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Applied and Environmental Microbiology, November 2006, p. 7418-7421, Vol. 72, No. 11
0099-2240/06/$08.00+0     doi:10.1128/AEM.01067-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Role of the ptsN Gene Product in Catabolite Repression of the Pseudomonas putida TOL Toluene Degradation Pathway in Chemostat Cultures{triangledown}

Isabel Aranda-Olmedo, Patricia Marín, Juan L. Ramos, and Silvia Marqués*

Consejo Superior de Investigaciones Científicas, Estación Experimental del Zaidín, Dept. of Environmental Protection, C/. Profesor Albareda no. 1, Granada E-18008, Spain

Received 9 May 2005/ Accepted 8 August 2006


   ABSTRACT
Top
 Abstract
Introduction
 References
 
The Pseudomonas putida KT2440 TOL upper pathway is repressed under nonlimiting conditions in cells growing in chemostat with succinate as a carbon source. We show that the ptsN gene product IIANtr participates in this repression. Crc, involved in yeast extract-dependent repression in batch cultures, did not influence expression when cells were growing in a chemostat with succinate at maximum rate.


   INTRODUCTION
Top
 Abstract
 Introduction
References
 
Pseudomonas putida KT2440 mt-2 is able to degrade and grow on aromatic substrates such as toluene and xylenes. The upper and meta-cleavage pathways required for complete degradation are coded by the TOL plasmid pWW0, where XylR is the master regulator of the concerted expression of the two pathways (21). The xylR gene is transcribed from two {sigma}70-dependent tandem promoters, PR1 and PR2 (Fig. 1). In the presence of substrates of the pathway, XylR protein becomes active (1) and induces transcription from the two {sigma}54-dependent promoters of this system, the upper-pathway promoter Pu and the xylS promoter PS1. XylR binding sites to activate PS1 overlap the two xylR tandem promoters, thus repressing their expression and consequently its own synthesis (3, 21, 23).


Figure 1
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  		FIG. 1. pWW0 TOL plasmid upper-pathway regulation. The diagram shows stimulation by the XylR protein of the two TOL pathway {sigma}54-dependent promoters, Pu and PS1. The TOL upper-pathway and regulatory genes xylS and xylR are shown as gray arrows. R is any substituent group in the aromatic ring. Elliptical and square boxes indicate the inactive and active forms of XylR, respectively. Plus and minus signs indicate transcription activation and repression by XylR, respectively. Black small boxes represent XylR binding sites to activate Pu and PS1 (UASs). Promoters are outlined as flags.

 
XylR induction of the TOL pathway is silenced under several growth conditions. Repression is exerted at the transcriptional level, the catabolite repression targets being the {sigma}54-dependent promoters Pu and PS1 (8, 17). Initially, this was referred to as "exponential silencing" (4), because TOL induction was repressed during exponential growth in rich medium and derepressed in the late exponential phase (6, 16). In contrast to the previous observation, in cells growing on minimal medium in the exponential phase, TOL upper-pathway activation occurred immediately after the addition of a XylR effector. This difference was attributed to the presence of inhibitory compounds in the rich medium (17). In addition, some carbon sources (glucose, gluconate, {alpha}-ketogluconate, lactate and acetate) but not others (succinate, citrate, pyruvate, glycerol, fructose, and arabinose) inhibited activation of Pu in batch cultures (4, 14, 25). Finally, full repression of the TOL pathway was observed in cells growing in continuous culture at a nonlimiting rate (µmax) at the expense of succinate (8). At a low growth rate, when an anabolic substrate (P, S, or N) was the growth-limiting factor (equivalent to a high-energy state in the cell) (12), the TOL degradation pathway was also repressed, whereas when a catabolic substrate (C or O2) was the growth-limiting factor (low energy state) (12), the pathway was functional (9). This suggests that catabolite repression is mediated by excess of carbon and/or energy rather than growth rate.

Some proteins have been involved in this process: IIANtr, the ptsN gene product, contributes to Pu repression by glucose or gluconate in batch cultures through a mechanism that would involve the phosphorylation of the conserved residue His68 (5). Conversely, IIANtr did not influence Pu down-regulation during exponential growth on LB (5). These results pointed to differences in the genetic factors used by the cell to adapt to environmental conditions. However, we have recently shown that IIANtr is also involved in the rich medium-dependent repression of Pu and PS1 (2). In addition, Crc, a protein involved in several catabolite repression mechanisms in Pseudomonas spp. (13, 20, 25, 26), also contributes to TOL {sigma}54-dependent promoter repression in rich medium. The role of Crc could be to "sense" the energy state and thus help the cell to launch an appropriate response (2).

Nevertheless, our knowledge of the genetic factors involved in TOL catabolite repression in continuous culture is limited. It is known that in batch cultures high concentrations of the preferred carbon source are required to produce catabolite repression (22). In contrast, repression in continuous cultures was observed with a lesser excess of the repressive carbon source, suggesting that the cell physiological state (i.e., nutritional excess) rather than the carbon source per se was the signal triggering the response. To clarify whether cells respond differently in terms of catabolite repression under optimal growth conditions, we investigated the extent to which TOL inhibition in cells growing in chemostat at a maximum growth rate (excess of all nutrients, including carbon; thus, a high-energy state) was affected by the inactivation of genes known to play a role in repression in batch culture.

