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Ítem Acceso Abierto HrpE, the major component of the Xanthomonas type three protein secretion pilus, elicits plant immunity responses(Springer Nature, 2018-06-29) Gottig, Natalia; Vranych, Cecilia Verónica; Sgro, Germán Gustavo; Piazza, Ainelén; Ottado, JorgelinaLike several pathogenic bacteria, Xanthomonas infect host plants through the secretion of effector proteins by the Hrp pilus of the Type Three Protein Secretion System (T3SS). HrpE protein was identified as the major structural component of this pilus. Here, using the Xanthomonas citri subsp. citri (Xcc) HrpE as a model, a novel role for this protein as an elicitor of plant defense responses was found. HrpE triggers defense responses in host and non-host plants revealed by the development of plant lesions, callose deposition, hydrogen peroxide production and increase in the expression levels of genes related to plant defense responses. Moreover, pre-infiltration of citrus or tomato leaves with HrpE impairs later Xanthomonas infections. Particularly, HrpE C-terminal region, conserved among Xanthomonas species, was sufficient to elicit these responses. HrpE was able to interact with plant Glycine-Rich Proteins from citrus (CsGRP) and Arabidopsis (AtGRP-3). Moreover, an Arabidopsis atgrp-3 knockout mutant lost the capacity to respond to HrpE. This work demonstrate that plants can recognize the conserved C-terminal region of the T3SS pilus HrpE protein as a danger signal to defend themselves against Xanthomonas, triggering defense responses that may be mediated by GRPs.Ítem Acceso Abierto The surfactin-like lipopeptides from Bacillus spp.: natural biodiversity and synthetic biology for a broader application range(Frontiers, 2021-03-02) Théatre, Ariane; Cano-Prieto, Carolina; Bartolini, Marco; Laurin, Yoann; Deleu, Magalí; Niehren, Joachim; Fida, Tarik; Gerbinet, Saïcha; Alanjary, Mohammad; Medema, Marnix H.; Léonard, Angélique; Lins, Laurence; Arabolaza, Ana Lorena; Gramajo, Hugo Cesar; Gross, Harald; Jacques, PhilippeSurfactin is a lipoheptapeptide produced by several Bacillus species and identified for the first time in 1969. At first, the biosynthesis of this remarkable biosurfactant was described in this review. The peptide moiety of the surfactin is synthesized using huge multienzymatic proteins called NonRibosomal Peptide Synthetases. This mechanism is responsible for the peptide biodiversity of the members of the surfactin family. In addition, on the fatty acid side, fifteen different isoforms (from C12 to C17) can be incorporated so increasing the number of the surfactin-like biomolecules. The review also highlights the last development in metabolic modeling and engineering and in synthetic biology to direct surfactin biosynthesis but also to generate novel derivatives. This large set of different biomolecules leads to a broad spectrum of physico-chemical properties and biological activities. The last parts of the review summarized the numerous studies related to the production processes optimization as well as the approaches developed to increase the surfactin productivity of Bacillus cells taking into account the different steps of its biosynthesis from gene transcription to surfactin degradation in the culture medium.Ítem Acceso Abierto Host-specific enzyme-substrate interactions in SPM-1 metallo-β-lactamase are modulated by second sphere residues(Public Library of Science (PLOS), 2014-01-02) González, Lisandro Javier; Moreno, Diego M.; Bonomo, Robert A.; Vila, Alejandro J.Pseudomonas aeruginosa is one of the most virulent and resistant non-fermenting Gram-negative pathogens in the clinic. Unfortunately, P. aeruginosa has acquired genes encoding metallo-β-lactamases (MβLs), enzymes able to hydrolyze most β-lactam antibiotics. SPM-1 is an MβL produced only by P. aeruginosa, while other MβLs are found in different bacteria. Despite similar active sites, the resistance profile of MβLs towards β-lactams changes from one enzyme to the other. SPM-1 is unique among pathogen-associated MβLs in that it contains “atypical” second sphere residues (S84, G121). Codon randomization on these positions and further selection of resistance-conferring mutants was performed. MICs, periplasmic enzymatic activity, Zn(II) requirements, and protein stability was assessed. Our results indicated that identity of second sphere residues modulates the substrate preferences and the resistance profile of SPM-1 expressed in P. aeruginosa. The second sphere residues found in wild type SPM-1 give rise to a substrate selectivity that is observed only in the periplasmic environment. These residues also allow SPM-1 to confer resistance in P. aeruginosa under Zn(II)-limiting conditions, such as those expected under infection. By optimizing the catalytic efficiency towards β-lactam antibiotics, the enzyme stability and the Zn(II) binding features, molecular evolution meets the specific needs of a pathogenic bacterial host by means of substitutions outside the active site.