Molecular and General Genetics MGG

, Volume 235, Issue 2–3, pp 406–412

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

  • Carmen Michán
  • Birgit Kessler
  • Victor de Lorenzo
  • Kenneth N. Timmis
  • Juan L. Ramos
Original Articles

DOI: 10.1007/BF00279387

Cite this article as:
Michán, C., Kessler, B., de Lorenzo, V. et al. Molec. Gen. Genet. (1992) 235: 406. doi:10.1007/BF00279387
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Summary

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.

Key words

TOL plasmid AraC/Xy1S family regulators Domain interactions 

Copyright information

© Springer-Verlag 1992

Authors and Affiliations

  • Carmen Michán
    • 1
  • Birgit Kessler
    • 2
  • Victor de Lorenzo
    • 2
  • Kenneth N. Timmis
    • 2
  • Juan L. Ramos
    • 1
  1. 1.C.S.I.C.Estación Experimental del ZaidínGranadaSpain
  2. 2.GBFNational Centre for BiotechnologyW-BraunschweigFRG
  3. 3.C.S.I.C.Centro Nacional de BiotecnologíaMadridSpain

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