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Functional domains of the TOL plasmid transcription factor XylS.

ABSTRACT: The alkylbenzoate degradation genes of Pseudomonas putida TOL plasmid are positively regulated by XylS, an AraC family protein, in a benzoate-dependent manner. In this study, we used deletion mutants and hybrid proteins to identify which parts of XylS are responsible for the DNA binding, transcriptional activation, and benzoate inducibility. We found that a 112-residue C-terminal fragment of XylS binds specifically to the Pm operator in vitro, protects this sequence from DNase I digestion identically to the wild-type (wt) protein, and activates the Pm promoter in vivo. When overexpressed, that C-terminal fragment could activate transcription as efficiently as wt XylS. All the truncations, which incorporated these 112 C-terminal residues, were able to activate transcription at least to some extent when overproduced. Intactness of the 210-residue N-terminal portion was found to be necessary for benzoate responsiveness of XylS. Deletions in the N-terminal and central regions seriously reduced the activity of XylS and caused the loss of effector control, whereas insertions into the putative interdomain region did not change the basic features of the XylS protein. Our results confirm that XylS consists of two parts which probably interact with each other. The C-terminal domain carries DNA-binding and transcriptional activation abilities, while the N-terminal region carries effector-binding and regulatory functions.

SUBMITTER: Kaldalu N

PROVIDER: S-EPMC94389 | biostudies-literature | 2000 Feb

REPOSITORIES: biostudies-literature

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Functional domains of the TOL plasmid transcription factor XylS.

Kaldalu N N Toots U U de Lorenzo V V Ustav M M

Journal of bacteriology 20000201 4

The alkylbenzoate degradation genes of Pseudomonas putida TOL plasmid are positively regulated by XylS, an AraC family protein, in a benzoate-dependent manner. In this study, we used deletion mutants and hybrid proteins to identify which parts of XylS are responsible for the DNA binding, transcriptional activation, and benzoate inducibility. We found that a 112-residue C-terminal fragment of XylS binds specifically to the Pm operator in vitro, protects this sequence from DNase I digestion identi ...[more]

PMID: 10648539

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Project description:The inducible Pm promoter together with its cognate positive transcription regulator XylS has been shown to be useful for recombinant protein production under high cell density conditions. Here we report directed evolution of XylS resulting in mutant proteins with increased ability to stimulate transcription in Escherichia coli from Pm. A first round of mutagenesis using error-prone PCR on xylS was used to construct a library consisting of about 430,000 clones, and this library could be efficiently screened with respect to stimulation of expression from Pm due to a positive correlation between the level of expression of the reporter gene, bla (encoding β-lactamase), and the ampicillin tolerance of the corresponding host cells. Fourteen different amino acid substitutions in XylS were found to separately lead to up to nearly a threefold stimulation of expression under induced conditions, relative to wild type. These mutations were all located in the part corresponding to the N-terminal half of the protein. Varying combinations of the mutations resulted in further stimulation, and the best results (about 10-fold stimulation under induced conditions) were obtained by using a random shuffling procedure followed by a new round of screening. The uninduced levels of expression for the same mutants also increased, but only about four times. Through in silico 3D modelling of the N-terminal domain of XylS, it was observed that the evolved mutant proteins contained substitutions that were positioned in different parts of the predicted structure, including a β-barrel putatively responsible for effector binding and a coiled coil probably important for dimerization. The total production of the host-toxic antibody fragment scFv-phOx expressed from Pm with the evolved XylS mutant protein StEP-13 was about ninefold higher than with wild-type XylS, demonstrating that directed evolution of transcription factors can be an important new tool to achieve high-level recombinant protein production.

Dataset Information

Functional domains of the TOL plasmid transcription factor XylS.

Publications

Functional domains of the TOL plasmid transcription factor XylS.

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