RESUMEN
Pseudomonas aeruginosa is a ubiquitous microorganism, capable of colonizing a wide range of habitats due to its metabolic versatility and wide adaptability to different conditions. Industrial and environmental research involving petroleum microbiology play a pivotal role in controlling many technical, operational, and environmental issues. P. aeruginosa PA1-Petro strain was isolated from oil production water in Northeastern Brazil. Herein we report the genomic sequencing and annotation of PA1-Petro, and a comparative genomics study against two widely used reference P. aeruginosa strains (PAO1 and PA14). PA1-Petro has a genome of 6,893,650 bp, the largest among the three analyzed in this study, with a 65.87% GC content. The analyzes resulted in a wide repertoire of 544 unique genes in PA1-Petro, and the highest copy numbers of common genes among the three strains (PA1-Petro, PAO1 and PA14). Unique sequences are hypothetical proteins, prophage sequences, mobile genetic elements, transcriptional regulators, metal resistance genes to copper, tellurium and arsenic, type IE CRISPR-Cas, Type VI Secretion System (T6SS)-associated proteins, and a toxin-antitoxin system. Taken together, these results provide intriguing insights on adaptive evolution within PA1-Petro genome, adding unprecedented information to the species' plasticity and ubiquitous characteristics.
RESUMEN
Cancer-related metabolic features are in part maintained by hexokinase 2 upregulation, which leads to high levels of glucose-6-phosphate (G6P) and is needed to provide energy and biomass to support rapid proliferation. Using a humanized model of the yeast Saccharomyces cerevisiae, we explored how human hexokinase 2 (HK2) behaves under different nutritional conditions. At high glucose levels, yeast presents aerobic glycolysis through a regulatory mechanism known as catabolic repression, which exerts a metabolic adaptation like the Warburg effect. At high glucose concentrations, HK2 did not translocate into the nucleus and was not able to shift the metabolism toward a highly glycolytic state, in contrast to the effect of yeast hexokinase 2 (Hxk2), which is a crucial protein for the control of aerobic glycolysis in S. cerevisiae. During the stationary phase, when glucose is exhausted, Hxk2 is shuttled out of the nucleus, ceasing catabolic repression. Cells harvested at this condition display low glucose consumption rates. However, glucose-starved cells expressing HK2 had an increased capacity to consume glucose. In those cells, HK2 localized to mitochondria, becoming insensitive to G6P inhibition. We also found that the sugar trehalose-6-phosphate (T6P) is a human HK2 inhibitor, like yeast Hxk2, but was not able to inhibit human HK1, the isoform that is ubiquitously expressed in almost all mammalian tissues. In contrast to G6P, T6P inhibited HK2 even when HK2 was associated with mitochondria. The binding of HK2 to mitochondria is crucial for cancer survival and proliferation. T6P was able to reduce the cell viability of tumor cells, although its toxicity was not impressive. This was expected as cell absorption of phosphorylated sugars is low, which might be counteracted using nanotechnology. Altogether, these data suggest that T6P may offer a new paradigm for cancer treatment based on specific inhibition of HK2.
Asunto(s)
Hexoquinasa , Fosfatos de Azúcar , Animales , Humanos , Hexoquinasa/metabolismo , Saccharomyces cerevisiae/metabolismo , Glucólisis , Glucosa/metabolismo , MamíferosRESUMEN
Pseudomonas aeruginosa is an opportunistic pathogen and an important model organism for the study of bacterial group behaviors, including cell motility and biofilm formation. Rhamnolipids play a pivotal role in biofilm formation and motility phenotypes in P. aeruginosa, possibly acting as wetting agents and mediating chemotactic stimuli. However, no biochemical mechanism or gene regulatory network has been investigated in regard to rhamnolipids' modulation of those group behaviors. Using DNA microarrays, we investigated the transcriptomic profiles in the stationary phase of growth of wild-type P. aeruginosa PAO1 and a rhlA-mutant strain, unable to produce rhamnolipids. A total of 134 genes were differentially expressed, comprising different functional categories, indicating a significant physiological difference between the rhamnolipid-producing and -non-producing strains. Interestingly, several flagellar genes are repressed in the mutant strain, which directly relates to the inability of the rhlA-minus strain to develop a swarming-motility phenotype. Supplementation with exogenous rhamnolipids has partially restored flagellar gene expression in the mutant strain. Our results show significant evidence that rhamnolipids, the major biosynthetic products of rhlABC pathway, seem to modulate gene expression in P. aeruginosa.
