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.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 (23). This halo results from the action of a non-hostspecific toxin known as phaseolotoxin [N ␦

No abstract

Pseudomonas syringae pv. phaseolicola synthesizes a non-host-specific toxin, phaseolotoxin, and also synthesizes a phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) to protect itself from its own toxin. ROCT is encoded by argK, which is expressed coordinately with phaseolotoxin synthesis at 18°C. To investigate the regulatory mechanisms of this system, null mutants were constructed for argK, argF (encoding the phaseolotoxin-sensitive OCTase [SOCT]), and amtA (encoding an amidinotransferase involved in phaseolotoxin synthesis). The argF mutant did not exhibit arginine auxotrophy when grown in M9 medium at 28°C, because under this condition SOCT was replaced by ROCT. This loss of thermoregulation of argK was apparently caused by accumulation of carbamoylphosphate, one of the substrates of SOCT. Carbamoylphosphate, which has a structure similar to that of the inorganic moiety of phaseolotoxin, was used in induction assays with wild-type P. syringae pv. phaseolicola and was shown to be able to induce argK expression in M9 medium at 28°C. These results indicate that argK expression is independent of temperature and is regulated directly by a compound resembling the inorganic moiety of phaseolotoxin.

BackgroundPseudomonas syringae pv. phaseolicola is a Gram-negative plant-pathogenic bacterium that causes "halo blight" disease of beans (Phaseolus vulgaris L.). This disease affects both foliage and pods, and is a major problem in temperate areas of the world. Although several bacterial genes have been determined as participants in pathogenesis, the overall process still remains poorly understood, mainly because the identity and function of many of the genes are largely unknown. In this work, a genomic library of P. syringae pv. phaseolicola NPS3121 was constructed and PCR amplification of individual fragments was carried out in order to print a DNA microarray. This microarray was used to identify genes that are differentially expressed when bean leaf extracts, pod extracts or apoplastic fluid were added to the growth medium.ResultsTranscription profiles show that 224 genes were differentially expressed, the majority under the effect of bean leaf extract and apoplastic fluid. Some of the induced genes were previously known to be involved in the first stages of the bacterial-plant interaction and virulence. These include genes encoding type III secretion system proteins and genes involved in cell-wall degradation, phaseolotoxin synthesis and aerobic metabolism. On the other hand, most repressed genes were found to be involved in the uptake and metabolism of iron.ConclusionThis study furthers the understanding of the mechanisms involved, responses and the metabolic adaptation that occurs during the interaction of P. syringae pv. phaseolicola with a susceptible host plant.

The bacterium Pseudomonas syringae pv. phaseolicola produces phaseolotoxin in a temperature dependent manner, being optimally produced between 18°C and 20°C, while no detectable amounts are present above 28°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.