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Department of Genetics, Institute of Molecular and Cell Biology, Tartu University and Estonian Biocentre, 51010 Tartu, Estonia
Correspondence
Maia Kivisaar
maiak{at}ebc.ee
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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, 2005; Marqués et al., 2006; Rojo & Dinamarca, 2004; Shingler, 2003). Several global factors such as the Crc protein (Hester et al., 2000; Morales et al., 2004; Yuste & Rojo, 2001), CyoB (Dinamarca et al., 2002; Petruschka et al., 2001) and the FtsH protease (Carmona & de Lorenzo, 1999; Sze et al., 2002) have been related to catabolite repression in Pseudomonas putida. As many aromatic compounds are toxic to living organisms, this also complicates their degradation. The toxicity of certain aromatic compounds such as toluene, xylenes and phenol occurs above a certain threshold, since they easily dissolve in the cell membrane, disorganizing its structure and impairing vital functions (Domínguez-Cuevas et al., 2006; Ramos et al., 2002; Sikkema et al., 1995). It has been demonstrated that sudden exposure of bacteria to sublethal inhibitory concentrations of phenol leads to reduction of growth rate of microbial culture until the bacteria are adapted to this concentration of phenol (Santos et al., 2004). During the adaptation to phenol, the fatty acid composition of the bacterial membranes changes (Heipieper et al., 2003; Sikkema et al., 1995). Also, the results of quantitative proteomics have revealed that the response of P. putida KT2440 to phenol-induced stress results in upregulation of many proteins, e.g. those involved in the oxidative stress response and transport of small molecules (Santos et al., 2004).
In P. putida, aromatic compounds are transformed by different enzymes to central intermediates such as protocatechuate and (substituted) catechols, which are further degraded either by a meta ring cleavage pathway or by an ortho ring cleavage pathway (Harayama & Timmis, 1989). The catBCA operon encodes three enzymes required for catechol degradation via the ortho pathway: cis,cis-muconate (CCM) lactonizing enzyme, muconolactone isomerase and catechol 1,2-dioxygenase (C12O), respectively (Houghton et al., 1995). The induction of this operon requires the LysR-family transcriptional activator CatR and an inducer molecule, CCM, which is an intermediate of the ortho pathway (Rothmel et al., 1991, 1990).
The pheB and pheA genes, originating from the plasmid DNA of Pseudomonas sp. EST1001, encode C12O and phenol monooxygenase (PMO), respectively (Kivisaar et al., 1990). When the pheBA operon is introduced into P. putida laboratory strain PaW85, the bacteria acquire the ability to degrade phenol (Kivisaar et al., 1991, 1990). The pheBA promoter resembles the catBCA promoter and is also activated by CatR (Kasak et al., 1993; Parsek et al., 1995; Tover et al., 2000). Comparative studies of the interaction of CatR at the promoters of the pheBA and catBCA operons have revealed that the CatR-mediated activation mechanism is well conserved, despite the different origins of these operons (Parsek et al., 1995; Tover et al., 2000). Transcriptional regulation of the pheBA operon in P. putida is illustrated in Fig. 1.
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). The pheBA operon was redetected in different bacterial species in watershed continuously polluted by phenols, but not in non-contaminated areas. In all isolates degrading phenol via the ortho pathway and harbouring the pheBA genes integrated either into other plasmids or in the chromosome, the original pheBA promoter was present as before and the pheBA genes were induced in the presence of phenol and benzoate (Peters et al., 1997). Thus, soil bacteria carrying the ortho pathway genes regulated by CatR may easily expand their substrate range via horizontal transfer of the pheBA genes without the need for subsequent extensive genetic rearrangements. Hence, the pheBA operon provides a good model for examining the regulation of phenol catabolic genes in soil bacteria.
Here, we have focused on mechanisms which negatively affect transcription from the pheBA promoter. We found that sudden exposure of P. putida to 2.5 mM phenol delayed the operon activation without affecting transcription in general, whereas micromolar concentrations allowed rapid induction of the phenol degradation pathway. Additionally, our previous studies have revealed that transcriptional activation from the pheBA and catBCA promoters is repressed in P. putida cells growing exponentially on minimal medium in the presence of amino acids (Tover et al., 2001). The results of the current study indicate that the Crc protein downregulates at the post-transcriptional level the expression of the pheBA genes, resulting in reduced amounts of phenol degradation enzymes PheA and PheB needed for the production of the pheBA operon inducer molecule, CCM.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. Escherichia coli cells were grown at 37 °C in Luria broth and P. putida cells were grown at 30 °C in M9 minimal medium (Adams, 1959). Antibiotics were added at the following final concentrations: for E. coli, ampicillin at 100 µg ml–1, tetracycline at 10 µg ml–1; for P. putida, carbenicillin at 1000–3000 µg ml–1, tetracycline at 80 µg ml–1; for both organisms, kanamycin at 50 µg ml–1, streptomycin at 100 µg ml–1 and potassium tellurite at 40 µg ml–1. E. coli was transformed with plasmid DNA as described by Hanahan (1983). P. putida was electrotransformed as described by Sharma & Schimke (1996). E. coli strains TG1 (Carter et al., 1985) and CC118 
pir (Herrero et al., 1990) were used for the DNA cloning procedures, and strain HB101 (Boyer & Roulland-Dussoix, 1969) was used as a host for helper plasmid pRK2013 (Figurski & Helinski, 1979), necessary for mobilization of non-conjugative plasmids.
