Análisis de la Estrategia de Internacionalización de MANGO

Màster Oficial d'Internacionalització, Facultat d'Economia i Empresa, Universitat de Barcelona, Curs: 2016-2018, Tutor: Fariza Achcaoucaou IallouchenEl presente trabajo analiza el proceso de internacionalización del grupo textil MANGO, una multinacional dedicada al diseño, fabricación, distribución y comercialización de prendas de vestir y complementos. En la actualidad, es la segunda empresa exportadora del sector textil español y una de las empresas textiles con mayor proyección internacional, estando presente en más de 100 países. Su rápida expansión se debe a los modelos de negocio y estrategias aplicadas, principalmente mediante dos vías de acceso al mercado: la franquicia y la tienda propia. Uno de los puntos clave para su crecimiento ha sido la adaptación de su producto, de calidad media-alta, a las necesidades de cada mercado, junto con el reforzamiento de la imagen de la marca mediante su política de marketing internacional. El objetivo de este trabajo, por tanto, es analizar todo lo comentado, determinar las claves de éxito de la compañía y profundizar en la situación y perspectivas de crecimiento internacional por áreas geográficas

Systematic Production of Inactivating and NonInactivating Suppressor Mutations at the relA Locus That Compensate the Detrimental Effects of Complete spoT Loss and Affect Glycogen Content in Escherichia coli

In Escherichia coli, ppGpp is a major determinant of growth and glycogen accumulation. Levels of this signaling nucleotide are controlled by the balanced activities of the ppGpp RelA synthetase and the dual-function hydrolase/synthetase SpoT. Here we report the construction of spoT null (DspoT) mutants obtained by transducing a DspoT allele from DrelADspoT double mutants into relA+ cells. Iodine staining of randomly selected transductants cultured on a rich complex medium revealed differences in glycogen content among them. Sequence and biochemical analyses of 8 DspoT clones displaying glycogen-deficient phenotypes revealed different inactivating mutations in relA and no detectable ppGpp when cells were cultured on a rich complex medium. Remarkably, although the co-existence of DspoT with relA proficient alleles has generally been considered synthetically lethal, we found that 11 DspoT clones displaying high glycogen phenotypes possessed relA mutant alleles with non-inactivating mutations that encoded stable RelA proteins and ppGpp contents reaching 45–85% of those of wild type cells. None of the DspoT clones, however, could grow on M9-glucose minimal medium. Both Sanger sequencing of specific genes and high-throughput genome sequencing of the DspoT clones revealed that suppressor mutations were restricted to the relA locus. The overall results (a) defined in around 4 nmoles ppGpp/g dry weight the threshold cellular levels that suffice to trigger net glycogen accumulation, (b) showed that mutations in relA, but not necessarily inactivating mutations, can be selected to compensate total SpoT function(s) loss, and (c) provided useful tools for studies of the in vivo regulation of E. coli RelA ppGpp synthetaseFil: Montero, Manuel. Gobierno de Navarra. Instituto de Agrobiotecnología; EspañaFil: Rahimpour, Mehdi. Gobierno de Navarra. Instituto de Agrobiotecnología; EspañaFil: Viale, Alejandro Miguel. Gobierno de Navarra. Instituto de Agrobiotecnología; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Almagro, Goizeder. Gobierno de Navarra. Instituto de Agrobiotecnología; EspañaFil: Eydallin, Gustavo. Gobierno de Navarra. Instituto de Agrobiotecnología; EspañaFil: Sevilla, Angel. Universidad de Murcia; EspañaFil: Canovas, Manuel. Universidad de Murcia; EspañaFil: Bernal, Cristina. Universidad de Murcia; EspañaFil: Lozano, Ana Belen. Universidad de Murcia; EspañaFil: Muñoz, Francisco Jose. Gobierno de Navarra. Instituto de Agrobiotecnología; EspañaFil: Bora Fernandez, Edurne. Gobierno de Navarra. Instituto de Agrobiotecnología; EspañaFil: Bahaji, Abdellatif. Gobierno de Navarra. Instituto de Agrobiotecnología; EspañaFil: Mori, Hirotada. Nara Institute of Science and Technology. Graduate School of Biological Sciences; JapónFil: Codoñer, Francisco M.. Lifesequencing SL. Valencia; EspañaFil: Potueza Romeo, Javier. Gobierno de Navarra. Instituto de Agrobiotecnología; Españ

