in a humidity chamber for two days at 28°C and the motility zones were measured. Results are the average of the motility zones of sixteen Petri dishes per strain. Data was statistically analyzed using one-way ANOVA (p < 0.05).
We thank Rodrigo Vena for assistance with the confocal microscopy facility, Microquin for the culture media, Catalina Anderson (INTA Concordia, Argentina), Gastón Alanis and Rubén Díaz Vélez (Proyecto El Alambrado) for the citrus plants, Sebastián Graziati and Diego Aguirre for plant technical assistance and the Proteomics laboratory from the Biosciences core laboratory, King Abdullah University of Science and Technology, for providing the facility and equipment for gel electrophoresis and mass spectrometry analyses.
This work was supported by grants from the Argentine Federal Government: ANPCyT (PICT2010-1507 to NG and PICT2010-0300 to JO) and CONICET (PIP2010-2012 to JO and NG), the Fundación Josefina Prats to CGG and FAF. JO and NG are staff members and TZ, GGS, CGG and FAF are fellows of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina).
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