Ítem Acceso Abierto Genomic comparative analysis of the environmental Enterococcus mundtii against enterococcal representative species(BMC, 2014-06-18) Repizo, Guillermo Daniel; Espariz, Martín; Blancato, Víctor Sebastián; Suárez, Cristian Alejandro; Esteban, Luis; Magni, ChristianBackground Enterococcus mundtii is a yellow-pigmented microorganism rarely found in human infections. The draft genome sequence of E. mundtii was recently announced. Its genome encodes at least 2,589 genes and 57 RNAs, and 4 putative genomic islands have been detected. The objective of this study was to compare the genetic content of E. mundtii with respect to other enterococcal species and, more specifically, to identify genes coding for putative virulence traits present in enterococcal opportunistic pathogens. Results An in-depth mining of the annotated genome was performed in order to uncover the unique properties of this microorganism, which allowed us to detect a gene encoding the antimicrobial peptide mundticin among other relevant features. Moreover, in this study a comparative genomic analysis against commensal and pathogenic enterococcal species, for which genomic sequences have been released, was conducted for the first time. Furthermore, our study reveals significant similarities in gene content between this environmental isolate and the selected enterococci strains (sharing an “enterococcal gene core” of 805 CDS), which contributes to understand the persistence of this genus in different niches and also improves our knowledge about the genetics of this diverse group of microorganisms that includes environmental, commensal and opportunistic pathogens. Conclusion Although E. mundtii CRL1656 is phylogenetically closer to E. faecium, frequently responsible of nosocomial infections, this strain does not encode the most relevant relevant virulence factors found in the enterococcal clinical isolates and bioinformatic predictions indicate that it possesses the lowest number of putative pathogenic genes among the most representative enterococcal species. Accordingly, infection assays using the Galleria mellonella model confirmed its low virulence.Ítem Acceso Abierto Staring at the cold sun: blue light regulation is distributed within the genus acinetobacter(Public Library of Science (PLOS), 2013-01-24) Golic, Adrián Ezequiel; Vaneechoutte, Mario; Nemec, Alexandr; Viale, Alejandro M.; Actis, Luis A.; Mussi, María AlejandraWe previously showed that the opportunistic nosocomial pathogen Acinetobacter baumannii is able to sense and respond to light via BlsA, a BLUF (Blue-Light-sensing Using FAD)-domain photoreceptor protein. Here, we extend our previous studies showing that light regulation is not restricted to A. baumannii, but rather widespread within the genus Acinetobacter. First, we found that blue light modulates motility and biofilm formation in many species of the genus, including members of the Acinetobacter calcoaceticus-A. baumannii complex. In many of these species blue light acts as a key factor guiding the decision between motility or sessility at 24°C, whereas in A. baumannii, light inhibits both motility and biofilm formation. We also show that light regulation of motility occurred not only at 24°C but also at 37°C in non-A. baumannii species, contrasting the situation of A. baumannii which only shows photoregulation at 24°C. Second, we show that Acinetobacter baylyi (strain ADP1) BLUF-photoreceptors can functionally replace in vivo the A. baumannii 17978 BlsA protein and that the pathways leading to biofilm formation are inversely regulated at 24°C between these two microorganisms. Finally, we found the presence of predicted genes coding BLUF-containing proteins in all Acinetobacter sequenced genomes, even though the copy number is variable among them. Phylogenetic analysis suggests a common origin for all BLUF domains present in members of this genus, and could distinguish well-differentiated clusters that group together BLUF homologs from different species, a situation particularly clear for members of the ACB complex. Despite a role played by these BLUF domain-containing proteins in the photoregulation observed in the members of the genus Acinetobacter is a likely scenario given our findings in A. baumannii and A. baylyi, further research will contribute to confirm this possibility.