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Journal of Bacteriology, April 2007, p. 2834-2843, Vol. 189, No. 7
0021-9193/07/$08.00+0     doi:10.1128/JB.01845-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Functional Characterization of the Gene Cluster from Pseudomonas syringae pv. phaseolicola NPS3121 Involved in Synthesis of Phaseolotoxin{triangledown}

Selene Aguilera,1 Karina López-López,1,2 Yudith Nieto,1 Rogelio Garcidueñas-Piña,1,{dagger} Gustavo Hernández-Guzmán,1 José Luis Hernández-Flores,1 Jesús Murillo,3 and Ariel Alvarez-Morales1*

Cinvestav, IPN Unidad Irapuato, Departamento de Ingeniería Genética, Irapuato, Gto., Apdo. Postal 629, CP 36500, Mexico,1 Departamento de Ciencias Agrícolas, Universidad Nacional de Colombia, Carrera 32 Chapinero, Palmira (Valle), Colombia,2 Depto. de Producción Agraria, Universidad Pública de Navarra, Pamplona, Spain3

Received 8 December 2006/ Accepted 15 January 2007

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.

* Corresponding author. Mailing address: Cinvestav, IPN Unidad Irapuato, Departamento de Ingeniería Genética, Irapuato, Gto., Apdo. Postal 629, CP 36500 Mexico. Phone: (52) 4626-239600. Fax: (52) 4626-245849. E-mail: aalvarez{at}ira.cinvestav.mx.

{triangledown} Published ahead of print on 19 January 2007.

{dagger} Present address: Facultad de Medicina Veterinaria y Zootecnia, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Mexico.

Journal of Bacteriology, April 2007, p. 2834-2843, Vol. 189, No. 7
0021-9193/07/$08.00+0     doi:10.1128/JB.01845-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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