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Journal of Bacteriology, June 2005, p. 3678-3686, Vol. 187, No. 11
0021-9193/05/$08.00+0     doi:10.1128/JB.187.11.3678-3686.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Levels and Activity of the Pseudomonas putida Global Regulatory Protein Crc Vary According to Growth Conditions

Ana Ruiz-Manzano, Luis Yuste, and Fernando Rojo*

Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, CSIC, Campus de la Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain

Received 12 January 2005/ Accepted 25 February 2005


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ABSTRACT
 
The global regulatory protein Crc is involved in the repression of several catabolic pathways for sugars, hydrocarbons, and nitrogenated and aromatic compounds in Pseudomonas putida and Pseudomonas aeruginosa when other preferred carbon sources are present in the culture medium (catabolite repression), therefore modulating carbon metabolism. We have analyzed whether the levels or the activity of Crc is regulated. Crc activity was followed by its ability to inhibit the induction by alkanes of the P. putida OCT plasmid alkane degradation pathway when cells grow in a complete medium, where the effect of Crc is very strong. The abundance of crc transcripts and the amounts of Crc protein were higher under repressing conditions than under nonrepressing conditions. The presence of crc on a high-copy-number plasmid considerably increased Crc levels, but this impaired its ability to inhibit the alkane degradation pathway. Crc shows similarity to a family of nucleases that have highly conserved residues at their catalytic sites. Mutation of the corresponding residues in Crc (Asp220 and His246) led to proteins that can inhibit induction of the alkane degradation pathway when present at normal or elevated levels in the cell. Repression by these mutant proteins occurred only under repressing conditions. These results suggest that both the amounts and the activity of Crc are modulated and support previous proposals that Crc may form part of a signal transduction pathway. Furthermore, the activity of the mutant proteins suggests that Crc is not a nuclease.


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INTRODUCTION
 
Expression of many bacterial catabolic pathways is controlled not only by the presence or absence of the compound to be assimilated (a specific control response) but also by several global regulatory proteins that link the induction of the pathway genes to the physiological status of the cell (a global control response). Global control is usually dominant over the specific control. In pseudomonads, this kind of global control has been studied for some pathways responsible for the assimilation of sugars, amino acids, hydrocarbons, and aromatic compounds (for reviews, see references 8, 10, 33, and 35). Although the molecular mechanisms responsible for these global regulatory processes are still not well understood, it is clear that there are several factors involved. Which global regulator, or combinations of regulators, controls a particular catabolic pathway depends not only on the signal to be transmitted but also on the characteristics of the promoters and of the specific transcriptional regulators of the pathway (33, 35). The signals sensed include nutrient or oxygen availability and the presence of alternative carbon sources in addition to that assimilated by the pathway considered. The factors involved include the alarmone (p)ppGpp (7, 37, 39), integration host factor (38, 40), components of the electron carbon chain (12, 13, 32), the FtsH chaperone/protease (6), and the Crc protein (18, 25, 29, 45).

Among these factors, Crc (catabolite repression control) is a master regulator of carbon metabolism in Pseudomonas. It is particularly important for catabolite repression, a complex regulatory response that allows the cell to preferentially use a particular carbon source over a mixture of several other potentially assimilable, but less preferred, compounds. Crc is involved in the catabolite repression generated by succinate or lactate on the expression of a number of genes implicated in the metabolism of several sugars and nitrogenated compounds in both Pseudomonas aeruginosa (10, 25, 43) and P. putida (18, 19). Crc also controls the assimilation of hydrocarbons and aromatic compounds. When cells grow in a complete medium, Crc inhibits the expression of the alkane degradation pathway encoded in the P. putida OCT plasmid (45) as well as that of the homogentisate, catechol, and protocatechuate pathways (29), which direct the assimilation of many aromatic compounds in P. putida (21). Available data suggest that Crc is a component of a signal transduction pathway modulating carbon metabolism and other phenomena such as biofilm development (19, 26, 30). Crc ultimately affects the expression of the target genes, although the precise molecular mechanism underlying this effect remains to be elucidated. The scarce information obtained so far suggests that Crc does not bind DNA, so it would not be a classical DNA-binding repressor (10, 19, 25). Its precise target is not known.

