Molecular analysis of thermoregulation of phaseolotoxin-resistant ornithine carbamoyltransferase (argK) from Pseudomonas syringae pv. phaseolicola

The phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) and phase olotoxin are produced by Pseudomonas syringae pv, phaseolicola at 18 degree s C but not at 28 degrees C, At 28 degrees C, the pathogen produces a prote in(s) that binds tin vitro) to a 485-bp fragment (thermoregulatory region, TRR) from a heterologous clone from the pathogen genomic library, which in multiple copies overrides thermoregulation of phaseolotoxin production in w ild-type cells (K, B, Rowley, D, E, Clements, M, Mandel, T Humphreys, and S . S, Patil, Mel. Microbiol, 8:625-635, 1993), We report here that DNase I p rotection analysis of the 485-bp fragment shows that a single site is prote cted from cleavage by the protein in the 28 degrees C extract and that this site contains two repeats of a core motif G/C AAAG separated by a. 5-bp sp acer. Partially purified binding protein forms specific complexes with a sy nthetic oligonucleotide containing four tandem repeats of this motif, A 492 -bp upstream fragment from argK encoding ROCT also forms specific complexes with the protein in the 28 degrees C crude extract, and a 260-bp subfragme nt from the TRR containing the binding site cross competes with the argk fr agment, indicating that the same protein binds to nucleotides in both fragm ents. DNase I protection analysis of the fragment from argK revealed four s eparate protected sequence elements, with element III containing half of th e core motif sequence (CTTTG), and the other elements containing similar se quences, Gel shift assays were done with DNA fragments from which one or al l of the sites were removed as competitor DNAs against the argK probe. The results of these experiments confirmed that the binding sites tin argK) are necessary for the protein to bind to the argK fragment in a specific manne r. Taken together, the results of studies presented here suggest that in ce lls of P, syringae pr. phaseolicola grown at high temperature argK may be n egatively regulated by the protein produced at this temperature.