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Journal of Bacteriology, November 2004, p. 7353-7363, Vol. 186, No. 21
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.21.7353-7363.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

TouR-Mediated Effector-Independent Growth Phase-Dependent Activation of the ⁵⁴ Ptou Promoter of Pseudomonas stutzeri OX1

Dafne Solera,^1, Fabio L. G. Arenghi,^1, Tanja Woelk,¹ Enrica Galli,¹ and Paola Barbieri^2*

Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Milan,¹ Dipartimento di Biologia Strutturale e Funzionale, Università dell'Insubria, Varese, Italy²

Received 29 April 2004/ Accepted 28 July 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
Transcription of the catabolic touABCDEF operon, encoding the toluene-o-xylene monooxygenase of Pseudomonas stutzeri OX1, is driven by the 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 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.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In microorganisms, the ability to readily activate or silence the expression of different metabolic routes is a fundamental ability for adapting to changes in nutrient availability. Many catabolic operons for the utilization of aromatic compounds, such as the phenol-degrading dmp system of Pseudomonas sp. strain CF600 (54), the methyl-benzene-degrading xyl system of Pseudomonas putida PaW1 (18, 48), or the tbu system of Burkholderia pickettii PKO1 for the catabolism of toluene (9), are regulated by means of ⁵⁴-dependent regulatory circuits. The RNA polymerase (RNAP) containing the alternative sigma factor ⁵⁴ recognizes and binds to a distinct class of promoters, which are characterized by invariant GG and GC motifs centered at positions –24 and –12, respectively (5). The core promoter is usually accompanied by enhancer-like elements (upstream activator sequences [UAS]) located 100 to 200 bp upstream of the –12/–24 region, which represent the binding site of the cognate regulatory proteins (32, 46). Unlike the ⁷⁰-RNAP, the ⁵⁴-RNAP forms extremely stable closed complexes and is unable to catalyze the isomerization to the transcription-competent open complex. Isomerization can occur only upon interaction with an NtrC-like transcriptional activator (8, 37). NtrC-like proteins share a typical three-domain structure, which includes the DNA-binding carboxy-terminal D domain; the highly conserved central C domain, which has ATPase activity and provides the surface contacting the holoenzyme; and the amino-terminal A domain (39, 50, 55, 62). The A domain represents the signal receiver module, as well as the regulatory domain of the transcription-promoting activity of the protein. In monocomponent ⁵⁴-dependent regulators, such as the XylR (30) and DmpR (54) proteins that control the expression of the xyl and dmp systems, respectively, the A domain is able to recognize and bind small effector molecules, which are usually the substrates or the intermediates of the regulated catabolic pathway (41, 50, 53). In the absence of effectors, the A domain acts as an intramolecular repressor, locking the regulator in a dimeric, inactive state. The direct interaction between the A domain and the specific effector alleviates this repression, leading to the oligomeric, transcriptionally competent state of the regulator (13, 20, 40, 44, 52, 60).

Whereas in in vitro transcription experiments with purified components the presence of the effector was sufficient to stimulate transcription from ⁵⁴-dependent promoters (3, 42), in vivo studies carried out with the two ⁵⁴-dependent xyl and dmp regulatory circuits showed that at least two overimposed levels of physiological control adjust the cognate Pu and Po promoter activities. In particular, the inducibility of the Pu and Po promoters appears to be down-regulated in cells growing exponentially in rich medium, a phenomenon known as exponential silencing (16, 29, 35, 58). A genetically separated physiological control is represented by the carbon source inhibition of the Pu promoter (11, 12, 28). In spite of the efforts aimed at finding a common mechanism underlying the physiological modulation of ⁵⁴-dependent regulatory circuits, it appeared that different global factors exploit system-specific characteristics to achieve the same final result, the silencing of the expression of alternative catabolic pathways when more readily utilizable carbon sources are available (51, 57).

The touABCDEF operon of Pseudomonas stutzeri OX1 codes for the multicomponent toluene-o-xylene-monooxygenase (ToMO) which catalyzes the first two steps of the toluene and o-xylene catabolic pathway (6, 7). ToMO expression is driven from the ⁵⁴-dependent Ptou promoter (previously des-ignated Ptomo), whose activity is controlled by the phenol-responsive monocomponent NtrC-like transcriptional activator TouR (2). In this paper we show that the tou regulatory circuit has the peculiar ability of being activated in the absence of effectors and in a growth phase-dependent manner, a feature that has not been described previously for the ⁵⁴-dependent regulatory circuits. The genetic elements specifically required and the physiological signal(s) triggering this phenomenon were investigated in this study.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains and plasmids. The bacterial strains and plasmids used in this work are listed in Table 1. Plasmids were isolated from P. putida PaW340 (22) as described by Hansen and Olsen (25) and from Escherichia coli JM109 (61) and E. coli S17-1 pir (27) by standard procedures (49) or by use of purification kits purchased from QIAGEN. Recombinant plasmids were constructed by standard procedures (49) and were introduced into the bacterial host strains by electroporation (19). E. coli JM109 was routinely used for plasmid construction and selection.

