XylS domain interactions can be deduced from intraallelic dominance in double mutants of Pseudomonas putida.
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 beta-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 His41 (Arg41-->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 Val288 (Asp288-->Val), and the double mutant was susceptible to activation by benzoates.(ABSTRACT TRUNCATED AT 250 WORDS)
Michán
C
C
C.S.I.C., Estación Experimental del Zaidín, Granada, Spain.
Kessler
B
B
de Lorenzo
V
V
Timmis
K N
KN
Ramos
J L
JL
D000483:Alleles; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D001565:Benzoates / Q000494:pharmacology*; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005809:Genes, Regulator; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016958:Pseudomonas putida / Q000187:drug effects / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D001565:Benzoates; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; C078720:XylS protein, Pseudomonas putida; D001616:beta-Galactosidase