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Ítem Acceso Abierto The stringent response plays a key role in Bacillus subtilis survival of fatty acid starvation(Wiley, 2017-02-07) Pulschen, André A.; Sastre, Diego Emiliano; Machinandiarena, Federico; Crotta Asis, Agostina; Albanesi, Daniela; De Mendoza, Diego; Gueiros-Filho, Frederico J.The stringent response is a universal adaptive mechanism to protect bacteria from nutritional and environmental stresses. The role of the stringent response during lipid starvation has been studied only in Gram-negative bacteria. Here, we report that the stringent response also plays a crucial role in the adaptation of the model Gram-positive Bacillus subtilis to fatty acid starvation. B. subtilis lacking all three (p)ppGpp-synthetases (RelBs, RelP and RelQ) or bearing a RelBs variant that no longer synthesizes (p)ppGpp suffer extreme loss of viability on lipid starvation. Loss of viability is paralleled by perturbation of membrane integrity and function, with collapse of membrane potential as the likely cause of death. Although no increment of (p)ppGpp could be detected in lipid starved B. subtilis, we observed a substantial increase in the GTP/ATP ratio of strains incapable of synthesizing (p)ppGpp. Artificially lowering GTP with decoyinine rescued viability of such strains, confirming observations that low intracellular GTP is important for survival of nutritional stresses. Altogether, our results show that activation of the stringent response by lipid starvation is a broadly conserved response of bacteria and that a key role of (p)ppGpp is to couple biosynthetic processes that become detrimental if uncoordinated.Ítem Acceso Abierto Unravelling the lipoyl-relay of exogenous lipoate utilization in Bacillus subtilis(Wiley, 2019-05-19) Rasetto, Natalí B.; Lavatelli, Antonela; Martin, Natalia; Mansilla, María Cecilia; https://orcid.org/0000-0002-1444-8661Lipoate is an essential cofactor for key enzymes of oxidative and one-carbon metabolism. It is covalently attached to E2 subunits of dehydrogenase complexes and GcvH, the H subunit of the glycine cleavage system. Bacillus subtilis possess two protein lipoylation pathways: biosynthesis and scavenging. The former requires octanoylation of GcvH, insertion of sulfur atoms and amidotransfer of the lipoate to E2s, catalyzed by LipL. Lipoate scavenging is mediated by a lipoyl protein ligase (LplJ) that catalyzes a classical two-step ATP-dependent reaction. Although these pathways were thought to be redundant, a ∆lipL mutant, in which the endogenous lipoylation pathway of E2 subunits is blocked, showed growth defects in minimal media even when supplemented with lipoate and despite the presence of a functional LplJ. In this study, we demonstrate that LipL is essential to modify E2 subunits of branched chain ketoacid and pyruvate dehydrogenases during lipoate scavenging. The crucial role of LipL during lipoate utilization relies on the strict substrate specificity of LplJ, determined by charge complementarity between the ligase and the lipoylable subunits. This new lipoyl-relay required for lipoate scavenging highlights the relevance of the amidotransferase as a valid target for the design of new antimicrobial agents among Gram-positive pathogens.Ítem Acceso Abierto Transfection of Capsaspora owczarzaki, a close unicellular relative of animals(The Company of Biologists, 2018) Parra Acero, Helena; Ros Rocher, Núria; Perez Posada, Alberto; Kozyczkowska, Aleksandra; Sánchez Pons, Núria; Nakata, Azusa; Suga, Hiroshi; Najle, Sebastián R.; Ruiz Trillo, Iñaki; https://orcid.org/0000-0003-0897-0186; https://orcid.org/0000-0001-6547-5304How animals emerged from their unicellular ancestor remains a major evolutionary question. New genome data from the closest unicellular relatives of animals have provided important insights into the evolution of animal multicellularity. We know that the unicellular ancestor of animals had an unexpectedly complex genetic repertoire, including many genes that are key to animal development and multicellularity. Thus, assessing the function of these genes among unicellular relatives of animals is key to understanding how they were co-opted at the onset of the Metazoa. However, such analyses have been hampered by the lack of genetic tools. Progress has been made in choanoflagellates and teretosporeans, two of the three lineages closely related to animals, whereas no tools are yet available for functional analysis in the third lineage: the filastereans. Importantly, filastereans have a striking repertoire of genes involved in transcriptional regulation and other developmental processes. Here, we describe a reliable transfection method for the filasterean Capsaspora owczarzaki. We also provide a set of constructs for visualising subcellular structures in live cells. These tools convert Capsaspora into a unique experimentally tractable organism to use to investigate the origin and evolution of animal multicellularity.