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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; Gonçalves, Camila Silva; Martinez Peralta, Gonzalo; Chesta, Maria Eugenia; Pezza, Alejandro; Tavernelli, Luis; Motta, Maria 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.Ítem Acceso Abierto HPV E6 and E7 oncoproteins cooperatively alter the expression of Disc Large 1 polarity protein in epithelial cells(BMC, 2020-04-07) Dizanzo, María Paula; Marziali, Federico Emanuel; Brunet Avalos, Clarise; Bugnon Valdano, Marina Paula; Leiva, Santiago Gabriel; Cavatorta, Ana Laura; Gardiol, DanielaBackground: Persistent infection with high-risk Human Papillomavirus (HPVs) is associated with the development of cervical cancer. The transforming capacity of these viruses relies on the cooperative action of the E6 and E7 viral oncoproteins. Among the oncogenic activities of E6, the interaction and interference with cell polarity PDZ proteins have been well established. One of the most characterized PDZ targets of HPV E6 is human Disc large 1 (DLG1), a scaffolding protein involved in the control of cell polarity and proliferation. Interestingly, in cervical squamous intraepithelial lesions, alterations in DLG1 expression were observed in association to tumour progression. Moreover, the expression of both HPV E6 and E7 proteins may be responsible for the changes in DLG1 abundance and cell localization observed in the HPV-associated lesions. Methods: Due to the relevance of DLG1 deregulation in tumour development, we have performed an in-depth investigation of the expression of DLG1 in the presence of the HPV oncoproteins in epithelial cultured cells. The effects of HPV E6 and E7 proteins on DLG1 abundance and subcellular localization were assessed by western blot and confocal fluorescence microscopy, respectively. Results: We demonstrated that the relative abundance of HPV-18 E6 and DLG1 is a key factor that contributes to defining the expression abundance of both proteins. We also show here that a high expression level of DLG1 may negatively affect HPV-18 E6 nuclear expression. Moreover, the co-expression of HPV-18 E6 and E7 produces a striking effect on DLG1 subcellular localization and a co-distribution in the cytoplasmic region. Interestingly, HPV-18 E7 is also able to increase DLG1 levels, likely by rescuing it from the E6-mediated proteasomal degradation. Conclusions: In general, the data suggest that HPV-18 E6 and E7 may have opposing activities in regards to the regulation of DLG1 levels and may cooperatively contribute to its subcellular redistribution in the HPV context. These findings constitute a step forward in understanding the differential expression of DLG1 during tumour progression in an HPV-associated model.Ítem Acceso Abierto A duo of Potassium-responsive Histidine Kinases govern the multicellular destiny of Bacillus subtilis(American Society for Microbiology, 2015-07-07) Grau, Roberto Ricardo; De Oña, Paula; Kunert, Maritta; Leñini, Cecilia; Gallegos Monterrosa, Ramses; Mhatre, Eisha; Vileta, Darío; Donato, Verónica; Hölscher, Theresa; Boland, Sebastian; Kuipers, Oscar P.; Kovács, Ákos T.; http://orcid.org/0000-0001-6430-7122; http://orcid.org/0000-0001-6784-2534Multicellular biofilm formation and surface motility are bacterial behaviors considered mutually exclusive. However, the basic decision to move over or stay attached to a surface is poorly understood. Here, we discover that in Bacillus subtilis, the key root biofilm-controlling transcription factor Spo0A~Pi (phosphorylated Spo0A) governs the flagellum-independent mechanism of social sliding motility. A Spo0A-deficient strain was totally unable to slide and colonize plant roots, evidencing the important role that sliding might play in natural settings. Microarray experiments plus subsequent genetic characterization showed that the machineries of sliding and biofilm formation share the same main components (i.e., surfactin, the hydrophobin BslA, exopolysaccharide, and de novo-formed fatty acids). Sliding proficiency was transduced by the Spo0A-phosphorelay histidine kinases KinB and KinC. We discovered that potassium, a previously known inhibitor of KinC-dependent biofilm formation, is the specific sliding-activating signal through a thus-far-unnoticed cytosolic domain of KinB, which resembles the selectivity filter sequence of potassium channels. The differential expression of the Spo0A~Pireporter abrB gene and the different levels of the constitutively active form of Spo0A, Sad67, in spo0A cells grown in optimized media that simultaneously stimulate motile and sessile behaviors uncover the spatiotemporal response of KinB and KinC to potassium and the gradual increase in Spo0A~Pi that orchestrates the sequential activation of sliding, followed