Dpto. Producción Agraria - Nekazaritza Ekoizpena Saila
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Browsing Dpto. Producción Agraria - Nekazaritza Ekoizpena Saila by Author "Aguilera, Selene"
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Publication Open Access Expression of the gene for resistance to phaseolotoxin (argK) depends on the activity of genes phtABC in Pseudomonas syringae pv. Phaseolicola(Public Library of Science, 2012) Aguilera, Selene; Torre Zavala, Susana de la; Hernández Flores, José Luis; Murillo Martínez, Jesús; Bravo, Jaime; Álvarez Morales, Ariel; Producción Agraria; Nekazaritza EkoizpenaThe bacterium Pseudomonas syringae pv. phaseolicola produces phaseolotoxin in a temperature dependent manner, being optimally produced between 18 degrees C and 20 degrees C, while no detectable amounts are present above 28 degrees C. Phaseolotoxin is an effective inhibitor of ornithine carbamoyltransferase (OCTase) activity from plant, mammalian and bacterial sources and causes a phenotypic requirement for arginine. To protect the cell from its own toxin, P. syringae pv. phaseolicola synthesizes a phaseolotoxin-resistant OCTase (ROCT). The ROCT is the product of the argK gene and is synthesized only under conditions leading to phaseolotoxin synthesis. The argK gene is included in a chromosomal fragment named Pht cluster, which contains genes involved in the synthesis of phaseolotoxin. The aim of the present work was to investigate the possible involvement of other genes included in the Pht cluster in the regulation of gene argK. We conducted transcriptional analyses of argK in several mutants unable to produce phaseolotoxin, transcriptional fusions and electrophoretic mobility shift assays, which allowed us to determine that genes phtABC, located within the Pht cluster, participate in the transcriptional repression of gene argK at temperatures not permissive for phaseolotoxin biosynthesis. This repression is mediated by a protein present in both toxigenic and nontoxigenic strains of P. syringae and in E. coli, and requires the coordinated participation of phtA, phtB and phtC products in order to carry out an efficient argK repression.Publication Open Access Functional characterization of the gene cluster from Pseudomonas syringae pv. phaseolicola NPS3121 involved in synthesis of phaseolotoxin(American Society for Microbiology, 2007) Aguilera, Selene; López-López, Karina; Nieto, Yudith; Garcidueñas-Piña, Rogelio; Hernández-Guzmán, Gustavo; Hernández Flores, José Luis; Murillo Martínez, Jesús; Álvarez Morales, Ariel; Producción Agraria; Nekazaritza EkoizpenaPseudomonas syringae pv. phaseolicola is the causal agent of halo blight disease of beans (Phaseolus vulgaris L.), which is characterized by water-soaked lesions surrounded by a chlorotic halo resulting from the action of a non-host-specific toxin known as phaseolotoxin. This phytotoxin inhibits the enzyme ornithine carbamoyltransferase involved in arginine biosynthesis. Different evidence suggested that genes involved in phaseolotoxin production were clustered. Two genes had been previously identified in our laboratory within this cluster: argK, which is involved in the immunity of the bacterium to its own toxin, and amtA, which is involved in the synthesis of homoarginine. We sequenced the region around argK and amtA in P. syringae pv. phaseolicola NPS3121 to determine the limits of the putative phaseolotoxin gene cluster and to determine the transcriptional pattern of the genes comprising it. We report that the phaseolotoxin cluster (Pht cluster) is composed of 23 genes and is flanked by insertion sequences and transposases. The mutation of 14 of the genes within the cluster lead to a Tox− phenotype for 11 of them, while three mutants exhibited low levels of toxin production. The analysis of fusions of selected DNA fragments to uidA, Northern probing, and reverse transcription-PCR indicate the presence of five transcriptional units, two monocistronic and three polycistronic; one is internal to a larger operon. The site for transcription initiation has been determined for each promoter, and the putative promoter regions were identified. Preliminary results also indicate that the gene product of phtL is involved in the regulation of the synthesis of phaseolotoxin.Publication Open Access Temperature-mediated biosynthesis of the phytotoxin phaseolotoxin by Pseudomonas syringae pv. phaseolicola depends on the autoregulated expression of the phtABC genes(Public Library of Science, 2017) Aguilera, Selene; Álvarez Morales, Ariel; Murillo Martínez, Jesús; Hernández Flores, José Luis; Bravo, Jaime; Torre Zavala, Susana de la; Producción Agraria; Nekazaritza EkoizpenaPseudomonas syringae pv. phaseolicola produces phaseolotoxin in a temperature dependent manner, being optimally synthesized between 18ºC and 20ºC, while no detectable amounts are present above 28ºC. The Pht cluster, involved in the biosynthesis of phaseolotoxin, contains 23 genes that are organized in five transcriptional units. The function of most of the genes from the Pht cluster is still unknown and little information about the regulatory circuitry leading to expression of these genes has been reported. The purpose of the present study was to investigate the participation of pht genes in the regulation of the operons coded into the Pht cluster. We conducted Northern blot, uidA fusions and reverse transcription- PCR assays of pht genes in several mutants unable to produce phaseolotoxin. This allowed us to determine that, in P. syringae pv. phaseolicola NPS3121, genes phtABC are essential to prevent their own expression at 28ºC, a temperature at which no detectable amounts of the toxin are present. We obtained evidence that the phtABC genes also participate in the regulation of the phtD, phtM and phtL operons. According to our results, we propose that PhtABC and other Pht product activities could be involved in the synthesis of the sulfodiaminophosphinyl moiety of phaseolotoxin, which indirectly could be involved in the transcriptional regulation of the phtA operon.