We have analyzed how Crc modulates gene expression using the alkane degradation pathway (alk pathway) encoded in the P. putida OCT plasmid as a model system (41, 42; Fig. 1). Induction of the pathway genes by alkanes is negatively modulated by a dominant global control when cells grow in a complete medium or in a minimal salts medium containing succinate as a carbon source (44). If citrate substitutes for succinate as a carbon source, inhibition does not occur. The global control observed in a complete medium, which is very strong, depends on the additive effects of Crc (45) and the cytochrome o ubiquinol oxidase (Cyo), a component of the electron transport chain (12, 13). In contrast, the inhibition imposed by succinate in a defined medium depends mainly on Cyo, with Crc playing a very minor role (12). At least in a complete medium, the final effect of the inhibition process is a strong decrease in the levels of the AlkS transcriptional activator, an unstable protein present in the cell in limiting amounts even under inducing conditions (45). Keeping AlkS levels below those required for maximal induction of the pathway allows down-modulation of the expression of the two clusters of the pathway in a simple and coordinated way (Fig. 1).



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  		FIG. 1. The P. putida OCT plasmid alkane degradation pathway. The genes are encoded in two clusters (alkBFGHJKL and alkST), both of which are regulated by the AlkS protein. In the absence of alkanes, alkS is expressed from promoter PalkS1. AlkS negatively modulates this promoter, allowing for a low expression. In the presence of alkanes (or of the nonmetabolizable analogue DCPK), AlkS activates transcription from promoters PalkB and PalkS2, which leads to a self-amplification of alkS expression. Activation of these two promoters is negatively modulated by a dominant global control, a process mediated by Crc and Cyo (see the text for details). Inhibition of PalkS2 leads to a decrease in AlkS levels, an unstable protein present in limiting amounts (modified from data in references 12 and 45).

In a complete medium, repression of the alk pathway is particularly strong during exponential growth but rapidly disappears when cells enter the stationary phase of growth. This can be explained in part by the decrease in Cyo levels that occurs upon entry into the stationary phase (12). However, the repression relief in stationary phase is still detected in cells in which Cyo has been inactivated (13). This suggests that either the levels or the activity of the Crc protein could be regulated, decreasing when cells enter the stationary phase of growth. To investigate this possibility, we have analyzed whether transcription of crc, the levels of the Crc protein, or its activity is modulated throughout the growth phase and in different growth mediums. The results indicate that Crc levels, and probably its activity, are modulated.


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MATERIALS AND METHODS
 
Bacterial strains and culture media. The strains and plasmids used are listed in Table 1. Cells were grown at 30°C in LB medium or in M9 minimal salts medium (34), with the latter supplemented with trace elements (4) and 30 mM citrate or succinate as the carbon source. Where indicated, a spent LB medium was used, which was obtained by growing cells to stationary phase, eliminating the cells by centrifugation and subsequent filtration, and sterilizing the medium after adjusting the pH to 7. Expression of promoter PalkB was induced by the addition of 0.05% (vol/vol) dicyclopropylketone (DCPK), a nonmetabolizable inducer that mimics the effect of alkanes (16). Antibiotics were added when appropriate at the following concentrations (in µg/ml): ampicillin, 100; kanamycin, 50; streptomycin, 50; tetracycline, 8. Cell growth was followed by measuring turbidity at 600 nm.


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  		TABLE 1. Strains and plasmids

P. putida PBS4 is derived from P. putida KT2442 (15) by insertion of a PalkB::lacZ transcriptional fusion and the alkS gene in its chromosome (44). P. putida PBS4C1 derives from strain PBS4 by inactivation of the crc gene (contains a crc::tet allele [45]). To obtain strain KT2442-C1, the crc gene of strain KT2442 was inactivated by marker exchange mutagenesis as described previously (45) by using plasmid pCRC10, which contains a crc::tet allele. Construction of all other strains is described further on in the text in the appropriate section.

S1 nuclease protection assays. Cells were collected by centrifugation and chilled, and total RNA was obtained using the phenol/guanidine thiocyanate mix Tri Reagent LS (Molecular Research Center, Inc.). S1 nuclease protection assays were performed as described previously (5), using equal amounts of RNA in each sample. The single-stranded DNA (ssDNA) probe used, which was added in excess to titrate the mRNA, was generated by linear PCR as described previously (5). The plasmid used as substrate, pCRC5 (45), contains the crc gene and upstream sequences and was linearized with BamHI. The oligonucleotide used as primer hybridized to positions 33 to 13 (5'->3' direction) relative to the crc translational start site. The size of the nondigested double-stranded DNA was determined by denaturing gel electrophoresis using a size ladder obtained by chemical sequencing (27) of the ssDNA used as a probe. Band intensities were quantitated by densitometry (QuantityOne software) after digitalizing the image with the VersaDoc imaging system (Bio-Rad).

