Two ornithine carbamoyltransferases (OCT 1 and OCT 2) were isolated from Pseudomonas syringae pv. phaseolicola and purified by precipitation with ammonium sulfate, heat denaturation, chromatography on DEAE-Sephadex A-50 and Sephadex G-200. Molecular weights of both enzymes: 110,000; optimal activity: pH 8.5 to 9.5 (OCT 1), pH 8.4 (OCT 2); apparent Km for ornithine: 7 X 10(-4) (both enzymes); apparent Km for carbamoyl-phosphate: 7 X 10(-4) (OCT 1), 2.8 X 10(-3) (OCT 2). Both enzymes possess only an anabolic function. OCT 1 is highly inhibited by low concentrations of phaseolotoxin and Orn-P(O)(NH2)-NH-SO3H, OCT 2 is insensitive to both compounds. The inhibition of OCT 1 is reversible.

The phaseolotoxin-producing Pseudomonas syringae pv. phaseolicola strain 1321 contains two ornithine carbamoyltransferases which differ in resistance to phaseolotoxin. Whereas ornithine carbamoyltransferase 1 (OCT 1) is inhibited at low concentrations of phaseolotoxin, ornithine carbamoyltransferase 2 (OCT 2) is insensitive to phaseolotoxin. The activity of the insensitive enzyme is correlated with the amount of toxin formed.

Rhizobium etli CFN42 bacteroids from bean nodules possessed an abundant 16-kDa protein (BacS) that was found in the membrane pellet after cell disruption. This protein was not detected in bacteria cultured in tryptone-yeast extract. In minimal media, it was produced at low oxygen concentration but not in a mutant whose nifA was disrupted. N-terminal sequencing of the protein led to isolation of a bacS DNA fragment. DNA hybridization and nucleotide sequencing revealed three copies of the bacS gene, all residing on the main symbiotic plasmid of strain CFN42. A stretch of 304 nucleotides, exactly conserved upstream of all three bacS open reading frames, had very close matches with the NifA and sigma 54 consensus binding sequences. The only bacS homology in the genetic sequence databases was to three hypothetical proteins of unknown function, all from rhizobial species. Mutation and genetic complementation indicated that each of the bacS genes gives rise to a BacS polypeptide. Mutants disrupted or deleted in all three genes did not produce the BacS polypeptide but were Nod+ and Fix+ on Phaseolus vulgaris.

Independently of their capacity to produce phytotoxins, strains of Pseudomonas syringae pv. phaseolicola contain two ornithine carbamoyltransferases (OCT, EC 2.1.3.3) which differ in resistance to phaseolotoxin and Orn-P(O) (NH2)-NH-SO3 H (PNSOrn). At 18 degrees C, the optimal temperature for product formation, the balance of the two types of OCT was shifted in favour of the insensitive type in phaseolotoxin producing strains, and in favour of the sensitive one in strains with little or no toxin production. The results suggest a causal relationship between the existence of an insensitive enzyme and the synthesis of toxins.

In contrast to the producer of phaseolotoxin and Orn-P(O)(NH2)-NH-SO3H (PNSOrn), Pseudomonas syringae pv. phaseolicola, which possesses a sensitive and an insensitive type of ornithine carbamoyltransferase (OCT, E.C. 2.1.3.3.), in Pseudomonas syringae pv. syringae W50, an organism which produce neither phaseolotoxin nor PNSOrn, only one type of OCT could be detected. This enzyme is highly sensitive to phaseolotoxin. This result supports our hypothesis that the existence of an insensitive ornithine carbamoyltransferase is an important prerequisite for the synthesis of phaseolotoxin in P. syringae pv. phaseolicola and that this enzyme does not occur generally in P. syringae spec.