Laboratory of Microbiology, The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako, Saitama 351-0198, Japan1
Author for correspondence: Toshiaki Kudo. Tel: +81 48 467 9544. Fax: +81 48 462 4672. e-mail: tkudo{at}postman.riken.go.jp
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ABSTRACT |
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Keywords: phenol, meta-pathway, biodegradation, Comamonas testosteroni
Abbreviations: 3HPP, 3-(3-hydroxyphenyl)propionate; 4OD, 4-oxalocrotonate decarboxylase; 4OI, 4-oxalocrotonate isomerase; ADA, acetaldehyde dehydrogenase (acylating); C23O, catechol 2,3-dioxygenase; CFE, cell-free extract; HMS, 2-hydroxymuconic semialdehyde; HMSD, HMS dehydrogenase; HMSH, HMS hydrolase; HOA, 4-hydroxy-2-oxovalerate aldolase; OEH, 2-oxopent-4-dienoate hydratase; pHB, p-hydroxybenzoate; PH, phenol hydroxylase
The DDBJ/EMBL/GenBank accession number for the sequence reported in this paper is AB029044.
a Present address: Department of Biotechnology, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan.
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INTRODUCTION |
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METHODS |
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Cloning of the region downstream of the aph gene cluster.
Because pYM50 did not contain the complete aph gene cluster, a 7 kb HindIII fragment (pYM70) containing the downstream region was cloned from total DNA of strain TA441 using a digoxigenin-labelled 0·6 kb HindIIIEcoRI fragment of pYM50 as a probe (Fig. 1).
Construction of mutant strains.
The mutant strains PDT, PDC, PDI, PDJI and PDHJ were constructed by insertion of a kanamycin-resistance (Kmr) gene into the chromosome of strain P1 according to the method described previously (Arai et al., 1998 ) using pDT1, pDC1, pDI1, pDI2 and pDH1, respectively (Fig. 1
). Strain TDT was constructed from strain TA441 using pDT1. The plasmids were constructed by subcloning of the indicated fragment into pUC19 and insertion of a blunt-ended 1·5 kb HindIIISalI fragment of pSUP5011 which contained the Kmr gene (Simon, 1984
) (Fig. 1
). Insertion of the Kmr gene into the chromosome of the mutant strains was confirmed by PCR (data not shown).
Preparation of cell-free extracts.
For preparation of cell-free extract (CFE), strains P1 and PDT were cultivated overnight. The cells were harvested by centrifugation, washed with 50 mM potassium phosphate buffer (pH 7·5), resuspended in the same buffer and disrupted by sonication. The cell debris was removed by centrifugation and the supernatant was used as CFE. Protein concentration was determined by the method of Bradford (1976) using the Bio-Rad protein assay kit, with bovine serum albumin as a standard.
Enzyme assays.
All enzyme assays were performed at 30 °C. HMSD and HMSH activities were determined by measuring the decrease in HMS. HMS was prepared from catechol using E. coli JM109 (pUC18xylE) cells in 50 mM potassium phosphate buffer (pH 7·5). After the ring-fission reaction was completed, the E. coli cells were removed by filtration, CFE of C. testosteroni strains was added and the decrease in A375 was monitored. NAD+ was added to 330 mM to the reaction mixture for the HMSD assay. One unit of enzymic activity is defined as the amount of enzyme required to consume 1 µmol HMS min-1. The molar extinction coefficient value for HMS at 375 nm was 36000 M-1 cm-1.
4-Oxalocrotonate for the enzymatic activity of 4OI and 4OD was produced from catechol as follows. HMS solution was prepared according to the method described above and diluted with 50 mM potassium phosphate buffer (pH 7·5) to an A375 of 1·5. HMS in the solution was converted to 4-oxalocrotonate by adding NAD+ (330 mM) and CFE of E. coli JM109 harbouring pUC18aphC, which was designed to express HMSD (the gene product of aphC). For the 4OI assay, CFE of C. testosteroni strains was added to the reaction mixture just after the decrease in A375 and the increase in A295 had stopped, and the activity was determined by measuring the initial rate of decrease in A295 due to the disappearance of the enol form of 4-oxalocrotonate. For the 4OD assay, MgSO4 (3·3 mM) and CFE of C. testosteroni strains were added after the reaction mixture reached an equilibrium state in terms of the levels of the keto and enol forms of 4-oxalocrotonate. The activity was determined by measuring the initial rate of decrease in A235 due to the disappearance of the keto form of 4-oxalocrotonate. One unit of activity is defined as the amount of enzyme required to convert 1 µmol substrate min-1. The molar extinction coefficient values for the keto form and the enol form of 4-oxalocrotonate were determined using the equations described by Harayama et al. (1989) . pUC18aphC was constructed as follows. A 2·5 kb fragment containing the aphC gene was amplified by PCR from a colony of strain TA441 with oligonucleotides Pri17 (5'-GGCGCGGAATTCACGTCCAGCACGCC-3') and Pri20 (5'-GTGTCCTGGATCCTGATCTTCCAGTC-3'), which were designed to introduce EcoRI and BamHI sites, respectively. The amplified fragment was digested with EcoRI and BamHI, and inserted into pUC18 between the EcoRI and BamHI sites in the plasmid.