Transcriptional fusions to lacZ. To obtain a transcriptional fusion of promoter Pcrc to the lacZ reporter gene, a 303-bp DNA fragment containing the promoter was PCR amplified using plasmid pCRC5 as a substrate and primers 5'-GCCGGATCCATAAATCTCGTGCGTGT and 5'-GGTTTTCCCAGTCACGACGT. The DNA fragment obtained was cloned into plasmid pGEM-T-Easy, sequenced to verify the absence of mutations generated through the amplification process, and excised as an EcoRI-BamHI fragment that was cloned between the same sites of plasmid pUJ8. The plasmid obtained was named pCRCR1. The Pcrc::lacZ transcriptional fusion was excised from pCRCR1 as a NotI DNA fragment and cloned into the suicide delivery plasmid pUT-mini-Tn5-Km, generating plasmid pCRCR1K. This plasmid was introduced into Escherichia coli CC118({lambda}pir) and used to deliver the Pcrc::lacZ fusion into the chromosome of KT2442 or of its crc-deficient strain, KT2442-C1, by triparental matings using plasmid pRK600 as a donor of transfer functions. Four isolates in which the mini-Tn5 containing the fusion had mobilized to the chromosome were selected and tested for expression of the lacZ gene. Representative strains were chosen and named CFR2 (derived from KT2442) or CFR2C1 (derived from KT2442-C1).

Assay for ß-galactosidase. A culture of the strain of interest which was grown overnight was diluted to a final turbidity (A600) of 0.04 in the appropriate medium. When turbidity reached 0.08, the nonmetabolizable inducer DCPK (0.05% [vol/vol]) was added, where indicated, to induce expression of the alk pathway promoters. Growth was continued at 30°C, and at different time points, aliquots were taken and ß-galactosidase activity was measured as described previously (28) by using o-nitrophenyl-ß-D-galactoside as a substrate. At least three independent assays were performed.

Crc derivatives (His6)crc, crc(H246E), and crc(D220A). To introduce a His6 tag at the N terminus of Crc, the crc gene was PCR amplified using pCRC5 as substrate and primers 5'-CACGGATCCATGCGGATCATCAGTGTG and 5'-GAACCATGGCCGATCAAATAACCA. The DNA fragment obtained, which contains the entire crc gene without its native promoter, was cloned into pGEM-T-Easy, generating pGHCRC. This plasmid was digested with BamHI and PstI, and the fragment containing crc was cloned between the equivalent sites of pQE30, generating pHCRC. A HindIII-XhoI DNA fragment containing (His6)crc was excised from pHCRC and cloned between the equivalent restriction sites of plasmid pKT231, generating pHCRC11.

Crc residues Asp-220 and His-246 were changed to Ala (D220A mutation) or to Glu (H246E mutation) by PCR, as described previously (9), using plasmid pCRC5 as a substrate. The final PCR product obtained was cloned into pGEM-T-Easy and sequenced. Plasmids with the appropriate sequence were named pGCRC-CA (D220A mutation) or pGCRCH246E (H246E mutation). A 420-bp DNA segment containing the mutagenized crc region was excised from pGCRCH246E with KpnI and NruI and used to substitute the equivalent segment in plasmid pCRC5, generating pCRC5H246E. The complete crc(D220A) and crc(H246E) genes, including their own promoters, were excised with BamHI from pGCRC-CA and pCRC5H246E, respectively, and cloned into plasmid pKT231, generating pCRC11-D220A and pCRC11H246E, respectively.

To insert crc or its mutant derivatives into the P. putida chromosome, the corresponding genes were cloned into the suicide delivery plasmid pUT-mini-Tn5Sm. Wild-type crc, including its own promoter, was excised from pCRC5 as an EcoRI-HindIII fragment and cloned between the EcoRI and HindIII sites of pUJ8, generating plasmid pUJ-CRC. A NotI DNA fragment containing crc was excised from pUJ-CRC and cloned at the NotI site of pUT-mini-Tn5Sm, generating pSMC1. The crc(D220A) gene, including its own promoter, was excised from pGCRC-CA as a NotI fragment and cloned into pUT-mini-Tn5Sm, generating pSMCRC-CA. The crc(H246E) gene, including its own promoter, was excised from pCRC5H246E as a BamHI fragment and cloned into the BamHI site of pUC18Not, generating plasmid pUC-E, from which it was recovered as a NotI DNA segment and cloned at the NotI site of pUT-mini-Tn5Sm. The plasmid obtained was named pSMC2-E. The (His6)crc gene was excised from pHCRC with EcoRI and cloned at the EcoRI site of pVTRAd, generating pVTR-HC. In this plasmid, (His6)crc is expressed from the Ptrc promoter of the vector. Plasmid pVTRC-HC was digested with NotI, and the fragment containing Ptrc-(His6)crc was cloned at the NotI site of pUT-mini-Tn5Sm, generating pSM-HC.

