Clonamiento y expresión heteróloga de tres genes que codifican para enzimas del tipo lipasa y proteasa, relacionadas con la actividad nematicida de Pseudomonas veronii R4
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2017
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es
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Universidad Andrés Bello
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Resumen
Pseudomonas veronii R4 es un nuevo aislado bacteriano que ha sido identificado previamente desde un panel de Pseudomonas ssp. fluorescentes con actividad nematicida. En particular, P. veronii R4, presenta actividad nematicida contra el fitopatógeno Xiphinema index, nematodo de importancia comercial en viticultura, debido a que es el principal vector del virus de la hoja en abanico de la vid (GFLV). Tras su caracterización y secuenciación de su genoma, se ha propuesto que esta actividad antagonista sobre X. index se debería a una batería enzimática que consiste en al menos una proteasa (AprA), una lipasa (LipA) y una fosfolipasa (ExoU). Con el objetivo de evaluar y confirmar la actividad nematicida de estas enzimas, en este trabajo de Tesis se clonaron los genes aprA, lipA y exoU (que codifican para las enzimas antes mencionadas) en vectores de entrada y expresión de la serie Gateway, utilizando como hospedero heterólogo la bacteria Escherichia coli BL21(DE3). La sobreexpresión de las proteínas recombinantes en el sistema de expresión produjo agregados de proteínas recombinantes, las cuales se debieron solubilizar y renaturalizar. Ensayos enzimáticos de extractos de enzimas recombinantes, AprA, LipA y ExoU, en los sustratos gelatina, tributirina y sangre, respectivamente, evidenciaron la funcionalidad de estas enzimas.
Finalmente, la exposición de X. index frente a los extractos de proteínas enriquecidos con las enzimas AprA (500 μg/mL), LipA (50 μg/mL) y ExoU (50 μg/mL) recombinantes provocaron pérdida de movilidad mayor a 90% después de 2 h de incubación a temperatura ambiente, evaluado a través de ensayos in vitro. Concordantemente, se observaron, mediante microscopia electrónica de barrido, daños estructurales en la cutícula, deshidratación y exposición de tejidos internos de los nematodos expuestos a los extractos de enzimas recombinantes.
Los resultados obtenidos permiten confirmar que la actividad nematicida de la bacteria es causada por al menos las enzimas AprA, LipA y ExoU de R4, lo que además representa proyecciones de estas enzimas para su aplicación biotecnológica.
Pseudomonas veronii R4 is a new bacterial isolate that has been previously identified from a panel of fluorescent Pseudomonas ssp. with nematicidal activity. In particular , P. veronii R4, has nematicidal activity against the phytopathogen Xiphinema index, a nematode of commercial importance in viticulture, because it is the main vector of the grapevine fanleaf virus in the vine (GFLV). After characterization and sequencing of its genome, it has been proposed that this antagonistic activity on X. index would be due to an enzymatic battery consisting of at least one protease (AprA), a lipase (LipA) and a phospholipase (ExoU). In order to evaluate and confirm the nematicidal activity of these enzymes, in this Thesis , the genes aprA, lipA and exoU (encoding the aforementioned enzymes) were cloned into input and expression vectors of the Gateway serie system , using the bacterium Escherichia coli BL21 (DE3) as an heterologous host .The overexpression of the recombinant proteins in the expression system produced aggregates of recombinant proteins, which had to be solubilized and renatured. Enzymatic assays of recombinant enzyme extracts, AprA, LipA and ExoU, on gelatin, tributrin and blood substrates, respectively, evidenced the functionality of these enzymes. Finally, the exposure of X. index to proteins enriched with the enzymes AprA (500 μg / mL), LipA (50 μg / mL) and ExoU (50 μg / mL recombinant resulted in loss of mobility greater than 90% after 2 h of incubation at room temperature, evaluated by in vitro assays. Accordingly, structural damage to the cuticle, dehydration and exposure of internal tissues of nematodes exposed to recombinant enzyme extracts were observed by scanning electron microscopy. The results obtained confirm that the nematicidal activity of the bacteria is caused by at least the enzymes AprA, LipA and ExoU of R4, which also represents projections of these enzymes for their biotechnological application.
Pseudomonas veronii R4 is a new bacterial isolate that has been previously identified from a panel of fluorescent Pseudomonas ssp. with nematicidal activity. In particular , P. veronii R4, has nematicidal activity against the phytopathogen Xiphinema index, a nematode of commercial importance in viticulture, because it is the main vector of the grapevine fanleaf virus in the vine (GFLV). After characterization and sequencing of its genome, it has been proposed that this antagonistic activity on X. index would be due to an enzymatic battery consisting of at least one protease (AprA), a lipase (LipA) and a phospholipase (ExoU). In order to evaluate and confirm the nematicidal activity of these enzymes, in this Thesis , the genes aprA, lipA and exoU (encoding the aforementioned enzymes) were cloned into input and expression vectors of the Gateway serie system , using the bacterium Escherichia coli BL21 (DE3) as an heterologous host .The overexpression of the recombinant proteins in the expression system produced aggregates of recombinant proteins, which had to be solubilized and renatured. Enzymatic assays of recombinant enzyme extracts, AprA, LipA and ExoU, on gelatin, tributrin and blood substrates, respectively, evidenced the functionality of these enzymes. Finally, the exposure of X. index to proteins enriched with the enzymes AprA (500 μg / mL), LipA (50 μg / mL) and ExoU (50 μg / mL recombinant resulted in loss of mobility greater than 90% after 2 h of incubation at room temperature, evaluated by in vitro assays. Accordingly, structural damage to the cuticle, dehydration and exposure of internal tissues of nematodes exposed to recombinant enzyme extracts were observed by scanning electron microscopy. The results obtained confirm that the nematicidal activity of the bacteria is caused by at least the enzymes AprA, LipA and ExoU of R4, which also represents projections of these enzymes for their biotechnological application.
Notas
Tesis (Magíster en Biotecnología)
proyecto “Nuevas Tecnologías de Mejoramiento”, código 502146-46, Laboratorio de Biotecnología, INIA La Platina
proyecto “Nuevas Tecnologías de Mejoramiento”, código 502146-46, Laboratorio de Biotecnología, INIA La Platina
Palabras clave
Pseudomonas, Enzimas