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; de Lorenzo et al., 1990), to integrate desired transcription fusion constructs stably into the genome of P. putida. In order to be sure that the acquisition of the selected phenotype by P. putida is due to the transposition event and not to the integration of the whole delivery plasmid into a recipient chromosome, the transconjugants were tested for resistance to carbenicillin. Only clones which were sensitive to this antibiotic were used for further examination whether we had obtained the desired strains (see below for details of testing). Additionally, all strains were confirmed by PCR analysis for the presence and correct organization of the inserted cassettes. We determined chromosomal locations of the integrated transcription fusion cassettes by sequencing the DNA flanking the insertion sites. None of the targeted genes (PP0806, PP1103, PP1308, PP1775, PP2872, PP3483, PP3956, PP4063 and PP5300) are related to the metabolism of aromatic compounds or encode general transcription factors. We have obtained reproducible results with strains carrying the same transcriptional fusions in different chromosomal locations. This also indicates that there are no additional regulatory effects caused by the insertional inactivation of certain chromosomal genes.
To study the regulation of transcription from the pheBA promoter, the pheBA promoter Pi was cloned upstream from the luxAB reporter system. The promoterless luxAB genes (these genes originated from Vibrio harveyi) were cloned within the 2050 bp HindIII and PaeI restriction fragment from plasmid pGP704L (Pavel et al., 1994) into vector pUC18Not (Herrero et al., 1990), cleaved with the same restriction enzymes, to obtain plasmid pUCNotluxAB. The 158 bp pheBA promoter region was excised from the plasmid carrying the pheBA promoter in pBluescript SK(+) vector (Tover et al., 2000) using restriction enzymes SmaI and RsaI, and inserted into the SmaI site of plasmid pUCNotluxAB. The pheBA promoter–luxAB transcription fusion Pi–luxAB was then inserted into the NotI site of plasmid mTn5SSgusA40 (Wilson et al., 1995) to generate plasmid pUTPiluxABSm. Finally, the plasmid pUTPiluxABSm, which was not able to replicate in hosts other than E. coli CC118 pir, was conjugatively transferred into P. putida PaW85 (Bayley et al., 1977) by using a helper plasmid, pRK2013, and transconjugants carrying random insertions of mini-transposon containing the Pi–luxAB fusion in the chromosome of PaW85 were isolated. We tested several independent clones in order to identify transconjugants whose luciferase expression was not affected by sequences flanking the inserted Pi–luxAB cassette [i.e. they expressed luciferase activity at a low basal level not exceeding 10 RLU (relative light units) per OD580 unit]. One of these clones was chosen for future research and was named PaWlux.
To construct a P. putida strain capable of degrading phenol, the pheBA operon was inserted into the chromosome of P. putida strain PaWlux. An approximately 6.3 kb DNA fragment, containing the pheBA genes under the control of the native inducible pheBA promoter Pi, was cloned from plasmid pAT1142 (Kasak et al., 1993) as a SacI- and EcoRI-generated DNA fragment into pUC18Not cleaved with same enzymes, to obtain plasmid pUCNotPipheBA. As a next step, the NotI fragment containing the inducible pheBA operon was subcloned from pUCNotPipheBA into plasmid pUTmini-Tn5 Km2 (de Lorenzo et al., 1990). The resulting plasmid, named pUTPipheBAKm, was used as a donor plasmid for the insertion of the pheBA operon into the chromosome of P. putida strain PaWlux to obtain strain PaWpheBA-lux. The procedure for isolation of transconjugants was the same as described above. Several independent clones were tested and the expression of the pheBA genes and the luxAB reporter in these clones was dependent on the presence of the pheBA operon inducer (greater than 100-fold induction appeared when the growth medium of bacteria contained phenol or benzoate, whereas the basal level of luciferase activity was the same as in PaWlux).