The suicide delivery plasmids pSMC1, pSM-HC, pSMCRC-CA, and pSMC2-E, which contain the crc, (His6)crc, crc(D220A), and crc(H246E) genes, respectively, were introduced into E. coli CC118({lambda}pir) and subsequently transferred to P. putida PBS4C1 (which contains an inactive crc::tet allele) by triparental matings using plasmid pRK600 as a donor of transfer functions. Selected isolates in which the mini-Tn5 containing either the wild-type crc, (His6)crc, crc(D220A), or crc(H246E) gene had been mobilized to the chromosome were named PBS4C1C, PBS4C1-HC, PBS4C1-CA, and PBS4C1-CE, respectively.

Protein purification. E. coli M15 containing plasmid pHCRC was grown in LB medium at 28°C. At a turbidity of 0.9 (A600), expression of (His6)Crc was induced by the addition of isopropyl-ß-D-thiogalactopyranoside up to 1 mM. After 3 h, cells were centrifuged; resuspended in 10 mM Tris-HCl, pH 8, 100 mM sodium phosphate, and 8 M urea; and disrupted by sonication. After elimination of cell debris, the supernatant was loaded onto a column containing Ni-NTA resin (QIAGEN Inc.) equilibrated in the same buffer. The column was washed with 20 volumes of Tris-HCl, pH 6.3, 100 mM sodium phosphate, and 8 M urea and 4 volumes of 10 mM Tris-HCl, pH 5.9, 100 mM sodium phosphate, and 8 M urea and eluted with 4 volumes of 10 mM Tris-HCl, pH 4.5, 100 mM sodium phosphate, and 8 M urea. The (His6)Crc protein was detected by immunoblotting with a His6 monoclonal antibody (Clontech).

Immunological techniques. Polyclonal antibodies against Crc were obtained by immunizing rabbits with purified (His6)Crc. The polyclonal antiserum obtained was purified by immunoadsorption to enrich it in antibodies directed towards Crc, essentially as described previously (34). Crc protein was detected by immunoblotting with the antiserum obtained using standard techniques (34). (His6)Crc was revealed by either the Crc antiserum or a monoclonal anti-His6 antibody (Clontech, Inc). Membranes were treated with a secondary antibody conjugated to peroxidase (anti-rabbit for the polyclonal antibody and anti-mouse for the monoclonal anti-His6 antibody; Amersham-Pharmacia Biotech). The blot was developed by chemiluminescence using the ECL+Plus system from Amersham-Pharmacia Biotech, as recommended by the supplier. To analyze and calibrate the linearity of the response of the Western blots, serial dilutions of PBS4 cell extracts (40, 20, 10, and 5 µg of total protein) were resolved in sodium dodecyl sulfate (SDS)-polyacrylamide gels, and the amounts of Crc protein were revealed with the polyclonal antiserum as indicated above.


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RESULTS
 
Expression of the crc gene under different growth conditions. The transcription start site of crc was determined by S1 nuclease protection assays performed with total RNA obtained from exponentially growing P. putida KT2442. Assays were performed with cells grown in rich LB medium or in minimal salts medium containing citrate as a carbon source and with samples collected at different moments of the growth curve. A single start site was observed (Fig. 2) and was located 23 nucleotides upstream from the predicted translation initiation codon. The –10 region of the promoter contained a sequence similar to the consensus recognized by the vegetative {sigma}70-RNA polymerase holoenzyme. Seventeen base pairs upstream from the –10 box, a moderately conserved –35 box was present (three out of six matches).



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  		FIG. 2. Characterization of the promoter for the crc gene. (A) P. putida KT2442 was grown in rich LB medium or in minimal salts medium supplemented with citrate as a carbon source; at different moments of the growth curve (A600 values in the figure), cells were collected and total RNA was purified. The transcription start site and the amounts of transcripts present were determined by S1 nuclease protection assays. A size ladder obtained by chemical sequencing (27) of the ssDNA used as a probe was run in parallel (lane M). (B) Sequence of the Pcrc promoter. The –10 and –35 consensus boxes for {sigma}70-RNA polymerase are indicated. The transcription start site (+1) is indicated with an arrow. (C, D) The signal obtained from promoter Pcrc in panel A was quantitated by densitometry and represented as a function of cell growth.