Ítem Embargo Light modulates important pathogenic determinants and virulence in ESKAPE pathogens Acinetobacter baumannii, Pseudomonas aeruginosa, and Staphylococcus aureus(American Society for Microbiology, 2021-02-08) Tuttobene, Marisel Romina; Pérez, J. F.; Pavesi, Estefanía S.; Pérez Mora, Bárbara; Biancotti, Daiana; Cribb, Pamela; Altilio, Matías; Müller, Gabriela Leticia; Gramajo, Hugo Cesar; Tamagno, G.; Ramírez, María Soledad; Diacovich, Lautaro; Mussi, María Alejandra; https://orcid.org/0000-0002-9904-7890; https://orcid.org/0000-0002-3339-0100; https://orcid.org/0000-0002-4168-3624; Rabinovich, Gabriel: provide HaCaT cells; Voyich, Jovanka: provide the hla and complemented hla mutantLight sensing has been extensively characterized in the human pathogen Acinetobacter baumannii at environmental temperatures. However, the influence of light on the physiology and pathogenicity of human bacterial pathogens at temperatures found in warm-blooded hosts is still poorly understand. In this work, we show that Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa (ESKAPE) priority pathogens, which have been recognized by the WHO and the CDC as critical, can also sense and respond to light at temperatures found in human hosts. Most interestingly, in these pathogens, light modulates important pathogenicity determinants as well as virulence in an epithelial infection model, which could have implications in human infections. In fact, we found that alpha-toxin-dependent hemolysis, motility, and growth under iron-deprived conditions are modulated by light in S. aureus. Light also regulates persistence, metabolism, and the ability to kill competitors in some of these microorganisms. Finally, light exerts a profound effect on the virulence of these pathogens in an epithelial infection model, although the response is not the same in the different species; virulence was enhanced by light in A. baumannii and S. aureus, while in A. nosocomialis and P. aeruginosa it was reduced. Neither the BlsA photoreceptor nor the type VI secretion system (T6SS) is involved in virulence modulation by light in A. baumannii. Overall, this fundamental knowledge highlights the potential use of light to control pathogen virulence, either directly or by manipulating the light regulatory switch toward the lowest virulence/persistence configuration. IMPORTANCE: Pathogenic bacteria are microorganisms capable of producing disease. Dangerous bacterial pathogens, such as Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, are responsible for serious intrahospital and community infections in humans. Therapeutics is often complicated due to resistance to multiple antibiotics, rendering them ineffective. In this work, we show that these pathogens sense natural light and respond to it by modulating aspects related to their ability to cause disease; in the presence of light, some of them become more aggressive, while others show an opposite response. Overall, we provide new understanding on the behavior of these pathogens, which could contribute to the control of infections caused by them. Since the response is distributed in diverse pathogens, this notion could prove a general concept.Ítem Acceso Abierto Osteomielitis por Scedosporium spp., a propósito de un caso(Asociación Argentina de Microbiología (AAM), 2019-06-13) Colombo, Laura G.R.; Gregorini, Eduardo R.; Dalmaso, Hernán; Podestá, María. V.; Luque, Alicia Graciela; Truccolo, Paula; Lerman Tenenbaum, DamiánScedosporium es un hongo de distribución mundial que se encuentra en el suelo y en aguas contaminadas. Raramente afecta tejido óseo y puede hacerlo por inoculación directa a través de traumatismos. Se presenta el caso clínico de un paciente de 54 años con antecedente de accidente acuático y fractura expuesta de tibia-peroné de ambos miembros inferiores, con diagnóstico de osteomielitis crónica bacteriana tratada con antibióticos de amplio espectro por 120 días. Luego de ocho meses iniciado el cuadro, se aísla Scedosporium spp. en colección de miembro afectado; por tal motivo, el paciente recibe terapia con voriconazol asociado a terbinafina.