We set up a number of continuous cultures of P. putida KT2440 (pWW0) and its ptsN and crc mutants (2). Custom-made reactors with working volumes of 100 ml of mineral salt medium (10) supplemented with 10 mM succinate, a primary carbon and energy source for pseudomonads (11), were used. Cultures were maintained at 30°C with a constant airflow. Cells were grown at a low dilution rate (D = 0.05 h–1, with succinate as the growth-limiting factor) to determine full induction level and at a high dilution rate (D = 0.85 h–1, with carbon excess) to set up catabolite repression conditions (8). Residual succinate concentrations at the high dilution rate were 1 to 6 mM, in similarity to previously reported values (10). At D = 0.05 h–1, no succinate was detected in the effluent. After 5 min induction with the nonmetabolizable effector o-xylene through the gas phase, 10-ml samples were taken for RNA isolation, primer extension assays, and routine analyses as described previously (2, 18).

Figure 2 compares the results of induction of Pu and PS1 at the two dilution rates. As shown before, transcription from the Pu and PS1 promoters in the wild-type strain was repressed under conditions of carbon excess (D = 0.85 h–1) compared to the results seen with cells growing with succinate as the growth-limiting factor (D = 0.05 h–1) (8). Pu transcription was reduced to about 10% of the level in a carbon and/or energy-limited medium, while PS1 transcription was reduced to about 25% of the values obtained under conditions of succinate limitation. Differences between the Pu and PS1 promoter regions (21) may account for the different repression levels seen in the two promoters under the same growth conditions.


Figure 2
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  		FIG. 2. Repression levels of Pu and PS1 promoters from P. putida (pWW0) and its ptsN and crc mutants. Cells were grown in continuous culture, at D = 0.05 h–1 (carbon limitation) and D = 0.85 h–1 (carbon excess), when o-xylene was added in the gas phase during 5 min. The presence of messengers was analyzed using reverse primer extension of equal amounts (10 or 20 µg) of total RNA and the corresponding labeled specific oligonucleotide (2). Samples were run in urea sequencing gels, which were exposed to a phosphor screen (Fuji Photo Film Co. Ltd.) for 5 to 12 h. Phosphor screens were scanned with a phosphorimaging instrument (Molecular Imager FX; Bio-Rad), and data were quantified with Quantity One software (Bio-Rad). The graph shows the mRNA levels of Pu (A) and PS1 (B) promoters in the three strains at D = 0.85 h–1 (repression conditions) as a percentage of the levels observed in each strain at a low growth rate (D = 0.05 h–1, full induction levels). (C) Representative urea-polyacrylamide gel electrophoresis results corresponding to mRNA-derived cDNA from the two promoters at each growth rate are shown. wt, wild type.

 
Analysis of the {sigma}70-dependent xylR tandem promoter transcription is a useful tool to indirectly track the efficiency of PS1 transcription. As mentioned above, XylR activation of PS1 blocks PR1 and PR2 transcription through binding to its upstream activation sites (UASs) overlapping PR1 and PR2 promoters (21). Therefore, when PS1 transcription is favored, PR1 and PR2 transcription is significantly reduced (3, 15). Figure 3 shows that in the wild-type strain, xylR promoter transcription was practically doubled in cells growing under conditions of carbon excess compared to the results seen with cells growing under conditions of carbon and/or energy limitation. This confirms a lower occupancy of XylR UAS in PS1 at high growth rates, in good correlation with the observed decrease in PS1 activity.


Figure 3
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  		FIG. 3. Level of mRNA derived from the PR1 and PR2 promoters from P. putida (pWW0) and its ptsN and crc mutants grown in continuous culture in response to o-xylene at D = 0.05 h–1 (carbon limitation) and D = 0.85 h–1 (carbon excess). The presence of messengers was analyzed by reverse primer extension of equal amounts (10 or 20 µg) of total RNA with the corresponding labeled specific oligonucleotide (2). Samples were run in urea sequencing gels, which were exposed to a phosphor screen (Fuji Photo Film Co. Ltd.) for 5 to 12 h. Phosphor screens were scanned with a phosphorimaging instrument (Molecular Imager FX; Bio-Rad). Data were quantified with Quantity One software (Bio-Rad). (A) The diagram at the top of the figure represents the xylR/xylS intergenic region (15). (B) The graph shows the mRNA levels of PR1 and PR2 promoters in the three strains at D = 0.85 h–1 as a percentage of the levels observed in each strain at a low growth rate (D = 0.05 h–1). (C) Representative urea-polyacrylamide gel electrophoresis results corresponding to mRNA-derived cDNA from the two promoters at each growth rate are shown. wt, wild type.