In order to construct a control strain expressing the luxAB genes under the control of the Ptac promoter, the BamHI-generated DNA fragment containing the lacIq–Ptac regulatory region was subcloned from pBRlacItac (Ojangu et al., 2000) into plasmid pUCNotluxAB to obtain plasmid pUCNottacluxAB. Then, the lacIq–Ptac–luxAB expression cassette was inserted into the NotI site of plasmid mTn5SSgusA40. Finally, the plasmid pUTtacluxABSm generated was used as a donor plasmid for the insertion of the lacIq–Ptac–luxAB expression cassette into the chromosome of P. putida strain PaW85 to obtain strain PaWtaclux. Similar procedures as described above were used to obtain mini-transposon insertion into the chromosome of P. putida, and several independent clones were tested to be sure that transcription from the Ptac promoter was inducible by IPTG in the constructed strain.
For the construction of P. putida strain PaWpheBA-lux-catR, carrying the catR gene under control of the Ptac promoter and the LacIq repressor, the lacIq–Ptac–catR expression cassette was inserted into the chromosome of P. putida strain PaWpheBA-lux. In contrast with the CatR-overexpression cassette constructed in our previous study (Tover et al., 2001), the new construct lacked the CatR auto-regulation site [i.e. an original catR gene is divergently transcribed from the catBCA promoter, which contains CatR binding sites overlapping the catR gene promoter region, thereby reducing transcription from its own gene (Rothmel et al., 1991)]. First, the 913 bp DNA fragment containing the catR gene, but lacking the CatR binding site, was PCR-amplified from P. putida PaW85 chromosome using the primers 5'-CCCACCATACCCTGGAGG-3' and 5'-GGAGCGCGAAGCTTTTCGGCCTGTTGTCAATCAA-3', complementary to the regions located at positions between –4 and +14 bp and +875 and +909 relative to the catR gene transcription start site, respectively. The PCR product was cloned into the EcoRV site of pBluescript KS(+) vector, yielding plasmid pKScatR. Then, the catR sequence was excised from pKScatR with EcoRI and HindIII, and inserted into the plasmid pBRlacItac, opened with the same enzymes. The resulting plasmid pBRtacCatR was cleaved with EcoRI and NheI to subclone the lacIq–Ptac–catR expression cassette into plasmid pUC18Not to generate plasmid pUCNottacCatR. Finally, the expression cassette lacIq–Ptac–catR was inserted into the NotI site of plasmid pUTmini-Tn5Tel (Sanchez-Romero et al., 1998) to obtain plasmid pUTtacCatRTel, which was used as donor plasmid for the insertion of the lacIq–Ptac–catR cassette into the chromosome of P. putida strain PaWpheBA-lux. We tested several independent P. putida PaWpheBA-lux transconjugants carrying random insertions of the lacIq–Ptac–catR expression cassette in the chromosome, and clones giving a luciferase expression pattern similar to that measured in the wild-type strain PaWpheBA-lux if IPTG was not added (this was tested in both the presence and absence of phenol) were chosen for future examination. The results of PCR analysis confirmed the presence of the lacIq–Ptac–catR expression cassette in the chromosome of transconjugants.
Inactivation of the catB gene in the chromosome of P. putida was performed by homologous recombination using plasmid pGPcatB : : tet carrying the catB sequence interrupted by the Tetr gene. To construct the plasmid pGPcatB : : tet, at first the 1122 bp DNA fragment containing the catB gene was PCR-amplified from the chromosome of P. putida PaW85 with the primers 5'-ATGACAAGCGTGCTGATTGA-3' and 5'-TCAGCGACGGGCGAAG-3', complementary to the regions containing the catB start and stop codons, respectively. The PCR product was cloned into the EcoRV site of plasmid pBluescript KS(+), resulting in plasmid pKScatB. An approximately 1.3 kb DNA fragment, containing the tetracycline resistance gene, was obtained from plasmid pBR322 (Bolivar et al., 1977) and inserted into the EheI site located 513 bp downstream from the catB translation start codon within plasmid pKScatB to obtain plasmid pKScatB : : tet. Finally, using the restriction enzymes Ecl136II and Acc65I, the DNA fragment containing the interrupted catB gene was cloned from pKScatB : : tet into plasmid pGP704del (a derivative of plasmid pGP704L obtained by deletion of the 1 kb EcoRI fragment from the luxA gene) to obtain plasmid pGPcatB : : tet. This plasmid, not able to replicate in hosts other than E. coli CC118 pir, was conjugatively transferred into P. putida PaWpheBA-lux by using the helper plasmid pRK2013. The integration of the whole delivery plasmid into a target site was excluded by testing transconjugants for resistance to carbenicillin (only those unable to grow in the presence of 1500 µg carbenicillin ml–1 were considered to be true recombinants, generated as a result of double recombination events). The catB-knockout strain PaWpheBA-lux-catB : : tet was verified by PCR analysis for the absence of the original catB gene and the presence of the catB : : tet allele. Additionally, we could confirm that this knockout strain was unable to grow on benzoate or phenol as a carbon source.
