Genomic Islands Of Brucella Instability Biology Essay

Brucellosis is one of the most of import zoonotic disease worldwide, impacting people from developing states and doing of import economical losingss [ 1 ] . The disease is produced by gm negative, intracellular facultative bacteriums of the genus Brucella [ 2 ] . Classically, Brucella species are recognized based on the discriminatory host scope and other phenotypic characteristics [ 3 ] : B. abortus affects chiefly cowss and other Bovidae every bit good as Cervidae ; B.

suis is isolated from swine and several signifiers of wild life ; B. melitensis infects chiefly sheep and caprine animals. B.

Canis is found in Canis familiariss ; B. neotomae is found in desert woodrats ; and B. Ovis is responsible for epididymitis in random-access memory.

In add-on, three new species have been proposed late: B. microti, isolated from common field mouses [ 4 ] and B. ceti and B. pinnipedialis, both isolated from marine mammals [ 5 ] .B. melitensis, B. suis and B. abortus [ 6-9 ] show a genome of 3.

3 Mb. No classical virulency factors such as capsules, exotoxins, cytolysins, pili or fimbriae, etc. have been found [ 10 ] . However, these bacteriums are capable of infecting host macrophages, hedging the innate immune system and set uping a replicative niche within cells [ 2 ] .

In malice of the restricted ecological niche occupied by these intracellular pathogens, there is grounds of horizontal cistron transportation ( HGT ) [ 11-14 ] .Genomic islands ( GI ) are among the DNA sequences transferred by HGT [ 15 ] . These sequences are big chromosomal parts that confer new phenotypic traits upon transfer-acquisition, frequently increasing bacterial fittingness under hostile environmental conditions. Most of the reported GI sequences contain an untypical base composing in comparing to their host chromosome and frequently they are found delimited by short direct repetition sequences ( DR ) [ 16 ] . Two venue involved in lipopolysaccharide ( LPS ) biogenesis with characteristics of GI have been described in B. melitensis [ 12, 14 ] and the presence of a GI besides related to LPS construction was reported late in B. abortus.

Interestingly, this part is absent in B. ovis [ 13 ] , proposing that GI might take into self-generated genome omissions through recombination events, doing genome instability and variableness [ 15 ] . At present, there is a individual recognized unstable GI in B. suis, B. Canis, B. neotomae and Marine isolates. This GI resides in the little chromosome and carries phage-related and tra cistrons, the latter related to conjugative machinery [ 17 ] . In add-on, a whole genome microarray comparing utilizing a bit based on B.

melitensis 16M genome showed the presence of extra GI-like parts in different Brucella species [ 11 ] . Nevertheless, no experimental grounds has been presented as yet that relates GI venue with instability in Brucella.In this survey, we performed a hunt for GI-like venue in B. abortus 9-941 utilizing a bioinformatic anticipation which combines the presence of genomic signature divergence and flanking repetitions. We identified eight GI-like venue runing from 4-21 kilobit, all flanked by direct repetitions, and found that these sequences are unstable in bacteriums grown in rich medium, let go ofing a round intermediate ( CI ) and go forthing behind a chromosomal cicatrix. Sequencing of CI-derived fragments suggested a site-specific recombination mechanism involved during GI deletion. Wholly, our observations provide grounds for a genome malleability linked to GI deletion events in species of the genus Brucella.

Consequences

Designation of the GI in the B.

abortus genome

The whole genome scanning of the B. abortus genome identified 10 putative parts with genomic signature divergence in chromosomes I and 6 in chromosomes II ( Figure 1, Additional file 1 ) , Although most of these parts showed GC % divergence, eight deficiency of flanking DR sequences. These venues corresponded to bunchs of ribosomal cistron, flagellar cistrons and GBAI3, a ( genomic island of Brucella abortus chromosome I ( for a complete description of the familial content of GI see the Additional File 1 ) .