Ítem Embargo Enterococcus faecalis uses a phosphotransferase system permease and a host colonization-related ABC transporter for maltodextrin uptake(American Society for Microbiology, 2017-04-11) Sauvageot, Nicolas; Mokhtari, Abdelhamid; Joyet, Philippe; Budin Verneuil, Aurélie; Blancato, Víctor Sebastián; Repizo, Guillermo Daniel; Henry, Céline; Pikis, Andreas; Thompson, John; Magni, Christian; Hartke, Axel; Deutscher, Josef; Fernández, María: provide plasmid pAGEntMaltodextrin is a mixture of maltooligosaccharides, which are produced by the degradation of starch or glycogen. They are mostly composed of α-1,4- and some α-1,6-linked glucose residues. Genes presumed to code for the Enterococcus faecalis maltodextrin transporter were induced during enterococcal infection. We therefore carried out a detailed study of maltodextrin transport in this organism. Depending on their length (3 to 7 glucose residues), E. faecalis takes up maltodextrins either via MalT, a maltose-specific permease of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), or the ATP binding cassette (ABC) transporter MdxEFG-MsmX. Maltotriose, the smallest maltodextrin, is primarily transported by the PTS permease. A malT mutant therefore exhibits significantly reduced growth on maltose and maltotriose. The residual uptake of the trisaccharide is catalyzed by the ABC transporter, because a malT mdxF double mutant no longer grows on maltotriose. The trisaccharide arrives as maltotriose-6″-P in the cell. MapP, which dephosphorylates maltose-6′-P, also releases Pi from maltotriose-6″-P. Maltotetraose and longer maltodextrins are mainly (or exclusively) taken up via the ABC transporter, because inactivation of the membrane protein MdxF prevents growth on maltotetraose and longer maltodextrins up to at least maltoheptaose. E. faecalis also utilizes panose and isopanose, and we show for the first time, to our knowledge, that in contrast to maltotriose, its two isomers are primarily transported via the ABC transporter. We confirm that maltodextrin utilization via MdxEFG-MsmX affects the colonization capacity of E. faecalis, because inactivation of mdxF significantly reduced enterococcal colonization and/or survival in kidneys and liver of mice after intraperitoneal infection. IMPORTANCE: Infections by enterococci, which are major health care-associated pathogens, are difficult to treat due to their increasing resistance to clinically relevant antibiotics, and new strategies are urgently needed. A largely unexplored aspect is how these pathogens proliferate and which substrates they use in order to grow inside infected hosts. The use of maltodextrins as a source of carbon and energy was studied in Enterococcus faecalis and linked to its virulence. Our results demonstrate that E. faecalis can efficiently use glycogen degradation products. We show here that depending on the length of the maltodextrins, one of two different transporters is used: the maltose-PTS transporter MalT, or the MdxEFG-MsmX ABC transporter. MdxEFG-MsmX takes up longer maltodextrins as well as complex molecules, such as panose and isopanose.Ítem Acceso Abierto Functional characterization of the first lipoyl-relay pathwayfrom a parasitic protozoan(Wiley, 2022-06) Scattolini, Albertina; Lavatelli, Antonela; Vacchina, Paola; Lambruschi, Daniel Andrés; Mansilla, María Cecilia; Uttaro, Antonio Domingo; https://orcid.org/0000-0001-7421-2039; https://orcid.org/0000-0001-7164-213X; https://orcid.org/0000-0001-5866-3657; https://orcid.org/0000-0001-5787-5711; https://orcid.org/0000-0002-1444-8661Lipoic acid (LA) is a sulfur-containing cofactor covalently attached to key enzymes of central metabolism in prokaryotes and eukaryotes. LA can be acquired by scavenging, mediated by a lipoate ligase, or de novo synthesized by a pathway requiring an octanoyltransferase and a lipoate synthase. A more complex pathway, referred to as “lipoyl-relay”, requires two additional proteins, GcvH, the glycine cleavage system H