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.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 ␦

Avoidance of suicide in antibiotic-producing microbes

“…In cells grown at 28-30°C, phaseolotoxin is not produced and OCT remains fully sensitive [216]. Two genes encoding OCT activity, argF and argK, are present in the producer [170] and one of them (argK, encoding ROCT) is located within the phaseolotoxin-biosynthetic cluster [1]. According to one model [126], temperature downshift is sensed in P. syringae pv.…”

Regional variations in the population structure of Pseudomonas syringae pathovar phaseolicola from Spain are revealed by typing with PmeI pulsed-field gel electrophoresis, plasmid profiling and virulence gene complement

“…phaseolicola, appears to increase virulence, and is responsible for the development of a yellow halo surrounding the watersoaked lesions characteristic of the disease (Bender et al, 1999;Patil et al, 1974). Resistance of the pathogen to its own toxin is due to production of a phaseolotoxinresistant OCTase, encoded by the argK gene, which is located, together with the biosynthetic genes, in a chromosomal region known as the argK-tox cluster (Aguilera et al, 2007;Genka et al, 2006;Mosqueda et al, 1990). Most isolates of P. syringae pv.…”

l -Amino Acid Ligase from Pseudomonas syringae Producing Tabtoxin Can Be Used for Enzymatic Synthesis of Various Functional Peptides

“…However, there are more reports on the biosynthesis of peptidic secondary metabolites involving nonribosomal peptide synthetase than reports describing Lals (26). Rhizocticin and phaseolotoxin are di-or tripeptide antibiotic and tripeptide phytotoxins, respectively, that contain an unusual amino acid, and two ATP-grasp enzymes were identified in each biosynthetic gene cluster (27,28). In rhizocticin biosynthesis, our results revealed that two Lals (RizA and RizB) combined three amino acids, yielding tripeptides similar to rhizocticin (19,20).…”