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

ABSTRACT: 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.

SUBMITTER: Aguilera S

PROVIDER: S-EPMC1855804 | biostudies-literature | 2007 Apr

REPOSITORIES: biostudies-literature

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

Aguilera Selene S López-López Karina K Nieto Yudith Y Garcidueñas-Piña Rogelio R Hernández-Guzmán Gustavo G Hernández-Flores José Luis JL Murillo Jesús J Alvarez-Morales Ariel A

Journal of bacteriology 20070119 7

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 ide ...[more]

PMID: 17237165

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Project description:P. syringae pv. phaseolicola, the causal agent of halo blight disease in bean, produces a toxin known as phaseolotoxin, whose synthesis involves the product of some of the genes found within the Pht region. This region, considered a pathogenicity island, comprises 23 genes arranged in five transcriptional units; two single-gene units (argK, phtL) and three arranged as operons (phtA, phtD, phtM), most with unknown function. In P. syringae pv. phaseolicola, maximal expression of most of the genes encoded in the Pht region and the synthesis of phaseolotoxin require the product of the phtL gene, which has been proposed to have a regulatory function. In order to evaluate the role of phtL gene in P. syringae pv. phaseolicola, we performed a comparative transcriptional analysis with the wild type and a phtL- mutant strains using microarrays. The microarray data analysis showed that PhtL regulates the expression not only of genes within the Pht region, but also alters the expression of genomic genes related with the iron-acquisition system, pathogenicity, oxidative stress and virulence. This study suggests the possible relation of the PhtL protein with the iron response genes and with the pathogenicity and or virulence of this bacterium. The microarrays used in this study correspond to an assembled DNA microarray of P. syringae pv. phaseolicola NPS3121 previously reported (GEO accession number GPL7115) (HernM-CM-!ndez-Morales et al., 2009). This microarray consist of 1911 probes, with an average length 2.4 Kbp, representing 1X the genome of P. syringae pv. phaseolicola NPS3121, as well as several PCR products corresponding to various known genes, which were printed as controls. Each microarray experiment was repeated six times (two technical replicates with the same RNA samples and three biological replicates using RNA isolated from a different culture). cDNA synthesis, labeling, hybridization, washing, and chip scanning were performed at the Microarray Core Facility at Cinvestav-Langebio. Hybridized microarray slides were scanned (GenePix 4000, Axon Instruments, Inc) at a 10 um resolution adjusting the laser and gain parameters to obtain similar levels of fluorescence intensity in both channels. The spot intensities were quantified using Axon GenePrix Pro 6.0 image analysis software. First, an automatic spot finding and quantification option of the software was used. Subsequently, all spots were inspected individually and in some cases, the spot diameters were corrected or the spots were removed from the analysis. The mean of the signals and the median of backgrounds were used for further analysis. Raw data were imported into the R.2.2.1 software. Background signals were subtracted using the Robust Multichip Analysis (RMA) whereas the normalization of the signal intensities within slides was carried out using M-bM-^@M-^\print-tip loessM-bM-^@M-^] method and the LIMMA package. Normalized data were log2 transformed and then fitted into mixed model ANOVAs using the Mixed procedure. The p-values of the phtL effects were adjusted for by the False Discovery Rate Method M-bM-^@M-^\FDRM-bM-^@M-^]. Changes in signal intensity of M-BM-11.5-fold or higher/lower between phtL- mutant and wild type were highly significant (FDR, p-value M-bM-^IM-$0.05), however we focused only in differential expressed genes that fell within the more traditional criteria, which is the cut-off threshold for up-regulated (M-bM-^IM-%1.8) and down-regulated genes (M-bM-^IM-$0.50).

Dataset Information

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

Publications

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

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