The staying eight venue showed a good anticipation value for flanking DR, with lengths that varied from 21 to 82 bp ( see Additional File 2 ) . The presence of DR was considered as index of instability since DR are musca volitanss for site- specific recombination events ( Figure 2, Additional file 2 ) . The anticipation consequences identified three putative GI antecedently reported by DNA microarray comparing among the six classical Brucellae species [ 11 ] Accordingly, the GI terminology was maintained ( Table 1 ) . The different GI are characterized by the type of sequence information that they carry. GI-1 codifications largely for conjectural proteins ( HP ) and mobility cistrons, such as a phage-like integrase and saccharase cistron. GI-2 includes the LPS wboA-wboB glycosiltransferases cistrons, a transcript of the IS711 transposase, several HP and a pseudogene putatively coding for the Omp25, an immunogenic protein, functional in B. melitensis [ 13 ] . Furthermore, GI-3, one of the largest ( of ~21 kb ) carry 28 ORF largely of HP and a protein with a TIR sphere [ 18 ] .

Although most of the GI identified were antecedently reported [ 19, 20 ] , the designation of flanking DR defined the boundaries of each GI. The chief features of the GI identified in B. abortus are summarized in Table I. GBAI1 comprises seven phage-related ORF including an active cistron for integrase.

GBAI2 matched with the antecedently reported wbk venue, involved in the biogenesis of LPS [ 12 ] . This GI is flanked by transposases and seaports eight ORF, six of which are related to LPS biogenesis. The GBAI4 venue encodes three ORF ( a transcriptional regulator, a putative amino acid transporter, and a little HP ) .

This venue is flanked by the interpolation sequence IS2020 and has already been considered a HGT acquisition [ 21 ] . The 2nd largest GI is GBAI5 with a length of 19.6 kilobit. This venue is peculiarly rich in information as it carries 14 ORF, a cistron for DNA fix enzyme XthA-2, a putative two-component system, a transcriptional regulator of the MerR household, a Cd transporter ( CadA ) , several HP and two ORF annotated as pseudogenes, one cryptography for an adhesin and the other for an extracellular Ser-protease. One putative unstable GI was identified in chromosome II, GBAII1.

This GI does non show a markedly deviated GC content and carries DR flanking sequences proposing its instability. Except for GBAI2 and GBAI5, all the GI described are found incorporate near to a transfer RNA cistrons ( Table 1 ) Searches carried out on GenBank showed that these GI are found entirely in brucellae.

Distribution of GI among mention strains

The genome sequences of representative strains of B. melitensis, B.

suis, B. abortus and B. ovis available at the Genebank, were scanned to place the location of the GI. Each GI was confirmed by long scope PCR ( Table 2 ) . All the GI were present in B. abortus 2308 and B. melitensis 16M. However, GI-1, GI-2 and GBAI4 were absent in B.

ovis 63/290, and GI-3 absent in B. suis 1330 ( informations non shown ) . , Of involvement is the determination of a PCR merchandise consistent with the presence of GBAI4 in the genome of the B. melitensis 16M strain. This GI is non included in the genome sequence deposited in GenBank for this strain [ 7 ] .

GI instability appraisal

The sensing of flanking DR and phage-like integrases encoded in the Brucella GI suggested that these venues may be unstable. In support of this possibility, long scope PCR was performed to observe both the complete GI and the chromosomal cicatrix left after deletion.

The consequences yielded non merely the expected GI amplicon, but besides a merchandise with a molecular size matching to the cicatrix ( informations non shown ) . To place the putative released GI, a PCR aiming the rejoining point of the chromosomal cicatrix was performed ( Figure 3A ) . Sequence analysis of PCR merchandises derived from cicatrixs, confirmed GI omissions ( Figure 3B ) . To corroborate the formation of circularized signifiers of GI, we carried out the CI sensing utilizing an opposite PCR scheme with primers braces oriented towards the right and left GI boundaries. As expected, a individual merchandise for each CI was obtained ( Figure 4A ) . In all instances, cloning and sequencing of these PCR merchandises showed that the CI maintain a transcript of the GI-flanking DR that confirm the circularisation A in writing representation of all CI detected is shown in Figure 4B.

Expression of integrase/recombinase cistrons

Comparative analysis of the GI venue indicates a similar cistron content and genomic organisation in all sequenced Brucella species Harmonizing to the notes, GI-1, GI-2, GI-3 and GBAI1 harbour a individual phage-like integrase/recombinase cistron ( int ) each, with the exclusion of the GI-1 of B. melitensis 16M that seems to encode an integrase of 82 aminoacid.

To find whether GI deletion is linked to the activity of the putative integrases, we performed rearward written text PCR analyses targeted the int cistrons. Using RNA extracted from three brucellae species grown under standard conditions, we detected integrase look for GI-2 and GI-3 but non for GI-1 and GBAI1 ( Figure 5A ) . This consequence suggests that there is variableness in the look degree of int cistrons.

