Functional characterization of the gene cluster from Pseudomonas syringae pv. phaseolicola NPS3121 involved in synthesis of phaseolotoxin.

Pseudomonas 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.

Importancia de la verificación-asignación de progenitores en sistemas extensivos de pie de cría / Importance of parentage verification-assignment in extensive multiple-sired breeding herds

Se aplicó un panel de nueve marcadores microsatélites para estructurar la genealogía de un hato de ganado Braford manejado bajo empadre múltiple y destinado a pié de cría, para evaluar las repercusiones de la adecuada asignación de progenitores, así como las implicaciones en su mejoramiento genético. Se logró la asignación de paternidad en el 100% de la progenie, mientras que en los ensayos de verificación de maternidad se estimó un porcentaje de error de asignación de aproximadamente 90%. Los resultados encontrados apoyan el uso de la asignación de paternidad para verificar la estructura genealógica (paternidad y maternidad) de hatos cuya certeza en el pedigrí es crítica para el mejoramiento genético de su raza, y en donde el sistema de manejo extensivo y empadre múltiple limitan el registro adecuado de la progenie al momento del parto.

To assess the implications of parentage assignation on herds-genetic improvement nine microsatellite markers were used in order to structure the genealogy of a multisired Braford herd. All progeny (100%) had satisfactory paternity assignment, conversely the maternity verification analysis showed assignation errors up to 90%. Our results support the use of molecular tools to verify the pedigree structure in those herds with management systems that limit the proper registration of progeny at calving.

The global arginine regulator ArgR controls expression of argF in Pseudomonas syringae pv. phaseolicola but is not required for the synthesis of phaseolotoxin or for the regulated expression of argK.

In Pseudomonas syringae pv. phaseolicola the enzyme ornithine carbamoyltransferase (OCTase), encoded by argF, is negatively regulated by argR, similar to what has been reported for Pseudomonas aeruginosa. However, production of the phaseolotoxin-resistant OCTase encoded by argK, synthesis of phaseolotoxin, and infectivity for bean pods occur independently of the ArgR protein.