Genetic evidence for activation of the positive transcriptional regulator Xy1R, a member of the NtrC family of regulators, by effector binding.

The Xy1R protein positively controls expression from the Pseudomonas putida TOL plasmid sigma 54-dependent Pu and Ps promoters, in response to the presence of aromatic effectors such as m-xylene, m-methylbenzyl alcohol, and p-chlorobenzaldehyde in the culture medium. Xy1R also autoregulates its own synthesis. A mutant Xy1R regulator called Xy1R7 was isolated after nitrosoguanidine mutagenesis of the wild-type gene and phenotypic selection for mutants that had acquired the ability to recognize m-nitrotoluene, a nitroarene that is not an effector for the wild-type regulator. The mutant regulator exhibited a single point mutation that resulted in a change in codon 172 (GAA-->AAA), which should result in a Glu-->Lys change in the polypeptide chain. The effector profile of the mutant regulator was determined by measuring beta-galactosidase from a fusion of the Pu promoter to a promoterless lacZ gene. The results showed that the mutant regulator had acquired the ability to recognize m-nitrotoluene, and retained the wild-type regulator's ability to recognize most of the wild-type effectors. Full transcriptional activation of the Pu promoter by Xy1R7, as with the wild-type Xy1R protein, requires its full modular structure, namely the sigma 54 recognition site, the integration host factor binding site, and the upstream activation sequences. The Xy1R7 regulator did not stimulate transcription from the Ps promoter in response to the presence of its effectors, and autoregulated its own synthesis at low levels.