Arrangement and regulation of the genes for meta-pathway enzymes required for degradation of phenol in Comamonas testosteroni TA441

Hiroyuki Araia,1, Tohru Ohishi1, Mee Young Chang1 and Toshiaki Kudo1

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


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Comamonas testosteroni TA441 degrades phenol by a meta-cleavage pathway after the occurrence of a spontaneous mutation that derepresses the aphKLMNOPQB operon encoding phenol hydroxylase and catechol 2,3-dioxygenase, the enzymes for the initial two steps of the degradation pathway. A gene cluster, aphCEFGHJI, encoding the meta-pathway enzymes for degradation of 2-hydroxymuconic semialdehyde (HMS) to TCA cycle intermediates was found downstream of the aphK operon. The upstream operon and the downstream gene cluster were found to be separated by two open reading frames of unknown function and an oppositely oriented aphT gene, which is similar to regulatory genes for ortho-cleavage of catechol or chlorinated catechols. A promoter assay using an aphC::lacZ transcriptional fusion plasmid revealed that the aphC promoter activity is induced by both phenol and HMS. The phenol-dependent induction was mediated by AphR and the HMS-dependent induction was mediated by AphT. The aphC promoter in strain TA441 was not silenced, unlike the cases of the aphK and aphR promoters, and was highly induced by HMS.

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.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Comamonas testosteroni TA441 does not grow on phenol as a sole carbon source, but it becomes able to utilize phenol after incubation with phenol for a few weeks in liquid medium or for several days on agar plates (Arai et al., 1998 , 1999a ). Strain TA441 has an operon (aphKLMNOPQB) which encodes phenol hydroxylase (PH) and catechol 2,3-dioxygenase (C23O), the enzymes for the initial two steps of the phenol-degradation pathway via meta-fission, i.e. conversion of phenol to 2-hydroxymuconic semialdehyde (HMS) (Fig. 1). The gene for a putative phenol-sensing activator, aphR, is located near aphK, in the opposite orientation. However, the genes are silent until adaptation occurs (Arai et al., 1998 ). This adaptation occurs through a mutation in the aphS gene, which is located downstream of aphR and which encodes a protein of the GntR family of transcriptional regulators. The aphS product binds to the intervening promoter region between aphK and aphR, and represses the expression of the aphK and aphR promoters (Arai et al., 1999a ).



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Fig. 1. Physical map of the aph gene cluster from C. testosteroni TA441. Boxes indicate the size and direction of transcription of the aph genes. Black boxes indicate regulatory genes. Upper bars labelled pYM50 and pYM70 indicate the fragments of chromosomal DNA of strain TA441 cloned. Lower bars indicate pUC19-derived plasmids used for construction of mutant strains by homologous recombination.

 
In Pseudomonas sp. strain CF600, a well-characterized phenol-degrading bacterium, the genes for PH and C23O are accompanied by those for the other meta-pathway enzymes, i.e. HMS dehydrogenase (HMSD), HMS hydrolase (HMSH), 2-oxopent-4-dienoate hydratase (OEH), acetaldehyde dehydrogenase (acylating) (ADA), 4-hydroxy-2-oxovalerate aldolase (HOA), 4-oxalocrotonate decarboxylase (4OD) and 4-oxalocrotonate isomerase (4OI) (Fig. 2) (Shingler et al., 1992 ). There are two routes for conversion of HMS to 2-oxopent-4-dienoate; one is the hydrolytic branch catalysed by HMSH and the other is the 4-oxalocrotonate branch catalysed by HMSD, 4OD and 4OI. The hydrolytic branch is required for degradation of 2- or 3-methylphenol and the 4-oxalocrotonate branch is required for degradation of phenol and 4-methylphenol. Because strain CF600 has both these branches, it can grow on methyl- and dimethylphenols. The genes for all meta-pathway enzymes of strain CF600 are transcribed as an extraordinarily large operon (Shingler, 1996 ). In this work, we show that the genes for the remaining steps of the complete phenol degradation pathway are clustered in a region downstream of the aphK operon in strain TA441. However, unlike the case of strain CF600, strain TA441 does not have HMSH and transcription of the downstream genes occurs in a manner independent of the upstream operon.



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Fig. 2. Phenol catabolic pathway in the adapted strains of C. testosteroni TA441. The metabolites are phenol (I), catechol (II), HMS (III), the enol form of 4-oxalocrotonate (IV), the keto form of 4-oxalocrotonate (V), 2-oxopent-4-dienoate (VI), 4-hydroxy-2-oxovalerate (VII), acetaldehyde (VIII), pyruvate (IX), acetyl CoA (X). Strain TA441 does not have the hydrolytic branch enzyme HMSH.

