Purificación y caracterización de deshidrogenasa gliceraldehído-3- fosfato de Pseudomonas syringae pv. tomato DC3000

1 página.Pseudomonas syringae pv. tomato causa frecuentemente una enfermedad en el tomate llamada técnicamente mancha bacteriana. Pero su mecanismo de infección sigue siendo muy poco conocido.Peer reviewe

Identification of an extracellular infection-induced glyceraldehyde-3-phosphate dehydrogenase of the phytopathogenic proteobacterium Pseudomonas syringae pv tomato DC3000

According to molecular biology, genomic and proteo- mic data, the phytopathogenic gamma-proteobacte- rium Pseudomonas syringae pv. tomato DC3000 pro-duces a number of proteins that may promote infec- tion and draw nutrients from plants. Remarkably, P. syringae DC3000 strain possesses three paralogous gap genes encoding glyceraldehyde-3-phosphate dehy- drogenase (GAPDH) enzymes with different predic- ted molecular sizes and metabolic functions. As GAPDH was shown to be a virulence factor in other microbial pathogens, in the current study, we analyzed the ex-pression levels of each paralogous gap gene by real- time PCR to understand the actual impact of their protein products on P. syringae virulence. We found that all of them were strongly induced during the in-fection process. Nevertheless, proteomic analysis of cul- ture supernatants revealed that only Class I GAPDH1 encoded by the gap1 gene was identified as an extra-cellular protein in infective cells. These results strongly suggest that this GAPDH should play a role in the infective process, including its well-know en-zymatic function in the glycolytic metabolic pathway.Peer Reviewe

Identification of an extracellular infection-induced glyceraldehyde-3-phosphate dehydrogenase of the phytopathogenic proteobacterium Pseudomonas syringae pv tomato DC3000

According to molecular biology, genomic and proteo- mic data, the phytopathogenic gamma-proteobacte- rium Pseudomonas syringae pv. tomato DC3000 pro-duces a number of proteins that may promote infec- tion and draw nutrients from plants. Remarkably, P. syringae DC3000 strain possesses three paralogous gap genes encoding glyceraldehyde-3-phosphate dehy- drogenase (GAPDH) enzymes with different predic- ted molecular sizes and metabolic functions. As GAPDH was shown to be a virulence factor in other microbial pathogens, in the current study, we analyzed the ex-pression levels of each paralogous gap gene by real- time PCR to understand the actual impact of their protein products on P. syringae virulence. We found that all of them were strongly induced during the in-fection process. Nevertheless, proteomic analysis of cul- ture supernatants revealed that only Class I GAPDH1 encoded by the gap1 gene was identified as an extra-cellular protein in infective cells. These results strongly suggest that this GAPDH should play a role in the infective process, including its well-know en-zymatic function in the glycolytic metabolic pathway.España AECID (MAEC) A1/043076/1

Assessment of the electrochemical behaviour of Nickel-Titanium-based orthodontic wires : effect of some natural corrosion inhibitors in comparison with fluoride

The aim of this study is to assess the corrosion resistance behaviour of Nickel-Titanium-based orthodontic wires (NiTi) in different concentrations of Sodium Fluoride (NaF) and the corrosion?s inhibitory effect of the extracts of some medicinal plants (essential oils, hydrosols and extract). In this study we used NiTi (3M) and CuNiTi (ORMCO, 35°C, California) orthodontic wires. The following electrolytes were prepared: Lactate Ringer solution with additions of 0.1%, 0.5% or 1% of Sodium Fluoride and the extracts of different plants: Artemisia, Syzygium aromaticum (Clove) and Celtis australis. Corrosion resistance was studied using anodic potentiodynamic polarisation and electrochemical impedance spectroscopy measurements. At the end of the experiment, microscopic images of wires were performed. ANOVA test with the comparison of Bonferroni and Tukey tests were performed to elucidate comparisons among all groups. The higher sodium fluoride concentration is related to negative corrosion potential for both NiTi and CuNiTi orthodontic wire. Hydrosols are associated to positive values of corrosion potential. CuNiTi has a lower corrosion resistance than NiTi. The prescription of toothpastes containing sodium fluoride should be reduced especially for patients wearing fixed orthodontic appliances. Eugenol may be considered as alternative of sodium fluoride for orthodontic patients for its anti-microbial and anti-corrosive effects

The ciliate protist tetrahymena pyriformis as a cellular adhesion model for the pathogenic bacterium staphylococcus aureus

Staphylococcus aureus is one of the main pathogenic agents responsible for nosocomial and community-acquired bacterial infections. The pathogenicity of this Gram-positive bacterium is ensured by its different adhesion factors. Collagen and the extracellular glycoprotein adhesin are among the Staphylococcus most important virulence factors. It has been shown that most of the S. aureus strains carry the ica operon, responsible for biofilm production. However, the coexpression of the icaA and the icaD genes is necessary for complete biofilm synthesis. The aim of our study was to study a collection of 15 clinical strains of S. aureus from different sources for the presence of cna and icaD genes coding intercellular adhesion proteins. We also intended to estimate the strains¿ ability to form biofilms by the red Cong method and to test the adhesion ability of S. aureus to the ciliated protist Tetrahymena pyriformis, which we used as a novel cellular adhesion model. Finally, we checked the adhesion¿s inhibition capacity of some plants extracts. The molecular detection of adhesion genes revealed that 80% of strains are cna positive, and 73% are icaD positive. Qualitative biofilm production of S. aureus revealed that 66.6% of strains were slime producers. The adhesion test revealed that 20% of strains are strongly adhering to T. pyriformis and that the Clematis cirrhosa extract has an anti-adhering effect of S. aureus to the ciliate T. pyriformis