In cells grown in complete LB medium, expression of crc was highest during exponential growth and decreased when the growth rate declined before cells entered into stationary phase (Fig. 2). In stationary-phase cells, transcript levels were three- to fourfold lower than those of cells in mid-exponential phase. When cells were grown in minimal salts medium with citrate as a carbon source, a substrate that does not induce catabolite repression on the alkane degradation pathway, expression of crc was two- to threefold lower than in LB-grown cells and also declined in stationary phase (Fig. 2).

A transcriptional fusion of promoter Pcrc to the lacZ reporter gene was constructed and inserted into the chromosome of P. putida KT2442 and its crc-deficient derivative, KT2442-C1, generating strains CFR2 and CFR2C1, respectively. Expression of the reporter gene was analyzed in cells cultivated in several growth media. At mid-exponential phase (turbidity of 0.5 to 0.6), transcription from promoter Pcrc in strain CFR2 was almost twofold higher in cells grown in LB (448 ± 37 Miller units [MU]) than in cells grown in minimal salts medium containing citrate as a carbon source (250 ± 32 MU). When citrate was substituted with succinate, ß-galactosidase levels increased to 360 ± 30 MU. It should be noted that succinate generates catabolite repression on the alkane degradation pathway, but this repression is signaled mainly by Cyo, with Crc having a very little role in it (12). When cells were grown in a spent LB medium (a medium already used for growth and filtered and sterilized), which generates little catabolite repression (44), expression of the Pcrc::lacZ transcriptional fusion was similar to that observed in minimal salts medium containing citrate as a carbon source (300 ± 25 MU). Inactivation of the crc gene (strain CFR2C1) did not affect the expression of promoter Pcrc in LB medium (402 ± 48 MU), indicating that Crc does not control the expression of its own gene under the conditions tested.

Levels of the Crc protein under different growth conditions. A Crc derivative with a His6 tag in its N terminus was constructed, purified, and used to obtain a polyclonal antiserum, which was used to analyze the levels of Crc in cells grown in different growth media and at different moments of the growth phase. The growth media used were LB, where Crc-mediated inhibition of alk pathway induction is very strong; minimal salts medium with citrate as carbon source, in which there is no inhibition of the alk pathway; and spent LB medium, where inhibition of alk pathway induction is very low (44). As shown in Fig. 3, Crc levels varied significantly as growth proceeded in the three media used. These levels increased at the start of the exponential phase of growth, reached a maximum at mid-exponential phase (turbidity of 0.3 to 0.6), and declined at turbidity values above 0.8 to 1. To compare Crc levels in cells grown in each of the three growth media, cells grown in each of them were collected at a turbidity of 0.6, and proteins were resolved by SDS-polyacrylamide gel electrophoresis on adjacent lanes of the gel. Probing Crc with the antiserum showed that Crc levels are about 2.5-fold higher in LB medium than in minimal salts or spent LB medium (not shown).



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  		FIG. 3. Crc levels along the growth phase in cells grown in different media. Strain PBS4 was grown in LB medium, in minimal salts medium containing citrate as carbon source (M9 + Cit), or in spent LB. At different time points, aliquots were taken, cellular proteins were resolved in SDS-polyacrylamide gels, and the amounts of Crc present were revealed by immunoblotting. The Western blots (top) show the Crc signal obtained at different turbidity values (A600); "C" corresponds to a control lane containing (His6)Crc. The graphs below show the Crc levels observed (squares) and the turbidity of the cultures (circles) as a function of time. Crc levels were calculated by dividing the signal intensity (densitometric values) obtained in each lane by the sum of the signal intensities corresponding to all lanes for a given growth medium. Since Crc levels were 2.5-fold lower in minimal salts medium and in spent LB medium than in LB medium (see the text), the values shown for these two media are corrected accordingly.

