EFFECTOR SPECIFICITY MUTANTS OF THE TRANSCRIPTIONAL ACTIVATOR NAHR OFNAPHTHALENE DEGRADING PSEUDOMONAS DEFINE PROTEIN SITES INVOLVED IN BINDING OF AROMATIC INDUCERS

This work reports a genetic analysis of the interactions between NahR, the LysR-type regulator of the NAH operons for biodegradation of naph thalene in Pseudomonas, and its aromatic effecters, Six mutants encodi ng NahR variants responsive to salicylate analogs such as benzoate, wh ich is not an inducer for the wild type regulator, were isolated with a polymerase chain reaction-based saturation mutagenesis protocol, Mos t mutants displaying a specific change of effector profile bore single amino acid substitutions within a short protein segment of 60 residue s located at the central portion of the NahR sequence, Some of the pro tein variants exhibited an increased affinity for salicylate and also for otherwise suboptimal effecters, with apparent K-s' values 5-100-fo ld lower than those of the wild type NahR protein, In addition, all mu tants were activated by inducers bearing novel substituents at positio ns 1 or 2 of the aromatic ring and displayed also an enhanced toleranc e to changes at positions 3 and 4, Correlation between mutations in Na hR, and the structures of the new effecters suggested that protein sit es Met(116), Arg(132), Asn(169), and Arg(248) are involved in effector recognition and binding during the earlier steps of the process leadi ng to transcriptional activation of cognate NAH promoters.