Inactivation of the crc gene in the chromosome of P. putida was performed by homologous recombination using plasmid pCRC10 carrying the P. putida crc gene interruption crc : : tet on plasmid pKNG101 (Yuste & Rojo, 2001). The plasmid pCRC10 was transferred to PaWpheBA-lux and sucrose-resistant colonies were selected according to the procedure described by Yuste & Rojo (2001) to isolate clones carrying the crc : : tet allele instead of the original crc gene. An isolated crc-defective strain was named PaWpheBA-lux-crc : : tet.
For the construction of P. putida strain PaWtaclux-pheB, where PheB is expressed in the presence of IPTG, the lacIq–Ptac–pheB expression cassette was inserted into the chromosome of P. putida strain PaWtaclux. A 1011 bp DNA fragment from plasmid pAT1442, containing the pheB gene, was excised with restriction enzymes Eco47II and HincII, and inserted into plasmid pBRlacItac cleaved with SmaI, yielding plasmid pBRlacItacpheB. The Ecl136II–BamHI fragment, containing the lacIq–Ptac–pheB expression cassette from plasmid pBRlacItacpheB, was cloned into the plasmid pUC18Not, yielding plasmid pUCNotPtacPheB. Finally, the expression cassette lacIq–Ptac</italic>–pheB was inserted into the NotI site of plasmid pUTmini-Tn5Tel, and the plasmid pUTPtacPheB generated was used as donor plasmid for the insertion of the lacIq–Ptac–pheB cassette into the chromosome of P. putida strain PaWtaclux. Inactivation of the crc gene in the chromosome of P. putida PaWtaclux-pheB was performed by homologous recombination by using the same strategy as already described for the construction of strain PaWpheBA-lux-crc : : tet. The resulting strain was named PaWtaclux-pheB-crc : : tet.
Growth and culture conditions.
To study the regulation of transcription from the pheBA promoter, all P. putida strains used in this study were grown in M9 minimal salts medium supplemented with 10 mM glucose. For enzyme assays, the bacterial cultures were grown overnight in M9 medium supplemented with 0.2 % casamino acids (CAA). To ensure that the bacterial culture was in exponential growth phase, the overnight culture was diluted 1 : 100 in fresh medium supplemented with 0.02 % CAA. After 3 h the exponential culture was diluted once more to obtain an OD580 of 0.05. To induce the transcription from Pi–pheBA and Pi–luxAB transcriptional fusions, 2.5 mM sodium benzoate or phenol at different final concentrations (0.0025–2.5 mM) was added to the growth medium. In parallel, control cultures lacking these substrates were sampled for enzyme assays. To investigate the role of amino acids on the transcription from the pheBA promoter, 0.2 % CAA solution was added when indicated. To investigate the effect of overexpression of the CatR protein on transcriptional activation from the pheBA promoter, 1 mM IPTG was added to the growth medium of strain PaWpheBA-lux-catR when overnight-grown cells were diluted the first and second time. To study the effect of Crc protein on PheB levels, strains carrying the lacIq–Ptac–pheB expression cassette were grown exponentially in M9 minimal medium supplemented with 0.02 % CAA until an OD580 of 0.3–0.4 was reached. The bacterial culture was divided in two and 1 mM IPTG was added to induce the expression of PheB. One of these cultures was supplemented with amino acids (0.2 % CAA).
Luciferase assay.
Samples for luciferase assay were taken from exponentially grown cultures. Luciferase assay was performed as follows. Phosphate buffer (990 µl; 100 mM Na2HPO4/KH2PO4, pH 7.5) and 10 µl decanal (5 mM decanal in ethanol) were mixed in a test tube. Bacterial culture (10 µl) was then added, and light emission was measured after 5 min incubation with a luminometer (TD-20/20; Turner BioSystems). The luciferase activities were measured in at least three separate experiments, where each experiment included two parallel cultures.
Immunoblotting of PheA and PheB.