The enzymic assay of OEH, HOA and ADA was performed according to the methods described by Shingler et al. (1992) . The activity of ß-galactosidase, the lacZ gene product, was measured by the protocol described by Miller (1992)
. The aphC::lacZ fusion plasmid pHAW943 was constructed by insertion of a 1·3 kb EcoRIBamHI fragment which contained the aphC promoter region into pRW2 between the EcoRI and BamHI sites in the plasmid (Lodge et al., 1990
). The fragment was prepared by PCR amplification with oligonucleotides Pri39 (5'-GGCCGGAATTCCACGCCGGGCAGGCG-3') and Pri18 (5'-CCGTAGTGGATCCCACCACGCCCGTC-3'), which were designed to introduce EcoRI and BamHI sites, respectively.
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RESULTS AND DISCUSSION |
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The genes for the 4-oxalocrotonate branch enzymes 4OD (aphH) and 4OI (aphI) followed aphG. An open reading frame of unknown function (aphJ) was located between aphH and aphI. aphJ encodes a protein consisting of 326 amino acid residues. The translated sequence of aphJ has a typical N-terminal signal sequence for membrane translocation, suggesting that the aphJ gene product is located in the periplasm. Strain TA441 has two other aphJ-like open reading frames (orf4 and orf5) in the mhp gene clusters for degradation of 3HPP (Arai et al., 1999b ). orf4 encodes a product with an N-terminal signal sequence, whereas orf5 does not encode a signal sequence. orf4 and orf5 are not necessary for utilization of 3HPP and their roles are still unclear (Arai et al., 1999b
). An aphJ-like gene has been found also in the gene cluster for the chlorocatechol ortho-degradation pathway in Pseudomonas sp. P51 (ORF3 of the tcb gene cluster) (van der Meer et al., 1991a
), and in the catabolic plasmid pAC27 (ORF3 of the clc gene cluster) (Frantz & Chakrabarty, 1987
), and in the gene cluster for biosynthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in Alcaligenes eutrophus H16 (ORF7) (Valentin et al., 1995
). ORF3 of the tcb gene cluster and ORF7 in A. eutrophus H16 encode putative proteins with N-terminal signal sequences, whereas ORF3 in the clc gene cluster does not encode a signal sequence. Similar genes have also been found in the gene clusters for metabolism of tartrate and urea (McMillan et al., 1998
; Salomone et al., 1996
). However, the roles of these genes have not yet been identified.
The gene for HMSH corresponding to dmpD of strain CF600 was not found in the aph gene cluster of strain TA441. Enzymic conversion of HMS by cells of this strain adapted to growth on phenol was completely dependent on NAD+, indicating that strain TA441 does not have the NAD+-independent HMSH and has only the NAD+-dependent HMSD (Table 3). The hydrolytic branch involving the step catalysed by HMSH is required for degradation of 2- and/or 3-methylated phenols (Shingler, 1996
). Strain P1 could not utilize methylphenols, confirming the deficiency in HMSH (data not shown). The lack of the hydrolytic branch was also confirmed by results indicating that 4-oxalocrotonate branch mutants, strain PDC (aphC-) and strain PDHJ (aphH-, aphJ-), were not able to grow through utilization of phenol (Fig. 4
). The 4OI mutant strains PDI (aphI-) and PDJI (aphJ-, aphI-) were able to grow on phenol although growth was delayed compared to that of the parent strain P1 (Fig. 4
). Our findings demonstrate that aphJ is not necessary for utilization of phenol; strains PDI and PDJI showed similar growth profiles (Fig. 4
) and strain PDHJ was able to grow on phenol when transformed with the aphH gene (data not shown). 4OI encoded by aphI catalyses the isomerization of the enol form of 4-oxalocrotonate to the keto form. The phenol-grown cells of strain PDI did not display 4OI enzymic activity (data not shown). The growth of strains PDI and PDJI was attributable to the spontaneous isomerization of 4-oxalocrotonate. This result differs from the cases of strain CF600 and P. putida U, in which the 4OI gene is essential for growth on phenol (Powlowski & Shingler, 1994
; Wigmore et al., 1974
).
