Bacteriology Group, International Centre for Genetic Engineering and Biotechnology, Area Science Park, Padriciano 99, 34012 Trieste, Italy1
Author for correspondence: Vittorio Venturi. Tel: +39 040 3757317. Fax: +39 040 226555. e-mail: venturi{at}icgeb.trieste.it
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ABSTRACT |
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Keywords: protocatechuic acid, pobA, pobC, aromatic acid
Abbreviations: PCA, protocatechuic acid; PHB, p-hydroxybenzoic acid
The GenBank/EMBL/DDBJ accession numbers for the pcaR, pobC-pobA and pcaHG sequences reported in this paper are AJ252090, AJ251792 and AJ295623, respectively.
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INTRODUCTION |
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In bacteria, diverse aromatic compounds are initially transformed to a limited number of central intermediates, namely catechol (or substituted derivatives) and PCA. These compounds are then channelled into two possible ring fission pathways, either the ortho- or meta-cleavage pathway, funnelling these compounds into the tricarboxylic acid cycle (van der Meer et al., 1992 ). In Gram-negative bacteria, ferulic acid is initially degraded to PCA via vanillic acid, whereas coumaric acid is degraded via PHB (Toms & Wood, 1970
; Venturi et al., 1998
) (Fig. 1
). These catabolic conversion steps require at least three genetic loci (Fig. 1
). The transformation of ferulic acid to vanillic acid involves an operon encoding an enoyl-CoA hydratase/aldolase, a vanillin dehydrogenase and another gene encoding feruloyl coenzyme A synthetase (Overhage et al., 1999b
; Priefert et al., 1997
; Venturi et a
l., 1998
). Vanillic acid is then degraded to PCA by a demethylase encoded by an operon consisting of two genes designated vanA and vanB (Brunel & Davison, 1988
; Priefert et al., 1997
; Venturi et al., 1998
). The degradation of p-coumaric acid to PHB also requires at least one locus that transforms ferulic acid to vanillic acid (Venturi et al., 1998
), whereas the conversion of PHB to PCA requires a hydroxylase encoded by the pobA gene (Wong et al., 1994
; Parke, 1996
; this study). All three loci involved in these conversion steps have been cloned and characterized in plant-growth-promoting P. putida WCS358 (Venturi et al., 1998
; this study).
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The regulation of pobA expression by PobR has been extensively studied in Acinetobacter sp. ADP1 at the genetic and molecular level, and the PHB response and the DNA target have been investigated (DiMarco & Ornston, 1994 ; Kok et al., 1997
). In this study we present genetic data on the regulation of pobA expression in plant-growth-promoting P. putida WCS358. The pobA-pobC locus has been identified. The genetically linked regulator exhibited low similarity to the other studied PobR regulators and belonged to the AraC family of regulators, hence it was designated PobC. PobC activated pobA expression in response to PHB. Interestingly, it also had weak activity in the presence of PCA. Finally, it was observed that the regulator PcaR was important for efficient activation of pobA expression and it was postulated that this effect was mediated via the expression of pcaK, the gene encoding a PHB transporter protein.
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METHODS |
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Reporter gene fusion assays and analysis of substrate metabolism.
ß-Galactosidase activity was determined essentially as described by Miller (1972) with the modifications of Stachel et al. (1985)
. The biodegradation of aromatic acids by P. putida WCS358 and Tn5 mutants was analysed by reverse-phase HPLC (RP-HPLC), using a Varian 9010 solvent delivery system equipped with a Varian 9050 UV/vis detector. Pseudomonas was grown either in LB medium supplemented with 0·15% (w/v) of the aromatic acid, or in M9 minimal medium supplemented with 0·1% and/or 0·05% of the aromatic acid. Samples were withdrawn from cultures after growth and centrifuged at 12000 g. The supernatant was diluted 100-fold into methanol and filtered through 0·2 µm filters; 10 µl samples were loaded on a 5 µm spherical C18 reverse-phase column (Supelcosil LC18 150x4·6 mm; Supelco) and eluted with 35% methanol and 65% water with 0·1% acetic acid at a flow rate of 0·8 ml min-1. The eluted metabolites were detected at 279 nm. The catabolic intermediates were identified by comparing their elution times with those of pure standards.
Isolation of mutants unable to use PHB as carbon source and their complementation with a P. putida WCS358 gene bank.