Cell suspensions used for separation of proteins by gel electrophoresis were prepared from the same cultures that were used for luciferase assays. Crude lysates, used for quantification of PheA and PheB proteins, were prepared from an amount of cells which expressed approximately 10 000 RLU luciferase activity per 10 s. Proteins were separated by sodium SDS-polyacrylamide (10 %) gel electrophoresis and transferred to a nitrocellulose membrane (Hybond ECL; Amersham Biosciences). For Western blotting, the membrane was probed simultaneously with mouse anti-PheA and anti-PheB polyclonal sera (the polyclonal antibodies against PheA and PheB proteins were prepared by LabAS) diluted 1 : 2000 and 1 : 1000 respectively, followed by alkaline phosphatase-conjugated goat anti-mouse IgG diluted 1 : 5000 (LabAS). The blots were developed using 5-bromo-4-chloro-indolyl phosphate/nitro blue tetrazolium. The polyclonal antibodies against PheA and PheB were obtained by using PheA and PheB proteins which were overproduced in E. coli with pUC19 derivatives pPU1930 for PheA (Nurk et al., 1991) and pBRlacItacPheB for PheB. These proteins were detectable as major visible bands in SDS-polyacrylamide gel. The PheA and PheB bands were cut from the gel. The gel slices were dialysed against 1x PBS, homogenized in the same buffer and the liquid was used for injection of mice. The polyclonal antibodies obtained were specific to PheA and PheB as we did not detect antibody reaction if cell lysates prepared from P. putida lacking the pheBA operon were analysed. We have controlled that PheA-specific antibodies do not react with polypeptide detected with PheB-specific antibodies and vice versa. To compare intracellular amounts of PheA and PheB in bacteria grown in the presence or absence of amino acids, gradual dilutions of crude lysates of PaWpheBA-lux-catR grown in the absence of amino acids were tested by Western blotting and compared with the amount of these enzymes detected in cells of the same strain grown in the absence of amino acids.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Parsek et al., 1995). Transcription from the pheBA promoter in P. putida carrying the functional pheBA operon is activated in the presence of either phenol or benzoate in the growth medium. Both phenol and benzoate are converted to catechol, which is subsequently oxidized by C12O to CCM, and the latter acts as an effector molecule on CatR-mediated transcriptional activation from the catBCA and pheBA promoters (Kasak et al., 1993; Parsek et al., 1995) (Fig. 1
). Our previous studies have shown that addition of benzoate into minimal growth medium of P. putida leads to rapid induction of the pheBA operon, but transcription from the pheBA promoter was severely impaired in exponentially growing bacteria when amino acids were present in the growth medium (Tover et al., 2001). In order to compare the activation of transcription regulation from the pheBA promoter in bacteria growing in the presence of either phenol or benzoate, we constructed P. putida strain PaWpheBA-lux (for construction details, see Methods). This strain harbours in the chromosome the pheBA genes under the control of their natural promoter Pi (enabling the bacteria to degrade phenol to CCM) and a Pi-luxAB reporter system (the pheBA promoter cloned upstream of the luxAB reporter).
Strain PaWpheBA-lux was grown exponentially in M9 minimal medium containing glucose in the presence of benzoate or phenol. Parallel experiments were carried out with cultures supplemented with amino acids (CAA solution). Bioluminescence was measured in cells sampled 1, 2 and 3 h after addition of 2.5 mM phenol or benzoate. As shown in Fig. 2, transcription from the pheBA promoter was significantly lower in the presence of amino acids in the growth medium irrespective of which source of inducer, phenol or benzoate, was used. At the same time, the transcription profile of the pheBA promoter was remarkably different in cells grown in the presence of either phenol or benzoate (Fig. 2). Interestingly, the negative effect of amino acids was much stronger when phenol was added as an inducer. Only a low basal level of transcription from the pheBA promoter (comparable with that obtained in non-induced cells) was detected during the 3 h examined, if phenol was added into the amino acid-containing growth medium. At the same time, bacteria grown in CAA-containing medium with benzoate expressed much higher luciferase activities. The differences appeared also in minimal medium lacking CAA. Addition of benzoate into the growth medium allowed rapid activation of transcription from the pheBA promoter whereas phenol did not. During the first hour of growth of bacteria in the presence of phenol, the level of transcription from the pheBA promoter did not increase significantly above that detected in non-induced culture. Only later, at 2 and 3 h, was the level of transcription from the pheBA promoter remarkably increased. This raised the question whether addition of 2.5 mM phenol into the growth medium could somehow impede transcription initiation from the pheBA promoter.
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). Thus, our results indicated that activation of the pheBA operon occurs rapidly in the presence of micromolar concentrations of phenol, but is severely delayed if bacteria are exposed to higher concentrations of this compound. At the same time, in the presence of amino acids in the growth medium, only basal level of transcription from the pheBA promoter occurred at any concentrations of phenol used (Fig. 3a).
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) revealed that transcription from this promoter is strongly dependent on IPTG (we have observed 100-fold induction). The presence of phenol in the growth medium did not affect IPTG-dependent transcriptional activation from the Ptac promoter (Fig. 3b). These data confirmed that the exposure of bacteria to a high concentration of phenol influences specifically transcription from the pheBA promoter, but not the expression of the luxAB reporter or transcription from the Ptac promoter, which is unrelated to phenol catabolism.