Ítem Acceso Abierto Transcriptional regulation of adhesive properties of Bacillus subtilis to extracellular matrix proteins through the fibronectin-binding protein YloA(John Wiley & Sons Ltd, 2017-03-27) Rodríguez Ayala, Facundo; Bauman, Carlos; Bartolini, Marco; Saball, Ester; Salvarrey, Marcela; Leñini, Cecilia; Cogliati, Sebastián; Strauch, Mark; Grau, Roberto Ricardo; http://orcid.org/0000-0002-6496-3696Bacterial adherence to extracellular matrix proteins (ECMp) plays important roles during host–pathogen interaction, however its genetic regulation remains poorly understood. yloA of the model bacterium Bacillus subtilis shows high homology to genes encoding fibronectin-binding proteins of Gram-positive pathogens. Here, we characterized the regulatory network of YloA-dependent adhesive properties of the probiotic B. subtilis natto (Bsn). YloA-proficient, but not YloA-deficient, Bsn specifically bound to ECMp in a concentration-dependent manner and were proficient in biofilm formation. yloA expression showed a continuous increase in activity during the growth phase and decreased during the stationary phase. The transcription factors AbrB and DegU downregulated yloA expression during the logarithmic and stationary growth phases respectively. Analysis of the yloA promoter region revealed the presence of AT-rich direct and inverted repeats previously reported to function as DegU-recognized binding sites. In spo0A cells, yloA expression was completely turned off because of upregulation of AbrB throughout growth. Accordingly, DNase I footprinting analysis confirmed that AbrB bound to the promoter region of yloA. Interestingly, Bsn bound fibronectin with higher affinity, lower Kd, than several bacterial pathogens and competitively excluded them from binding to immobilized-fibronectin, a finding that might be important for the anti-infective properties of B. subtilis and its relatives.Ítem Acceso Abierto Overexpression of Trypanosoma cruzi high mobility group B protein (TcHMGB) alters the nuclear structure, impairs cytokinesis and reduces the parasite infectivity(Springer Nature, 2019-01-17) Tavernelli, Luis Emilio; Motta, María Cristina M.; Silva Gonçalves, Camila; Santos da Silva, Marcelo; Elías, María Carolina; Alonso, Victoria Lucía; Serra, Esteban Carlos; Cribb, Pamela; Dr. De Gaudenzi, Javier: provide the pTcINDEX-eGFP strainKinetoplastid parasites, included Trypanosoma cruzi, the causal agent of Chagas disease, present a unique genome organization and gene expression. Although they control gene expression mainly post-transcriptionally, chromatin accessibility plays a fundamental role in transcription initiation control. We have previously shown that High Mobility Group B protein from Trypanosoma cruzi (TcHMGB) can bind DNA in vitro. Here, we show that TcHMGB also acts as an architectural protein in vivo, since the overexpression of this protein induces changes in the nuclear structure, mainly the reduction of the nucleolus and a decrease in the heterochromatin:euchromatin ratio. Epimastigote replication rate was markedly reduced presumably due to a delayed cell cycle progression with accumulation of parasites in G2/M phase and impaired cytokinesis. Some functions involved in pathogenesis were also altered in TcHMGB-overexpressing parasites, like the decreased efficiency of trypomastigotes to infect cells in vitro, the reduction of intracellular amastigotes replication and the number of released trypomastigotes. Taken together, our results suggest that the TcHMGB protein is a pleiotropic player that controls cell phenotype and it is involved in key cellular processes.