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J. Bacteriol. doi:10.1128/JB.01845-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

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

Cinvestav, IPN Unidad Irapuato, Departamento de Ingeniería Genética, Irapuato, Gto., Apdo. Postal 629, CP 36500 Mexico; Departamento de Ciencias Agrícolas, Universidad Nacional de Colombia, Carrera 32 Chapinero, Palmira (Valle), Colombia; Depto. de Producción Agraria, Universidad Pública de Navarra, Pamplona, Spain

^* To whom correspondence should be addressed. Email: aalvarez{at}ira.cinvestav.mx.

	Abstract

Pseudomonas syringae pv. phaseolicola is the causal agent of halo blight disease of beans (Phaseolus vulgaris L.), characterized by water-soaked lesions surrounded by a chlorotic halo resulting from the action of a nonhost-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 involved in immunity of the bacterium to its own toxin, and amtA involved in the synthesis of homoarginine. We sequenced the region from P. syringae pv. phaseolicola NPS3121 around argK and amtA in order 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 it is flanked by insertion sequences and transposases. 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 RT-PCR indicates the presence of five transcriptional units, two monocistronic and three polycistronic, one of them internal to a larger operon. The site for transcription initiation has been determined for each promoter and the putative promoter regions identified. Preliminary results also indicate that the gene product of phtL is involved in regulation of the synthesis of phaseolotoxin.