Appl Microbiol Biotechnol (2001) 56:724–730 DOI 10.1007/s002530100698

O R I G I N A L PA P E R

K. Brandt · S. Thewes · J. Overhage · H. Priefert A. Steinbüchel

Characterization of the eugenol hydroxylase genes (ehyA /ehyB) from the new eugenol-degrading Pseudomonas sp. strain OPS1 Received: 10 January 2001 / Received revision: 14 February 2001 / Accepted: 5 April 2001 / Published online: 17 July 2001 © Springer-Verlag 2001

Abstract During the screening for bacteria capable of converting eugenol to vanillin, strain OPS1 was isolated, which was identified as a new Pseudomonas species by 16 s rDNA sequence analysis. When this bacterium was grown on eugenol, the intermediates, coniferyl alcohol, ferulic acid, vanillic acid, and protocatechuic acid, were identified in the culture supernatant. The genes encoding the eugenol hydroxylase (ehyA, ehyB), which catalyzes the first step of this biotransformation, were identified in a genomic library of Pseudomonas sp. strain OPS1 by complementation of the eugenol-negative mutant SK6165 of Pseudomonas sp. strain HR199. EhyA and EhyB exhibited 57% and 85% amino acid identity to the eugenol hydroxylase subunits of Pseudomonas sp. strain HR199 and up to 34% and 54% identity to the corresponding subunits of p-cresol methylhydroxylase from P. putida. Moreover, the amino-terminal sequences of the α- and β-subunits reported recently for an eugenol dehydrogenase of P. fluorescens E118 corresponded well with the appropriate regions of EhyA and EhyB. Downstream of ehyB, an open reading frame was identified, whose deduced amino acid sequence exhibited up to 71% identity to azurins, representing most probably the gene (azu) of the physiological electron acceptor of the eugenol hydroxylase. The eugenol hydroxylase genes were amplified by PCR, cloned, and functionally expressed in Escherichia coli.

Introduction Eugenol (4-allyl-2-methoxyphenol) is the main component of the essential oil of the clove tree, Syzygium aromaticum. It is used for the production of flavor compositions for foods and fragrances for perfumes because of its oriental and spicy clove odor. Since it has an antibacK. Brandt · S. Thewes · J. Overhage · H. Priefert (✉) A. Steinbüchel Institut für Mikrobiologie der Westfälischen Wilhelms-Universität Münster, Corrensstrasse 3, 48149 Münster, Germany e-mail: [email protected] Tel.: +49-251-8339830, Fax: +49-251-8338388

terial property, it is also frequently used in dental medicine. Nevertheless, eugenol is degraded by bacteria, which was first shown for a Corynebacterium sp. by Tadasa (1977), who also proposed the formation of the epoxide eugenol oxide as the initial reaction of eugenol catabolism in a Pseudomonas sp. (Tadasa and Kayanara 1983). In contrast, a p-quinone propenide was shown to be an intermediate in the conversion of eugenol to coniferyl alcohol, catalyzed by vanillyl alcohol oxidase of Penicillium simplicissimum (Fraaije and van Berkel 1997). Recently, eugenol was discovered to be a cheap substrate for a biotransformation process to produce substituted methoxyphenols (Rabenhorst 1996) and natural vanillin (4-hydroxy-3-methoxybenzaldehyde) with Pseudomonas sp. strain HR199 (Rabenhorst and Hopp 1990; Steinbüchel et al. 1998). Investigation of this biotransformation process revealed that eugenol is converted via coniferyl alcohol (4-hydroxy-3-methoxycinnamyl alcohol), coniferyl aldehyde (4-hydroxy-3-methoxycinnamyl aldehyde), ferulate (4-hydroxy-3-methoxycinnamate), vanillin, and vanillate (4-hydroxy-3-methoxybenzoate) to the key intermediate protocatechuate (3,4-dihydroxybenzoate) (Rabenhorst 1996; Achterholt et al. 1998; Gasson et al. 1998; Overhage et al. 1999b; Priefert et al. 1999), which is further metabolized by ortho-cleavage (Overhage et al. 1999a). The initial reaction is catalyzed by an eugenol hydroxylase, which is encoded by the genes ehyA and ehyB (Priefert et al. 1999), and which belongs to the family of flavocytochromes c, as also reported recently for an eugenol dehydrogenase of P. fluorescens E118 (Furukawa et al. 1998). The strong sequence similarities to the p-cresol methylhydroxylase from P. putida, whose three-dimensional structure is known (Mathews et al. 1991), suggests analogous reaction mechanisms, including a p-quinone methide intermediate for the p-cresol methylhydroxylase and the eugenol hydroxylase. In the present study, we describe a new eugenol-degrading bacterial species, referred to as Pseudomonas sp. strain OPS1, and the molecular characterization and heterologous expression of the eugenol hydroxylase genes