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TABLE 1. Bacterial strains and plasmids

To obtain the Ptou-lacZ transcriptional fusion, the ToMO promoter region spanning positions –185 to 23 was amplified from plasmid pFB1112 (6) as a 208-bp SacI fragment by using primers PTOMO5S (5'-CGGAGCTCGATCAGGCGACAACTATCCTGA-3'; SacI site underlined) and PTOMO3S (5'-CGGAGCTCTTGTCTCTGTTTGTTGTTATGA-3'; SacI site underlined) and was cloned into the corresponding site of plasmid pUC18 (61), giving pPT1821. From pPT1821 the Ptou promoter was subsequently excised as an EcoRI-HindIII fragment and cloned into the corresponding sites of the promoter-probing vector pUJ8 (17) upstream of a lacZ reporter gene, which resulted in a plasmid designated pUPT218. The whole Ptou-lacZ transcriptional fusion generated in pUPT218 was excised as a NotI cassette and cloned into the corresponding site of the broad-host-range vector pKGB4N, giving pTN1021. The same NotI cassette was also cloned into the corresponding site of a mini-Tn5 transposon which carried tellurite resistance as a selection marker (14). The plasmid obtained, designated pPTZ, was stably maintained in the E. coli S17-1 pir strain and used to integrate the Ptou-lacZ fusion into the chromosome of P. putida PaW340. To obtain the Po-lacZ transcriptional fusion, the Po promoter region from position –215 to position 18 was amplified from plasmid pVI671 (57) with primers Po5'Eco (5'-CCCCGAATTCGACGGCAGTGATTTTAGTATTAG-3'; EcoRI site underlined) and Po3'Bam (5'-CCCCGGATCCAGGTTGGCGGCTTGCGCAGGAC-3'; BamHI site underlined) and cloned into the EcoRI-BamHI sites of pUJ8, giving plasmid pUME. The NotI cassette containing the Po-lacZ fusion was excised from pUME and cloned in pKGB4N, giving plasmid pKUME. pKMAD was obtained by cloning into the NotI site of pKGB4N a Pu-lacZ cassette, kindly provided by V. de Lorenzo, equivalent to the Ptou-lacZ and Po-lacZ transcriptional fusions.

Plasmids pKR210 and pKDmpR were constructed as follows. A 2.1-kb DNA fragment encompassing the touR regulatory gene and its native promoter was amplified from plasmid pFB1112 (6) with primers PREGBam (5'-CGGGATCCGAGCAACCAGCTATAAGCCCACA-3'; BamHI site underlined) and TouHind (5'-CCCAAGCTTTCAGGCTTCAGAAAAAATGCC-3'; HindIII site underlined). Similarly, a 1.9-kb fragment spanning the dmpR gene and its promoter was amplified from pVI401 (43) with primers PRDBam (5'-CGGGATCCCCGTCGATTGATCATTTGGTTG-3'; BamHI site underlined) and DmpHind (5'-CCCAAGCTTCTAGCCTTCGATGCCGATTTTCTT-3'; HindIII site underlined). The regulatory genes were then cloned as BamHI-HindIII fragments in pUC18rrnB, a pUC18Not (27) derivative containing the strong rrnB operon terminator downstream of the polylinker, giving plasmids pRBH210 (touR⁺) and pDmpRrrnB (dmpR⁺). From these constructs, the NotI cassette containing the regulatory gene and the terminator was subsequently excised and cloned in pKGB4N, giving plasmids pKR210 and pKDmpR, respectively.

Mobilization and transposition of the Ptou-lacZ transcriptional fusion into the chromosome of P. putida PaW340. Plasmid pPTZ was transferred from the donor E. coli S17-1 pir into the P. putida PaW340 target strain by mobilization by using a filter mating technique (14, 27) and a mixture of donor and recipient cells at a ratio of 1:2. Filters were incubated overnight at 30°C on the surface of Luria broth (LB) plates, and cells were then suspended in 1 ml of 10 mM MgSO₄. Appropriate dilutions of the mating mixture were then plated on selective mineral medium containing 20 mM malate, 0.05 mM tryptophan, and 40 µg of tellurite per ml. To verify whether the tellurite-resistant P. putida PaW340 exconjugants arose from authentic transposition into the host chromosome rather than from cointegration of the whole delivery plasmid, which harbored the ampicillin-piperacillin resistance gene, their piperacillin sensitivity was also verified.

