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, 186 (21), 7353-63

TouR-mediated Effector-Independent Growth Phase-Dependent Activation of the sigma54 Ptou Promoter of Pseudomonas Stutzeri OX1

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TouR-mediated Effector-Independent Growth Phase-Dependent Activation of the sigma54 Ptou Promoter of Pseudomonas Stutzeri OX1

Dafne Solera et al. J Bacteriol.

Abstract

Transcription of the catabolic touABCDEF operon, encoding the toluene-o-xylene monooxygenase of Pseudomonas stutzeri OX1, is driven by the sigma(54)-dependent Ptou promoter, whose activity is controlled by the phenol-responsive NtrC-like activator TouR. In this paper we describe for the first time a peculiar characteristic of this system, namely, that Ptou transcription is activated in a growth phase-dependent manner in the absence of genuine effectors of the cognate TouR regulator. This phenomenon, which we named gratuitous activation, was observed in the native strain P. stutzeri OX1, as well as in a Pseudomonas putida PaW340 host harboring the reconstructed tou regulatory circuit. Regulator-promoter swapping experiments demonstrated that the presence of TouR is necessary and sufficient for imposing gratuitous activation on the Ptou promoter, as well as on other sigma(54)-dependent catabolic promoters, whereas the highly similar phenol-responsive activator DmpR is unable to activate the Ptou promoter in the absence of effectors. We show that this phenomenon is specifically triggered by carbon source exhaustion but not by nitrogen starvation. An updated model of the tou regulatory circuit is presented.

Figures

FIG. 1.
FIG. 1.
Northern analysis of ToMO transcripts in P. stutzeri OX1 during growth in the absence of effectors. (A) Growth curve in mineral medium supplemented with 20 mM malate. (B) Northern blot. Total RNA (20 μg loaded in each lane) was extracted from cells collected at different times and was probed with touA. The arrows indicate the time when the culture entered the stationary phase. RNA for the positive control (lane +) was extracted from an exponentially growing P. stutzeri OX1 culture exposed to 2 mM o-cresol for 3 h. OD600nm, optical density at 600 nm.
FIG. 2.
FIG. 2.
Transcriptional profiles of Ptou (Ptomo) and other σ54-dependent promoters in the presence of either touR or dmpR regulatory genes. The growth curves (open symbols) and β-galactosidase activities (closed symbols) measured in 20 mM malate-mineral medium in the presence (triangles) or in the absence (squares) of 1 mM o-cresol are shown. (A to C) P. putida PaW340::touR harboring the following transcriptional fusions: Ptou-lacZ (pTN1021) (A), Pu-lacZ (pKMAD) (B), and Po-lacZ (pKUME) (C). (D and E) P. putida PaW340::Ptou-lacZ harboring either plasmid pKR210 (touR+) (D) or plasmid pKDmpR (dmpR+) (E). The dotted lines indicate the growth curve (no symbols) and the β-galactosidase activity (multiplication signs) measured in the corresponding strain devoid of the regulatory gene. Note that different scales were used to facilitate the comparison among strains. OD600nm, optical density at 600 nm.
FIG. 3.
FIG. 3.
Western analysis of TouR during growth in 20 mM malate-mineral medium in the absence of effectors. (A) Growth curves (open symbols) and β-galactosidase activities (closed symbols) of strains PaW340::Ptou-lacZ(pKR210) (triangles) and PaW340::touR(pTN1021)(squares). (B) Western analysis. Crude extracts were prepared from both strains at 1-h intervals from the exponential phase (3 h) until the stationary phase (7 h). At the bottom of the panel the times and the strains from which the crude extracts were prepared are indicated. Lane M contained purified His-TouR (solid arrow) and His-ΔA-TouR (open arrow) (40 ng each). OD600nm, optical density at 600 nm.
FIG. 4.
FIG. 4.
Effects of nitrogen (A and B) and carbon (C) exhaustion on gratuitous activation in P. putida PaW340::touR(pTN1021). The growth curves (lines) and β-galactosidase activities (bars) are shown. (A and B) PaW340::touR(pTN1021) was grown in a modified M9 mineral medium containing 20 mM malate as the carbon source and a limiting concentration (2 mM) of NH4Cl (open symbols and open bars). At the onset of the stationary phase, the culture was split into three subcultures, and 20 mM NH4Cl (saturating concentration) (A) (solid symbols and solid bars) or 1 mM o-cresol (B) (solid symbols and grey bars) was added to two of the subcultures. (C) PaW340::touR(pTN1021) was grown in M9 mineral medium containing 5 mM malate as the carbon source (open symbols and open bars). At the onset of the stationary phase, the culture was divided into two subcultures, and 15 mM malate was added to one of these subcultures (solid symbols and striped bars). OD600nm, optical density at 600 nm.
FIG. 5.
FIG. 5.
β-Galactosidase activity profile of the Ptou promoter in P. putida PaW340::touR(pTN1021) grown on different carbon sources. (A to D) Growth curves (open symbols) and β-galactosidase activities (closed symbols) during growth in the absence of TouR effectors on mineral medium supplemented with 10 mM glucose (A), 20 mM malate (B), 20 mM succinate (C), or 40 mM pyruvate (D). (E) β-Galactosidase activity measured in P. putida PaW340::touR(pTN1021) exponentially growing in mineral medium supplemented with the carbon sources described above in the absence of effectors (open bars) or after 2 h of exposure to 2 mM o-cresol (solid bars). OD600nm, optical density at 600 nm.
FIG. 6.
FIG. 6.
Model of the tou regulatory circuit. TouR activates transcription from the Ptou promoter (Ptomo) either in response to phenolic effectors or, in the absence of effectors, by means of an unknown mechanism upon carbon exhaustion. In the presence of hydrocarbons, gratuitous expression of the tou operon would ensure a ToMO basal activity sufficient to convert the substrates into the phenolic intermediates, which in turn could be recognized by TouR and stimulate the expression of the enzymatic activities at high levels by virtue of a positive feedback mechanism.

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