by sessile biofilm formation and finally sporulation in the same population. Overall, these results provide insights into how multicellular behaviors formerly believed to be antagonistic are coordinately activated in benefit of the bacterium and its interaction with the host. IMPORTANCE Alternation between motile and sessile behaviors is central to bacterial adaptation, survival, and colonization. However, how is the collective decision to move over or stay attached to a surface controlled? Here, we use the model plantbeneficial bacterium Bacillus subtilis to answer this question. Remarkably, we discover that sessile biofilm formation and social sliding motility share the same structural components and the Spo0A regulatory network via sensor kinases, KinB and KinC. Potassium, an inhibitor of KinC-dependent biofilm formation, triggers sliding via a potassium-perceiving cytosolic domain of KinB that resembles the selectivity filter of potassium channels. The spatiotemporal response of these kinases to variable potassium levels and the gradual increase in Spo0A~Pi levels that orchestrates the activation of sliding before biofilm formation shed light on how multicellular behaviors formerly believed to be antagonistic work together to benefit the population fitness.Ítem Acceso Abierto Systematic production of inactivating and non-inactivating suppressor mutations at the relA locus that compensate the detrimental effects of complete spoT loss and affect glycogen content in Escherichia coli(Public Library of Science, 2014-09-04) Montero, Manuel; Rahimpour, Mehdi; Viale, Alejandro M.; Almagro, Goizeder; Eydallin, Gustavo; Sevilla, Ángel; Cánovas, Manuel; Bernal, Cristina; Muñoz, Francisco José; Baroja Fernández, Edurne; Bahaji, Abdellatif; Mori, Hirotada; Codoñer, Francisco M.; Pozueta Romero, JavierIn 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 médium 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 synthetase.Ítem Acceso Abierto Phage biocontrol of enteropathogenic and shiga toxin-producing Escherichia coli in meat products(Frontiers Media, 2013-06-06) Tomat, David Damián; Migliore, Leonel; Aquili, Virginia; Quiberoni, Andrea; Balagué, Claudia ElizabethTen bacteriophages were isolated from faeces and their lytic effects assayed on 103 pathogenic and non-pathogenic Enterobacteriaceae. Two phages (DT1 and DT6) were selected based on their host ranges, and their lytic effects on pathogenic E. coli strains inoculated on pieces of beef were determined. We evaluated the reductions of viable cells of Escherichia coli O157:H7 and non-O157 Shiga toxigenic E. coli strains on meat after exposure to DT6 at 5 and 24◦C for 3, 6, and 24 h and the effect of both phages against an enteropathogenic E. coli strain. Significant viable cell reductions, compared to controls without phages, at both temperatures were observed, with the greatest decrease taking place within the first hours of the assays. Reductions were also influenced by phage concentration, being the highest concentrations, 1.7 × 10 10 plaque forming units per milliliter (PFU/mL) for DT1 and 1.4 × 10 10 PFU/mL for DT6, the most effective. When enteropathogenic E. coli and Shiga toxigenic E. coli (O157:H7) strains were tested, we obtained viable cell reductions of 0.67 log (p = 0.01) and 0.77 log (p = 0.01) after 3 h incubation and 0.80 log (p = 0.01) and 1.15 log (p = 0.001) after 6 h. In contrast, all nonpathogenic E. coli strains as well as other enterobacteria tested were resistant. In addition, phage cocktail was evaluated on two strains and further reductions were observed. However, E. coli bacteriophage insensitive mutants (BIMs) emerged in meat assays. BIMs isolated from meat along with those isolated by using the secondary culture method were tested to evaluate resistance phenotype stability and reversion. They presented low emergence frequencies (6.5 × 10−7 –1.8 × 10−6 ) and variable stability and reversion. Results indicate that isolated phages were stable on storage, negative for all the virulence factors assayed, presented lytic activity for different E. coli virotypes and could be useful in reducing Shiga toxigenic E. coli and enteropathogenic E. coli viable cells in meat products.