This reading is supported by the fact that the GI-1 and GBAI-1 int merchandises were merely detected utilizing existent clip RT-PCR ( Figure 5B ) . Noteworthy, although the note of GI-1 int in B. melitensis 16M ( the BMEI0907, a HP ) predicts a protein a little protein and reciprocally oriented, a 400 bp amplicon was besides detected. Therefore, this experimental consequence demonstrates an mistake in the note of BMEI0907.

Discussion

Lateral transportation of GI is portion of the complex mechanism that determines the pathogenicity of bacteriums, depending on the ecological niche where the bacterium lives in.

The authoritative illustration is the GI-encoded attachment factors ( fimbriae ) of Escherichia coli which in the kidney and urinary piece of land contribute to the pathogenicity of the bacterium ; nevertheless, in the enteric vegetation these factors are non-pathogenic and E. coli becomes portion of the normal vegetations [ 22 ] . Therefore, unless alterations occur in the bacteria or the host that leads to pathogenicity, HGT events contribute to a successful symbiotic endurance.Our purpose was to seek for the GI in the genome of B.

abortus to analyze its part to genome stableness and pathogen physiology. We ab initio used a bioinformatic hunt based on the difference on GC content between the genomic island and the host DNA and the difference on genomic signature ( based on dinucleotide frequences values ) . Another criterium was the interpolation of the GI in a tRNA cistron and the presence of flanking DR. To avoid betterment, that is, the doomed of a GI due to its similarity in GC content and codon use to that of the host, we used all of the above mentioned standards for GI designation.Our consequences showed 16 conjectural GI in B. abortus of which, eight contained DR ( Fig 1 ) .

Among the GI lacking DR, three corresponded to ribosomal cistron bunchs. These cistrons show a typical codon use, different to the host DNA, suggestive of upregulated look. Ribosomal cistrons along with other cistrons e.g transcriptional factors and chaperones are often classified as “ foreign ” by compositional analysis techniques, but due to biological significance, it is improbable that they are capable to HGT [ 23, 24 ] . The other five GI might hold originated from antediluvian HGT which became stable sequences as consequence of mutants at GI boundaries or in cistrons related to DNA mobility ( 15 ) . Yet, some of these parts, i. e.

the virB venue and the cistrons for flagellar proteins, outer membrane protein, DNA fix enzymes and oxidation-reduction composite ( 27-39 ) , are indispensable to Brucella virulency. Likewise, the DR-flanked GI might be considered as recent acquisitions. In add-on, the sinteny displayed by these venues among brucellae suggests that HGT events must hold occurred before speciation.Brucella, like other intracellular parasites, exhibits considerable clonality.

Clonality, nevertheless, does non intend familial stableness and, in this work we describe 8 parts exposing instability. Three venue, GI-1, GI-2 and GI-3, matched GI reported antecedently [ 11 ] . Although they do non represent a prophage, GI-1 encodes phage related cistrons, and mutant analysis performed on several cistrons show a deficiency of engagement in virulency [ 25 ] . GI-2 carries two cistrons which participate in O-chain LPS biogenesis: wboA and wboB.

It has been shown that most cistrons in GI-3 encode HP and, with the exclusion of the integrase, are expressed [ 26 ] . Therefore far, merely one cistron in this island has been associated with pathogenicity: BruAb1_0274 or btp1, which presents a TIR sphere and is implicated in the downregulation of the NF-i?«B signaling cascade, avoiding the ripening of host dendritic cells [ 18 ] . GBAI1, like GI-1, encodes several phage-related cistrons and HP. Transposon mutagenesis identified an ORF of this island in B. suis ( BMEI1658 ) as indispensable for intracellular reproduction [ 27 ] . GBAI2 encodes six ( gmd, per, wbkB, wzm, wzt, wbkC ) of the 10 wbk cistrons necessary for LPS O-chain biogenesis and conveyance across the envelope. Interestingly, the sensing of the corresponding CI demonstrated the instability of a tandem of the six wbk cistrons in GBAI2. This CI besides included the 3 ‘ terminal interpolation sequence but non the flaking wbkA gen.