Asunto(s)
Glucolípidos , Pseudomonas aeruginosa , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Glucolípidos/genética , Glucolípidos/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismoRESUMEN
The inability of the yeast Saccharomyces cerevisiae to produce ethanol from xylose has hampered the biofuel production from lignocellulosic biomass. However, prior studies reveal that functional expression of xylose isomerase (XI) from Burkholderia cenocepacia (XylABc) in S. cerevisiae has remarkably improved xylose consumption and ethanol productivity. Yet, little is known about kinetic and structural properties of this enzyme. Hereby, a purified recombinant XylA was assayed in vitro, showing optimal enzyme activity at 37 °C and pH 7.2. The Km of XylA for D-xylose was at least threefold lower than the Km results for any XI published to date (e.g. XylA from Piromyces sp.). In addition, oligomerization behavior as a tetramer was observed for XylA in solution. Functional and structural comparative analyses amongst three microbial XIs were further performed as theoretical models, showing that xylose orientation at the active site was highly conserved among the XIs. Mg2+ ions anchor the sugar and guide its pyranoside oxygen towards a histidine residue present at the active site, allowing an acid-base reaction, linearizing xylose. Electrostatic surface analyses showed that small variations in the net charge distribution and dipole moment could directly affect the way the substrate interacts with the protein, thus altering its kinetic properties. Accordingly, in silico modeling suggested the tetramer may be the major functional form. These analyses and the resulting model promote a better understanding of this protein family and pave the way to further protein engineering and application of XylA in the ethanol industry.
RESUMEN
Burkholderia harbors versatile Gram-negative species and is ß-Proteobacteria. Recently, it was proposed to split the genus in two main branches: one of animal and plant pathogens and another, Paraburkholderia, harboring environmental and plant-beneficial species. Currently, Paraburkholderia comprises more than 70 species with ability to occupy very diverse environmental niches. Herein, we sequenced and analyzed the genome of Paraburkholderia kururiensis type strain KP23T , and compared to P. kururiensis M130, isolated in Brazil, and P. kururiensis susbp. thiooxydans, from Korea. This study focused on the gene content of the three genomes with special emphasis on their potential of plant-association, biocontrol, and bioremediation. The comparative analyses revealed several genes related to plant benefits, including biosynthesis of IAA, ACC deaminase, multiple efflux pumps, dioxygenases, and degradation of aromatic compounds. Importantly, a range of genes for protein secretion systems (type III, IV, V, and VI) were characterized, potentially involved in P. kururiensis well documented ability to establish endophytic association with plants. These findings shed light onto bacteria-plant interaction mechanisms at molecular level, adding novel information that supports their potential application in bioremediation, biofertilization, and biocontrol of plant pathogens. P. kururiensis emerges as a promising model to investigate adaptation mechanisms in different ecological niches.
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Biodegradación Ambiental , Burkholderiaceae/genética , Genómica , Control Biológico de Vectores , Animales , Brasil , Burkholderiaceae/aislamiento & purificación , Genoma Bacteriano , Corea (Geográfico) , Redes y Vías Metabólicas/genética , Enfermedades de las Plantas/prevención & control , Plantas/microbiología , Análisis de Secuencia de ADN , Factores de Virulencia/genéticaRESUMEN
RhlR is a 241-residue quorum sensing receptor that controls the expression of a myriad of virulence genes in Pseudomonas aeruginosa. Here, the DNA sequence encoding the carboxi-terminal DNA-binding domain of RhlR was cloned into the pET-RP1B plasmid and expressed as an N-terminal fusion protein to the expression/purification Thio6His6 tag. The fusion construct expressed insolubly in Escherichia coli BL21 (DE3) cells. The recombinant protein was extracted from the bacterial inclusion bodies and refolded in the presence of the charged amino acids l-arginine and l-glutamate. The refolded protein was purified by a combination of Ni(+2)-affinity and size exclusion chromatography, allowing the production of 2 mg of highly purified protein (>95% purity) per 5 mg of wet cells derived from 1 L culture. (1)H 1D NMR analysis revealed that the recombinant protein is folded. Moreover, a fluorescence anisotropy DNA-binding assay showed that the refolded protein is functional, as it recognizes the rhlAB promoter. This is the first time that a domain of the quorum sensing regulator RhlR was produced in sufficient amounts for structural studies, enabling the investigation of the molecular basis for RhlR specific interaction with DNA promoters.