 
Repression of Pu transcription in the ptsN mutant when the culture was subjected to carbon and/or energy excess was lower than in the wild-type strain. In this mutant, Pu transcription was reduced by about 50% compared to transcription in the nonlimited medium. A similar behavior was observed for PS1 transcription, although PS1 derepression was less apparent because of the weaker repressive effect on this promoter in the wild type (Fig. 2). As expected, the increase in PR1 and PR2 transcription was smaller when PS1 transcription was more markedly enhanced under nonlimiting growth conditions (Fig. 3).

When we analyzed transcription of Pu and PS1 in the crc mutant as described above, we found no significant difference from the wild-type strain results (Fig. 2). Accordingly, PR1 and PR2 transcription remained unchanged (Fig. 3). In the light of these results, a question mark hangs over the role of Crc in TOL catabolite repression in batch cultures when glucose is the repressor agent. Crc has been shown to contribute to TOL regulation in rich medium-mediated repression (2), but no data are available on its potential role in glucose-mediated repression in batch cultures. To investigate this issue, we compared the ß-galactosidase activity of wild-type and crc mutant strains carrying a Pu::lacZ fusion and xylR gene in the pS10 plasmid (2) in the presence of 75 mM (1.5% [wt/vol]) glucose in the medium. The P. putida ptsN mutant was used as a positive control of derepression of the Pu promoter (5). Fresh mineral salt medium cultures (5) were divided in two fractions. One was supplemented with both the XylR inducer o-xylene and with 1.5% glucose; only the inducer was added to the second fraction. When cultures reached the exponential phase, ß-galactosidase activity was determined (19) (Fig. 4). Pu promoter expression was repressed in the presence of 1.5% (wt/vol) glucose in the wild-type strain P. putida KT2440 (4), but, as expected, this repression was weaker in the ptsN mutant (5). In the crc mutant, glucose repression of Pu was similar to wild-type results. This finding shows that despite its role in Pu repression in rich medium (2), Crc does not participate in the Pu repression caused by an excess of carbon either in continuous or in batch cultures (Fig. 4). It has been shown that Crc levels vary with growth medium composition. While Crc expression significantly increases when yeast extract is added to cells growing on glucose minimal medium (2), its levels are low in continuous cultures with succinate excess (24). This suggests that the role of Crc in catabolite repression of the TOL plasmid is related to its concentration in the cell, which seems to be modulated by the composition of the medium (i.e., the carbon source). This is consistent with previous findings showing no contribution of Crc to succinate-dependent catabolite repression of the P. putida GPo1 alkane degradation pathway (7).


Figure 4
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  		FIG. 4. Effect of glucose on Pu expression. ß-galactosidase activity (in Miller units [MU]) was measured in o-xylene-induced batch cultures of P. putida strain KT2440 and its ptsN and crc mutants carrying the pS10 plasmid with Pu fused to the lacZ gene and the regulator gene xylR (2) in the presence or absence of glucose.

 
Taken together, these results help clarify some aspects of the processes controlling the toluene degradation pathway of the TOL plasmid pWW0 in P. putida KT2440. Bacterial growth in continuous culture helps maintain a physiological steady state at a selected growth rate in a closed culture system. In this study we analyzed how knockout of ptsN and crc, the two genes known to play an important role in TOL repression under batch culture conditions (2, 5), affected the intensity of repression of the TOL upper pathway in continuous culture with carbon and/or energy excess. Primer extension analyses showed that, in contrast with findings in batch cultures, the ptsN gene product but not Crc protein was involved in the phenomenon of TOL catabolite repression under these conditions. Subsequent analysis of Pu expression in the crc mutant grown in batch cultures confirmed that Crc did not play a role in TOL repression by carbon excess. These results point to a difference in the genetic factors used by the cell to adapt to different environmental conditions that cause TOL catabolite repression. We conclude that no single response mechanism is suitable for all environmental conditions; instead, cells may resort to more than one strategy to react appropriately to a changing environment. Catabolite repression triggered by carbon and/or energy excess and repression produced by some components of rich medium are sensed differently. In the first case, IIANtr (PtsN), hence, a mechanism involving {sigma}54-dependent transcriptional machinery, plays a central role, while Crc protein seems to be involved only in the negative modulation of catabolic pathways when cells are growing exponentially on a complex rich medium.


   ACKNOWLEDGMENTS
 
This work was supported by European Communities grant QLK3-CT-2002-01923, grant BCM 2001-0515 from the Spanish Ministry of Science and Education, and grant VEM2004-08560 from Spanish Ministry of the Environment.

We thank K. Shashok for checking the English in the manuscript.


   FOOTNOTES
 
* Corresponding author. Mailing address: Consejo Superior de Investigaciones Científicas, Estación Experimental del Zaidín, Dept. of Environmental Protection, C/. Profesor Albareda no. 1, Granada E-18008, Spain. Phone: 34 958 181600, ext. 285. Fax: 34 958 135740. E-mail: silvia.marques{at}eez.csic.es. Back

{triangledown} Published ahead of print on 22 September 2006. Back


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Applied and Environmental Microbiology, November 2006, p. 7418-7421, Vol. 72, No. 11
0099-2240/06/$08.00+0     doi:10.1128/AEM.01067-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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