 

   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and growth conditions.
The bacterial strains and plasmids used in this study are listed in Table 1. C. testosteroni strains were cultivated in LB medium or C medium at 30 °C. C medium was supplemented with phenol, p-hydroxybenzoate (pHB), succinate or HMS. HMS was prepared enzymically from catechol using Escherichia coli JM109 cells harbouring pUC18–xylE; the xylE gene in this plasmid encodes C23O and is derived from the TOL plasmid (Arai et al., 1999b ). Growth was monitored by measuring the OD550. E. coli JM109 was cultivated at 37 °C. The medium composition and concentration of antibiotics have been described previously (Arai et al., 1998 ).


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Table 1. Bacterial strains and plasmids

 
Recombinant DNA techniques.
DNA manipulations were performed by standard methods (Sambrook et al., 1989 ) or as described previously (Arai et al., 1998 ). An ABI 373A DNA sequencer (Applied Biosystems) was used for sequence determination. A dye primer cycle sequence kit or a dye terminator cycle sequence kit (Perkin-Elmer) was used for dideoxy chain-termination reactions.

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 HindIII–EcoRI 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 HindIII–SalI 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 (pUC18–xylE) 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 pUC18–aphC, 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) . pUC18–aphC 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 EcoRI–BamHI 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.


   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Cloning, sequencing and characterization of the downstream aph gene cluster
In Pseudomonas CF600, the genes for phenol degradation (dmpKLMNOPQBCDEFGHI) are transcribed as an extremely large operon (Shingler, 1996 ). Expression of the operon is regulated by a phenol-sensing transcriptional activator, DmpR, which is encoded divergently from dmpK (Shingler et al., 1993 ). We have reported the genes for multi-component PH and C23O (aphKLMNOPQB), and the gene for a putative phenol-sensing regulator (aphR) in C. testosteroni TA441 (Arai et al., 1998 ). The aph genes are similar to the corresponding dmp genes in strain CF600 and the order of the genes is conserved between the two strains. The genes for degradation of HMS, which is produced from phenol through the action of PH and C23O, to TCA cycle intermediates were expected to be located in the vicinity of the PH and C23O genes in strain TA441 as is often the case with Gram-negative bacteria. We cloned and sequenced the region downstream of aphB and identified ten open reading frames (Fig. 1 and Table 2). In the case of strain CF600, the HMSD gene (dmpC) is located just downstream of the C23O gene (dmpB). However, in the case of strain TA441, the HMSD gene (aphC) was found about 2·7 kb downstream of the C23O gene (aphB). The genes aphB and aphC were found to be separated by two open reading frames of unknown function (orfX, orfY) and a putative regulatory gene (aphT). orfX encodes a protein consisting of 296 amino acid residues. The translated sequence of orfX has a typical N-terminal signal sequence for membrane translocation, suggesting that the orfX gene product is located in the periplasm. No protein that has overall similarity with the translated sequence of orfX was found in the protein databases, but a TFASTA search of DNA databases revealed that an OrfX-like protein is encoded in the region downstream of tdnC in Pseudomonas putida UCC2 (GenBank accession no. X59790) and in the region downstream of cdoE in Comamonas sp. JS765 (GenBank accession no. U93090) (Parales et al., 1997 ). tdnC and cdoE encode 3-methyl catechol 2,3-dioxygenase and C23O, respectively, and are similar to aphB. orfY encodes a relatively hydrophobic protein consisting of 150 amino acid residues. Genes similar to orfY have been found in some gene clusters encoding meta-pathway enzymes for degradation of aromatic compounds such as cbzX of P. putida GJ31, nahX of P. putida G7 and cmpX of Sphingomonas sp. HV3 (Grimm & Harwood, 1999 ; Mars et al., 1999 ; Yrjälä et al., 1997 ). A similar gene was also found in the gene cluster for glycerol metabolism in Citrobacter freundii (Daniel et al., 1995 ). The role of these orfY-like genes has not been identified. Our preliminary results showed that an orfXY mutant strain grew poorly on phenol and accumulated yellow HMS in the medium, indicating that the genes are involved in the degradation of HMS (data not shown).


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Table 2. Gene products of the downstream aph gene cluster of strain TA441 and homology with other proteins

 
aphT encodes a protein consisting of 298 amino acid residues. Analysis of the translated sequence of aphT has shown that this gene product belongs to the LysR family of transcriptional regulators. AphT shows high similarity to CdoR from Comamonas sp. JS765 (Parales et al., 1997 ). cdoR is located adjacent to cdoTE, in the opposite orientation, and the latter correspond to aphQB of strain TA441 encoding a ferredoxin-like protein and C23O. AphT is also similar to CatR-type and ClcR-type regulators, which regulate the genes for the ortho-cleavage pathway for degradation of catechol and 3-chlorocatechol, respectively (Table 2). The compounds recognized as sensing signals by CatR and ClcR are cis,cis-muconate and 2-chloro-cis,cis-muconate, the ortho-cleavage compounds produced from catechol and 3-chlorocatechol, respectively (McFall et al., 1997 ; Parsek et al., 1992 ). Phylogenetic analysis of LysR-family regulators revealed that AphT is grouped with the ClcR-type regulators (Fig. 3).