Ítem Acceso Abierto The dual nature of trehalose in citrus canker disease: a virulence factor for Xanthomonas citri subsp. citri and a trigger for plant defence responses(Oxford University, 2015-03-15) Piazza, Ainelén; Zimaro, Tamara; Garavaglia, Betiana Soledad; Ficarra, Florencia Andrea; Thomas, Ludivine; Marondedze, Claudius; Feil, Regina; Lunn, John E.; Gehring, Chris; Ottado, Jorgelina; Gottig, NataliaXanthomonas citri subsp. citri (Xcc) is a bacterial pathogen that causes citrus canker in susceptible Citrus spp. The Xcc genome contains genes encoding enzymes from three separate pathways of trehalose biosynthesis. Expression of genes encoding trehalose-6-phosphate synthase (otsA) and trehalose phosphatase (otsB) was highly induced during canker development, suggesting that the two-step pathway of trehalose biosynthesis via trehalose-6-phosphate has a function in pathogenesis. This pathway was eliminated from the bacterium by deletion of the otsA gene. The resulting XccΔotsA mutant produced less trehalose than the wild-type strain, was less resistant to salt and oxidative stresses, and was less able to colonize plant tissues. Gene expression and proteomic analyses of infected leaves showed that infection with XccΔotsA triggered only weak defence responses in the plant compared with infection with Xcc, and had less impact on the host plant’s metabolism than the wild-type strain. These results suggested that trehalose of bacterial origin, synthesized via the otsA–otsB pathway, in Xcc, plays a role in modifying the host plant’s metabolism to its own advantage but is also perceived by the plant as a sign of pathogen attack. Thus, trehalose biosynthesis has both positive and negative consequences for Xcc. On the one hand, it enables this bacterial pathogen to survive in the inhospitable environment of the leaf surface before infection and exploit the host plant’s resources after infection, but on the other hand, it is a tell-tale sign of the pathogen’s presence that triggers the plant to defend itself against infection.Ítem Acceso Abierto Resistance to citrus canker induced by a variant of Xanthomonas citri ssp. citri is associated with a hypersensitive cell death response involving autophagy-associated vacuolar processes(Wiley, 2017-11-08) Roeschlin, Roxana Andrea; Favaro, María Alejandra; Chiesa, María Amalia; Alemano, Sergio Gabriel; Vojnov, Adrián Alberto; Castagnaro, Atilio Pedro; Filippone, María Paula; Gmitter, Frederick George; Gadea, José; Marano, María RosaXanthomonas citri ssp. citri (X. citri) is the causal agent of Asiatic citrus canker, a disease that seriously affects most commercially important Citrus species worldwide. We have identified previously a natural variant, X. citri AT, that triggers a host-specific defence response in Citrus limon. However, the mechanisms involved in this canker disease resistance are unknown. In this work, the defence response induced by X. citri AT was assessed by transcriptomic, physiological and ultrastructural analyses, and the effects on bacterial biofilm formation were monitored in parallel. We show that X. citri AT triggers a hypersensitive response associated with the interference of biofilm development and arrest of bacterial growth in C. limon. This plant response involves an extensive transcriptional reprogramming, setting in motion cell wall reinforcement, the oxidative burst and the accumulation of salicylic acid (SA) and phenolic compounds. Ultrastructural analyses revealed subcellular changes involving the activation of autophagy-associated vacuolar processes. Our findings show the activation of SA-dependent defence in response to X. citri AT and suggest a coordinated regulation between the SA and flavonoid pathways, which is associated with autophagy mechanisms that control pathogen invasion in C. limon. Furthermore, this defence response protects C. limon plants from disease on subsequent challenges by pathogenic X. citri. This knowledge will allow the rational exploitation of the plant immune system as a biotechnological approach for the management of the disease.Ítem Acceso Abierto Comparative genomic and phylogenetic analyses of gammaproteobacterial glg genes traced the origin of the Escherichia coli glycogen glgBXCAP pperon to the last common ancestor of the sister orders enterobacteriales and pasteurellales(Public Library of Science, 2015-01-21) Almagro, Goizeder; Viale, Alejandro M.; Montero, Manuel; Rahimpour, Mehdi; Muñoz, Francisco José; Baroja Fernández, Edurne; Bahaji, Abdellatif; Zúñiga, Manuel; González Candelas, Fernando; Pozueta Romero, JavierProduction of branched α-glucan, glycogen-like polymers is widely spread in the Bacteria domain. The glycogen pathway of synthesis and degradation has been fairly well characterized in the model enterobacterial species Escherichia coli (order Enterobacteriales, class Gammaproteobacteria), in which the cognate genes (branching enzyme glgB, debranching enzyme glgX, ADP-glucose pyrophosphorylase glgC, glycogen synthase glgA, and glycogen phosphorylase glgP) are clustered in a glgBXCAP operon arrangement. However, the evolutionary origin of this particular arrangement and of its constituent genes is unknown. Here, by using 265 complete gammaproteobacterial genomes we have carried out a comparative analysis of the presence, copy number and arrangement of glg