subunit, and an amidotransferase. This route was described so far in Bacillus subtilis and related Gram-positive bacteria, Saccharomyces cerevisiae, Homo sapiens, and Caenorhabditis elegans. Using collections of S. cerevisiae and B. subtilis mutants, defective in LA metabolism, we gathered evidence that allows us to propose for the first time that lipoyl-relay pathways are also present in parasitic protozoa. By a reverse genetic approach, we assigned octanoyltransferase and amidotransferase activity to the products of Tb927.11.9390 (TblipT) and Tb927.8.630 (TblipL) genes of Trypanosoma brucei, respectively. The B. subtilis model allowed us to identify the parasite amidotransferase as the target of lipoate analogs like 8-bromo-octanoic acid, explaining the complete loss of protein lipoylation and growth impairment caused by this compound in T. cruzi. This model could be instrumental for the screening of selective and more efficient chemotherapies against trypanosomiases.Ítem Acceso Abierto On the offensive: the role of outer membrane vesicles in the successful dissemination of New Delhi Metallo-β-lactamase (NDM-1)(American Society for Microbiology, 2021-09-28) Martínez, Melina María Belén; Bonomo, Robert A.; Vila, Alejandro J.; Maffía, Paulo César; González, Lisandro Javier; https://orcid.org/0000-0002-4007-8721; https://orcid.org/0000-0002-3299-894X; https://orcid.org/0000-0002-7978-3233; https://orcid.org/0000-0001-7423-2646; https://orcid.org/0000-0002-0575-1810The emergence and worldwide dissemination of carbapenemase-producing Gram-negative bacteria are a major public health threat. Metallo-β-lactamases (MBLs) represent the largest family of carbapenemases. Regrettably, these resistance determinants are spreading worldwide. Among them, the New Delhi metallo-β-lactamase (NDM-1) is experiencing the fastest and largest geographical spread. NDM-1 β-lactamase is anchored to the bacterial outer membrane, while most MBLs are soluble, periplasmic enzymes. This unique cellular localization favors the selective secretion of active NDM-1 into outer membrane vesicles (OMVs). Here, we advance the idea that NDM-containing vesicles serve as vehicles for the local dissemination of NDM-1. We show that OMVs with NDM-1 can protect a carbapenem-susceptible strain of Escherichia coli upon treatment with meropenem in a Galleria mellonella infection model. Survival curves of G. mellonella revealed that vesicle encapsulation enhances the action of NDM-1, prolonging and favoring bacterial protection against meropenem inside the larva hemolymph. We also demonstrate that E. coli cells expressing NDM-1 protect a susceptible Pseudomonas aeruginosa strain within the larvae in the presence of meropenem. By using E. coli variants engineered to secrete variable amounts of NDM-1, we demonstrate that the protective effect correlates with the amount of NDM-1 secreted into vesicles. We conclude that secretion of NDM-1 into OMVs contributes to the survival of otherwise susceptible nearby bacteria at infection sites. These results disclose that OMVs play a role in the establishment of bacterial communities, in addition to traditional horizontal gene transfer mechanisms. IMPORTANCE: Resistance to carbapenems, last-resort antibiotics, is spreading worldwide, raising great concern. NDM-1 is one of the most potent and widely disseminated carbapenem-hydrolyzing enzymes spread among many bacteria and is secreted to the extracellular medium within outer membrane vesicles. We show that vesicles carrying NDM-1 can protect carbapenem-susceptible strains of E. coli and P. aeruginosa upon treatment with meropenem in a live infection model. These vesicles act as nanoparticles that encapsulate and transport NDM-1, prolonging and favoring its action against meropenem inside a living organism. Secretion of NDM-1 into vesicles contributes to the survival of otherwise susceptible nearby bacteria at infection sites. We propose that vesicles play a role in the establishment of bacterial communities and the dissemination of antibiotic resistance, in addition to traditional horizontal gene transfer mechanisms.