Modulation of the function of the signal receptor domain of XylR, a member of a family of prokaryotic enhancer-like positive regulators

Abstract

The XylR protein controls expression from the Pseudomonas putida TOL plasmid upper pathway operon promoter (Pu) in response to aromatic effectors. XylR-dependent stimulation of transcription from a Pu::lacZ fusion shows different induction kinetics with different effectors. With toluene, activation followed a hyperbolic curve with an apparent K of 0.95 mM and a maximum beta-galactosidase activity of 2,550 Miller units. With o-nitrotoluene, in contrast, activation followed a sigmoidal curve with an apparent K of 0.55 mM and a Hill coefficient of 2.65. m-Nitrotoluene kept the XylR regulator in an inactive transcriptional form. Therefore, upon binding of an effector, the substituent on the aromatic ring leads to productive or unproductive XylR forms. The different transcriptional states of the XylR regulator are substantiated by XylR mutants. XylRE172K is a mutant regulator that is able to stimulate transcription from the Pu promoter in the presence of m-nitrotoluene; however, its response to m-aminotoluene was negligible, in contrast with the wild-type regulator. These results illustrate the importance of the electrostatic interactions in effector recognition and in the stabilization of productive and unproductive forms by the regulator upon aromatic binding. XylRD135N and XylRD135Q are mutant regulators that are able to stimulate transcription from Pu in the absence of effectors, whereas substitution of Glu for Asp135 in XylRD135E resulted in a mutant whose ability to recognize effectors was severely impaired. Therefore, the conformation of mutant XylRD135Q as well as XylRD135N seemed to mimic that of the wild-type regulator when effector binding occurred, whereas mutant XylRD135E seemed to be blocked in a conformation similar to that of wild-type XylR and XylRE172K upon binding to an inhibitor molecule such as m-nitrotoluene or m-aminotoluene.

FIG. 1

Domains of the XylR regulator and locations of point mutations. The organization of the XylR domains is according to Inouye et al. (17). The mutations located at the N-terminal end of this regulator are shown.

FIG. 2

Dose dependence of the activation of transcription from Pu by XylR and XylRE172K in response to toluene and nitrotoluenes. E. coli ET8000(pRD579 Pu::′lacZ) also bearing pTS174 (encoding wild-type XylR) or pAD1 (encoding XylRE172K) was grown for 5 h with vigorous shaking in the absence or presence of increasing concentrations of toluene or nitrotoluenes. The β-galactosidase activity was assayed in permeabilized cells as described in Materials and Methods. The data are the averages of at least five independent determinations. The results were fitted to hyperbolic or sigmoidal equations, and the values of K_app and Hill’s coefficient were deduced from these equations. (A) Dose dependence of the activation of transcription from Pu by toluene. Open symbols, wild-type XylR; closed symbols, XylRE172K. (B) Dose dependence of the activation of transcription from Pu by nitrotoluenes. Triangles, wild-type XylR; squares, XylRE172K; open symbols, o-nitrotoluene; closed symbols, m-nitrotoluene.

FIG. 3

Inhibition of XylR mediated by m-nitrotoluene. (A) Inhibition of expression from Pu mediated by XylR with o-nitrotoluene and m-nitrotoluene. E. coli ET8000(pRD579, pTS174) was grown for 5 h with vigorous shaking in the presence of 0.5 mM o-nitrotoluene (o-NT) and increasing concentrations of m-nitrotoluene (m-NT) (0 to 2.5 mM). After incubation, β-galactosidase activity was assayed in permeabilized cells as described in Materials and Methods. The data are the averages and standard deviations of at least five independent determinations. (B) mRNA synthesis from Pu mediated by XylRP85S in the absence of an aromatic (lane 1) and in the presence of m-methylbenzyl alcohol (lane 2) or m-nitrotoluene (lane 3). E. coli ET8000(pERD401, pAD6) was grown overnight on Luria-Bertani medium supplemented with appropriate antibiotics. Bacterial cells were diluted 1/100 in the same medium, and after 1 h of cell growth, the samples were supplemented with m-methylbenzyl alcohol or m-nitrotoluene to a final concentration of 1 mM. After 30 min, samples were withdrawn for mRNA analysis. The extended products (arrow) were 134 nucleotides long.