The reason why the pheBA promoter is rapidly activated in the presence of 0.025–0.25 mM phenol in the growth medium, but the initiation of transcription is severely delayed when bacteria are exposed to 2.5 mM phenol, is not clear. It is possible that after sudden exposure of bacteria to a high concentration of phenol, the intracellular concentrations of this chemical may rise too high, leading to inactivation of the CatR activator protein or phenol degradation enzymes. However, the results of our studies do not support this explanation, because we have not seen inactivation of the phenol degradation enzymes even in the presence of 5 mM phenol in the reaction buffer (data not shown). Inactivation of CatR by phenol is also less plausible, because transcription from the pheBA promoter was rapidly induced at any concentration of phenol in a CatB-deficient strain expressing CatR at a natural level (see below). Alternatively, one may also speculate that phenol could be rapidly extruded from cells before the bacteria are adapted to a toxic concentration of this compound, thereby making intracellular amounts of phenol limiting for the production of CCM, which is needed for the operon activation. Several studies have demonstrated that efflux pumps are induced to extrude deleterious compounds like toluene and other aromatic compounds (Ramos et al., 2002; Santos et al., 2004). However, although P. putida contains many efflux systems (Domínguez-Cuevas et al., 2006; Ramos et al., 2002), no efflux pumps for phenol export have been studied so far.
Negative effect of amino acids in the growth medium on transcription from the pheBA promoter is suppressed either by overproducing CatR or by increasing the cellular amount of CCM
Study of the effects of growth medium composition on the expression of the pheBA operon revealed that, despite the availability of the source of the pheBA operon inducer CCM (benzoate or phenol), the activation of transcription from the pheBA promoter is impeded if bacteria are grown exponentially in the presence of amino acids. The molecular basis of physiological control mechanisms that are superimposed on transcriptional activation of catabolic operons by specific regulators in the presence of effector molecules varies largely, depending on the particular operon studied. In some cases, this control operates through the level of expression of a transcription activator protein (see e.g. Müller et al., 1996; Sze et al., 1996; Yuste et al., 1998). For example, expression of the n-alkanes catabolic pathway is repressed when cells grow exponentially in rich medium even if exposed to the inducer, but overproduction of the regulatory protein AlkS relieves the repression of transcription (Yuste et al., 1998).
In order to test whether CatR would limit transcription from the pheBA promoter in bacteria grown in rich medium, the catR gene under the control of the IPTG-inducible Ptac promoter was introduced into the chromosome of strain PaWpheBA-lux. The resulting P. putida strain PaWpheBA-lux-catR was grown exponentially in the presence or absence of amino acids, and 1 mM IPTG was added to overexpress the catR gene. Luciferase activities were measured in cells sampled after 1 h of growth in the presence of different concentrations of phenol. The control samples analysed from the cultures grown in the absence of phenol revealed that the overexpression of CatR itself (in the absence of an external source of CCM) did not induce pheBA promoter expression. Although we observed somewhat higher levels of luciferase activity compared with those measured in the wild-type strain (compare Fig. 3a and Fig. 4a), this change was not significant compared with the activity measured in the presence of phenol. When phenol was added into the amino acid-containing growth medium, the overexpression of CatR protein resulted in more than 100-fold higher level of the pheBA promoter activity (compare Fig. 3a and Fig. 4a). Notably, up to 20-fold positive effect of the CatR overexpression could be detected in minimal-medium-grown cells as well. This indicated that the intracellular amount of CatR is limiting the transcriptional activation from the pheBA promoter not only in the presence of amino acids, but also in bacteria grown in minimal medium.
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). When the catB gene is inactivated in P. putida, the phenol or benzoate degradation pathways end up with the formation of CCM. To investigate whether the quantity of CCM in cells grown in the presence of amino acids may limit the rate of transcriptional activation from the pheBA promoter, we constructed the catB-deficient mutant PaWpheBA-lux-catB : : tet by interrupting the chromosomal catB gene in strain PaWpheBA-lux with the tetracycline-resistance-encoding gene. Interestingly, when bacteria were grown in the absence of phenol, the transcription from the pheBA promoter occurred at about a 10-fold higher level in the catB-defective mutant than in the wild-type strain (compare Fig. 3a and Fig. 4b). One possible explanation for this phenomenon is that a small amount of CCM is formed endogenously, most likely as a result of metabolism of aromatic amino acids in P. putida. However, here we wish to note that the pheBA promoter was still about 100-fold inducible in PaWpheBA-lux-catB : : tet (Fig. 4b).