Media, culture conditions, and ß-galactosidase assays. P. putida and E. coli strains were routinely grown in LB (49) at 30 and 37°C, respectively. Ampicillin, kanamycin, tetracycline, sodium tellurite, and piperacillin were used in selective media at concentrations of 100, 30, 25, 40, and 150 µg/ml, respectively. To study the Ptou promoter activity, P. putida derivatives were grown (unless specified otherwise) in M9 medium (31) containing 20 mM malate and the appropriate antibiotic(s). When P. putida PaW340 was used, 0.05 mM tryptophan was added to satisfy the strain auxotrophy. In induction experiments o-cresol was supplied at the beginning of the experiment (unless indicated otherwise) at a final concentration of 1 or 2 mM. ß-Galactosidase assays were performed with cells permeabilized with chloroform and sodium dodecyl sulfate as described by Miller (38) under the conditions specified below. The ß-galactosidase activity values given throughout this paper are the averages of at least two independent experiments, each of which was conducted in duplicate, with standard deviations of less than 20%.

RNA purification and Northern analysis. RNA was isolated from the wild-type P. stutzeri OX1 cells as described previously (2). Cells from 1.5-ml samples were collected by centrifugation (8,000 x g, 4°C), resuspended in 200 µl of lysis buffer (20 mM sodium acetate [pH 5.5], 0.5% sodium dodecyl sulfate, 1 mM EDTA), and extracted for 5 min at 65°C with 200 µl of prewarmed phenol saturated with 20 mM sodium acetate (pH 5.5). After centrifugation, the aqueous phase was extracted with an equal volume of chloroform-isoamyl alcohol (24:1, vol/vol), and the nucleic acids were precipitated by addition of 0.1 volume of 3 M sodium acetate (pH 6.0) and 3 volumes of absolute ethanol. The precipitate was suspended in 50 µl of diethylpyrocarbonate-treated water and incubated for 15 min at 37°C with 20 U of RNase-free DNase I (Boehringer Mannheim). The RNA was subsequently phenol extracted twice, ethanol precipitated, and dissolved in diethylpyrocarbonate-treated water. The RNA concentration was estimated by measuring the optical densities at 260 and 280 nm.

In Northern hybridization analyses, 20-µg portions of total RNA samples were denatured with formamide and formaldehyde, analyzed at 4°C on a 1.2% agarose gel containing formaldehyde, transferred to a Hybond-N⁺ membrane (Amersham) (49), and then probed with touA. The probe was isolated from pMZ1201 (7) as a 1,330-bp SalI-MluI fragment and labeled with [-³²P]dATP by using a random primer DNA labeling kit (Boehringer Mannheim).

Western analysis. Cells from 5 to 50 ml of culture, depending on the optical density at 600 nm, were collected, resuspended in 20 mM Tris (pH 7.5)-5 mM EDTA, and sonicated. Cell debris was removed by centrifugation to obtain the crude extract used in the assay. The protein concentration was determined with a Bradford protein assay kit (Bio-Rad). Twenty-five micrograms of crude extract from each sample was separated by sodium dodecylsulfate-10% polyacrylamide gel electrophoresis along with purified His-tagged TouR derivatives (3). Proteins were transferred to nitrocellulose membranes and Western blotted with a polyclonal anti-ATouR mouse antiserum, prepared as previously described (21). Immunodecorated bands were revealed by using Amersham's ECF fluorescence reagents as directed by the supplier. Detection and quantification of Western blots were performed by using a Typhoon analytical scanner and software (Molecular Dynamics).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effector-independent, growth phase-dependent transcription of the ToMO-encoding operon in P. stutzeri OX1. The P. stutzeri OX1 ToMO-encoding operon, touABCDEF, is transcribed from the ⁵⁴-dependent Ptou promoter under the control of the phenolic compound-responsive, NtrC-like transcriptional activator TouR (2). By studying the ToMO expression in P. putida PaW340 harboring plasmid pFB1112, which contains the entire tou operon and its regulator, touR, the presence of tou transcripts in cells grown in the absence of TouR effectors was observed (2). This was rather unexpected and in contrast to the characteristics of the ⁵⁴-dependent catabolic operons, which are known to require, besides the ⁵⁴-RNAP, an effector-activated form of the cognate regulatory protein to be transcribed. Moreover, in the absence of TouR effectors, the transcription of the tou operon appeared not to be constitutive, as it became evident only at some time during the growth of the culture and seemed to increase with time (2). To verify these observations, tou transcripts were analyzed in P. stutzeri OX1 throughout growth in the absence of any TouR effectors. Thus, RNA was extracted from malate-grown cells collected at 1-h intervals and subjected to Northern analysis by using touA as the probe. Part of the P. stutzeri OX1 culture was exposed during exponential growth to the powerful TouR effector o-cresol, and the RNA extracted after 3 h of exposure served as the positive control. As shown in Fig. 1, in cells growing in the absence of TouR effectors tou transcripts were almost undetectable during the exponential phase, whereas they became evident in the stationary phase, although the levels were much lower than those in the cells exposed to o-cresol. The presence of multiple bands has been observed previously in P. putida PaW340(pFB1112) (2) and might be ascribed to the processing of the transcripts. However, the presence of tou transcripts in uninduced samples strongly suggested that effector-independent transcription of the ToMO-encoding operon, which was first observed in P. putida PaW340(pFB1112), also occurs in the native strain P. stutzeri OX1, thus ruling out the possibility that it was determined by cloning procedure artifacts. Furthermore, it was found that this effector-independent transcription was also dependent on the growth phase and occurred only when cells entered the stationary phase (Fig. 1). To the best of our knowledge, this was the first time that effector-independent, growth phase-dependent transcription of a ⁵⁴-dependent catabolic system was observed. For brevity, we named this phenomenon gratuitous activation.