This cistron is besides indispensable for LPS biogenesis and it is found near to an IS711 transcript. Both, wbkA and IS711 are flanked by imperfect interpolation sequences incorporating a putative DR brace, proposing that the wbk venue has resulted from more than one acquisition event. The smooth LPS is a deciding virulency factor of B. abortus, B.

melitensis and B. suis, and the dissociation of the smooth bacteriums into unsmooth attenuated mutations missing the O-chain is a frequent event in vitro. Therefore, self-generated omissions of GI related to LPS might be involved in the outgrowth of unsmooth phenotype.On the other manus, GBAI4 encodes phage related cistrons, and both GBAI4 and GBAII1 have putative transporters.

These cistrons may be involved in metabolic fittingness inside the host. Finally, Wattam et gap [ 20 ] have reported the being of a putative island named SAR 1-17, which corresponds to the GBAI5 identified in this work. This island harbors cistrons with similarity to those encoding virulency factors such as a putative adhesin and extracellular peptidase. However, these cistrons are inactivated in B. abortus. One possibility is that these pseudogenes in GI venue of B.

abortus but non in B. melitensis relate to the differences in the pathobiology of these two species. In this context, those inactivations impacting conjectural virulency cistrons may stand for advantageous familial alterations that positively impact on a better version to a specific host. Further surveies with a big figure of strains would be necessary to measure if these cistrons are really pseudogenes derived from selective inactivations or events related to a familial decay of GI.Refering the instability, the experimental grounds suggests the deletion of the four GI encoding int cistrons is likely to be mediated by the blood relation integrases. Even though look of GI-3 int in B. abortus and B. melitensis grown on trypticase soy broth has non been detected before [ 26 ] , we demonstrated the look of the int cistrons of GI-2 and GI-3 by RT-PCR utilizing RNA from bacteriums grown under standard conditions.

On the other manus, the look of GI-1 and GBAI1 integrases could merely be detected by existent clip RT-PCR. These different degrees of look suggest that some integrases are expressed constitutively whereas others may be downregulated. Another interesting consequence was the designation of the misannotation for BMEI0907, the GI-1 integrase in B. melitensis. Automatic ORF anticipation by Glimmer utilizing the sequence of B.

melitensis 16M as templet showed an ORF located on the +3 reading frame embracing 1305 bp ( Additional File 3 ) . The cistron would be similar in both size an orientation to its B. abortus orthologue, as shown by the sensing of a PCR merchandise derived from complementary DNA with indistinguishable molecular size to the amplicons obtained for B. abortus and B. suis.

The look of int cistrons in other brucellae suggests that GI circularisation ( and omission ) may non be restricted to B. abortus genome.The instability of the four GI non encoding int cistrons should be explained by alternate mechanisms. Recombination between DR is a conserved mechanism that contributes to phenotypic diverseness in procaryotes [ 28 ] . It has been demonstrated that RecA catalyzes the homologous recombination between long DNA sections exposing sequence similarity [ 29 ] . GBAI4 is flanked by DR which are portion of IS2020. Thus, GBAI2 is flanked by several transcripts of degenerated IS.

Nevertheless, sequencing of PCR merchandises that include putative attP sites derived from the corresponding CI indicates site-specific recombination events. One possibility is that there is a similarity between flanking DR of unrelated islands such that offers recombination sites recognized by unrelated integrases, as reported in other bacteriums [ 29 ] . However, with the exclusion of GBAI5, sequence analyses of all DR of Brucella GI resulted negative for the presence of a similarity that could account for the necessary short canonical inverted repetitions sequences. In fact, the DR of GBAI5 island contains internal palindromic repetitions of 7 bp separate by 11 bp which resembles a point of recombination.

Interestingly, sequence analysis indicates that such DR belongs to Bru-RS repetition household reported antecedently as a hot-spot interpolation for IS711 component [ 30 ] . Further mutational analysis of int cistrons and quantitative surveies utilizing recA mutations will be necessary to find the specificity of Brucella integrases and the possible function of RecA in GI instability.From an evolutionary point of position, the genomic instability displayed by the GI explains several omissions reported among the genomes of the six different brucellae [ 11 ] . GI-1, GI-2 and GBAI4 are absent in B. Ovis and GI-3 is non present in B. suis and B. Canis.

