, Volume 235, Issue 2-3, pp 406-412

XylS domain interactions can be deduced from intraallelic dominance in double mutants of Pseudomonas putida

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The XylS protein is the positive regulator of the TOL plasmid-encoded meta-cleavage pathway for the metabolism of alkylbenzoates in Pseudomonas putida. This protein is activated by a variety of benzoate analogues. To elucidate the functional domains of the regulator and their interactions, several fusions of the XylS C-terminus to MS2 polymerase and of the N-terminus to β-galactosidase were constructed but all are inactive. In addition, 15 double mutant xylS genes were constructed in vitro by fusing parts of various mutant genes to produce mutant regulators exhibiting C-terminal and N-terminal amino acid substitutions. The phenotypic properties of the parental single mutant genes, and those of the double mutant genes, suggest that the C-terminal region is involved in binding to DNA sequences at the promoter of the meta-cleavage pathway operon, and that the benzoate effector binding pocket includes critical residues present at both the N-terminal and C-terminal ends of the protein. The intraallelic dominance of the Ile229 (Ser229 → Ile) and Val274 (Asp274 → Val) substitutions over the N-terminal His4l (Arg4l → His) substitution, and the intraallelic dominance of Thr45 (Arg45 → Thr) over Ile229 and Val274, support the proposal that these two regions of the regulator interact functionally. Combination of the Leu88 (Trp88 → Leu) and Arg256 (Pro256 → Arg) substitutions did not suppress the semiconstitutive phenotype conferred by Leu88, but resulted in a protein with altered ability to recognize benzoates. In contrast, the Leu88 semiconstitutive phenotype was suppressed by Va1288 (Asp288 → Val), and the double mutant was susceptible to activation by benzoates. The results suggest that intramolecular interactions between the C- and N-terminal regions of XylS are critical for activation of the regulator by the effector.

Communicated by H. Hennecke