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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.

Gratuitous activation requires the minimal regulatory sequences. In order to analyze the genetic elements involved in gratuitous activation, we constructed a reporter system containing the lacZ gene fused to the minimal Ptou promoter (i.e., the region spanning the putative UAS, the –12/–24 consensus, and the transcriptional start previously mapped by primer extension) (2). This reporter system was cloned in the low-copy-number vector pKGB4N, giving plasmid pTN1021, and introduced into P. putida PaW340::touR, which harbored a single copy of touR under the control of its native promoter integrated in the chromosome. Transcription from the Ptou promoter was thus monitored during growth by measuring ß-galactosidase activity, both in the absence and in the presence of o-cresol. As shown in Fig. 2A, during the exponential growth phase in the absence of any TouR effectors, ß-galactosidase activity was barely detectable, whereas at the transition from the exponential phase to the stationary phase strong activation of the Ptou promoter occurred, with the same pattern observed in P. stutzeri OX1 (Fig. 1). The presence of touR was required, as the control strain devoid of the regulatory gene showed no gratuitous activation (Fig. 2A), hence ruling out the possibility that another transcriptional regulator takes over control of the Ptou promoter in the stationary phase. Gratuitous activation was thus reproduced in the heterologous P. putida PaW340 host with the minimal tou regulatory sequences, suggesting that no other P. stutzeri OX1-specific genetic elements were required for gratuitous activation to occur and ruling out the possibility that this phenomenon could be driven by an alternative tou operon promoter.

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FIG. 2. Transcriptional profiles of Ptou (Ptomo) and other 54-dependent promoters in the presence of either touR or dmpR regulatory genes. The growth curves (open symbols) and ß-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 ß-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.

Gratuitous activation is specifically mediated by TouR. Since the ⁵⁴-dependent promoters are known to be transcriptionally inactive in the absence of an activated form of their cognate regulators, the regulator-promoter swapping experiments described below were carried out to elucidate whether the Ptou gratuitous activation represented an exception rather than a common, hitherto unknown feature shared by other catabolic operon ⁵⁴-dependent regulatory systems that somehow became evident only under our experimental conditions. To ascertain whether in our hands another ⁵⁴-dependent regulator could activate the Ptou promoter in an effector-independent manner, the reporter strain P. putida PaW340::Ptou-lacZ was constructed and transformed with either pKR210 or pKDmpR, which harbored the touR gene and the dmpR gene, respectively, from Pseudomonas sp. strain CF600 (54) under the control of their native promoters. We used DmpR because among the aromatic compound-responsive NtrC-like transcriptional regulators it is the most similar (83%) to TouR and recognizes the same phenolic effectors (2). ß-Galactosidase activity was monitored during growth in mineral M9 medium supplemented with 20 mM malate containing 1 mM o-cresol as the effector or not containing o-cresol. As shown in Fig. 2D and E, both regulators could induce ß-galactosidase activity after exposure to o-cresol, which indicates that DmpR could activate transcription from the noncognate Ptou promoter. However, in the absence of effectors only TouR could induce ß-galactosidase activity at the phase transition, whereas in the presence of dmpR the promoter activity was always low, regardless of the growth phase.

To elucidate whether the Ptou promoter was specifically required, we tested the ability of TouR to activate heterologous ⁵⁴-dependent promoters in the absence of effectors. Therefore, plasmid pKMAD, carrying the lacZ gene fused to the xyl upper operon Pu promoter, and plasmid pKUME, harboring the lacZ gene fused to the dmp operon Po promoter, were introduced into P. putida PaW340::touR. The promoter output was monitored by measuring the ß-galactosidase activity throughout growth in the presence and in the absence of o-cresol and was compared to that of Ptou. All the promoters tested were regulated by TouR in response to o-cresol and were strongly activated in the absence of TouR effectors at the transition from the exponential phase to the stationary phase, although with different absolute outputs (Fig. 2A to C). Taken together, the data from the regulator-promoter swapping experiments indicated that (i) the gratuitous activation of the Ptou promoter is specifically mediated by TouR, which possesses peculiar capabilities that are not shared by the highly similar DmpR protein, and (ii) the presence of touR is necessary and sufficient for imposing, under our experimental conditions, gratuitous activation on the three ⁵⁴-dependent promoters tested.