Our work besides draws attending on the being of important fluctuations in the genomes of aggregation strains. We found that GBAI4 was present in the B. melitensis 16M strain conserved at SAG and besides detected the cicatrix ensuing from its deletion. This instability can account for the absence of the corresponding sequence in the 16M genome sequence available in GenBank, an observation that stresses the variableness of Brucella in vitro. Despite the life style that keeps the bacteriums restricted to an ecological niche genetically isolated from foreign DNA acquisitions, Brucella has the possible to circularise several GI as shown by the CI sensing.

GI circularisation has been reported in free life micro-organisms and it is considered the first measure in the possible horizontal extension, therefore lending to genome malleability and bacterial development [ 15, 31 ] . An challenging inquiry arises from the consequences ; what is the biological significance of GI circularisation in an intracellular parasite like Brucella, if any? Since the brucellae are non of course susceptible to transmutation and neither transduction nor junction have been reported [ 32 ] , it is likely the circularisation is non associated to HGT of GI. Probably, the selective force per unit area to keep GI is lessened under rich growing conditions, prefering the formation of CI. Quantitative experiments done on biological samples derived from infected animate beings will assist to find whether island deletion contributes to virulence fading in vivo.

Decisions

The consequences show that the intracellular pathogen Brucella carries several unstable GI. The instability displayed by the GI strongly suggests that HGT has been a procedure by which Brucella has gained infective traits.

More interestingly, genome malleability and putative selective inactivations within the GI found in B. abortus are in understanding with the thought that these parts can play a function in host specificity and version to an intracellular life style. Future surveies on conjectural cistrons with unknown maps carried on the GI will let clarifying their function in the pathogenicity of Brucella.

Methods

Bacterial strains and civilization conditions

All strains used in this survey are listed in Table 2. Bacterial civilizations were obtained by reassigning a loopfull of frozen glycerin stock ( conserved at 70°C ) to Brucella agar home bases and incubation for 48 hour at 37A°C. Liquid civilizations from a individual settlement were grown on 20 milliliter of Brucella stock, maintained for 24 hour at 37°C and 100 revolutions per minute until the stationary stage was reached.

For E. coli JM109 growing, Luria-Bertani stock ( LB ) , supplemented with Principen ( 100 Aµg/ml ) was used.

Bioinformatic scheme

Complete genome sequences in GenBank format for Brucella abortus 9-941 ( accession Numberss AE017223 and AE017224 ) were retrieved from NCBI and used to execute genomic island anticipation analysis utilizing the Artemis package [ 33 ] at hypertext transfer protocol: //www.sanger.ac.uk/Software/Artemis. Briefly, we used Karlin signature difference ( i?¤ a‰? 0.05 ) and GC % content secret plans to observe divergence extremums, seting to 10 kb the sliding window through full sequences.

Boundaries of these GI were defined by DR hunts utilizing the REputer web waiter ( http: //bibiserv.techfak.uni-bielefeld.de/reputer/submission.html ) with default values and FASTA sequence sections retrieved from the genome sequence [ 34 ] . Predicted flanking repetitions pairs with E values & lt ; 10-10 were considered probably putative GI boundaries. Oligonucleotides were designed sing 500-1000 bp upstream and downstream of theoretical bounds to magnify the full parts by long scope PCR.

Long elaboration merchandises were analyzed foremost by computational simulation of limitation digestion, and so confirmed by experimentation. PCR merchandises from chromosomal junction ( which include the attB site ) of GI were cloned and sequenced. Because GI show built-in instability mediated by a site-specific recombination mechanism and are frequently found transiently excised from the chromosome as CI, putative CI were simulated utilizing Vector NTI package [ 35 ] ( available at Invitrogen website hypertext transfer protocol: //www.invitrogen.com ) , taking history predicted flanking DR as cutting and rejoining points ( attL and attR sites ) .

These round sequences were used as templets for primer design to magnify a specific section generated by deletion mechanism ( the attP site ) .

Deoxyribonucleic acid extraction and PCR analysis

Genomic DNA was extracted from heat inactivated ( 30 min at 90°C ) pure cultures utilizing lysozime-SDS-proteinase K lysis, followed by DNA-CTAB extraction and isopropanol precipitation [ 36 ] . To find the presence of GI, long scope PCR checks were performed in a concluding volume of 25 ul incorporating 25-50 nanogram of gDNA, 0.4 millimeter dNTPs, 12.5 pmol primer and 1 U of Long Range Enzyme Mix ( Fermentas ) . The PCR conditions were: initial denaturation at 95A°C for 1 min ; 30 rhythms of denaturation at 95A°C for 20 sec ; tempering at 60A°C for 30 sec ; extension at 68A°C for 1 min/kb merchandise ; concluding extension measure of 10 min at 68A°C. PCR were followed by limitation analysis to corroborate the merchandise individuality.

