Karina López-López

Functional Characterization of the Gene Cluster from Pseudomonas syringae pv. phaseolicola NPS3121 Involved in 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 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.

In Pseudomonas syringae pv. phaseolicola, Expression of the argK Gene, Encoding the Phaseolotoxin-Resistant Ornithine Carbamoyltransferase, Is Regulated Indirectly by Temperature and Directly by a Precursor Resembling Carbamoylphosphate

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 degrees 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 degrees 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 degrees C. These results indicate that argK expression is independent of temperature and is regulated directly by a compound resembling the inorganic moiety of phaseolotoxin.

The global arginine regulator ArgR controls expression of argF in Pseudomonas syringae pv. phaseolicola but is not required for the synthesis of phaseolotoxin or for the regulated expression of argK.

In Pseudomonas syringae pv. phaseolicola the enzyme ornithine carbamoyltransferase (OCTase), encoded by argF, is negatively regulated by argR, similar to what has been reported for Pseudomonas aeruginosa. However, production of the phaseolotoxin-resistant OCTase encoded by argK, synthesis of phaseolotoxin, and infectivity for bean pods occur independently of the ArgR protein.

Current Ideas on the Genetics and Regulation of the Synthesis of Phaseolotoxin in Pseudomonas Syringae PV. Phaseolicola

The bacterium Pseudomonas syringae pv. phaseolicola is the causal agent of the “halo blight” disease of beans (Phaseolus vulgaris L.). This bacterium shares wide evolutionary relatedness with Pseudomonas savastonoi, and it was proposed that its taxonomic status be changed to P. savastanoi pv. phaseolicola; however, this proposal has been rejected and the organism has been maintained within the P. syringae group66. The disease attacks both foliage and pods, and is a major problem in temperate areas of the world. Leaf symptoms appear several days after infection as small water-soaked spots on the lower surface. At 7-10 days after infection, the lesions appear as greasy water-soaked points of infections. Pods can also be infected and lesions appear as brown or red water-soaked spots.Seeds may also become infected and the disease may be transmitted through the infected seed. At temperatures between 18°C and 23°C and high humidity, a zone of yellow-green tissue may develop around the initial site of infection after 3–8 days, giving rise to what is known as the chlorotic halo. In cases of severe infection, the chlorosis may become systemic. In fact, halo blight is considered to be a low-temperature disease and epidemic potential is greatest at temperatures ranging between 18°C and 22°C.

In Pseudomonas syringae pv. phaseolicola the Synthesis of Phaseolotoxin and the Concurrent Expression of the argK Gene Coding for the Phaseolotoxin-Resistant Ornithyl-Carbamoyl Transferase Occur Independent of the Global Arginine Regulator ArgR

Pseudomonas syringae pv. phaseolicola, the causal agent of halo blight in beans produces a non-host specific toxin capable of inhibiting the enzyme ornithine carbamoyltransferase (OCTase) of plant, bacterial and mammalian origin. When P. s. pv. phaseolicola grown under conditions leading to the synthesis of phaseolotoxin, synthesises a phaseolotoxin-resistant OCTase, the product of the argK gene. Under any other growth conditions, the synthesis of arginine is mediated by a phaseolotoxin-sensitive OCTase encoded by the argF gene. In P. aeruginosa genes involved in the catabolism of arginine, the synthesis of carbamoylphosphate and the expression of argF are regulated by arginine and the ArgR protein. We isolated and characterised the argR gene of P. s. pv phaseolicola, and constructed a null argR mutant. Northern blot analysis using this mutant showed that in this bacterium regulated expression of argF is dependent on the ArgR product, but argK expression and phaseolotoxin synthesis occur independent of this regulatory protein. Also, we have shown that exogenous arginine has an effect on the expression of argF but not on argK. These results strongly indicate a lack of metabolic coordination between the synthesis of phaseolotoxin and the system regulating arginine metabolism mediated by ArgR