Monitoring TouR protein during growth. Since TouR displays effector-independent behavior only when cells enter the stationary phase but it completely relies on the presence of an effector to stimulate transcription during the exponential phase (Fig. 2), we wondered whether it was somehow modified at the phase transition. It is known that monocomponent ⁵⁴-dependent regulator derivatives devoid of the A domain (A) can activate transcription, both in vivo and in vitro, regardless of the presence of effectors, since they lack the intramolecular repressor module (3, 20, 45, 52). Production or accumulation of a A derivative of TouR at the phase transition could explain its dual behavior in the exponential and stationary phases. To monitor the physical form of TouR, a Western analysis of strains PaW340::Ptou-lacZ(pKR210) and PaW340::touR(pTN1021) growing on malate-containing mineral medium in the absence of effectors was performed along with measurement of the ß-galactosidase activity. As shown in Fig. 3, only one band specifically corresponding to TouR could be detected at any time in both strains, regardless of the growth phase and of the Ptou activity observed in vivo. This band was the size of full-length TouR (67 kDa), whereas no A derivatives could be detected by this method. The protein levels in the two strains reflected the copy number of the touR gene; in PaW340::Ptou-lacZ(pKR210), in which the touR gene was plasmid borne, the TouR levels were higher than they were in PaW340::touR(pTN1021), which harbored a single copy of touR integrated into the chromosome (Fig. 3B). Moreover, in PaW340::Ptou-lacZ(pKR210), but not in PaW340::touR(pTN1021), the level of TouR appeared to increase with time, reaching in the late stationary phase a value that was approximately threefold the level observed in the exponential phase, a phenomenon that could be ascribed to an increase in the plasmid copy number in the stationary phase. However, the fact that gratuitous activation was observed in both strains with the same growth phase-dependent pattern (Fig. 3A) despite the different levels of the regulatory protein indicated that the occurrence of this phenomenon at the transition phase did not rely on accumulation of TouR. Indeed, the low TouR level observed in PaW340::touR(pTN1021) was sufficient to stimulate gratuitous activation to the same extent as, or even to a greater extent than, that in PaW340::Ptou-lacZ cells harboring the touR⁺ pKR210 plasmid.

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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 ß-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-A-TouR (open arrow) (40 ng each). OD600nm, optical density at 600 nm.

Physiological control of gratuitous activation. The fact that gratuitous activation specifically occurs at the onset of the stationary phase, when the cells are in a precise physiological state, suggests that higher-order, global control is superimposed on the specific, TouR-mediated regulatory circuit. Hence, several factors and parameters that are characteristic of the stationary phase were systematically analyzed to ascertain their possible involvement in this phenomenon.

It was demonstrated that gratuitous activation is independent of the stationary-phase sigma factor, ^S (reviewed in reference 34), since the Ptou promoter could be activated by TouR in the absence of effectors in the ^S-deficient strain P. putida KT2442 5.2 with the same growth phase-dependent pattern observed in the KT2442 wild-type counterpart (data not shown) and in the P. putida PaW340 reporter strains described above. To verify the influence of the growth rate and the optical density, these two parameters were manipulated by growing the reporter strains at a suboptimal temperature (21°C) and at a low concentration of a carbon source (5 mM malate), respectively. However, gratuitous activation of the Ptou promoter occurred at the onset of the stationary phase regardless of the growth rate of the culture and the optical density reached (data not shown). The hypothesis that the tou regulatory circuit might be subservient to the quorum sensing (reviewed in references 24 and 26) or that TouR might recognize as effectors some other diffusible molecules produced in the stationary phase was also addressed by exposing exponentially growing P. putida PaW340::Ptou-lacZ(pKR210) cells to the filter-sterilized medium from a stationary-phase culture of the same strain; negative results were obtained (data not shown).