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Strain PDT was able to grow on phenol, indicating that aphT is not necessary for degradation of phenol (Fig. 4). In strain P1, the activity of each of the meta-pathway enzymes except HOA was induced when phenol was being used as a carbon source, indicating that the expression of these enzymes is under the control of a regulatory system responsive to phenol or its metabolites. The HOA activity was expressed even when the strain was grown on succinate, and the activity was highest when pHB was being used as a carbon source. The HOA activity in cells grown on pHB or succinate was probably attributable to the expression of isofunctional enzymes such as MhpE and TIP4, derived from genes other than aphG. The levels of HMSD, 4OI, 4OD, OEH and ADA activity in strain PDT were lower than those in strain P1 when phenol was added, suggesting that AphT may be involved in the induction of these enzymes. However, the role of the aphT gene in the phenol-grown cells was not so important because induction of the enzymes by phenol was still operative and growth on phenol was not affected by mutation in aphT. The levels of HMSD, 4OI, 4OD, OEH and ADA activity in strain PDT grown in the presence of pHB or succinate were higher than those in strain P1. This was probably because of read-through from the Kmr gene that was inserted in the aphT gene.
Dual regulation of the aphC promoter by AphR and AphT
The transcriptional activity of the aphC promoter was assessed by monitoring the expression of ß-galactosidase from the lacZ-fusion plasmid pHAW943 (Fig. 5). Interestingly, strains TA441 and P1 showed quite different induction patterns. The aphC promoter activity was significantly increased in strain TA441 upon incubation with HMS (Fig. 5a
). The promoter activity in strain P1 was lower than that in strain TA441 for some unknown reason. The induction by HMS was not observed in the aphT-deficient strains TDT and PDT, indicating that the HMS-mediated activation of the aphC promoter was under the control of AphT. The activity in strain PR921, an aphR derivative of strain P1 (Arai et al., 1998
), was comparable to that in strain P1, indicating that AphR is not involved in the activation by HMS. The compound recognized as a sensing signal by AphT is probably HMS, because the activity in strain PDC, in which HMS was not further metabolized, was also comparable to that in strain P1.
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The results of the present study indicate that the aphC promoter is subject to dual regulation by AphR and AphT. Phenol-sensing regulation by AphR is dominant in the adapted strain P1. On the contrary, in strain TA441, HMS-sensing regulation by AphT occurs exclusively because the expression of AphR is repressed by AphS. Interestingly, the aphC promoter was not silenced by AphS as in the case of the upstream aphR promoter, suggesting that strain TA441 could degrade HMS not derived from phenol.
The distance between aphG and aphH is a relatively long stretch (697 bp), but no transcriptional activity was detected in the aphGaphH intervening region (data not shown). Because the downstream meta-pathway enzymes except HOA were found to be similarly regulated (Table 3), the aphCEFGHJI genes might be transcribed as an operon. Our preliminary data showed that AphT binds to the aphGaphH intervening region. 4OI and 4OD, encoded by aphI and aphH, catalyse the isomerization and decarboxylation of 4-oxalocrotonate, respectively (Fig. 2
). The structure of the enol form of 4-oxalocrotonate, the substrate of 4OI, is very similar to that of 2-chloro-cis,cis-muconate (Fig. 3
). Considering that AphT is phylogenetically close to regulators known to recognize 2-chloro-cis,cis-muconate, AphT might recognize 4-oxalocrotonate. There is a possibility that AphT is involved in 4-oxalocrotonate-sensing fine regulation of the aphHJI subcluster in addition to playing a role in HMS-sensing activation of the aphC promoter.
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ACKNOWLEDGEMENTS |
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Received 11 January 2000;
revised 27 March 2000;
accepted 4 April 2000.