GI instability appraisal was carried out by sensing of chromosomal junctions and CI, utilizing conventional PCR check in a concluding volume of 25 ul incorporating 0.5-1.0 Aµg of gDNA, 10 pmol of each primer, 0.2 millimeter of dNTPs, 2 millimeter of MgCl2 and 1U of Taq Platinum ( Invitrogen ) . For this PCR, the conditions were: pre-incubation at 95A°C ; 30 rhythms at 95A°C for 20 sec: 60A°C for 30 sec ; and 72A°C for 1 min with a concluding extension at 72A°C for 5 min. To corroborate individuality, PCR merchandises from chromosomal junctions and CI-derived amplicons were purified and cloned in pGEMT-easy vector ( Promega ) and sequenced ( Macrogen ) .

All PCR unit of ammunitions were amplified in a GeneAmp PCR System 9700 ( Applied Biosystems ) . Amplicons and limitation fragments were resolved by cataphoresis on 1.0-2.0 % TAE agarose gels.

All primers used are listed in Additional File 4.

RNA extraction and RT-PCR analysis of int cistrons

Entire RNA was prepared with bacteriums harvested from liquid civilizations utilizing RNA purification system ( Qiagen ) . Following RNA isolation, samples were treated with DNAse free RNAse Turbo ( Ambion ) and the output was quantified spectrophotometrically. After that, 100 nanogram of entire RNA were rearward transcribed to bring forth complementary DNA utilizing Transcriptor RT ( Roche ) with random hexamers at 55A°C for 30 min.

PCR elaboration unit of ammunitions were performed on 2 Aµl of complementary DNA templet utilizing 12.5 pmol of forward and contrary primers of each int gen in detached experiments with 0.4 millimeters dNTPs, 2 millimeter MgCl2, Buffer 1x and 1 U of Taq Platinum in a concluding volume of 25 Aµl. Samples were incubated at 95A°C for 2 min followed by 30 rhythms of 95A°C for 20 sec, 60A°C for 30 sec and 72A°C for 30 sec with a concluding extension for 5 min at 72A°C.

RT-PCR unit of ammunitions were besides performed in a GeneAmp 9700 thermocycler ( Applied Biosystems ) .For int cistrons that were non detected by the conventional attack, existent clip PCR unit of ammunitions were performed on a LightCycler 1.5 PCR machine ( Roche ) utilizing the same sum of RNA samples, 0.3 i?­M of forward and contrary primers, MgCl2 4 millimeter, 1 i?­l of LightCycler FastStart DNA Master SYBR Green I mix and PCR grade H2O in a concluding volume of 20 i?­l. After enzyme activation for 10 min at 95A°C, 45 rhythms ( denaturation at 95A°C for 8 sec, tempering at 65A°C at 8 sec and extension at 72A°C for 20 sec ) were performed to magnify a specific fragment derived from int complementary DNA.

Then the runing curve of PCR merchandises was analyzed with LightCycler3 package to verify PCR specificity. Data was acquired by heating the amplicons to 95A°C for 1 sec, 15 sec at 70A°C and so easy heating at 0.1A°C/sec to 99A°C under uninterrupted fluorescence monitoring. Negative control without DNA was included for each unit of ammunition.

Writers ‘ parts

MM conceived the survey, participated in its design, accomplished computational analysis, carried out PCR checks, performed RT-PCR of integrases and drafted the manuscript. AMZ participated in the design, coordination and fiscal support of the survey and helped to outline the manuscript.

Recognitions

We are thankful to Minie Villarroel from Servicio Agricola Y Ganadero, SAG, for providing bacterial strains. We thank besides to Dr. Ignacio Moriyon from Universidad de Navarra for his valuable aid to outline the manuscript. This work was supported by doctorial thesis undertaking D-2005-17 from Direccion de Investigacion y Desarrollo, Universidad Austral de Chile and FONDEF D02I1111 grant.

MM was the receiver of a doctorial family from CONICYT-Chile.