Expression of Crc over its physiological levels decreases its repressing effect. If regulation of Crc activity relies on adjusting its levels in the cell, an artificial increase in the amount of Crc may lead to a much stronger inhibition of the alk pathway. To test this possibility, the crc gene including its own promoter was cloned into the broad-host-range high-copy-number plasmid pKT231, which has about 15 to 20 copies per cell (2). The resulting plasmid, named pCRC11, was introduced into strains PBS4 and PBS4C1. PBS4 is an indicator strain that contains a transcriptional fusion of the alk pathway PalkB promoter to the lacZ reporter gene. The strain also harbors alkS, which encodes the transcriptional regulator that activates PalkB in the presence of alkanes. Strain PBS4C1 is derived from P. putida PBS4 by inactivation of its crc gene. When strain PBS4 is grown in LB medium containing alkanes (or the nonmetabolizable analog DCPK), the activity of promoter PalkB remains very low during exponential growth due to the strong inhibition imposed by Crc and Cyo (44; Fig. 4B). Inhibition ceases at the onset of stationary phase, when ß-galactosidase levels abruptly increase. Inactivation of the crc gene (strain PBS4C1) partially relieves this inhibitory effect (45; Fig. 4B). Introduction of crc (including its own promoter) into the chromosome of strain PBS4C1 via a minitransposon (strain PBS4C1C) restored normal crc expression levels (Fig. 4A) as well as the inhibitory effect on PalkB induction (Fig. 4B). Introduction of the multicopy plasmid pCRC11 (which contains crc) into strain PBS4C1 led to a considerable increase in the Crc levels (Fig. 4A) but did not restore the inhibitory effect (Fig. 4B). The amount of Crc detected in the Western blots for strain PBS4C1 containing pCRC11 was about 20-fold higher than that detected in the wild-type strain, which fits well with the copy number of the vector. The protein remained soluble, since after disrupting the cells, it was detected in the soluble fraction rather than in the particulate fraction (not shown). Introduction of plasmid pCRC11 into strain PBS4, which has a wild-type copy of crc in the chromosome, severely interfered with normal Crc function, although a partial repression was still observed during the early exponential phase (Fig. 4A and C). Altogether, the results suggest that the presence of Crc at high levels impairs its inhibitory effect on PalkB induction. Alternatively, the effect may result from a mutation in the crc copy present in pCRC11 or from an unpredicted negative influence of the plasmid vector used (pKT231). The crc gene present in pCRC11 was sequenced, and no mutations were observed. Plasmid pKT231 was introduced into strain PBS4, but it did not interfere with normal Crc function (Fig. 4C). To further analyze this problem, the crc gene was excised from pCRC11 as a BamHI DNA fragment and cloned into the BglII site of the low-copy-number broad-host range plasmid pRK2501, which has about three to four copies per cell (22). Introduction of the resulting plasmid (pCRC21) into strain PBS4C1 led to Crc levels very similar to those of wild-type strain PBS4, as judged by Western blot assays (not shown). Repression of the alk pathway in the presence of plasmid pCRC21 was as efficient as that in strain PBS4 (Fig. 4C). We conclude that the presence of 3 to 4 copies of the crc gene does not interfere with its function, while increasing the copy number to about 15 to 20 leads to a loss of its repression efficiency. In other words, the inhibitory effect of Crc is reduced or lost when the protein is present in amounts that exceed a certain level.



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  		FIG. 4. Effect of an increased gene dosage on the ability of Crc to inhibit induction of the alk pathway. (A) Levels of Crc in strains PBS4 (wild type for crc; contains a PalkB::lacZ fusion and the alkS gene in the chromosome), PBS4C1 (derived from PBS4 by inactivation of crc), PBS4C1C (derived from PBS4C1 by insertion of a wild-type crc gene in its chromosome), PBS4C1 containing plasmid pCRC11 (a multicopy plasmid with a wild-type crc gene), and PBS4 containing plasmid pCRC11. Cells were grown in LB medium, collected at mid-exponential phase (A600 of 0.6), and disrupted by sonication; proteins were resolved in an SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane, and the presence of Crc was revealed with and antiserum directed against (His6)Crc. (B, C) Activity of Crc in cells containing different Crc levels. Cells of the indicated strains were grown in duplicate flasks in rich LB medium, and at a turbidity of 0.08, DCPK was added up to 0.05% (vol/vol) to one of the flasks to allow the AlkS activator to induce expression of lacZ from promoter PalkB. The levels of ß-galactosidase were determined at different time points as indicated by Miller (28). ß-Galactosidase levels in the absence of DCPK were very low and are not indicated.