The lack of GlnB and YejB compensates the detrimetal effect of spoT deletion in E. coli cells expressing wild type relA

Trabajo presentado en el XXXVII Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celbrado en Granada del 9 al 12 de septiembre de 2014.In Escherichia coli, intracellular ppGpp content in response to nutritional deficiency is controlled by the balanced action of RelA (synthetase I) and the dual-function (synthetase/hydrolase) SpoT. Generally ascribed to the detrimental effects of high (p)ppGpp levels, the co-existence of spoT null (DspoT) with relA proficient alleles has been considered synthetically lethal. However, in a recent work we have reported the construction of DspoT mutants in a relA+background accumulating nearly wild type (WT) ppGpp levels when cells were cultured on a rich complex medium. Sequencing of the genomes from various selected DspoT clones identified in all of them suppressor mutations located in relA. In two of them, additional inactivating mutations were found in glnB and yejB, two genes that have not been characterized as genetically linked to relA. To know whether mutations resulting in the total abolishment of the glnB and yejB genes could compensate the detrimental effects of spoT deletions in E. coli in this work we obtained multiple independent DspoTDglnB and DspoTDyejB clones. Importantly, these clones expressed WT RelA, displayed a nearly WT growth phenotype, and accumulated nearly WT ppGpp and glycogen contents when cultured in a rich complex medium. None of these mutants, however, could grow on glucose minimal medium. The overall data (a) show that different mutations other than in relA can be selected in E. coli cells compensating the detrimental effects of spoT deletion, and (b) point to the occurrence in E. coli of mechanism(s), other than SpoT-mediated ppGpp hydrolytic breakdown, that prevent ppGpp over-accumulation. To our knowledge this is the first report describing the production of spoT null mutants of E. coli expressing WT RelA.Peer Reviewe

Glycogen contents and expression of chromosomal <i>glgB::lacZY</i> fusions in the 19 selected Δ<i>spoT</i> clones.

<p>Glycogen content and expression of chromosomal <i>glgB::lacZY</i> fusion at the stationary phase (after 7 h of culture) in WT cells, and in the 19 selected Δ<i>spoT</i> clones cultured in KM. Data are referred to as percentage of glycogen measured in WT cells. In “A”, average glycogen content in WT cells was equivalent to 257 nmol glucose/mg protein. In “B”, average level of <i>glgB::lacZY</i> expressions in WT cells was 312 Miller units <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106938#pone.0106938-Miller1" target="_blank">[31]</a>. The results are the mean ± SE of 3 independent experiments.</p

ppGpp contents in WT cells and in the 19 selected Δ<i>spoT</i> clones.

<p>Cells were cultured in liquid KM and harvested (A) at the stationary phase (after 7 h of culture) or (B) at the exponential growth phase (after 100 min of culture). The results are the mean ± SE of 3 independent experiments. Growth curves of WT and four representative Δ<i>spoT</i> clones are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106938#pone-0106938-g005" target="_blank"><b>Figure 5A</b></a>.</p

Schematic representation and sequence characterization of the different <i>relA</i> mutations identified in the Δ<i>spoT</i> clones studied here.