Cell stress by phosphate of two protozoa tetrahymena thermophila and tetrahymena pyriformis

Phosphorus is one of the bioelements most needed as a compound cell by living organisms. Phosphorus is involved in several pathologies: in human with bone and kidney diseases, in mammals with metabolism disorder (glucose, insulin···), in microorganisms whose phosphorus is involved in cell growth. Phosphorus has various forms including pyrophosphate, a by-product of multiple pathways of biosynthesis. Enzymes that hydrolyze pyrophosphate are called inorganic pyrophosphatases (PPases). Two major types of inorganic pyrophosphatases are distinguished: the soluble pyrophosphatases (sPPases) and the membrane pyrophosphatases (mPPases or H+/Na+-PPases). They play a key role in the control of intracellular inorganic pyrophosphate level and produce an important ions gradient (H+ or Na+) to the cells. In this work, we primarily focused on the physiological study in a phosphate-poor medium of two models Tetrahymena thermophile and Tetrahymena pyriformis, following the mobility, the growth and the morphology of cells. Secondly, we evaluated the enzymatic activity of soluble and membrane pyrophosphatases in both species grown in the same complex medium. A decrease of cell growth is correlated with unusual morphologies and different mobility in the stress medium. The measurement of soluble and membrane inorganic pyrophosphatases activities also shows a decrease which illustrates the lack of phosphate found in the stress medium. Deficiency of phosphate is a limiting factor for protozoan growth. These results indicate that Tetrahymena can be used as a model of cellular stress and consists of a target to study inorganic pyrophosphatases for a better understanding of phosphate cycle in higher organisms

Inorganic Pyrophosphatases: Study of Interest

Inorganic pyrophosphatases are enzymes that catalyze the hydrolysis of inorganic pyrophosphate to orthophosphate. These enzymes are divided into two groups: the soluble pyrophosphatases and the membrane pyrophosphatases. They vary in structure and each has a determined catalysis mechanism. Soluble pyrophosphatases are ubiquitous enzymes and play a key role in regulating the rate of pyrophosphate and balance in this sense, the biosynthetic reactions. Membrane pyrophosphatases are ion pumps, producing a proton or sodium gradient, and provide critical energy reserves to organisms, especially during stress conditions. Several studies have shown that these enzymes are involved in numerous disorders (diseases, fault cell growth∙∙∙). However they are potential targets for the development of agents against parasites. This article consists of a description of the different types, structures, catalytic properties of inorganic pyrophosphatases and their involvement in cellular metabolism

Cloning and heterologous overexpression of three gap genes encoding different glyceraldehyde-3-phosphate dehydrogenases from the plant pathogenic bacterium Pseudomonas syringae pv. tomato strain DC3000

The gammaproteobacterium Pseudomonas syringae pv. tomato DC3000 is the causal agent of bacterial speck, a common disease of tomato. The mode of infection of this pathogen is not well understood, but according to molecular biological, genomic and proteomic data it produces a number of proteins that may promote infection and draw nutrients from the plant. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a major enzyme of carbon metabolism that was reported to be a surface antigen and virulence factor in other pathogenic microorganisms, but its possible role in the infection process of P. syringae has so far not been studied. Whole-genome sequence analyses revealed the occurrence in this phytopathogenic bacterium of three paralogous gap genes encoding distinct GAPDHs, namely two class I enzymes having different molecular mass subunits and one class III bifunctional D-erythrose-4-phosphate dehydrogenase/GAPDH enzyme. By using genome bioinformatics data, as well as alignments of both DNA and deduced protein sequences, the three gap genes of P. syringae were one-step cloned with a His-Tag in pET21a vector using a PCR-based strategy, and its expression optimized in E. coli BL21 to achieve high yield of the heterologous proteins. In accordance with their distinct molecular phylogenies, these bacterial gap genes encode functional GAPDHs of diverse molecular masses and nicotinamide-coenzyme specificities, suggesting specific metabolic and/or cellular roles.Agencia Española de Cooperación Internacional y Desarrollo (MAEC) A1/043076/11 A/030965/10Junta de Andalucía BIO-26

Antibacterial activity of plant methanolic extracts on a field isolate of Pseudomonas syringae pv tomato from the Casablanca region (Morocco)

A bacterial field isolate recovered from infected to- mato plants in a green-house at Sidi Rehal, a region near Casablanca city (Morocco), was identified as the gammaproteobacterium Pseudomonas syringae pv. to- mato DC3000 strain, the causal agent of bacterial speck. The bacterial isolate was characterized by morphological, biochemical and molecular biological tests, its growth curves carried out in various culture media, and its phytopathogenicity verified by infec- tion tests. A screening was performed to evaluate the antibacterial activity of methanolic extracts of 12 se- lected Moroccan plants against the P. syringae pv. tomato DC3000 isolate, and Agar-well diffusion and Broth microdilution methods were used to determine minimum inhibitory and minimum bactericidal con- centrations. Among the methanolic extracts tested, only those of Nigella sativa, Geranuim robertianum, Aizoon canariense and Rubia peregrine showed clear inhibitory and bactericidal activities, although the highest values were achieved with N. sativa, a plant used in Morocco as a spice, condiment and medicinal treatment.Peer Reviewe