As shown in Fig. 4(b), transcription from the pheBA promoter was rapidly induced in the catB-deficient mutant strain in the presence of phenol, even when amino acids were added into the growth medium. The level of transcription observed in this strain in the presence of amino acids exceeded up to ten times the pheBA promoter activity measured in the strain overexpressing the CatR protein. Additionally, our results indicate that CCM almost always limits the transcription from the CatR-regulated promoters: even if bacteria were grown in a nutritionally poor environment, transcription from the pheBA promoter was more than fivefold higher with excess of CCM compared with that measured in the wild-type strain (compare Fig. 3a and Fig 4b). Taken together, the results of the experiments with the CatB-defective strain indicate that the intracellular amounts of CCM primarily limit the level of transcription from the pheBA promoter. In the light of these data, the positive effect of CatR overproduction on the pheBA promoter activation might be interpreted as follows: if there is more CatR protein, then a limiting number of CCM molecules available at an early stage of induction of the phenol degradation pathway are more likely bound by CatR to activate transcription.
We also tried to increase the intracellular amounts of CCM by overproduction of the enzymes PheA and PheB, required for the degradation of phenol to CCM. Indeed, artificial overexpression of these enzymes on the medium-copy plasmid pAT1142 carrying the pheBA operon enabled rapid activation of the pheBA promoter in both the presence and absence of amino acids in the growth medium. The overexpression of these enzymes was verified by Western blot analysis (data not shown). Overproduction of PheA and PheB almost completely relieved the repressive effect of the presence of amino acids (Fig. 4c). However, the plasmid pAT1142-harbouring cells always exhibited a lower level of expression of the pheBA promoter compared with the level that was detected when bacteria carried the pheBA operon as a single copy in the bacterial chromosome. Plasmid pAT1142 is an RSF1010 derivative which has more than 10 copies per cell. As already discussed above, the cellular amount of CatR is always below the level which allows maximal level of transcription from its target promoters. Therefore, we suppose that the presence of multiple binding sites for CatR in cells carrying the plasmid pAT1142 could titrate out this activator protein, resulting in a reduced level of expression of the chromosomal Pi–luxAB reporter system used to measure the pheBA promoter activity. Indeed, this reporter system expressed much higher luciferase activity when the catR gene was also present in the pheBA operon-carrying medium-copy plasmid (data not shown). However, the results of the overproduction of PheA and PheB also implied that the intracellular amount of CCM, but not CatR, is the most limiting factor determining the rate of transcription from the pheBA promoter, when bacteria are grown in the presence of amino acids. At the same time, we cannot exclude the possibility that amino acids reduce catR expression as well.
The importance of intracellular CCM concentration for the regulation of expression of catabolic operons is also seen in Acinetobacter baylyi ADP1 (Collier et al., 1998; Cosper et al., 2000; Ezezika et al., 2006). At the same time, the accumulation of high levels of CCM is toxic to the cells (Gaines et al., 1996). A complex regulatory circuit involving two LysR-family transcriptional regulators BenM and CatM has evolved to allow optimal expression of benzoate and catechol degradation genes, while at the same time keeping the concentration of potentially harmful CCM sufficiently low (Ezezika et al., 2006).
Repression of transcription from the pheBA promoter in the presence of amino acids is caused by a mechanism affecting expression of the pheBA operon at the post-transcriptional level
There are several possible mechanisms which may reduce intracellular levels of CCM in the presence of amino acids: the intracellular amount of precursor molecule phenol may be too low, CCM is degraded fast, the enzymes for CCM production are inhibited or the levels of enzymes needed for the production of CCM are reduced. Here, we decided to compare the intracellular amounts of PheA and PheB under different nutritional conditions (i.e. in the presence or absence of amino acids). In order to separate the possible effects of growth medium composition on the cellular amounts of the PheA and PheB proteins from those which directly influence transcription initiation of the corresponding genes, we quantified these enzymes by Western blot analysis with anti-PheB and anti-PheA polyclonal antibodies by examining cell lysates prepared from such amounts of bacteria which gave the same value of transcription from the pheBA promoter. More precisely, the level of transcription of the pheBA genes was monitored by luciferase activity as described above, and the samples of bacteria taken for quantification of PheA and PheB proteins expressed approximately 10 000 RLU/OD580 luciferase activities per 10 s. The catR-overexpressing strain PaWpheBA-luxAB-catR was used in these assays to obtain high levels of transcription from the pheBA promoter in cells growing in the presence of amino acids. As shown in Fig. 5(a), despite the fact that bacteria derived from different growth conditions expressed comparable luciferase activities (which could reflect equal transcription of the pheBA genes from their promoter), the cellular amounts of the PheA and PheB proteins were remarkably affected by the growth medium composition. About four- and eight-fold lower amounts of the PheA and PheB proteins, respectively, were detected if bacteria were grown in the presence of amino acids (Fig. 5a, compare lanes 1 and 2). Thus, our results indicated that the cellular amounts of PheA and PheB enzymes are negatively controlled by amino acids at the post-transcriptional level of expression of the corresponding genes.