In mineral medium, which was used in all the experiments described in this paper, the stationary phase is usually entered because of exhaustion of nutrients. To address the question of whether nutrient limitation could be the signal triggering gratuitous activation, P. putida PaW340::touR(pTN1021) was used to assay the effect on gratuitous activation of nitrogen or carbon starvation. P. putida PaW340::touR(pTN1021) was grown in a modified M9 medium containing a limiting concentration of NH₄Cl (2 mM instead of the standard 20 mM) and 20 mM malate (Fig. 4A and B), and the transcription from the Ptou promoter was monitored during growth by determining the ß-galactosidase activity. At the onset of the stationary phase, the culture was split into three subcultures; one of these subcultures was allowed to enter the stationary phase without any addition, whereas either NH₄Cl (final concentration, 20 mM) (Fig. 4A) or o-cresol (final concentration, 1 mM) (Fig. 4B) was added to the other subcultures. As shown in Fig. 4A and B, when the primary culture entered the stationary phase, which was due to nitrogen exhaustion, no gratuitous activation was observed. However, in nitrogen-limiting conditions the system was still fully functional, as the exposure to o-cresol induced an immediate transcriptional response from the Ptou promoter (Fig. 4B). The subculture supplemented with saturating NH₄Cl in the stationary phase resumed exponential growth until the second stationary phase, which was likely due to carbon exhaustion, when gratuitous activation occurred (Fig. 4A). To test the effect of carbon exhaustion, P. putida PaW340::touR(pTN1021) was grown in mineral medium containing 20 mM NH₄Cl and 5 mM malate as the carbon source, and the ß-galactosidase activity was measured during growth. When the stationary phase was reached due to carbon exhaustion, gratuitous activation of the Ptou promoter occurred (Fig. 4C). The culture was then divided into two subcultures, and malate (final concentration, 15 mM) was added to one of them. The latter culture started growing again, and, conversely, ß-galactosidase activity decreased until the second transition phase, when the Ptou promoter was activated for the second time. These data indicate that gratuitous activation was specifically triggered at the phase transition by carbon exhaustion and not by nitrogen limitation, as no increase in ß-galactosidase activity was observed when the stationary phase was entered because of nitrogen exhaustion. However, the results shown in Fig. 4 did not rule out the possibility that TouR might be repressed by binding to malate, like, for example, fumarate represses the LysR-type ClcR transcriptional activator of P. putida (36). Therefore, to ascertain whether gratuitous activation occurred at the phase transition in the presence of carbon sources other than malate, P. putida PaW340::touR(pTN1021) was grown in mineral M9 medium containing diverse carbon sources that enter different pathways of the central metabolism, including glucose (10 mM), pyruvate (40 mM), succinate (20 mM), and malate (20 mM). ß-Galactosidase activity was monitored during growth in the absence of TouR effectors, and gratuitous activation occurred in all cases at the phase transition (Fig. 5). When cells reached an optical density at 600 nm of 0.4 to 0.5, part of each exponentially growing culture was exposed to 2 mM o-cresol. After 2 h of exposure, when the cultures were still exponentially growing, the ß-galactosidase activities were compared. The data indicated that the different carbon sources exerted different degrees of repression on the output from the Ptou promoter. In particular, pyruvate was the least repressive compound, succinate had a mild effect, and glucose and malate were the most repressive substrates, reducing the ß-galactosidase activity to one-half that measured in pyruvate-grown cells (Fig. 5E). However, gratuitous activation always occurred at the onset of the stationary phase regardless of the carbon source used and the magnitude of the repression that the carbon source exerted on effector-induced activity during exponential growth. Therefore, the results obtained from the experiments shown in Fig. 4 can be generalized, and it can be concluded that exhaustion of the carbon source is the specific signal that triggers the TouR-mediated gratuitous activation of the Ptou promoter at the phase transition.

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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 ß-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.

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FIG. 5. ß-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 ß-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) ß-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.

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The ⁵⁴-RNAP requires interaction with an activated form of an NtrC-like transcriptional activator to initiate transcription; thus, ⁵⁴-dependent aromatic compound catabolic operons are usually completely silent in the absence of the appropriate effectors. In this paper we show that the ⁵⁴-dependent tou regulatory circuit of P. stutzeri OX1 is activated in the absence of known effectors of the cognate regulatory protein TouR at the onset of the stationary phase.

The similarity among the UAS of different ⁵⁴-dependent promoters (2) often allows cross-activation among heterologous regulatory circuits (15). We exploited this feature of ⁵⁴-dependent systems to perform regulator-promoter swapping experiments in order to dissect the contributions of distinct genetic elements within the tou regulatory circuit to the gratuitous activation. Unexpectedly, effector-activated TouR stimulated a higher transcriptional response from the heterologous promoters than from its own target promoter (Fig. 2A to C). This might have been due to differences in the intrinsic promoter strength (i.e., in the ability to recruit the ⁵⁴-RNAP) or in the binding affinity of TouR for the UAS at different promoters. TouR would be expected to have the highest affinity for its binding site at Ptou. However, a binding affinity lower than expected could be accounted for by the fact that this promoter is likely not the original target for TouR (1, 2). Also, a greater difference between effector-induced and gratuitous activation was observed at Po and Pu than at the Ptou promoter. Although the reasons for this behavior remain unclear, the behavior seems to indicate that the form of TouR that is active in the absence of effectors functions better at its native target promoter. Nevertheless, in spite of the differences in promoter output, the regulator-promoter swapping experiments demonstrated that TouR is the sole element within the specific regulatory circuit that is required for the effector-independent transcription to occur and that its presence is sufficient to impose the same behavior on two ⁵⁴-dependent promoters other than the Ptou promoter. On the other hand, the growth phase dependence of gratuitous activation clearly indicated that this phenomenon is also subject to global regulation, and in particular, our data showed that it is triggered by carbon starvation.