Ítem Acceso Abierto Revisiting the coupling of fatty acid to phospholipid synthesis in bacteria with FapR regulation(Wiley, 2020-07-16) Machinandiarena, Federico; Nakamatsu, Leandro; Schujman, Gustavo Enrique; De Mendoza, Diego; Albanesi, Daniela; https://orcid.org/0000-0003-4380-9152A key aspect in membrane biogenesis is the coordination of fatty acid to phospholipid synthesis rates. In most bacteria, PlsX is the first enzyme of the phosphatidic acid synthesis pathway, the common precursor of all phospholipids. Previously, we proposed that PlsX is a key regulatory point that synchronizes the fatty acid synthase II with phospholipid synthesis in Bacillus subtilis. However, understanding the basis of such coordination mechanism remained a challenge in Gram-positive bacteria. Here, we show that the inhibition of fatty acid and phospholipid synthesis caused by PlsX depletion leads to the accumulation of long-chain acyl-ACPs, the end products of the fatty acid synthase II. Hydrolysis of the acyl-ACP pool by heterologous expression of a cytosolic thioesterase relieves the inhibition of fatty acid synthesis, indicating that acyl-ACPs are feedback inhibitors of this metabolic route. Unexpectedly, inactivation of PlsX triggers a large increase of malonyl-CoA leading to induction of the fap regulon. This finding discards the hypothesis, proposed for B. subtilis and extended to other Gram-positive bacteria, that acyl-ACPs are feedback inhibitors of the acetyl-CoA carboxylase. Finally, we propose that the continuous production of malonyl-CoA during phospholipid synthesis inhibition provides an additional mechanism for fine-tuning the coupling between phospholipid and fatty acid production in bacteria with FapR regulation.Ítem Acceso Abierto A genetic screen for mutations affecting temperature sensing in Bacillus subtilis(Microbiology Society, 2018-11-15) Díaz, Alejandra Raquel; Porrini, Lucía; De Mendoza, Diego; Mansilla, María CeciliaTwo component systems, composed of a receptor histidine kinase and a cytoplasmic response regulator, regulate pivotal cellular processes in microorganisms. Here we describe a new screening procedure for the identification of amino acids that are crucial for the functioning of DesK, a prototypic thermosensor histidine kinase from Bacillus subtilis. This experimental strategy involves random mutagenesis of the membrane sensor domain of the DesK coding sequence, followed by the use of a detection procedure based on changes in the colony morphogenesis that take place during the sporulation programme of B. subtilis. This method permitted us the recovery of mutants defective in DesK temperature sensing. This screening approach could be applied to all histidine kinases of B. subtilis and also to kinases of other bacteria that are functionally expressed in this organism. Moreover, this reporter assay could be expanded to develop reporter assays for a variety of transcriptionally regulated systems.Ítem Acceso Abierto Structural determinant of functionality in acyl lipid desaturases(Elsevier, 2018-10) Sastre, Diego Emiliano; Saita, Emilio Adolfo; Uttaro, Antonio Domingo; De Mendoza, Diego; Altabe, Silvia GracielaLittle is known about the structure-function relationship of membrane-bound lipid desaturases. Using a domain-swapping strategy, we found that the N terminus (comprising the two first transmembrane segments) region of Bacillus cereus DesA desaturase improves Bacillus subtilis Des activity. In addition, the replacement of the first two transmembrane domains from Bacillus licheniformis inactive open reading frame (ORF) BL02692 with the corresponding domain from DesA was sufficient to resurrect this enzyme. Unexpectedly, we were able to restore the activity of ORF BL02692 with a single substitution (Cys40Tyr) of a cysteine localized in the first transmembrane domain close to the lipidwater interface. Substitution of eight residues (Gly90, Trp104, Lys172, His228, Pro257, Leu275, Tyr282, and Leu284) by site-directed mutagenesis produced inactive variants of DesA. Homology modeling of DesA revealed that His228 is part of the metal binding center, together with the canonical His boxes. Trp104 shapes the hydrophobic tunnel, whereas Gly90 and Lys172 are probably involved in substrate binding/recognition. Pro257, Leu275, Tyr282, and Leu284 might be relevant for the structural arrangement of the active site or interaction with electron donors. This study reveals the role of the N-terminal region of 5 phospholipid desaturases and the individual residues necessary for the activity of this class of enzymes.