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ehyA and ehyB. So far these genes, which are essential for the initial conversion of eugenol to coniferyl alcohol, have been described only once (Priefert et al. 1999). Comparative sequence analysis of the eugenol hydroxylase genes of Pseudomonas sp. strain OPS1 and Pseudomonas sp. strain HR199 and the p-cresol methylhydroxylase genes of P. putida (Kim et al. 1994; Cronin et al. 1999) suggests that eugenol hydroxylases belong to the family of flavocytochromes c.

corresponding spectra, using a diode-array detector (WellChrom Diodenarray Detektor K-2150; Knauer, Berlin, Germany). Proteins were separated under non-denaturing conditions in 7.4% (w/v) polyacrylamide gels, as described by Stegemann et al. (1973), and under denaturing conditions in 11.5% (w/v) polyacrylamide gels, according to Laemmli (1970). The proteins in the gels were stained with Serva Blue R. To stain gels for eugenol hydroxylase activity, the gels were incubated at 30 °C in the dark in 100 mM potassium phosphate buffer, pH 7.0, containing 0.04% (w/v) p-nitroblue tetrazolium chloride, 0.003% (w/v) phenazine methosulfate, and 1 mM eugenol.

Materials and methods

Enzyme assay

Bacterial strains and culture conditions The strains of Pseudomonas sp. and Escherichia coli and the plasmids used in this study are listed in Table 1. Cells of E. coli were grown at 37 °C in Luria-Bertani (LB) or in M9 minimal medium (Sambrook et al. 1989). Cells of Pseudomonas sp. strains were grown at 30 °C, either in a nutrient broth (NB) medium (0.8%, w/v; Bacto, Difco), in mineral salts medium (MM; Schlegel et al. 1961), or in yeast extract-supplemented mineral salts medium (HR-MM; Rabenhorst 1996), with carbon sources as indicated in the text. Ferulic acid, vanillin, vanillic acid, and protocatechuic acid were dissolved in dimethyl sulfoxide and were added to the medium at final concentrations of 0.1% (w/v). Eugenol was directly added to the medium at a final concentration of 0.1% (v/v). Tetracycline and kanamycin were used at final concentrations of 25 µg/ml and 300 µg/ml, respectively, for Pseudomonas sp. Growth of the bacteria was monitored with a Klett-Summerson photometer. Samples were taken from the cultures and cells were removed by centrifugation. The obtained culture supernatants were analyzed by HPLC with respect to the appearance or disappearance of catabolic intermediates as described below. Analytical methods Culture supernatants were analyzed for excreted intermediates derived from eugenol by liquid chromatography without prior extraction, using a Knauer HPLC apparatus. Intermediates were separated by reversed-phase chromatography on Nucleosil-100 C-18 (5-µm particles; column 250.0×4.0 mm) with a gradient of 0.1% (v/v) formic acid (eluant A) and acetonitrile (eluant B), using 20–100% (v/v) eluant B and a flow rate of 1 ml/min. For quantification, all intermediates were calibrated with external standards. The compounds were identified by their retention times and the Table 1 Bacterial strains and plasmids used in this study

Isolation, manipulation, analysis, and transfer of DNA Plasmid DNA, DNA restriction fragments, and PCR products were isolated and analyzed by standard methods (Sambrook et al. 1989). Competent cells of E. coli were prepared and transformed by the CaCl2 procedure (Sambrook et al. 1989). Conjugations of E. coli S17-1 (donor)-harboring hybrid plasmids and Pseudomonas sp. (recipient) were performed as described by Priefert et al. (1997). For the construction of a genomic library of Pseudomonas sp. strain OPS1, genomic DNA partially digested with EcoRI was ligated with EcoRI-linearized cosmid pVK100. The ligation mixtures were packaged in λ particles and subsequently transduced into E. coli S17-1. Transductants (1,500) were selected on LB/tetracycline agar plates; and the hybrid cosmids of these strains were transferred to the eugenol-negative mutant SK6165 of Pseudomonas sp. strain HR199 by conjugation. DNA restriction fragments were separated electrophoretically in 0.8% (w/v) agarose gels in