The ability of TouR to gratuitously activate transcription appears not to be a general feature of the XylR/DmpR subclass of NtrC-like regulators since effector-independent growth phase-dependent transcription from the cognate promoter was never described for the well-studied XylR and DmpR models, nor under our experimental conditions could the gratuitous activation of Ptou be observed in the presence of DmpR. Transcriptional activation in the absence of genuine effectors was reported for the PhcR protein of the phenol-degrading strain Comamonas testosteroni R5 (59). However, it is not clear whether PhcR is a fully constitutive regulator, and the authors did not investigate if this gratuitous transcription was dependent on the growth phase; thus, the tou and phc regulatory systems cannot be directly compared. Another transcriptional activator that can function in the absence of effectors is the AraC-like protein XylS, the transcriptional activator of the ⁷⁰/^s-dependent Pm promoter of the xyl meta operon. XylS can function in two modes; at low levels, it needs to interact with a specific effector(s) to become active, but when it is expressed at high levels, it can activate transcription even in the absence of effectors (48). Western analysis data indicated that in the system which we investigated, gratuitous activation was not due to increased levels of TouR in the stationary phase (Fig. 3).

We observed different activities from Ptou depending on the reporter strain used; the output was higher when TouR was expressed at lower levels (Fig. 2A and D). This could be explained by the fact that in P. putida PaW340::touR(pTN1021) the reporter system is on a multicopy plasmid and thus more copies of lacZ are available for transcription than in P. putida PaW340::Ptou-lacZ. This implies that in P. putida PaW340::touR the pool of TouR is large enough to saturate more than one promoter and that the fraction of this pool that is active in the absence of the effector is probably the factor limiting the maximal transcription output in noninduced conditions. On the other hand, in P. putida PaW340::Ptou-lacZ(pKR210) the factor limiting the transcriptional output is likely the single-copy reporter system, which, together with the fact that TouR is overexpressed with respect to its native levels (Fig. 3B), might explain the smaller difference between effector-induced and gratuitous activation observed in this strain.

TouR appears to be unique among the well-studied transcriptional regulators of the XylR/DmpR subclass in terms of its ability to stimulate in vivo a transcriptional response in different modes depending on the growth phase. In fact, TouR does not appear to be a fully constitutive activator, since it exhibits constitutive or semiconstitutive behavior only in carbon-starved stationary phase cells but displays an effector-responsive behavior, typical of the other XylR/DmpR-like proteins, during the exponential growth phase. Consistently, in in vitro transcription experiments performed with purified components TouR-mediated transcription from the Ptou promoter was previously shown to be strictly dependent on the presence of the effector (3). Taken together, these observations suggest that TouR activity is regulated in vivo in response to the nutritional status of the cell.

Since it is known that A derivatives of a monocomponent ⁵⁴-dependent regulator can activate transcription regardless of the presence of effectors (3, 20, 45, 52), the possible accumulation of A-TouR during the phase transition was investigated, but no major changes in the physical form of TouR were detected. Although the accumulation of very small amounts of a constitutive A derivative which were not detectable by Western analysis cannot be completely excluded, the levels of full-length TouR and A-TouR would be expected to be similar at least in P. putida PaW340::Ptou-lacZ (pKR210), in which the ß-galactosidase activity measured in the absence of effectors was almost as high as the effector-induced activity (Fig. 2D).

Since the activity state of the XylR/DmpR-like regulators is dictated by the mode of interaction between the A and C domains (50, 60, 62), it may be envisaged that in TouR the intramolecular repression is released or becomes intrinsically weaker when the molecule encounters specific physiological conditions. Although gratuitous activation does not appear to be under the control of quorum sensing, the possibility that in carbon-starved stationary-phase cells intracellular metabolites accumulate and function as TouR effectors cannot be excluded. Alternatively, the A domain-C domain repressive interactions may be somehow relaxed at all growth phases, but the partial release of the repression would result in effective stimulation of transcription only in the stationary phase because in this phase the ⁵⁴-bound holoenzyme is more abundant or more active or because the interactions between the holoenzyme and the regulator at the promoter are facilitated. Indeed, the accumulation of the stringent response alarmone (p)ppGpp at the phase transition was shown to favor ⁵⁴ in the competition with the housekeeping ⁷⁰ for the limiting core RNAP, thus improving the performance of ⁵⁴-dependent promoters in the stationary phase (33, 56). The in vitro TouR-mediated transcription experiments cited above (3) are in contrast with the latter hypothesis, but it must be considered that the in vitro conditions do not match those of the intracellular environment and might be very different from the physiological status of stationary-phase cells in which gratuitous activation occurs in vivo. Further work is needed to ascertain whether and under what conditions gratuitous activation can be reproduced in vitro.