<p>A. The designation of each Δ<i>spoT</i> clone characterized in this work is indicated below the <i>E. coli relA</i> gene representation. The RelA region encoding the N-terminal 455 amino acid residues bearing ppGpp synthetase activity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106938#pone.0106938-Cashel1" target="_blank">[9]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106938#pone.0106938-Gropp1" target="_blank">[45]</a> is shadowed. B. Amino acid sequence comparisons of the N-terminal regions (∼400 amino acid residues) of <i>S. equisimilis</i> Rsh, <i>E. coli</i> RelA, and SpoT. The alignments of the corresponding regions were constructed by using ClustalW (<a href="http://www.genome.jp/tools/clustalw" target="_blank">http://www.genome.jp/tools/clustalw</a>) using default parameters, and subsequently refined to maximize secondary structure similarities between <i>S. equisimilis</i> Rsh <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106938#pone.0106938-Hogg1" target="_blank">[46]</a> and RelA (as predicted by Jpred3; <a href="http://www.compbio.dundee.ac.uk" target="_blank">http://www.compbio.dundee.ac.uk</a>). The corresponding amino acid sequence positions are indicated at the right. Identical (*) or conserved (:) amino acids at a given position among the three sequences are indicated below the alignments, and deletions/insertions by hyphens (−). The sequence regions spanning the ppGpp hydrolase (⌜ ⌝) and synthetase (⌜ ⌝) domains of <i>S. equisimilis</i> Rsh, as well as the central-3 helix bundle region joining these domains defined by the ⌝ and boundaries <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106938#pone.0106938-Hogg1" target="_blank">[46]</a> are indicated above the alignments. The different amino acid substitutions and the Trp39 deletion (Δ) identified in <i>E. coli</i> RelA in Δ<i>spoT</i> clones 8–19 (this work) are indicated below the alignments. Conservation of equivalent residues in <i>S. equisimilis</i> Rsh and <i>E. coli</i> SpoT is indicated by highlighting the corresponding amino acids in bold. Conserved residues whose site-directed mutagenesis has been found by other authors to promote loss (G251E) or reductions (H354Y) in <i>E. coli</i> RelA ppGpp synthesis capability <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106938#pone.0106938-Gropp1" target="_blank">[45]</a> are indicated by closed arrowheads below the alignments. The HDXXED motif typical of metal-dependent pyrophosphohydrolases located in the hydrolase domain of bifunctional enzymes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106938#pone.0106938-Hogg1" target="_blank">[46]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106938#pone.0106938-Sajish1" target="_blank">[55]</a> is underlined. In turn, the synthetase domain motif distinguishing monofunctional (EXDD) from bifunctional (RFKD) enzymes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106938#pone.0106938-Hogg1" target="_blank">[46]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106938#pone.0106938-Sajish1" target="_blank">[55]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106938#pone.0106938-Sajish2" target="_blank">[56]</a> is double underlined. The sources of the sequences were: <i>S. equisimilis</i> Rsh, ref. 46; <i>E. coli</i> K-12 MG1655 SpoT, GenBank accession NP_418107.1; <i>E. coli</i> RelA BW25113: this work. The latter is identical to <i>E. coli</i> K-12 MG1655 RelA, GenBank accession NP_417264.1.</p

Growth behavior of Δ<i>relA</i> single mutants, Δ<i>relA</i>Δ<i>spoT</i> double mutants, and four representative Δ<i>spoT</i> clones cultured in M9-glucose medium.

<p>Growth in glucose-containing M9 minimal medium of Δ<i>relA</i> and Δ<i>relA</i>Δ<i>spoT</i> mutants as well as of representative ppGpp-less (3 and 7) and ppGpp-containing (9 and 11) Δ<i>spoT</i> clones. All Δ<i>spoT</i> cells tested were transformed with the empty plasmid vector pCA24N-EV or with pCA24N-<i>spoT</i> expressing the cloned <i>spoT</i> gene (p-<i>spoT</i>) as indicated in the inset. Aliquots were taken at the indicated times for Abs₆₀₀ measurements. For details see Materials and Methods.</p

Kinetics of cell growth, glycogen accumulation, and expression of chromosomal <i>glgB::lacZY</i> fusions in representative ppGpp-less and ppGpp-containing Δ<i>spoT</i> clones cultured in liquid KM.

<p>Time-course of (A) cell growth, (B) expression of chromosomal <i>glgB::lacZY</i> fusions, and (C) glycogen accumulation of WT cells and representative Δ<i>spoT</i> clones displaying either no detectable ppGpp (clones 3 and 7) or significant ppGpp accumulation (clones 9 and 11).</p

<i>E. coli</i> strains used in this study.

<p>Spc^R, spectinomycin resistance. Km^R, kanamycin resistance. Δ<i>spoT</i> clones that were subjected to high-throughput genome sequencing are indicated in <b>bold.</b></p><p><i>E. coli</i> strains used in this study.</p