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

Abstract

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.

FIG. 1.

Gel retardation assay performed with extracts from the P. syringae pv. phaseolicola wild-type strain and the argR mutant UILH13R. Lane 1, free argF promoter probe; lane 2, argF promoter probe plus extract from the wild-type strain grown in rich medium at 28°C; lane 3, argF promoter probe plus extract from the wild-type strain grown in minimal medium at 28°C; lane 4, argF promoter probe plus extract from UILH13R grown in rich medium at 28°C; lane 5, competition with nonlabeled argF promoter probe; lane 6, competition with nonlabeled argK. “B” indicates the position of retardation signals observed in lanes 2 and 6 and very weak signals in lanes 3 and 5; no retardation signal was observed in lane 4. “A” indicates the position of free probe.

FIG. 2.

Phaseolotoxin bioassay. The assay was performed by using supernatants from P. syringae pv. phaseolicola (spot 1) and UILH13R (spot 2) grown in minimal medium at 18°C (A) and 28°C (B).

FIG. 3.

Pod inoculation assay. Fully developed green pods from susceptible bean plants (P. vulgaris cv. Flor de Mayo) were inoculated by puncturing them with toothpicks soaked in fresh cultures of the UILH13R mutant (spot 1), P. syringae pv. phaseolicola (spot 2), P. syringae pv. tomato DC3000 (spot 3), and sterile distilled water (spot 4). A typical hypersensitive response was observed in spot 3, and no difference in lesion formation was observed between wild-type bacteria (spot 2) and the argR null mutant (spot 1). Inoculated pods were incubated inside a sealed plastic container with a wet paper towel in a growth room at 28°C. At this temperature, phaseolotoxin production was not expected to occur, but lesion development was clear. The lesions were examined 4 days postinoculation.