PURIFICATION AND CHARACTERIZATION OF AN ARGININE REGULATORY PROTEIN, ARGR, FROM PSEUDOMONAS-AERUGINOSA AND ITS INTERACTIONS WITH THE CONTROL REGIONS FOR THE CAR, ARGF, AND ARU OPERONS

Pseudomonas aeruginosa ArgR, a regulatory protein that plays a major r ole in the control of certain biosynthetic and catabolic arginine gene s, was purified to homogeneity, ArgR was shown to be a dimer of two eq ual subunits, each with a molecular mass of 37,000 Da, Determination o f the amino-terminal amino acid sequence showed it to be identical to that predicted from the derived sequence for the argR gene, DNase I fo otprinting sbowed that ArgR protects a region of 45 to 47 bp that over laps the promoters for the biosynthetic car and argF operons, indicati ng that ArgR exerts its negative control on the expression of these op erons by steric hindrance, Studies were also carried out with the aru operon, which encodes enzymes of the catabolic arginine succinyl-trans ferase pathway. Quantitative SI nuclease experiments showed that expre ssion of the first gene in this operon, araC, is initiated from an arg inine-inducible promoter, Studies with an aruC::lacZ fusion showed tha t this promoter is under the control of ArgR. DNase I experiments indi cated that ArgR protects two 45-bp binding sites upstream of aruC; the 3' terminus for the downstream binding site overlaps the -35 region f or the identified promoter, Gel retardation experiments yielded appare nt dissociation constants of 2.5 x 10(-11), 4.2 x 10(-12), and 7.2 x 1 0(-11) M for carA, argF, and aruC operators, respectively, Premethylat ion interference and depurination experiments with the car and argF op erators identified a common sequence, 5'-TGTCGC-3', which may be impor tant for ArgR binding, Alignment of ArgR binding sites reveals that th e ArgR binding site consists of two half-sites, in a direct repeat arr angement, with the consensus sequence TGTCGCN(8)AAN(5).