genes in all lineages of the Gammaproteobacteria. These analyses revealed large variations in glg gene presence, copy number and arrangements among different gammaproteobacterial lineages. However, the glgBXCAP arrangement was remarkably conserved in all glg-possessing species of the orders Enterobacteriales and Pasteurellales (the E/P group). Subsequent phylogenetic analyses of glg genes present in the Gammaproteobacteria and in other main bacterial groups indicated that glg genes have undergone a complex evolutionary history in which horizontal gene transfer may have played an important role. These analyses also revealed that the E/P glgBXCAP genes (a) share a common evolutionary origin, (b) were vertically transmitted within the E/P group, and (c) are closely related to glg genes of some phylogenetically distant betaproteobacterial species. The overall data allowed tracing the origin of the E. coli glgBXCAP operon to the last common ancestor of the E/P group, and also to uncover a likely glgBXCAP transfer event from the E/P group to particular lineages of the Betaproteobacteria.Ítem Acceso Abierto Unsaturated long chain free fatty acids are input signals of the Salmonella enterica PhoP/PhoQ regulatory system(American Society for Biochemistry and Molecular Biology, 2013-08-02) Viarengo, Gastón; Sciara, Mariela Inés; Salazar, Mario Oscar; Kieffer, Pablo M.; Furlán, Ricardo Luis Eugenio; García Véscovi, EleonoraThe Salmonella enterica serovar Typhimurium PhoP/PhoQ system has largely been studied as a paradigmatic two-component regulatory system not only to dissect structural and functional aspects of signal transduction in bacteria but also to gain knowledge about the versatile devices that have evolved allowing a pathogenic bacterium to adjust to or counteract environmental stressful conditions along its life cycle. Mg2+ limitation, acidic pH, and the presence of cationic antimicrobial peptides have been identified as cues that the sensor protein PhoQ can monitor to reprogram Salmonella gene expression to cope with extra- or intracellular challenging conditions. In this work, we show for the first time that long chain unsaturated free fatty acids (LCUFAs) present in Salmonella growth medium are signals specifically detected by PhoQ. We demonstrate that LCUFAs inhibit PhoQ autokinase activity, turning off the expression of the PhoP-dependent regulon. We also show that LCUFAs exert their action independently of their cellular uptake and metabolic utilization by means of the β-oxidative pathway. Our findings put forth the complexity of input signals that can converge to finely tune the activity of the PhoP/PhoQ system. In addition, they provide a new potential biochemical platform for the development of antibacterial strategies to fight against Salmonella infections.Ítem Acceso Abierto The phosphatidic acid pathway enzyme PlsX plays both catalytic and channeling roles in bacterial phospholipid synthesis(American Society for Biochemistry and Molecular Biology, 2020-01-09) Sastre, Diego Emiliano; Pulschen, André A.; Basso , Luis G.M.; Benites Pariente, Jhonathan S.; Marques Netto, Caterina G.C.; Machinandiarena, Federico; Albanesi, Daniela; Navarro, Marcos V.A.S.; De Mendoza, Diego; Gueiros-Filho, Frederico J.PlsX is the first enzyme in the pathway that produces phosphatidic acid in Gram-positive bacteria. It makes acylphosphate from acyl-acyl carrier protein (acyl-ACP) and is also involved in coordinating phospholipid and fatty acid biosyntheses. PlsX is a peripheral membrane enzyme in Bacillus subtilis, but how it associates with the membrane remains largely unknown. In the present study, using fluorescence microscopy, liposome sedimentation, differential scanning calorimetry, and acyltransferase assays, we determined that PlsX binds directly to lipid bilayers and identified its membrane anchoring moiety, consisting of a hydrophobic loop located at the tip of two amphipathic dimerization helices. To establish the role of the membrane association of PlsX in acylphosphate synthesis and in the flux through the phosphatidic acid pathway, we then created mutations and gene fusions that prevent PlsX's interaction with the membrane. Interestingly, phospholipid synthesis was severely hampered in cells in which PlsX was detached from the membrane, and results from metabolic labeling indicated that these cells accumulated free fatty acids. Because the same mutations did not affect PlsX transacylase activity, we conclude that membrane association is required for the proper delivery of PlsX's product to PlsY, the next enzyme in the phosphatidic acid pathway. We conclude that PlsX plays a dual role in phospholipid synthesis, acting both as a catalyst and as a chaperone protein that mediates substrate channeling into the pathway.