Ítem Acceso Abierto Prevalence of Acinetobacter baumannii strains expressing the type 6 secretion system in patients with bacteremia(Taylor & Francis, 2017-07-31) Repizo, Guillermo DanielAcinetobacter baumannii (Ab) is an important nosocomial pathogen, of major concern worldwide due to its multi-drug resistance and the recent appearance of hyper-virulent strains in the clinical setting. Ab multi-drug resistant (MDR) strains are frequently associated to different types of infections, such as pneumonia, skin burns, endocarditis, meningitis and septicemia, prevalent in intensive care units. For these reasons, Ab has just been included by the World Health Organization in the list of critical priority pathogens for further studies and development of novel therapeutic approaches. In this respect, advanced knowledge of Ab physiology and mechanisms involved in environmental persistence, host colonization and virulence, all of which could be included in what is known as the physiopathology of the microorganism, is required to reduce the socio-economic impact caused by Ab infections. [...]Ítem Acceso Abierto TcHRG plays a central role in orchestrating heme uptake in Trypanosoma cruzi epimastigotes(Wiley, 2023-04-05) Tevere, Evelyn; Di Capua, Cecilia Beatriz; Chasen, Nathan Michael; Etheridge, Ronald Drew; Cricco, Julia Alejandra; https://orcid.org/0000-0001-9215-5015; https://orcid.org/0000-0002-8982-3489; https://orcid.org/0000-0002-9144-287X; https://orcid.org/0000-0002-8993-3880Trypanosoma cruzi, a heme auxotrophic parasite, can control intracellular heme content by modulating TcHRG expression when a free heme source is added to axenic culture. Herein, we explore the role of TcHRG protein in regulating the uptake of heme derived from hemoglobin in epimastigotes. It was found that the parasite’s endogenous TcHRG (protein and mRNA) responds similarly to bound (hemoglobin) and free (hemin) heme. Additionally, the overexpression of TcHRG leads to an increase in intracellular heme content. The localization of TcHRG is also not affected in parasites supplemented with hemoglobin as the sole heme source. Endocytic null epimastigotes do not show a significant difference in growth profile, intracellular heme content and TcHRG protein accumulation compared to WT when feeding with hemoglobin or hemin as a source of heme. These results suggest that the uptake of hemoglobin-derived heme likely occurs through extracellular proteolysis of hemoglobin via the flagellar pocket, and this process is governed by TcHRG. In sum, T. cruzi epimastigotes controls heme homeostasis by modulating TcHRG expression independently of the source of available heme.Ítem Acceso Abierto Assessing different ways of Bacillus subtilis spreading over abiotic surfaces(Bio-protocol L.L.C., 2019-11-20) Bartolini, Marco; Grau, Roberto RicardoSurface-associate motility on biotic and abiotic environments is a key mechanism used by the model bacterium Bacillus subtilis and its closest relatives (i.e., B. amyloliquefaciens, B. thuringiensis, B. cereus, B. pumilus) for surface colonization and spreading across surfaces. The study of this mechanism in a research, industrial or clinic laboratory is essential; however, precautions should be taken for the reproducibility of the results, for example, the procedure to inoculate the bacteria on the testing plate, the humidity of the plate and the agar concentration. In this protocol, we describe, using Bacillus subtilis, how to perform these assays and, in addition, we show how by varying the agar concentration in the plate, you can make a first approximation of what type of motility has other bacterial species.Ítem Acceso Abierto Dynamic state of plasmid genomic architectures resulting from XerC/D-mediated site-specific recombination in Acinetobacter baumannii Rep_3 superfamily resistance plasmids carrying blaOXA-58- and TnaphA6-resistance modules(Frontiers Media, 2023-02-09) Giacone, Lucía; Cameranesi, María Marcela; Sánchez, Rocio Inés; Limansky, Adriana S.; Morán Barrio, Jorgelina ; Viale, Alejandro M.The acquisition of blaOXA genes encoding different carbapenem-hydrolyzing class-D β-lactamases (CHDL) represents a main determinant of carbapenem resistance in the nosocomial pathogen Acinetobacter baumannii. The blaOXA-58 gene, in particular, is generally embedded in similar resistance modules (RM) carried by plasmids unique to the Acinetobacter genus