Ítem Acceso Abierto Expression of the agmatine deiminase pathway in Enterococcus faecalis is activated by the AguR regulator and repressed by CcpA and PTS(Man) systems(Public Library of Science, 2013-10-14) Suárez, Cristian Alejandro; Espariz, Martín; Blancato, Víctor Sebastián; Magni, ChristianAlthough the agmatine deiminase system (AgDI) has been investigated in Enterococcus faecalis, little information is available with respect to its gene regulation. In this study we demonstrate that the presence of exogenous agmatine induces the expression of agu genes in this bacterium. In contrast to the homologous and extensively characterized AgDI system of S. mutants, the aguBDAC operon in E. faecalis is not induced in response to low pH. In spite of this, agmatine catabolism in this bacterium contributes by neutralizing the external medium while enhancing bacterial growth. Our results indicate that carbon catabolic repression (CCR) operates on the AgDI system via a mechanism that involves interaction of CcpA and PSer-HPr with a cre site found in an unusual position considering the aguB promoter (55 nt upstream the +1 position). In addition, we found that components of the mannose phosphotransferase (PTSMan) system also contributed to CCR in E. faecalis since a complete relief of the PTS-sugars repressive effect was observed only in a PTSMan and CcpA double defective strain. Our gene context analysis revealed that aguR is present in oral and gastrointestinal microorganisms. Thus, regulation of the aguBDAC operon in E. faecalis seems to have evolved to obtain energy and resist low pH conditions in order to persist and colonize gastrointestinal niches.Ítem Acceso Abierto Revisiting the cell biology of the acyl-ACP:phosphate transacylase PlsX suggests that the phospholipid synthesis and cell division machineries are not coupled in Bacillus subtilis(Wiley, 2016-05-06) Sastre, Diego Emiliano; Bisson-Filho, Alexandre; De Mendoza, Diego; Gueiros-Filho, Frederico J.PlsX is a central enzyme of phospholipid synthesis in bacteria, converting acyl-ACP to acyl-phosphate on the pathway to phosphatidic acid formation. PlsX has received attention because it plays a key role in the coordination of fatty acid and phospholipid synthesis. Recently, PlsX was also suggested to coordinate membrane synthesis with cell division in Bacillus subtilis. Here, we have re-investigated the cell biology of PlsX and determined that the enzyme is uniformly distributed on the membrane of most cells, but occasionally appears as membrane foci as well. Foci and homogenous patterns seem freely interconvertible but the prevalence of the uniform staining suggests that PlsX does not need to localize to specific sites to function correctly. We also investigated the relationship between PlsX and the divisome. In contrast to previous observations, PlsX's foci showed no obvious periodicity of localization and did not colocalize with the divisome. Furthermore, depletion of PlsX did not affect cell division if phospholipid synthesis is maintained by an alternative enzyme. These results suggest that coordination between division and membrane synthesis may not require physical or functional interactions between the divisome and phospholipid synthesis enzymes.Ítem Acceso Abierto CRISPR-based platform for carbapenemases and emerging viruses detection using Cas12a (Cpf1) effector nuclease(Taylor & Francis, 2020-06-02) Curti, Lucía Ana; Pereyra Bonnet, Federico; Repizo, Guillermo Daniel; Fay, Jessica Vannina; Salvatierra, Karina; Blariza, María José; Ibáñez Alegre, Macarena; Rinflerch, Adriana Raquel; Miretti, Marcos; Giménez, Carla AlejandraCRISPR-Cas12a (also called Cpf1) has been commonly used for genomic editing, based on its ability to generate precise double-stranded DNA (dsDNA) breaks. Recently, it was demonstrated that Cas12a exhibits unspecific ssDNAse activity upon target recognition. This feature allows CRISPR-Cas to be coupled with a ssDNA reporter and generate a fast, accurate and ultrasensitive molecular detection method. Here, we demonstrate that Cas12a was able to detect DNA target sequences corresponding to carbapenemases resistance genes such as KPC, NDM and OXA. Also, with the addition of a reverse-transcription step, we were able to detect viral RNA sequences from DENV, ZIKV and HANTV genomes. In all cases, assay run time was less than two hours. Additionally, we report attomolar levels of detection. This methodology was validated using clinical samples from patients infected with Dengue virus. Reactions were visualized by detection of a fluorescent signal, as well as by the use of a simple lateral flow strip. These results indicate that Cas12a is able to detect both DNA and RNA targets, making it an appropriate and convenient tool to detect all types of pathogens.