Strain or plasmid

Relevant characteristics

Source or reference

Pseudomonas sp. OPS1

Wild type, eugenol-positive, ferulic acid-positive

HR199

Wild type, eugenol-positive, ferulic acid-positive

SK6165a

eugenol-negative, ferulic acid-positive

DSM 14291, this study DSM 7063 (Rabenhorst 1996) Priefert et al. (1999)

Escherichia coli XL1-blue S17-1

a SK6165 is a mutant of Pseudomonas sp. strain HR199

Cells were disrupted either by a two-fold French press passage at 96 MPa, or by sonication (1 min/ml of cell suspension with an amplitude of 40 µm) with a Bandelin Sonopuls GM200 ultrasonic disintegrator. The soluble fractions of crude extracts were obtained by centrifugation at 100,000 g at 4 °C for 1 h. The eugenol hydroxylase activity was assayed photometrically at 30 °C in a total volume of 1 ml, containing 5 µmol eugenol, 0.67 µmol phenazine methosulfate, 0.1 µmol 2,6-dichlorophenol-indophenol (DCPIP), and an appropriate amount of enzyme in 100 mM potassium phosphate buffer (pH 7.0). The reaction was followed by measuring the initial absorbency changes at 600 nm, due to the reduction of DCPIP (ε=21 cm2/µmol). The activities determined with the spectrophotometric test were confirmed, using HPLC analysis to measure the eugenol and coniferyl alcohol concentrations in corresponding stopped-reaction tests. The amount of soluble protein was determined as described by Bradford (1976).

Plasmids pVK100 pBBR1MCS-5 pBluescript SK–

recA1 endA1 gyrA96 thi hsdR17, (rK– mK+) Bullock et al. (1987) supE44relA1, λ–, lac, [F′ proAB lacIqZ∆M15, Tn10(Tet)] recA; harboring the tra genes of plasmid RP4 in the Simon et al. (1983) chromosome, proA, thi-1 Tcr, Kmr, cosmid, broad host range Gmr, lacPOZ′, mob, broad host range Apr lacPOZ′, T7 and T3 promoter

Knauf and Nester (1982) Kovach et al. (1995) Stratagene, San Diego, Calif.

726 50 mM Tris/50 mM borate/1.25 mM EDTA buffer (pH 8.5) (Sambrook et al. 1989). Transfer of denatured DNA to positively charged nylon membranes (0.45-µm pores; Pall Filtrationstechnik, Dreieich, Germany) and hybridization with digoxigenin (DIG)-labeled probes was done by using standard procedures (Sambrook et al. 1989). The 16 S rRNA gene of Pseudomonas sp. strain OPS1 was amplified by PCR from genomic DNA, using oligonucleotides as primers as described by Rainey et al. (1996). The nucleotide sequence of the PCR product was determined, employing the primers described in the same study (Rainey et al. 1996). The genes ehyA and ehyB were amplified together with open reading frame 2 (ORF2) in a PCR reaction, using the primers PCRup (5′-AAAAGAGCTCTACTCTCCACAAAAA CAATTAGGAGACC-3′, which corresponded to the sequence at positions 2,182–2,209; accession no. AF368761) and PCRdown (5′-AAAAGCGGCCGCGCAGAGCAG AGGGTT AAAATATCAGTC-3′, which was complementary to the sequence at positions 4,836–4,862; accession no. AF368761). For DNA sequence determination, the dideoxy chain-termination method (Sanger et al. 1977) was applied. The nucleotide sequences were determined with a 4000L DNA sequencer (Li-Cor, Lincoln, Neb., USA). A Thermo Sequenase fluorescent-labeled primer cycle-sequencing kit with 7-deaza-dGTP (Amersham Life Science, Little Chalfont, UK) was used, as specified by the manufacturer, together with synthetic, fluorescent-labeled oligonucleotides as primers. Nucleotide and amino acid sequences were analyzed with the Genetics Computer Group sequence analysis software package (GCG Package, ver. 6.2, June 1990), as described by Devereux et al. (1984) and with the BLAST database search programs (Altschul et al. 1997). Materials NB was from Oxoid (Basingstoke, UK). The BBL Oxi/Ferm Tube II system was obtained from Becton Dickinson (Heidelberg, Germany). Restriction endonucleases, T4 DNA ligase, lambda DNA, DIG DNA labeling kit, and the enzymes and substrates used in the enzyme assays were obtained from Roche Molecular Biochemicals (Mannheim, Germany) or from GibcoBRL Life Technologies (Karlsruhe, Germany). Agarose type NA was purchased from Amersham Pharmacia Biotech (Freiburg, Germany). Synthetic oligonucleotides were purchased from MWG-Biotech (Ebersberg, Germany). All other chemicals were from Merck Eurolab (Darmstadt, Germany), Serva Feinbiochemica (Heidelberg, Germany), or Sigma-Aldrich Fine Chemicals (Deisenhofen, Germany). Nucleotide sequence accession numbers The nucleotide and amino acid sequence data reported in this paper have been submitted to the EMBL, GenBank, and DDBJ nucleotide sequence databases. The sequence of the 16 S rRNA gene is listed under accession no. AF368760 and the data for the eugenol catabolism genes are listed under accession no. AF368761. Fig. 1A–C Localization of ehyA, ehyB, ORF2, eugR, and azu. A Restriction map of the sequenced region. B Relevant subfragments used in this study. C Structural genes of the eugenol hydroxylase (ehyA, ehyB), azurin (azu), and the putative, positive transcriptional regulator (eugR)