Besides the specific induction systems, expression of ⁵⁴-dependent catabolic operons is subjected to higher levels of regulation. The most extensive studies have been performed on global controls that adjust the gene expression to nutrient availability, such as glucose repression in the xyl system (11, 12, 28) or exponential silencing in the dmp system (56, 58). We observed carbon source-mediated repression of the o-cresol-induced activity of the Ptou promoter during exponential growth; glucose and malate were the most repressive substrates, followed by succinate and pyruvate (Fig. 5). However, with all the carbon sources used, the profile of Ptou induction in the absence of effectors was the same, and also the absolute outputs were very similar in all cases. Therefore, gratuitous activation, unlike the exponential silencing and the carbon repression phenomena, does not appear to rely on the availability of specific, preferred carbon sources. Since it is triggered by the general signal of carbon starvation, it is possible that the effector-independent transcription of the tou system correlates to the metabolic or energy flow within the cell. Interestingly, in LB the effector-independent activation of the Ptou promoter did not occur at the phase transition and exponential silencing was observed in effector-exposed cultures (data not shown). We infer that in rich media such as LB the entry into the stationary phase is not due to exhaustion of the carbon source and therefore the signal triggering gratuitous activation is not provided. Further work is under way to clarify this point.

Although it is known that at least some of the global regulation controls of ⁵⁴-dependent transcription are channeled through the transcriptional machinery, such as the (p)ppGpp-mediated sigma factor competition (33), it is still not known whether the activity of specific transcriptional regulators may be modulated as well. In this paper we show that some kind of global control related to the general nutrient availability is channeled, at least in part, through the specific regulatory protein TouR. In this respect it is worth noting that in the (p)ppGpp-dependent dmp system the overexpression of DmpR alone was sufficient to overcome the exponential silencing (58), indicating that regulators may play a role in physiological control.

On the basis of the data presented here, the model of the tou regulatory circuit can be refined as shown in Fig. 6. It was proposed previously that the touABCDEF operon was acquired upon a transposition event in an otherwise dmp-like system (1, 2). By virtue of the low specificity of regulator-UAS recognition within ⁵⁴-dependent regulatory systems (15), the preexisting regulator TouR would have been recruited for transcriptional control of the Ptou promoter. This scenario would explain the high level of similarity between TouR and DmpR and the fact that TouR is phenol but not toluene responsive. We believe that gratuitous activation evolved in order to exploit the genetic information harbored by the tou operon and to expand the catabolic capabilities of P. stutzeri OX1, which otherwise would be able to use hydrocarbons as carbon sources only when phenols are also found in the environment. Carbon exhaustion control would have the physiological role of limiting the costly synthesis of the multicomponent ToMO to conditions where no other carbon sources are available. Notably, the gratuitous expression observed in the P. putida host was much stronger than that in the native P. stutzeri OX1 strain if the data were compared to the effector-induced transcription (Fig. 1 and 2), indicating that in P. stutzeri OX1 there may be some other mechanisms that keep the expression at a level high enough to start the conversion of hydrocarbons into phenols but low enough to limit energy waste.

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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.

Although the mechanism by which TouR becomes capable of effector-independent transcriptional activation and how this capability is modulated in response to the metabolic status of the cell remain elusive, we believe that gratuitous activation may prove to be a useful tool for dissecting the interactions between the specific and global regulatory circuits that control the expression of ⁵⁴-dependent systems.

 
This work was supported by Consiglio Nazionale delle Ricerche (Rome), by grant 01.00714.PF49 from the Target Project on Environmental Biotechnology, and by the Ministero dell'Università e della Ricerca Scientifica e Tecnologica (Rome) under the Programma di Ricerca di Interesse Nazionale (contract "Biodegradative Enzymatic Systems: Oxidases and Oxygenases. Characterization and Integrated Utilization" of P.B.).

We are grateful to Victor de Lorenzo and Victoria Shingler for useful discussions and for providing some plasmids used in this work. We are also grateful to A. Baratto and R. Macchi for collaboration on the experimental work.

 
* Corresponding author. Mailing address: Dipartimento di Biologia Strutturale e Funzionale, Via J. H. Dunant, 3, 21100 Varese, Italy. Phone: (39) 0332 421420. Fax: (39) 0332 421330. E-mail: paola.barbieri{at}uninsubria.it.

Present address: Department of Molecular Biology, Umeå University, S-901 87 Umeå, Sweden.

Present address: ACS Dobfar, 20067 Tribiano, Italy.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Bacteriology, November 2004, p. 7353-7363, Vol. 186, No. 21
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.21.7353-7363.2004
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