Ítem Acceso Abierto MapB, the Brucella suis TamB homologue, is involved in cell envelope biogenesis, cell division and virulence(Springer Nature, 2019-02-15) Bialer, Magalí Graciela; Ruiz-Ranwez, Verónica; Sycz, Gabriela; Estein, Silvia Marcela; Russo, Daniela Marta; Altabe, Silvia Graciela; Sieira, Rodrigo; Zorreguieta, Ángeles; De Bolle, Xavier: provide the anti-OMPs and anti-LPS antibodies; Ugalde, Juan: provide the anti-OMPs and anti-LPS antibodies; Cassataro, Juliana: provide the anti-OMPs and anti-LPS antibodies; Spera, Juan Manuel: provide the PQE31-3xFLAG plasmid; Bravo, Marta: DNA sequencingBrucella species are Gram-negative, facultative intracellular pathogens responsible for a worldwide zoonosis. The envelope of Brucella exhibits unique characteristics that make these bacteria furtive pathogens and resistant to several host defence compounds. We have identified a Brucella suis gene (mapB) that appeared to be crucial for cell envelope integrity. Indeed, the typical resistance of Brucella to both lysozyme and the cationic lipopeptide polymyxin B was markedly reduced in a ∆mapB mutant. MapB turned out to represent a TamB orthologue. This last protein, together with TamA, a protein belonging to the Omp85 family, form a complex that has been proposed to participate in the translocation of autotransporter proteins across the outer membrane (OM). Accordingly, we observed that MapB is required for proper assembly of an autotransporter adhesin in the OM, as most of the autotransporter accumulated in the mutant cell periplasm. Both assessment of the relative amounts of other specific outer membrane proteins (OMPs) and a proteome approach indicated that the absence of MapB did not lead to an extensive alteration in OMP abundance, but to a reduction in the relative amounts of a protein subset, including proteins from the Omp25/31 family. Electron microscopy revealed that ∆mapB cells exhibit multiple anomalies in cell morphology, indicating that the absence of the TamB homologue in B. suis severely affects cell division. Finally, ∆mapB cells were impaired in macrophage infection and showed an attenuated virulence phenotype in the mouse model. Collectively, our results indicate that the role of B. suis TamB homologue is not restricted to participating in the translocation of autotransporters across the OM but that it is essential for OM stability and protein composition and that it is involved in cell envelope biogenesis, a process that is inherently coordinated with cell division.Ítem Acceso Abierto The role of cell-envelope synthesis for envelope growth and cytoplasmic density in Bacillus subtilis(Oxford University Press, 2022-07-26) Kitahara, Yuki; Oldewurtel, Enno R.; Wilson, Sean; Sun, Yingjie; Altabe, Silvia Graciela; De Mendoza, Diego; Garner, Ethan C.; Van Teeffelen, Sven; http://orcid.org/0000-0001-8034-9058; http://orcid.org/0000-0002-2813-0259; http://orcid.org/0000-0002-7216-9804; http://orcid.org/0000-0002-1309-753X; http://orcid.org/0000-0003-0141-3555; http://orcid.org/0000-0002-0877-1294All cells must increase their volumes in response to biomass growth to maintain intracellular mass density within physiologically permissive bounds. Here, we investigate the regulation of volume growth in the Gram-positive bacterium Bacillus subtilis. To increase volume, bacteria enzymatically expand their cell envelopes and insert new envelope material. First, we demonstrate that cell-volume growth is determined indirectly, by expanding their envelopes in proportion to mass growth, similarly to the Gram-negative Escherichia coli, despite their fundamentally different envelope structures. Next, we studied, which pathways might be responsible for robust surface-to-mass coupling: We found that both peptidoglycan synthesis and membrane synthesis are required for proper surface-tomass coupling. However, surprisingly, neither pathway is solely rate-limiting, contrary to wide-spread belief, since envelope growth continues at a reduced rate upon complete inhibition of either process. To arrest cell-envelope growth completely, the simultaneous inhibition of both envelope-synthesis processes is required. Thus, we suggest that multiple envelope-synthesis pathways collectively confer an important aspect of volume regulation, the coordination between surface growth, and biomass growth.