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Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , Percepción de Quorum/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , Pliegue de Proteína , Pseudomonas aeruginosa/genéticaRESUMEN
Rhamnolipids have emerged as a very promising class of biosurfactants in the last decades, exhibiting properties of great interest in several industrial applications, and have represented a suitable alternative to chemically-synthesized surfactants. This class of biosurfactants has been extensively studied in recent years, aiming at their large-scale production based on renewable resources, which still require high financial costs. Development of non-pathogenic, high-producing strains has been the focus of a number of studies involving heterologous microbial hosts as platforms. However, the intricate gene regulation network controlling rhamnolipid biosynthesis represents a challenge to metabolic engineering and remains to be further understood and explored. This article provides an overview of the biosynthetic pathways and the main gene regulatory factors involved in rhamnolipid production within Pseudomonas aeruginosa, the prototypal producing species. In addition, we provide a perspective view into the main strategies applied to metabolic engineering and biotechnological production.
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Biotecnología/métodos , Regulación Bacteriana de la Expresión Génica , Glucolípidos/biosíntesis , Ingeniería Metabólica , Vías Biosintéticas/genética , Glucolípidos/químicaRESUMEN
Biosurfactants are a class of functional molecules produced and secreted by microorganisms, which play important roles in cell physiology such as flagellum-dependent or -independent bacterial spreading, cell signaling, and biofilm formation. They are amphipathic compounds and comprise a variety of chemical structures, including rhamnolipids, typically produced by Pseudomonas spp. and also reported within other bacterial genera. The present study is focused on Burkholderia kururiensis KP23(T), a trichloroethylene (TCE)-degrading, N-fixing, and plant growth-promoting bacterium. Herein, we describe the production of rhamnolipids by B. kururiensis, and its characterization by LTQ-Orbitrap Hybrid Mass Spectrometry, a powerful tool that allowed efficient identification of molecular subpopulations, due to its high selectivity, mass accuracy, and resolving power. The population of rhamnolipids produced by B. kururiensis revealed molecular species commonly observed in Pseudomonas spp. and/or Burkholderia spp. In addition, this strain was used as a platform for expression of two Pseudomonas aeruginosa biosynthetic enzymes: RhlA, which directly utilizes ß-hydroxydecanoyl-ACP intermediates in fatty acid synthesis to generate the HAA, and RhlB, the rhamnosyltransferase 1, which catalyzes the transfer of dTDP-L-rhamnose to ß-hydroxy fatty acids in the biosynthesis of rhamnolipids. We show that rhamnolipid production by the engineered B. kururiensis was increased over 600 % when compared to the wild type. Structural analyses demonstrated a molecular population composed mainly of monorhamnolipids, as opposed to wild-type B. kururiensis and P. aeruginosa in which dirhamnolipids are predominant. We conclude that B. kururiensis is a promising biosurfactant-producing organism, with great potential for environmental and biotechnological applications due to its non-pathogenic characteristics and efficiency as a platform for metabolic engineering and production of tailor-made biosurfactants.
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Burkholderia/genética , Burkholderia/metabolismo , Glucolípidos/química , Glucolípidos/metabolismo , Ingeniería Metabólica , Redes y Vías Metabólicas/genética , Clonación Molecular , Expresión Génica , Espectrometría de Masas , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tensoactivos/química , Tensoactivos/metabolismoRESUMEN
Pseudomonas aeruginosa is associated with increased mortality in cystic fibrosis (CF) patients, and expresses type III secretion system proteins (TTSP), which is a common mechanism used by gram-negative pathogens for delivery of anti-host factors. Our aim was to investigate whether or not these antigens (TTSP) would be recognized by CF sera, by Western blot reaction. We have showed herein that all patients (n = 11) not chronically infected by P. aeruginosa had their first serum positive for TTSP (ExoS, ExoT, PopB, and/or PopD). All chronic patients had a strong positive serology to TTSP, although relatively weak reactions to TTSP were observed for some individuals in the negative control group. Therefore, TTSP that were early produced in P. aeruginosa infected CF patients, induced a detectable antibody response in those patients and were easily detected by Western-blot reaction.