Results Isolation, identification and characterization of the eugenol-degrading bacterium Pseudomonas sp. strain OPS1 HR-MM (50 ml) supplemented with 0.05% (v/v) eugenol was inoculated with soil samples collected from areas beneath a clove tree in the botanical garden of Münster (Germany). After 3 days of incubation at 30 °C, a sample of this culture was used as inoculum for 50 ml of HR-MM supplemented with 0.1% (v/v) eugenol. From this culture, 100-µl samples were taken and spread on MM agar plates; and 100 µl eugenol were applied to a filter-paper deposited in the lid of the plates. Pure cultures were obtained from growing colonies by separation on MM agar plates with eugenol as sole carbon and energy source. One fast-growing strain (OPS1) was a motile, Gram-negative, oxidase- and catalase-positive bacterium, which was classified as a member of the genus Pseudomonas by the BBL Oxi/Ferm Tube II system. For further taxonomic characterization, the sequence of the 16 S rRNA gene of strain OPS1 was determined. The highest similarity (99.1%; 1,467 bp/1,480 bp) was obtained with Pseudomonas sp. strain IpA-2 (accession no. X96788.1). The highest similarity to strains of known species (98.6%; 1,478 bp/1,499 bp) was obtained with P. jessenii (accession no. AF068259.1). In conclusion, strain OPS1 does not belong to any validly typed species and the name P. eugenolovorans is proposed for isolate OPS1. Pseudomonas sp. strain OPS1 has been deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany (DSMZ) under no. DSM 14291. Cloning of the genes involved in the eugenol degradation pathway A nitrosoguanidine-induced mutant of Pseudomonas sp. strain HR199 (SK6165), which was unable to grow on eugenol but retained the ability to grow on ferulic acid

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Fig. 2 Relationship between the α subunit of the eugenol hydroxylase from Pseudomonas sp. strain OPS1 and cytochrome c subunits of flavocytochromes c from different sources. The dendrogram was constructed using the CLUSTAL program, based on pairwise alignments of the amino acid sequences of the cytochrome c subunit of the eugenol hydroxylase from Pseudomonas sp. strain OPS1 deduced from ehyA, with the amino acid sequences of the corresponding subunits of the eugenol hydroxylase from Pseudomonas sp. strain HR199 (Priefert et al. 1999), the p-cresol methylhydroxylase from P. putida NCIMB 9866 (McIntire et al.