Mutations at Crc residue Asp220 or His246 generate active proteins. The use of BLAST (1) and PFAM (3) software (http://ncbi.nlm.nih.gov/BLAST/) shows that Crc is related to the endonuclease-exonuclease-phosphatase family of proteins, which includes Mg2+-dependent endonucleases and a large number of phosphatases involved in intracellular signaling (14). The highest similarity scores correspond to exonuclease III from several bacterial species (67%, 54%, and 43% similarity, respectively, to that of Coxiella burnetii, Bacillus subtilis, and E. coli). Proteins from this family have a striking conservation of the catalytic residues of apurinic/apyrimadinic endonucleases (14), which correspond to Asp220 and His246 in Crc. To investigate the role of these residues, Asp220 was changed to Ala by site-directed mutagenesis, while His246 was changed to Glu. The genes encoding these mutant proteins, named crc(D220A) and crc(H246E), were individually introduced into the chromosome of the crc-deficient strain PBS4C1 via the mini-Tn5Sm transposon. The strains obtained were named PBS4C1CA and PBS4C1CE, respectively. Induction of promoter PalkB in these two strains by alkanes was severely inhibited during exponential growth in LB medium, indicating that crc(D220A) and crc(H246E) can complement the crc::tet mutation present in PBS4C1 (Fig. 5B). Therefore, these two residues are not essential for Crc function. Interestingly, mutant proteins Crc(D220A) and Crc(H246E) were also active when their corresponding genes were introduced into PBS4C1 via a multicopy plasmid, which led to elevated levels of the two proteins (Fig. 5A). It should be noted, however, that mutant protein Crc(D220A) appeared to be less stable than the wild-type Crc, since the protein levels detected were lower and some degradation bands were detected (Fig. 5A). Introduction of the multicopy plasmids expressing these mutant proteins into strain PBS4, which contains a wild-type crc allele, did not interfere with the normal function of wild-type Crc, since inhibition of PalkB induction during exponential growth was efficient (Fig. 5D). However, inhibition ceased when cells entered into the stationary phase (at turbidity values above 1.5), in spite of the fact that the levels of the mutant Crc proteins were still high. As will be discussed below, these results suggest that repression by Crc depends not only on its levels in the cell but also on its activity and/or on its cooperation with additional factors.



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  		FIG. 5. Ability of mutant proteins Crc(H246E) and Crc(D220A) to inhibit the induction of promoter PalkB. (A) The levels of Crc in strain PBS4 and strain PBS4C1 containing plasmid pCRC11, pCRC11H246E, or pCRC11D220A were determined by Western blots as indicated in Fig. 4. (B, C, D) Strains PBS4 (wild type for crc), PBS4C1 (crc derivative of PBS4), PBS4C1CE [PBS4C1 containing crc(H246E) inserted into the chromosome], PBS4C1CA [PBS4C1 containing crc(D220A) inserted in the chromosome], and PBS4C1 containing the multicopy plasmid pCRC11H246E [which bears the crc(H246E) gene] or pCRC11D220A [which bears the crc(D220A) gene] were grown in LB medium. At a turbidity of 0.08, DCPK was added to 0.05% (vol/vol) to induce expression of lacZ from promoter PalkB. The levels of ß-galactosidase were determined at different time points as indicated by Miller (28). ß-Galactosidase levels in the absence of DCPK were very low and are not represented.

To analyze whether Crc(H246E) could inhibit induction of the alk pathway in a nonrepressing medium, strain PBS4C1CE, which contains crc(H246E) inserted in the chromosome, or PBS4C1 containing plasmid pCRC11H246E was grown in a minimal salts medium with citrate as a carbon source. Expression of ß-galactosidase from the PalkB promoter after the addition of an inducer of the alk pathway was very efficient in the two strains, reaching levels similar to those observed in strain PBS4 grown in the same medium (not shown). Therefore, mutant protein Crc(H246E) cannot repress the alk pathway in this nonrepressing medium.


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DISCUSSION
 
Previous reports have shown that induction of the alk pathway by alkanes is severely inhibited when cells grow exponentially in rich LB medium, repression that is partly dependent on Crc and which rapidly diminishes when cells reach stationary phase (44, 45). The results presented here show that in cells growing in rich LB medium, transcription of the crc gene varies according to the growth phase, being three- to fourfold more efficient during exponential growth than in stationary phase. Similarly, the levels of the Crc protein are four- to fivefold higher in mid-exponential phase than in the stationary phase of growth. Therefore, Crc levels are high during the phase of growth in which there is a high repression of the alk pathway and decrease when repression is no longer observed. When cells were grown in minimal salts medium containing citrate as a carbon source, where there is no repression of the alk pathway, transcription of crc and the amounts of Crc protein observed were also higher during exponential growth and decreased at the onset of the stationary phase. However, maximal Crc levels in this growth medium were about 2.5-fold lower than that in rich LB medium. A similar result was observed when cells were grown in spent LB medium, in which catabolic repression is very weak (44). Therefore, under the conditions analyzed, there is a clear correlation between the Crc levels and the degree of repression of the alk pathway.