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Fig. 3. Phylogenetic tree for AphT and the LysR family of transcriptional regulators. Multiple sequence alignment was done using CLUSTAL W. Tree topology and evolutionary distance estimations were done by the neighbour-joining method (PHYLIP 3.5). The numbers indicated at the nodes are bootstrap values calculated from 100 replications using the SEQBOOT, PROTDIST, NEIGHBOR and CONSENSE programs of the PHYLIP 3.5 program package. cis,cis-Muconate and 2-chloro-cis,cis-muconate are the effectors for the CatR-type and the ClcR-type regulators, respectively.

 
The HMSD gene (aphC) was found to be followed by the genes aphEFG encoding OEH, ADA, and HOA, respectively. These three enzymes catalyse the final three steps of the meta-pathway and these reactions are common to other meta-degradation pathways for various aromatic compounds. The N-terminal sequence of the aphG gene product is highly similar (26 of 28 amino acids identical) to that of a protein (TIP4), which is induced by testosterone in C. testosteroni (ATCC 11996) (Möbus et al., 1997 ). However, aphG mutants of strains TA441 and of the adapted strain P1 were found to be capable of growth with testosterone as a carbon source (data not shown). An aphG mutant of strain P1 was also able to grow on phenol, indicating that the aphG gene is not necessary for utilization of phenol (data not shown). Strain TA441 has another set of genes (mhpDFE) encoding the enzymes for the final three steps of the meta-pathway corresponding to aphEFG (Arai et al., 1999b ). The mhpDFE genes are involved in degradation of 3-(3-hydroxyphenyl)propionate (3HPP). Probably, mhpE or another unidentified isofunctional gene, which might encode TIP4, complements the mutation in aphG.

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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Table 3. Activity of the meta-pathway enzymes

 


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Fig. 4. Growth of C. testosteroni strains on phenol. The strains were grown in 100 ml C medium supplemented with 5 mM phenol. {bullet}, P1; {blacktriangleup}, PDT; {blacksquare}, PDC; {bigtriangledown}, PDHJ; {lozenge}, PDJI; {square}, PDI.

 
Regulation of the meta-pathway enzymes
Strain TA441 could grow on phenol when the recombinant aphKLMNOPQB genes were expressed, driven by a tac promoter, indicating that transcription of the downstream meta-pathway genes is independent of the upstream aphK operon. (Arai et al., 1998 ). In the case of strain CF600, the genes for all the enzymes required for degradation of phenol are transcribed as an operon (Shingler, 1996 ). However, strain TA441 must have an additional regulatory system specific for the downstream aph gene cluster. The arrangement of the oppositely oriented aphT gene upstream of the aphC gene suggests that AphT may be concerned with regulation of transcription directed by the aphC promoter (Fig. 1). For investigation of the role of aphT, we constructed the aphT mutant strain PDT from strain P1, and compared the levels of activity of the meta-pathway enzymes in strains P1 and PDT (Table 3).

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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Fig. 5. Transcriptional activity of the aphC promoter in C. testosteroni strains. The strains were transformed with pHAW943 carrying the transcriptional fusion construct aphC::lacZ. Cultures of the strains grown overnight in C medium with pHB as a carbon source were diluted 10-fold with C medium containing 0·4 mM HMS (a) or 5 mM phenol (b). The changes in the levels of activity of ß-galactosidase, the lacZ gene product, were monitored for 8 h. The data are representative of at least two experiments. {circ}, TA441; {bullet}, P1; {triangleup}, TDT; {blacktriangleup}, PDT; {blacksquare}, PDC; {diamondsuit}, PR921.

 
The aphC promoter activity in strain P1 was significantly induced upon incubation with phenol (Fig. 5b). The induction was also observed in strain PDT although the level of expression was significantly lower than in strain P1. Induction of the downstream meta-pathway genes by phenol in strain PDT was confirmed through assay of the levels of enzyme activity (Table 3). The induction by phenol was poor in strains PR921 or TA441, in which aphR was disrupted or expression of aphR was repressed, respectively. These results indicated that the phenol-induced activation of the aphC promoter was mediated by AphR. The high activity in strain P1 was probably a synergistic effect of AphR and AphT, considering that HMS is produced during the degradation of phenol.

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 aphG–aphH 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 aphG–aphH 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.


   ACKNOWLEDGEMENTS
 
This work was partially supported by Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency of the Japanese Government and by a grant for the Eco Molecular Sciences Research Program from RIKEN.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Arai, H., Akahira, S., Ohishi, T., Maeda, M. & Kudo, T. (1998). Adaptation of Comamonas testosteroni TA441 to utilize phenol: organization and regulation of the genes involved in phenol degradation. Microbiology 144, 2895-2903.[Abstract]

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Received 11 January 2000; revised 27 March 2000; accepted 4 April 2000.