lacking self-transferability. The ample variations in the immediate genomic contexts in which blaOXA-58-containing RMs are inserted among these plasmids, and the almost invariable presence at their borders of nonidentical 28-bp sequences potentially recognized by the host XerC and XerD tyrosine recombinases (pXerC/D-like sites), suggested an involvement of these sites in the lateral mobilization of the gene structures they encircle. However, whether and how these pXerC/D sites participate in this process is only beginning to be understood. Here, we used a series of experimental approaches to analyze the contribution of pXerC/D-mediated site-specific recombination to the generation of structural diversity between resistance plasmids carrying pXerC/D-bounded blaOXA-58- and TnaphA6-containing RM harbored by two phylogenetically- and epidemiologicallyclosely related A. baumannii strains of our collection, Ab242 and Ab825, during adaptation to the hospital environment. Our analysis disclosed the existence of different bona fide pairs of recombinationally-active pXerC/D sites in these plasmids, some mediating reversible intramolecular inversions and others reversible plasmid fusions/resolutions. All of the identified recombinationally-active pairs shared identical GGTGTA sequences at the cr spacer separating the XerC- and XerD-binding regions. The fusion of two Ab825 plasmids mediated by a pair of recombinationally-active pXerC/D sites displaying sequence differences at the cr spacer could be inferred on the basis of sequence comparison analysis, but no evidence of reversibility could be obtained in this case. The reversible plasmid genome rearrangements mediated by recombinationally-active pairs of pXerC/D sites reported here probably represents an ancient mechanism of generating structural diversity in the Acinetobacter plasmid pool. This recursive process could facilitate a rapid adaptation of an eventual bacterial host to changing environments, and has certainly contributed to the evolution of Acinetobacter plasmids and the capture and dissemination of blaOXA-58 genes among Acinetobacter and non-Acinetobacter populations co-residing in the hospital niche.Ítem Acceso Abierto Culturing bacteria from Caenorhabditis elegans gut to assess colonization proficiency(Bio-protocol L.L.C., 2017-06-20) Rodríguez Ayala, Facundo; Cogliati, Sebastián; Bauman, Carlos; Leñini, Cecilia; Bartolini, Marco; Villalba, Juan Manuel; Argañaraz, Federico; Grau, Roberto RicardoDetermining an accurate count of intestinal bacteria from Caenorhabditis elegans is one critical way to assess colonization proficiency by a given bacteria. This can be accomplished by culturing appropriate dilutions of worm gut bacteria on selective or differential agarized media. Because of the high concentration of bacteria in gut worm, dilution is necessary before plating onto growth media. Serial dilutions can reduce the concentration of the original intestinal sample to levels low enough for single colonies to be grown on media plates, allowing for the calculation of the initial counts of bacteria in the intestinal sample.Ítem Acceso Abierto A cAMP/CRP-controlled mechanism for the incorporation of extracellular ADP-glucose in Escherichia coli involving NupC and NupG nucleoside transporters(Nature Research, 2018-10-19) Almagro, Goizeder; Viale, Alejandro M.; Montero, Manuel; Muñoz, Francisco José; Baroja Fernández, Edurne; Mori, Hirotada; Pozueta Romero, JavierADP-glucose is the precursor of glycogen biosynthesis in bacteria, and a compound abundant in the starchy plant organs ingested by many mammals. Here we show that the enteric species Escherichia coli is capable of scavenging exogenous ADP-glucose for use as a glycosyl donor in glycogen biosynthesis and feed the adenine nucleotide pool. To unravel the molecular mechanisms involved in this process, we screened the E. coli single-gene deletion mutants of the Keio collection for glycogen content in ADP-glucose-containing culture medium. In comparison to wild-type (WT) cells, individual ∆nupC and ∆nupG mutants lacking the cAMP/CRP responsive inner-membrane nucleoside transporters NupC and NupG displayed reduced glycogen contents and slow ADP-glucose incorporation. In concordance, ∆cya and ∆crp mutants accumulated low levels of glycogen and slowly incorporated ADP-glucose. Two-thirds of the glycogen-excess mutants identifed during screening lacked functions that underlie envelope biogenesis and integrity, including the RpoE specifc RseA anti-sigma factor. These mutants exhibited higher ADP-glucose uptake than WT cells. The incorporation of either ∆crp, ∆nupG or ∆nupC null alleles sharply reduced the ADP-glucose incorporation and glycogen content initially witnessed in ∆rseA cells. Overall, the data showed that E. coli incorporates extracellular ADP-glucose through a cAMP/ CRP-regulated process involving the NupC and NupG nucleoside transporters that is facilitated under envelope stress conditions.