Mutants unable to use PHB as carbon source but retaining the ability to use PCA, were screened as follows. Colonies from a Tn5 genomic mutant bank (Marugg et al., 1985 ) were grown at 30 °C in duplicate on minimal M9 plates containing kanamycin and either PHB or PCA as sole carbon source. Mutants were selected which were unable to grow on PHB but retained the ability to grow on PCA. Complementation was performed via triparental matings between a cosmid gene library of P. putida WCS358, an E. coli(pRK2013) mobilizer (about 4x109 cells each) and the WCS358 Tn5 mutant (2x108 cells) were set up on a 0·45 µm membrane filter (Millipore) on an LB plate. After 12 h incubation at 30 °C, cells were resuspended and plated on minimal M9 plates containing PHB (0·15%) with tetracycline.
Cloning of the pcaHG and pcaR loci of P. putida WCS358.
The pcaHG genes of strain WCS358 were cloned using the pcaH gene from P. aeruginosa PAO1 as probe against the cosmid gene library. A 736 bp EcoRIBamHI fragment from pPAOH1 was used as probe and a cosmid designated pCOSHG1 was identified that harboured the pcaHG locus on a 5 kb HindIII fragment. The genes were then further localized on a 1·8 kb EcoRIHindIII subfragment. This fragment was cloned in pBluescript to yield pBGH18. The 5 kb HindIII fragment containing the pcaHG locus was cloned in pLAFR3 to yield pCOSHG5.
The pcaR gene of P. putida WCS358 was also cloned using the pcaR gene of P. aeruginosa PAO1 as probe to screen a cosmid bank of P. putida WCS358. An 875 bp EcoRIHindIII fragment of pPAOPCAR was used as probe and a cosmid, designated pCOS272, was identified which harboured the pcaR gene on a 6·5 kb HindIII fragment. This fragment was cloned in pBluescript KS to yield p2726.5. The pcaR gene was further localized in a 2·2 KpnI subfragment (Fig. 2b) which was also cloned in pBluescript KS to yield pBKP2.2.
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E. coli HB101 cells containing Tn5 insertions within the plasmid p2726.5 were identified by purifying plasmid DNA from HB101::Tn5 (p2726.5) which was subsequently used to transform E. coli DH5. Transformants were selected for ampicillin and kanamycin resistance. Transposon insertions in the pcaR gene were then mapped by restriction and Southern analyses and by DNA sequencing. One Tn5 insertion in the pcaR gene in p2726.5 was identified (plasmid designated p27266.5::Tn5) and used in a marker exchange experiment to transfer this Tn5 insertion into the pcaR gene harboured in pCOS272, yielding pCOS272::Tn5. Tn5 was located at position 604 of the sequence (accession no. AJ252090) where position 135 is the ATG of pcaR.
Site-specific exchange of mutations with the Pseudomonas chromosome.
Plasmids pCOS272::Tn5 and pCOSHG5::Tn5 carrying a Tn5 insertion in the pcaR and pcaH genes, respectively, were homogenized with the corresponding target region of the genome of P. putida WCS358 by a marker exchange procedure described by Corbin et al. (1982) and Venturi et al. (1998)
. pPH1JI was used as the incoming IncP1 incompatible plasmid and selections were made on LB plates containing nalidixic acid, ampicillin, gentamicin and kanamycin. Putative marker-exchanged mutants were streaked on LB plates containing tetracycline to confirm loss of IncP1 recombinant plasmids. By this method genomic Tn5 mutants were generated, one called P. putida VBPC, harbouring a Tn5 insertion in pcaR, and another designated VBHG, harbouring a Tn5 insertion in the pcaH gene of P. putida WCS358. The fidelity of each marker exchange event was confirmed by purifying chromosomal DNA of the mutants, which were analysed by digestion with restriction enzymes and via Southern hybridization, verifying that pcaR and pcaH, respectively, were mutated in the chromosome.
DNA sequence determination and analysis.
A 2·2 kb KpnI fragment harbouring pcaR (pBKP2.2), a 4·7 kb KpnINotI fragment harbouring pobA-pobC (pB4.7KN) and a 1·8 kb EcoRIHindIII fragment harbouring pcaHG (pBGH18) were subcloned from pCOS272, pCOSHB and pCOSHG5, respectively, and utilized for sequencing. The constructs were either encapsidated as single-stranded DNA upon infection with helper phage VCSM13 (Stratagene) or used directly for DNA sequencing. Several oligonucleotides were synthesized and used in sequencing reactions. Nucleotide sequences were determined by the dideoxy chain-termination method (Sanger et al., 1977 ) using [
-35S]dATP for labelling and 7-deaza-dGTP (Pharmacia) instead of dGTP. The nucleotide sequence was determined in both directions and across all restriction sites.