Ítem Acceso Abierto Enterococcus faecalis MalR acts as a repressor of the maltose operons and additionally mediates their catabolite repression via direct interaction with seryl-phosphorylated-HPr(Wiley, 2019-11-22) Grand, Maxime; Blancato, Víctor Sebastián; Espariz, Martín; Deutscher, Josef; Pikis, Andreas; Hartke, Axel; Magni, Christian; Sauvageot, Nicolas; https://orcid.org/0000-0003-3945-2859; https://orcid.org/0000-0002-4090-515X; https://orcid.org/0000-0002-2537-0062; https://orcid.org/0000-0002-5179-9700; https://orcid.org/0000-0002-7595-4330; Brückner, Reinhold: provide plasmid pRB473; Thompson, John: provide phosphorylated sugarsEnterococci are gram-positive pathogens and lead to cause hospital-acquired infections worldwide. Central carbon metabolism was shown as highly induced in Enterococcus faecalis during infection context. Metabolism of α-polysaccharides was previously described as an important factor for host colonisation and biofilm formation. A better characterisation of the adaptation of this bacterium to carbohydrate availabilities may lead to a better understanding of the link between carbohydrate metabolism and the infection process of E. faecalis. Here we show that MalR, a LacI/GalR transcriptional regulator, is the main factor in the regulation of the two divergent operons involved in maltose metabolism in this bacterium. The malR gene is transcribed from the malP promoter, but also from an internal promoter inside the gene located upstream of malR. In the absence of maltose, MalR acts as a repressor and in the presence of glucose, it exerts efficient CcpA-independent carbon catabolite repression. The central PTS protein P-Ser-HPr interacts directly with MalR and enhances its DNA binding capacity, which allows E. faecalis to adapt its metabolism to environmental conditions.Ítem Acceso Abierto Alpha-tubulin acetylation in Trypanosoma cruzi: a dynamic instabilityof microtubules is required for replication and cell cycle progression(Frontiers Media, 2021-03-11) Alonso, Victoria Lucía; Carloni, Mara Emilia; Silva Gonçalves, Camila; Martínez Peralta, Gonzálo; Chesta, Maria Eugenia; Pezza, Alejandro; Tavernelli, Luis Emilio; Motta, María Cristina M.; Serra, Esteban CarlosTrypanosomatids have a cytoskeleton arrangement that is simpler than what is found in most eukaryotic cells. However, it is precisely organized and constituted by stable microtubules. Such microtubules compose the mitotic spindle during mitosis, the basal body, the flagellar axoneme and the subpellicular microtubules, which are connected to each other and also to the plasma membrane forming a helical arrangement along the central axis of the parasite cell body. Subpellicular, mitotic and axonemal microtubules are extensively acetylated in Trypanosoma cruzi. Acetylation on lysine (K) 40 of a-tubulin is conserved from lower eukaryotes to mammals and is associated with microtubule stability. It is also known that K40 acetylation occurs significantly on flagella, centrioles, cilia, basal body and the mitotic spindle in eukaryotes. Several tubulin posttranslational modifications, including acetylation of K40, have been cataloged in trypanosomatids, but the functional importance of these modifications for microtubule dynamics and parasite biology remains largely undefined. The primary tubulin acetyltransferase was recently identified in several eukaryotes as Mec-17/ATAT, a Gcn5-related N-acetyltransferase. Here, we report that T. cruzi ATAT acetylates a-tubulin in vivo and is capable of autoacetylation. TcATAT is located in the cytoskeleton and flagella of epimastigotes and colocalizes with acetylated a-tubulin in these structures. We have expressed TcATAT with an HA tag using the inducible vector pTcINDEX-GW in T. cruzi. Over-expression of TcATAT causes increased levels of the alpha tubulin acetylated species, induces morphological and ultrastructural defects, especially in the mitochondrion, and causes a halt in the cell cycle progression of epimastigotes, which is related to an impairment of the kinetoplast division. Finally, as a result of TcATAT over-expression we observed thatÍtem Acceso Abierto A coiled coil switch mediates cold sensing by the thermosensory protein DesK(Wiley, 2015-10-08) Saita, Emilio Adolfo; Abriata, Luciano Andrés; Tsai, Yi-Ting; Trajtenberg, Felipe; Lemmin, Thomas; Buschiazzo, Alejandro; Dal Peraro, Matteo; De Mendoza, Diego; Albanesi, DanielaThe