Functional domains of the TOL plasmid transcription factor XylS.
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.
Kaldalu
N
N
Department of Microbiology and Virology, Institute of Molecular and Cell Biology, Tartu University, Estonian Biocentre, 51010 Tartu, Estonia.
Toots
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U
de Lorenzo
V
V
Ustav
M
M
D000595:Amino Acid Sequence; D051777:AraC Transcription Factor; D001426:Bacterial Proteins; D001565:Benzoates / Q000378:metabolism; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D016958:Pseudomonas putida / Q000235:genetics; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors; D015533:Transcriptional Activation
D051777:AraC Transcription Factor; C497371:AraC protein, E coli; D001426:Bacterial Proteins; D001565:Benzoates; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C078720:XylS protein, Pseudomonas putida