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RESULTS |
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Gene promoter activity of the pobA promoter
The promoter of pobA was cloned upstream from a promoterless ß-galactosidase (lacZ) gene in the promoter probe vector pMP220 as described in Methods. The transcriptional fusion, called pPPOBA, was conjugated into P. putida WCS358 and mutant derivatives and ß-galactosidase activities were determined in response to several phenolic acids (Fig. 3).
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Phenotype of pobC::Tn5 genomic mutants
As mentioned above, these mutants could not grow on PHB as sole carbon source but could grow on PCA, thus they were impaired only in the hydroxylation step. Gene promoter studies in the pobC::Tn5 mutant VBHB are presented in Fig. 3. They showed that the pobA promoter, harboured in pPPOBA, in the parent strain was very strong and inducible by PHB, reaching a value of approximately 30000 Miller units; this activity was not observed in the pobC mutant VBHB (Fig. 3a
). As mentioned above, activation by PCA occurred to a lesser extent in P. putida WCS358(pPPOBA) (1100 Miller units). This activity was not observed in the pobC::Tn5 mutant (Fig. 3b
), demonstrating that this PCA induction was occurring via PobC.
PCA weakly induces pobA gene promoter activity
It was observed that PCA weakly but significantly induces pobA promoter activity (see above). It was possible, however, that this induction was occurring via another compound resulting from the stepwise transformation of PCA in the ß-ketoadipate pathway (Nichols & Harwood, 1995 ; Romero-Steiner et al., 1994
). To establish that PCA was responsible for the observed pobA promoter activity we determined ß-galactosidase levels in a P. putida WCS358 genomic mutant which was blocked in the first transformation step of PCA catabolism. This mutant was constructed via the cloning and characterization of the pcaHG locus, performing transposon mutagenesis on the cloned locus and generating a genomic mutant via a marker exchange technique (see Methods). This led to the construction of a mutant designated P. putida VBHG exhibiting a Tn5 insertion in the pcaH gene in the chromosome of P. putida WCS358. The pcaHG operon encodes two proteins (Fig. 2c
) (accession no. AJ295623): PcaH (26·7 kDa), the ß-subunit, and PcaG (22·4 kDa), the
-subunit of protocatechuate 3,4-dioxygenase which transforms PCA to carboxymuconate (Nichols & Harwood, 1995
; Romero-Steiner et al., 1994
). As expected, these two proteins have very high identities (approx. 80%) with homologues identified in other Pseudomonas spp. (data not shown; Overhage et al., 1999a
). It was verified that the P. putida VBHG mutant was unable to transform PCA since when present in the medium after overnight growth, the compound was not transformed. In addition this mutant could not grow in minimal medium when PCA was sole carbon source. This mutant could be complemented for these two phenotypes by pCOSHG5, carrying a 5 kb HindIII fragment with the pcaHG genes.
We determined pobA promoter activity in mutant P. putida VBHG(pPPOBA) in the presence of PCA and it was observed that in this mutant the weak but significant response to PCA was retained (Fig. 3b). Thus, it was concluded that it was PCA that could act as a weak inducer of pobA gene expression via PobC.
Expression of pobA is influenced by PHB concentration
The PHB-induced activity of the pobA promoter via PobC was determined. pobA promoter activity was determined in P. putida WCS358(pPPOBA) grown in the presence of various concentrations of PHB through ß-galactosidase assays. As shown in Fig. 4, it was established that pobA gene promoter activity varied according to the amount of PHB present: the activity rose with increasing concentrations of the phenolic compound and the highest level was achieved at 0·05%. At higher concentrations the activity began to decrease and at approximately 0·15% there was a sharp drop in ß-galactosidase activity, most probably because higher concentrations of the phenolic compound were bacteriostatic.