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Fibrosis Quística/microbiología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/aislamiento & purificación , Infecciones del Sistema Respiratorio/microbiología , Adolescente , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/sangre , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/sangre , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Niño , Preescolar , Estudios Transversales , Fibrosis Quística/sangre , Fibrosis Quística/metabolismo , Humanos , Infecciones por Pseudomonas/sangre , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa/metabolismo , Infecciones del Sistema Respiratorio/inmunología , Infecciones del Sistema Respiratorio/metabolismo , Estudios RetrospectivosRESUMEN
One of the most interesting aspects of Trypanosoma cruzi is its adaptation to obtain sialic acid from its host, fulfilling this need exclusively through the reaction catalyzed by enzymatically active trans-sialidase (aTS), thought to play an important role in the pathogenesis of Chagas' disease. Herein, we report that 2-difluoromethyl-4-nitrophenyl-3,5-dideoxy-d-glycero-alpha-d-galacto-2-nonulopyranosid acid (NeuNAcFNP) inactivates aTS time- and dose-dependently, and this inhibition was not relieved removing the inhibitor. Also, NeuNAcFNP causes a decrease in infection of mammalian cells. Characterization of labeled aTS by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry revealed that inactivation of the enzyme occurs through formation of a covalent bond between Arg245 and Asp247 and the inhibitor aglycone. Participation of Asp247 in the catalytic mechanism was proved by constructing a TSD247A mutant, which presents only residual activity. Molecular dynamic simulations indicate that the D247A mutation results in a more open catalytic cleft. In summary, NeuNAcFNP is the first reported mechanism-based inhibitor of aTS, representing a new template for drug design and opening new possibilities for chemotherapy of Chagas' disease, as well as for the elucidation of aTS function in T. cruzi pathogenesis and biology.
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Inhibidores Enzimáticos/farmacología , Glicoproteínas/antagonistas & inhibidores , Interacciones Huésped-Parásitos/efectos de los fármacos , Neuraminidasa/antagonistas & inhibidores , Ácidos Siálicos/farmacología , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/patogenicidad , Animales , Biocatálisis/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Glicoproteínas/química , Glicoproteínas/metabolismo , Simulación de Dinámica Molecular , Estructura Molecular , Neuraminidasa/química , Neuraminidasa/metabolismo , Ácidos Siálicos/química , Relación Estructura-Actividad , Trypanosoma cruzi/efectos de los fármacosRESUMEN
Burkholderia kururiensis is a diazotrophic bacterium originally isolated from a polluted aquifer environment and presents a high level of similarity with the rice endophyte "B. brasilensis" species. This work assessed the ability of B. kururiensis to endophytically colonize rice plantlets by monitoring different tissues of root-inoculated plants for the presence of bacterial growth in different media, electron microscopy and by 16S rDNA analysis. Observations of roots, stems and leaves of inoculated rice plantlets by electron microscopy revealed B. kururiensis colonization predominantly on root hair zones, demonstrating endophytic colonization primarily through the endodermis, followed by spreading into xylem vessels, a possible pathway leading to aerial parts. Although indifferent for the bacterial growth itself, addition of a nitrogen source was a limiting factor for endophytic colonization. As endophytic colonization was directly associated to an enhanced plant development, production of phytohormone auxin/indole-3-acetic acid by B. kururiensis was assayed with transgenic rice plantlets containing an auxin-responsive reporter (DR5-GUS). Our findings suggest the ability of auxin production by plant-associated B. kururiensis which may have a stimulatory effect on plant development, as evidenced by activation of DR5-GUS. We hereby demonstrate, for the first time, the ability of B. kururiensis to endophytically colonize rice, promoting both plant growth and rice grain yield.