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revealed the presence of three protein bands which reacted with PheA-specific antibodies, two of which migrated slower than the predicted pheA gene product. We did not detect these bands when cell lysate prepared from P. putida lacking the pheBA operon was analysed (Fig. 5a, lanes 5 and 6). These data exclude the possibility that some bands are derived from reaction of these antibodies with other proteins. The presence of three protein bands reacting with PheA-specific antibodies also appeared in the case of Western blot analysis of cell lysates prepared from E. coli cells expressing the pheA gene alone, but not when pheA was absent (data not shown). At present, the nature of the multiple PheA-specific bands remains unclear.
Crc protein is involved in regulation of expression of PMO and C12O
The Crc protein is involved in repression of several catabolic pathways responsible for degradation of aromatic compounds or other carbon sources in Pseudomonas when other preferred carbon sources are present in the culture medium (Rojo & Dinamarca, 2004). It has been demonstrated that the expression of P. putida chromosomal genes for the homogentisate, catechol and protocatechuate pathways is inhibited by Crc when cells grow in a complete medium (Morales et al., 2004). The precise function and mechanism of Crc are still unknown, but it has been suggested that Crc modulates the expression of target genes posttranscriptionally (Hester et al., 2000; Rojo & Dinamarca, 2004). The most recently published studies suggest that Crc acts through generating a metabolic signal (Aranda-Olmedo et al., 2005; Morales et al., 2004).
Comparison of the results of the Western blot analysis of the PheA and PheB enzymes in P. putida wild-type strain and its Crc-defective derivative grown in the presence or absence of amino acids revealed that, in contrast to the differences in cellular amounts of the phenol degradation enzymes in the wild-type strain (Fig. 5a, lanes 1 and 2), the abundance of these proteins was not affected by the growth medium composition in the Crc mutant strain (Fig. 5a, lanes 3 and 4). These data indicate that Crc may negatively affect the expression of the pheBA operon at the post-transcriptional level in the presence of amino acids. To verify this hypothesis, we performed an additional control experiment by monitoring intracellular amounts of PheB in wild-type and Crc-defective bacteria expressing the pheB gene under the control of the IPTG-inducible Ptac promoter, which is not dependent on levels of CCM. The results of the Western blot analysis presented in Fig. 5(b) revealed lower amounts of PheB in Crc-proficient cells grown in amino acid-containing medium compared with the minimal-medium-grown bacteria. At the same time, no negative effect of the amino acids on the levels of PheB could be detected in bacteria lacking the Crc protein, which is in accordance with the results presented in Fig. 5(a).
Based on the results of the Western blot analysis indicating that Crc controls negatively the intracellular amounts of the PheA and PheB proteins in bacteria growing in the presence of amino acids, we expected to see that transcription from the pheBA promoter was elevated in the Crc-deficient background. Indeed, data presented in Fig. 4(d) revealed remarkable differences between the level of transcription from the pheBA promoter in the wild-type and Crc-deficient P. putida strains grown in the presence of the amino acids solution (compare Fig. 3a and Fig. 4d). The most remarkable effects of Crc appeared in the presence of 0.05–0.5 mM phenol in the amino acid-containing growth medium. Whereas the transcription from the pheBA promoter occurred at a very low level under such growth conditions in the wild-type strain, an up to 150 times higher level of transcription was detected in the Crc-defective strain PaWpheBA-lux-crc : : tet. As lower intracellular amounts of phenol degradation enzymes in Crc-proficient background lead to decreased production of the pathway inducer CCM, this might be a mechanism by which this protein indirectly interferes with the transcription of the pheBA genes.
The lack of induction of the phenol degradation pathway in the presence of amino acids cannot be explained only by the action of the Crc protein, as the expression level of the promoter of the pheBA genes was still about four times lower in rich growth medium, compared with that when bacteria were grown in minimal medium (Fig. 4d). This indicates that some additional control mechanisms operating through transcriptional activation of the pheBA promoter may also exist. As a transfer of bacteria from minimal to rich growth medium recruits transcription machinery to promoters whose expression is greatly needed with an excess of nutrients (e.g. stable RNA promoters), it is possible that some other promoters, like the pheBA promoter, are less efficiently transcribed due to competition for free RNA polymerase.
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ACKNOWLEDGEMENTS |
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Edited by: M. A. Kertesz
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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