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To investigate the possible role of PcaR in pobA gene regulation, promoter fusion pPPOBA was conjugated in mutant VBPC and activation by PHB was determined. In mutant VBPC the promoter had only 15% of promoter activity as compared to the wild-type (Fig. 3a) and it was concluded that pcaR played an important role in pobA gene expression. This observed effect was probably indirect via the regulation of other gene(s) important for PHB degradation. It was previously observed that in P. putida, pcaR regulates the PHB transporter gene pcaK (Nichols & Harwood, 1995
). We observed that the same was true in strain WCS358. The pcaK gene was genetically linked to pcaR (Fig. 2b
) and contained a putative pcaR binding region in its promoter. The pcaK promoter of strain WCS358 was also cloned upstream from a promoterless ß-galactosidase gene in construct pPPCAK and this showed that in strain WCS358 this promoter was activated in the presence of PCA; this activity disappeared in the pcaR mutant VBPC (Fig. 3c
). It was concluded that pcaR regulated the expression of pcaK; thus in a pcaR mutant there is less efficient transport of PHB, most probably leading to a less efficient pobA activation in response to PHB.
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DISCUSSION |
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The pobA promoter was regulated by PobC and it was activated very efficiently by PHB; the levels of ß-galactosidase activity obtained using the pobA promoter lacZ fusion indicated that this promoter is very strong, considerably stronger than the pobA promoter of Acinetobacter sp. ADP1 (DiMarco et al., 1993 ). A weak but significant level of pobA promoter activity was also observed in response to PCA and this was confirmed to be most probably due to a PobC-PCA interaction since in pobC::Tn5 mutants (VBHB) this protocatechuate-dependent activity was not observed (Fig. 3b
). It was verified that it was PCA which was responsible for this weak activation and not another intermediate in its catabolism since in a pcaH::Tn5 mutant, which cannot further catabolize PCA, this activation was not significantly altered. To our knowledge this is the first pobA regulator reported to have a response activity on PCA. The co-inducer response domain of the P. putida WCS358 PobC, which recognizes PHB, probably also responds to protocatechuate which is very similar in structure, the only difference being a hydroxyl group at the meta position. This property could be helpful in future work designed to precisely define the inducer response domain via mutations which alter the response to a new effector (e.g. PCA).
The PcaR protein has been identified and characterized in P. putida PRS2000 as a regulator of several loci of the ortho cleavage pathway, e.g. pcaBDC, pcaIJ, pcaF and pcaK (Nichols & Harwood, 1995 ; Romero-Steiner et al., 1994
). The PcaR of P. putida WCS358 had 95% identity with PcaR of strain PRS2000 and the pcaR::Tn5 mutant of strain WCS358, VBPC, could no longer utilize all of the aromatic compounds shown in Fig. 1
, thus it is very likely that in strain WCS358 PcaR regulates the same loci as in strain PRS2000. In strain WCS358 pcaR is genetically linked to pcaK, just like in strain PRS2000, the two genes most likely being independently transcribed. Promoter studies revealed that in the pcaR::Tn5 mutant there is considerable reduction in pobA promoter activity, displaying approximately one-sixth of the activity (Fig. 3a
). This effect of PcaR on pobA gene expression most likely occurs indirectly. It has been demonstrated in P. putida PRS2000 that PcaK transports p-hydroxybenzoate and that pcaK expression is controlled by PcaR (Nichols & Harwood, 1995
). In P. putida WCS358, just like in P. putida PRS2000, a pcaR-binding site has been observed in the pcaK promoter region (TGTTCGATAAACGGACAAT-247 bp-ATG; data not shown) and it was demonstrated that in pcaR::Tn5 mutants (VBPC) there was no pcaK expression (Fig. 3c
). Consequently, in pcaR::Tn5 mutants there is less efficient transport of PHB, possibly resulting in a decrease of pobA promoter activity (Fig. 3a
). In fact it was observed that pobA expression varies considerably with PHB concentration (Fig. 4
). It cannot be excluded, however, that PcaR might regulate the expression of pobC which would result in the observed decreased pobA expression in the pcaR::Tn5 mutant. We have cloned the pobC promoter upstream of a promoterless lacZ and have observed that this promoter is weak and constitutive with respect to genetic background and presence of aromatic acids (data not shown).
This study has provided data on the regulation of pobA expression in P. putida WCS358 via PobC and PcaR, with PHB and PCA being identified as effectors. Interestingly, this demonstrated that in P. putida the genetically linked regulator was different in primary structure, that it responded efficiently and strongly to PHB, weakly to PCA and that PcaR was important via pcaK gene expression for complete pobA activation (Fig. 5). Future work will more precisely define the mode of action of these regulatory responses which are summarized in Fig. 5
and the role that these genes and proteins might play in signalling gene expression in the rhizosphere in this plant-growth-promoting Pseudomonas strain.
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ACKNOWLEDGEMENTS |
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Received 30 October 2000;
revised 27 February 2001;
accepted 5 March 2001.