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Burkholderia/fisiología , Ácidos Indolacéticos/análisis , Oryza/microbiología , Burkholderia/ultraestructura , Recuento de Colonia Microbiana , Microscopía Electrónica , Oryza/crecimiento & desarrollo , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16SRESUMEN
Burkholderia kururiensis is a diazotrophic bacterium originally isolated from a polluted aquifer environment and presents a high level of similarity with the rice endophyte "B. brasilensis" species. This work assessed the ability of B. kururiensis to endophytically colonize rice plantlets by monitoring different tissues of root-inoculated plants for the presence of bacterial growth in different media, electron microscopy and by 16S rDNA analysis. Observations of roots, stems and leaves of inoculated rice plantlets by electron microscopy revealed B. kururiensis colonization predominantly on root hair zones, demonstrating endophytic colonization primarily through the endodermis, followed by spreading into xylem vessels, a possible pathway leading to aerial parts. Although indifferent for the bacterial growth itself, addition of a nitrogen source was a limiting factor for endophytic colonization. As endophytic colonization was directly associated to an enhanced plant development, production of phytohormone auxin/indole-3-acetic acid by B. kururiensis was assayed with transgenic rice plantlets containing an auxin-responsive reporter (DR5-GUS). Our findings suggest the ability of auxin production by plant-associated B. kururiensis which may have a stimulatory effect on plant development, as evidenced by activation of DR5-GUS. We hereby demonstrate, for the first time, the ability of B. kururiensis to endophytically colonize rice, promoting both plant growth and rice grain yield.
Burkholderia kururiensis é uma bactéria diazotrófica, originalmente isolada de um ambiente aquático poluído e apresenta alto nível de similaridade com a espécie endofítica "B. brasilensis" encontrada na planta de arroz. Este artigo demonstrou a habilidade de B. kururiensis colonizar endofiticamente plântulas de arroz, após esta bactéria ter sido inoculada na raiz das plantas. Esta capacidade foi confirmada pelo crescimento bacteriano em diferentes tecidos da planta, por microscopia eletrônica e pela análise do 16S rADN. Observação por microscopia eletrônica das raízes, caule e folhas das plântulas de arroz inoculadas, revelou predominância da colonização de B. kururiensis na zona pilífera da raiz, demonstrando que a colonização endofítica inicia-se na endoderme, espalha-se pelo xilema, sendo esta a possível via para a bactéria alcançar as partes aéreas. A adição de uma fonte de nitrogênio, embora não tenha influenciado no crescimento bacteriano, foi um fator limitante para a colonização endofítica. Como a colonização endofítica mostrou-se diretamente associada ao aumento no desenvolvimento da planta, a produção do fitohormônio auxina/ácido 3-indolacético pela B. kururiensis foi verificada utilizando uma plântula de arroz transgênica, contendo o repórter responsivo para auxina (DR5-GUS). Nossos resultados sugerem que a produção de auxina pela B. kururiensis é responsável pelo estímulo no desenvolvimento da planta verificado pela ativação do DR5-GUS. Neste trabalho demonstramos, pela primeira vez, a habilidade de B. kururiensis colonizar endofiticamente a planta de arroz, promovendo tanto o aumento no crescimento da planta como a produção de sementes de arroz.
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Burkholderia/fisiología , Ácidos Indolacéticos/análisis , Oryza/microbiología , Burkholderia/ultraestructura , Recuento de Colonia Microbiana , Microscopía Electrónica , Oryza/crecimiento & desarrollo , Reacción en Cadena de la PolimerasaRESUMEN
The LEE 4 genes sepL, espA, espD, espB, and espF were detected in 50 strains of typical and atypical enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli by PCR. sepL was amplified in 90%, espA in 94%, espB in 50%, espD in 40%, and espF in 78% of all strains, employing prototype EPEC-based primers. With O26:H(-)-based primers, espB was detected in all O26 strains, and O157:H7-specific primers amplified espD and espB among all O55:H7 and O157:H7 strains. Our results indicated that espA and sepL should be more conserved between different EPEC and EHEC serotypes, while espB, espD, and espF should be more diverse. Apparently this variation is related to serogroup or serotype, but sequencing assays are necessary to confirm such conservation/diversity and their association with serogroup or serotype. Secreted protein analyses of espA, espD, and espB PCR-negative strains demonstrated that their encoded proteins present distinct immunological types, reflecting the genetic variability of those genes.