Abstract
From crude protein extracts of Pseudomonas putida KT2440, we identified a small protein, TurA, able to bind to DNA fragments bearing the entire Pu promoter sequence of the TOL plasmid. The knock-out inactivation of the turA gene resulted in enhanced transcription initiation from the Pu promoter, initially suggesting a negative regulatory role of TurA on Pu expression. Ectopic expression of TurA both in P. putida and in Escherichia coli reporter strains and transcription in vitro of the Pu promoter in the presence of purified TurA confirmed the TurA repressor role on Pu activity. turA gene inactivation did not significantly alter two well characterized physiological regulations of the Pu expression in routine conditions of cultivation, exponential silencing, and carbon-mediated repression, respectively. However, the growth at suboptimal temperatures resulted in a TurA-dependent increase of Pu repression. These results strongly suggest that a physiological significance of the negative role of TurA on Pu activity could be limitation of the expression of the toluene-degrading enzymes at suboptimal growth temperatures. Therefore, the identification of TurA as Pu-binding protein revealed a novel physiological modulation of Pu promoter that is different from those strictly nutritional described previously.
Highlights
From crude protein extracts of Pseudomonas putida KT2440, we identified a small protein, TOL upper operon repressor A” (TurA), able to bind to DNA fragments bearing the entire putida strain MAD1 (Pu) promoter sequence of the TOL plasmid
Screening for Novel Regulatory Proteins of the Host Strain P. putida KT2440 Able to Bind to the Pu Promoter of the TOL Plasmid—To identify P. putida KT2440 proteins involved in Pu co-regulation, we aimed to screen for novel Pu DNA-protein interactions by protein chromatography coupled with SouthWestern blotting using a radioactive Pu probe
integration host factor (IHF) seems to provide a uniquely structural aid for overcoming these limiting steps and is not a key regulator involved in the physiological control of Pu expression [53]
Summary
From crude protein extracts of Pseudomonas putida KT2440, we identified a small protein, TurA, able to bind to DNA fragments bearing the entire Pu promoter sequence of the TOL plasmid. TurA gene inactivation did not significantly alter two well characterized physiological regulations of the Pu expression in routine conditions of cultivation, exponential silencing, and carbon-mediated repression, respectively. To reach such physiological control, specific carbon regulatory devices have supposedly integrated in global networks able to sense the environmental stimuli One example of this is the degradation of the aromatic hydrocarbons, toluene and m-/p-xylene, by the Pseudomonas putida, encoded by two large operons, upper and meta, harbored by the TOL mega-plasmid (for a review, see Ref. 4). In addition to the specific response to aromatic substrates, it is conceivable that the TOL system had integrated additional co-regulatory devices to avoid the expression of its energetically expensive pathway, under unfavorable physiochemical conditions (temperature, pH, osmolarity, etc.) and/or in the presence of more appetizing carbon and energy sources than toluenes. To date, only protein regulatory components involved in the specific response to the aromatic inducers have been described
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