thermosensor histidine kinase DesK from Bacillus subtilis senses changes in membrane fluidity initiating an adaptive response. Structural changes in DesK have been implicated in transmembrane signaling, but direct evidence is still lacking. On the basis of structure-guided mutagenesis, we now propose a mechanism of DesK-mediated signal sensing and transduction. The data indicate that stabilization/destabilization of a 2-helix coiled coil, which connects the transmembrane sensory domain of DesK to its cytosolic catalytic region, is crucial to control its signaling state. Computational modeling and simulations reveal couplings between protein, water and membrane mechanics. We propose that membrane thickening is the main driving force for signal sensing and that it acts by inducing helix stretching and rotation prompting an asymmetric kinase-competent state. Overall, the known structural changes of the sensor kinase, as well as further dynamic rearrangements that we now predict, consistently link structure determinants to activity modulation.Ítem Acceso Abierto The pleiotropic transcriptional regulator NlpR contributes to the modulation of nitrogen metabolism, lipogenesis and triacylglycerol accumulation in oleaginous rhodococci(Wiley, 2016-11-25) Hernández, Martín Alejandro; Lara, María Julia; Gago, Gabriela; Gramajo, Hugo Cesar; Álvarez, Héctor ManuelThe regulatory mechanisms involved in lipogenesis and triacylglycerol (TAG) accumulation are largely unknown in oleaginous rhodococci. In this study a regulatory protein (here called NlpR: Nitrogen lipid Regulator), which contributes to the modulation of nitrogen metabolism, lipogenesis and triacylglycerol accumulation in oleaginous rhodococci was identified. Under nitrogen deprivation conditions, in which TAG accumulation is stimulated, the nlpR gene was significantly upregulated, whereas a significant decrease of its expression and TAG accumulation occurred when cerulenin was added. The nlpR disruption negatively affected the nitrate/nitrite reduction as well as lipid biosynthesis under nitrogen-limiting conditions. In contrast, its overexpression increased TAG production during cultivation of cells in nitrogen-rich media. A putative ‘NlpR-binding motif’ upstream of several genes related to nitrogen and lipid metabolisms was found. The nlpR disruption in RHA1 strain led to a reduced transcription of genes involved in nitrate/nitrite assimilation, as well as in fatty acid and TAG biosynthesis. Purified NlpR was able to bind to narK, nirD, fasI, plsC and atf3 promoter regions. It was suggested that NlpR acts as a pleiotropic transcriptional regulator by activating of nitrate/nitrite assimilation genes and others genes involved in fatty acid and TAG biosynthesis, in response to nitrogen deprivation.Ítem Acceso Abierto Construction of three new Gateway® expression plasmids for Trypanosoma cruzi(Instituto Oswaldo Cruz, 2014-12) Alonso, Victoria Lucía; Ritagliati, Carla; Cribb, Pamela; Serra, Esteban CarlosWe present here three expression plasmids for Trypanosoma cruzi adapted to the Gateway® recombination cloning system. Two of these plasmids were designed to express trypanosomal proteins fused to a double tag for tandem affinity purification (TAPtag). The TAPtag and Gateway® cassette were introduced into an episomal (pTEX) and an integrative (pTREX) plasmid. Both plasmids were assayed by introducing green fluorescent protein (GFP) by recombination and the integrity of the double-tagged protein was determined by western blotting and immunofluorescence microscopy. The third Gateway adapted vector assayed was the inducible pTcINDEX. When tested with GFP, pTcINDEX-GW showed a good response to tetracycline, being less leaky than its precursor (pTcINDEX).Ítem Acceso Abierto Overexpression of bromodomain factor 3 in Trypanosoma cruzi (TcBDF3) affects differentiation of the parasite and protects it against bromodomain inhibitors(Wiley, 2016-06-06) Alonso, Victoria Lucía; Ritagliati, Carla; Cribb, Pamela; Cricco, Julia Alejandra; Serra, Esteban Carlos; Glaxo Smith Kline (GSK): provide I-BET151The bromodomain is the only protein domain known to bind acetylated lysine. In the last few years many bromodomain inhibitors have been developed in order to treat diseases such as cancer caused by aberrant acetylation of lysine residues. We have previously characterized Trypanosoma cruzi bromodomain factor 3 (TcBDF3), a bromodomain with an atypical localization that binds acetylated α-tubulin. In the present work we show that parasites overexpressing TcBDF3 exhibit altered differentiation patterns and are less susceptible to treatment with bromodomain inhibitors. We also demonstrate that recombinant TcBDF3 is able to bind to these inhibitors in vitro in a concentration-dependant manner. In parallel, the overexpression of a mutated version of TcBDF3 negatively affects growth of epimastigotes. Recent results, including the ones presented here, suggest that bromodomain inhibitors can be conceived as a new type of anti-parasitic drug against trypanosomiasis.