High Crc levels, however, do not guarantee an efficient repression of the alk pathway. The mutant crc::tet allele present in strain PBS4C1 could be complemented by a wild-type crc allele if the gene was introduced into the chromosome of this strain via a minitransposon or a low-copy-number plasmid. However, no complementation was observed if the crc gene was introduced by means of a high-copy-number plasmid, which led to Crc protein levels about 20-fold higher than when the gene was present in just one copy. Interestingly, introduction of the crc-containing multicopy plasmid into strain PBS4, which has a wild-type crc allele in the chromosome, interfered with the normal Crc function, considerably reducing the repression effect. In other words, the inhibitory effect of Crc is reduced or lost when the protein is present in amounts that greatly exceed its normal levels. It has been proposed that Crc is a component of a signal transduction pathway that senses nutritional signals (30). Taking this into account, the above-mentioned results can be explained if we assume that Crc needs to be activated by a signal, or an upper element of the pathway, to inhibit induction of the alk pathway, the activating mechanism being a bottleneck that is overloaded or titrated out when large amounts of Crc are present. In this model, Crc would exist in two forms, one active and the other one inactive. When present in high amounts, the inactive form would predominate. Competition of both forms for its target under conditions in which the inactive form is more abundant would result in poor or no inhibition of the alk pathway. An Ala-for-Asp substitution at Crc position 220, or a Glu-for-His substitution at Crc position 246, yields mutant proteins that can inhibit induction of the alk pathway even when present in high amounts. The behavior of these Crc derivatives suggests that they no longer need to be activated to achieve their function. Alternatively, the levels of these mutant proteins, albeit high, do not reach levels high enough to disturb signal transduction. When present at high levels, these two mutant proteins could efficiently inhibit the alk pathway only during exponential growth but not when cells reached the stationary phase, where Crc(D220A) and Crc(H246E) levels were still very high. In addition, Crc(H246E) was active only in cells growing exponentially in LB medium but not in cells grown in a nonrepressing medium. It is clear that under nonrepressing conditions, Crc cannot exert its function even if it contains mutations at residue D220 or H246. This would be an expected behavior if Crc has not received a proper signal under these conditions or if it needs to transmit a signal to another component of the transmission pathway that is not present or is not active under nonrepressing conditions, or if Crc does not work alone at the final target. The nature of this hypothetical factor or signal is at present unknown.

The role of residues Asp220 and His246 in Crc activity is not clear at present. Similarity searches place Crc in a family that includes Mg2+-dependent endonucleases and a divergent set of prokaryotic and eukaryotic proteins that include phosphatases involved in cell cycle regulation and signal transduction (14, 25). It has been proposed that this protein superfamily has an ancient phosphoesterase fold which served as a scaffold for a large set of functionally diverse proteins (14). Some residues are highly conserved in domains believed to surround the catalytic pocket. Crc residues Asp220 and His246 align with the catalytic residues of the Mg2+-dependent endonucleases, which are invariably conserved in the family. However, our results clearly show that these residues are not essential for Crc function, suggesting that although Crc may have kept the folding characteristic of these endonucleases, it does not conserve an endonuclease activity. The similarity to endonucleases suggests that Crc may either bind to or act on nucleic acids. Purified Crc does not seem to bind DNA (10, 25). Its effect on the mRNAs generated at the PalkB and PalkS2 promoters has not been studied in detail.

Crc similarity to phosphatases involved in regulation and signal transduction suggests that it may form part of a signal transmission cascade that includes components activated by phosphorylation or dephosphorylation. Protein kinase cascades are widely used in bacteria to sense and respond to external and internal signals (20). However, Crc shows no obvious similarity to known components of these transmission pathways, such as the classical two-component and His-Asp phosphorelay systems (20), the transcriptional regulators controlled by components of the PTS system through phosphorylation of a histidine residue (36), and the HWE histidine kinases (23). Therefore, the precise role of Crc residues Asp220 and His246, as well as the molecular mode of action of Crc, cannot be predicted at this time.

In summary, our results indicate that the levels of Crc fluctuate according to growth conditions, although this cannot fully explain the changes in Crc activity observed. Available information suggests that Crc must receive a signal and probably transmit it to another unknown factor or must work in combination with this factor to repress gene expression.


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ACKNOWLEDGMENTS
 
We are grateful to J. L. Martínez and G. Morales for critical reading of the manuscript.

This work was supported by grants BMC2003-00063 and GEN2001-4698-C05-01 from the Spanish Ministry of Education and Science. A.R.-M. was a recipient of a predoctoral fellowship from the Government of the Basque Country.


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FOOTNOTES
 
* Corresponding author. Mailing address: Centro Nacional de Biotecnología, CSIC, Campus de la Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain. Phone: (34) 91 585 45 39. Fax: (34) 91 585 45 06. E-mail: frojo{at}cnb.uam.es. Back


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Journal of Bacteriology, June 2005, p. 3678-3686, Vol. 187, No. 11
0021-9193/05/$08.00+0     doi:10.1128/JB.187.11.3678-3686.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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