Ítem Acceso Abierto High cell density production of multimethyl-branched long-chain esters in Escherichia coli and determination of their physicochemical properties(BMC (part of Springer Nature), 2016-10-14) Menendez Bravo, Simón M.; Roulet, Julia; Sabatini, Martín; Comba, Santiago; Dunn, Robert; Gramajo, Hugo Cesar; Arabolaza, Ana LorenaBackground: microbial synthesis of oleochemicals derived from native fatty acid (FA) metabolism has presented significant advances in recent years. Even so, native FA biosynthetic pathways often provide a narrow variety of usually linear hydrocarbons, thus yielding end products with limited structural diversity. To overcome this limitation, we took advantage of a polyketide synthase-based system from Mycobacterium tuberculosis and developed an Escherichia coli platform with the capacity to synthesize multimethyl-branched long-chain esters (MBE) with novel chemical structures. Results: with the aim to initiate the characterization of these novel waxy compounds, here, we describe the chassis optimization of the MBE producer E. coli strain for an up-scaled oil production. By carrying out systematic metabolic engineering, we improved the final titer to 138.1 ± 5.3 mg MBE L−1 in batch cultures. Fed-batch microbial fermentation process was also optimized achieving a maximum yield of 790.2 ± 6.9 mg MBE L−1 with a volumetric productivity of 15.8 ± 1.1 mg MBE (L h)−1. Purified MBE oil was subjected to various physicochemical analyses, including differential scanning calorimetry (DSC) and pressurized-differential scanning calorimetry (P-DSC) studies. Conclusions: the analysis of the pour point, DSC, and P-DSC data obtained showed that bacterial MBE possess improved cold flow properties than several plant oils and some chemically modified derivatives, while exhibiting high oxidation stability at elevated temperatures. These encouraging data indicate that the presence of multiple methyl branches in these novel esters, indeed, conferred favorable properties which are superior to those of linear esters.Ítem Acceso Abierto Xanthomonas citri ssp. citri requires the outer membrane porin OprB for maximal virulence and biofilm formation(Wiley, 2017-06) Ficarra, Florencia Andrea; Grandellis, Carolina; Galván, Estela M.; Ielpi, Luis; Feil, Regina; Lunn, John E.; Gottig, Natalia; Ottado, JorgelinaXanthomonas citri ssp. citri (Xcc) causes canker disease in citrus, and biofilm formation is critical for the disease cycle. OprB (Outer membrane protein B) has been shown previously to be more abundant in Xcc biofilms compared with the planktonic state. In this work, we showed that the loss of OprB in an oprB mutant abolishes bacterial biofilm formation and adherence to the host, and also compromises virulence and efficient epiphytic survival of the bacteria. Moreover, the oprB mutant is impaired in bacterial stress resistance. OprB belongs to a family of carbohydrate transport proteins, and the uptake of glucose is decreased in the mutant strain, indicating that OprB transports glucose. Loss of OprB leads to increased production of xanthan exopolysaccharide, and the carbohydrate intermediates of xanthan biosynthesis are also elevated in the mutant. The xanthan produced by the mutant has a higher viscosity and, unlike wild-type xanthan, completely lacks pyruvylation. Overall, these results suggest that Xcc reprogrammes its carbon metabolism when it senses a shortage of glucose input. The participation of OprB in the process of biofilm formation and virulence, as well as in metabolic changes to redirect the carbon flux, is discussed. Our results demonstrate the importance of environmental nutrient supply and glucose uptake via OprB for Xcc virulence.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.