Ítem Acceso Abierto Implications of the expression of Enterococcus faecalis citrate fermentation genes during infection(Public Library of Science, 2018-10-18) Martino, Gabriela Paula; Pérez, Cristian E.; Magni, Christian; Blancato, Víctor Sebastián; http://orcid.org/0000-0003-0508-260X; http://orcid.org/0000-0003-3945-2859; López, Paloma: provide the pTLGR plasmid.; Requena, Teresa: provide the pTLGR plasmid.; Chapo, Gustavo: provide the defibrinated blood.; González, Fabián: provide the defibrinated blood.Citrate is an ubiquitous compound in nature. However, citrate fermentation is present only in a few pathogenic or nonpathogenic microorganisms. The citrate fermentation pathway includes a citrate transporter, a citrate lyase complex, an oxaloacetate decarboxylase and a regulatory system. Enterococcus faecalis is commonly present in the gastro-intestinal microbiota of warm-blooded animals and insect guts. These bacteria can also cause infection and disease in immunocompromised individuals. In the present study, we performed whole genome analysis in Enterococcus strains finding that the complete citrate pathway is present in all of the E. faecalis strains isolated from such diverse habitats as animals, hospitals, water, milk, plants, insects, cheese, etc. These results indicate the importance of this,metabolic preservation for persistence and growth of E. faecalis in different niches. We also analyzed the role of citrate metabolism in the E. faecalis pathogenicity. We found that an E. faecalis citrate fermentation-deficient strain was less pathogenic for Galleria mellonella larvae than the wild type. Furthermore, strains with deletions in the oxaloacetate decarboxylase subunits or in the α-acetolactate synthase resulted also less virulent than the wild type strain. We also observed that citrate promoters are induced in blood, urine and also in the hemolymph of G. mellonella. In addition, we showed that citrate fermentation allows E. faecalis to grow better in blood, urine and G. mellonella. The results presented here clearly indicate that citrate fermentation plays an important role in E. faecalis opportunistic pathogenic behavior.Ítem Acceso Abierto Catalases of the polyextremophylic andean isolate Acinetobacter sp. Ver 3 confer adaptive response to H2O2 and UV radiation(Wiley, 2020-02-09) Sartorio, Mariana Gabriela; Repizo, Guillermo Daniel; Cortez, Néstor; https://orcid.org/0000-0003-1318-8738; Perdomo, Virginia: assistance with qRT-PCR measurements; Dr. Carrillo, Néstor: review of the manuscriptThe polyextremophilic strain Acinetobacter sp. Ver3 isolated from high-altitude Andean lakes exhibits elevated tolerance to UV-B radiation and to pro-oxidants, a feature that has been correlated to its unusually high catalase activity. The Ver3 genome sequence analysis revealed the presence of two genes coding for monofunctional catalases: AV3KatE1 and AV3KatE2, the latter harboring an N-terminal signal peptide. We show herein that AV3KatE1 displays one of the highest catalytic activities reported so far and is constitutively expressed at relatively high amounts in the cytosol, acting as the main protecting catalase against H2O2 and UV-B radiation. The second catalase, AV3KatE2, is a periplasmic enzyme strongly induced by both peroxide and UV, conferring supplementary protection against pro-oxidants. The N-terminal signal present in AV3KatE2 was required not only for transport to the periplasm via the twin-arginine translocation pathway, but also for proper folding and subsequent catalytic activity. The analysis of catalase distribution among 114 Acinetobacter complete genomes revealed a great variability in the catalase classes, with A. baumannii clinical isolates exhibiting higher numbers of isoenzymes and the most variable profiles.