1986; Kim et al. 1994), and a p-cresol methylhydroxylase-related flavocytochrome (plasmid pNL1-encoded; PchC) from Sphingomonas aromaticivorans F199 (Romine et al. 1999). The pairwise similarity scores were generated by the method of Wilbur and Lipman (1983), with the following parameters: k-tuple length = 1, gap penalty = 3, number of diagonals = 5, diagonal window size = 5, gap-opening penalty = 10, gap-extension penalty = 0.10, and protein weight matrix blosum. Relatedness is represented by the branch length (distances are given as 0.01% divergence in parentheses)

Fig. 3 Relationship between the β subunit of the eugenol hydroxylase from Pseudomonas sp. strain OPS1 and flavoprotein subunits of flavocytochromes c from different sources and vanillyl-alcohol oxidase from Penicillium simplicissimum. The dendrogram was constructed based on pairwise alignments of the amino acid sequence of the flavoprotein subunit of the eugenol hydroxylase from Pseudomonas sp. strain OPS1, deduced from ehyB, with the amino acid sequences of the corresponding subunits of the eugenol hydroxylase from Pseudomonas sp. strain HR199 (Priefert et al. 1999), the p-cresol methylhydroxylase from P. putida NCIMB 9866 (Kim et al. 1994), two p-cresol methylhydroxylase-related flavocytochromes (plasmid pNL1 encoded; PchFa and PchFb) from Sphingomonas aromaticivorans F199 (Romine et al. 1999), and vanillyl-alcohol oxidase (VaoA) from Penicillium simplicissimum (Benen et al. 1998). The dendrogram was constructed using the same program and parameters as for Fig. 2

eugenol-negative mutant SK6165 and complementation was achieved with a 2,200-bp BamHI fragment (B22). This fragment was cloned in the vector pBluescript SK– (pSKB22) and sequenced. DNA fragments overlapping with fragment B22 were identified by hybridization of B22 to pVO23-1260 DNA, digested with different restriction endonucleases.

(Priefert et al. 1997, 1999), was chosen as recipient for a genomic library of Pseudomonas sp. strain OPS1. One transconjugant was isolated, which was complemented by the received hybrid cosmid pVO23-1260, and which grew again on eugenol. Different subfragments of pVO23-1260 were cloned in pBBR1MCS-5. The resulting hybrid plasmids were conjugatively transferred to the

Molecular characterization of the eugenol hydroxylase genes The nucleotide sequences of fragments B22, E18, P20, S22, and X24 (Fig. 1) were determined after cloning in the vector pBluescript SK–. Adjacent regions were sequenced using pVO23-1260 as template DNA, together with specific synthetic oligonucleotides as primers, applying the primer hopping strategy (Fig. 1). The major part of an ORF of 378 bp (ehyA), whose putative translational product exhibited 57% identity to the ehyA gene product of Pseudomonas sp. strain HR199 and 34% to the cytochrome c subunit of the p-cresol meth-

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Fig. 4 The relationship of the azu gene product from Pseudomonas sp. strain OPS1 with azurins from different sources. The dendrogram was constructed based on pairwise alignments of the amino acid sequence of the azurin from Pseudomonas sp. strain OPS1, deduced from azu, with the amino acid sequences of the azurin precursor from Pseudomonas aeruginosa (Arvidsson et al. 1989), the azurin from Pseudomonas putida (Barber et al. 1993), the azurin from Pseudomonas fluorescens bv. C (Ambler 1971), the azurin-I precursor from Achromobacter xylosoxidans subsp. xylosoxidans (accession no. BAA33677), the azurin precursor from Achromobacter xylosoxidans subsp. denitrificans (Hoitink et al. 1990), and the azurin iso-1 from Methylomonas sp. strain J (Taguchi et al. 1998). The dendrogram was constructed using the same program and parameters as for Fig. 2

ylhydroxylase of P. putida (Fig. 2), was identified on fragment B22. The amino acid sequence deduced from ehyA exhibited a typical leader peptide structure, comprising the amino acids at positions 1–37. The stop codon of ehyA overlapped with the start codon of a second ORF of 687 bp (ORF2). At 15 bp downstream of ORF2, the start codon of a third ORF of 1,554 bp (ehyB) was identified, whose putative translational product exhibited 85% identity to the ehyB gene product of Pseudomonas sp. strain HR199 and 54% to the β subunit of the p-cresol methylhydroxylase of P. putida (Fig. 3). At 113 bp downstream of ehyB, another ORF of 447 bp was identified on the complementary strand, which was referred to as azu because its putative translational product exhibited up to 71% identity to azurins from different sources (Fig. 4). Since the amino acid sequence exhibited also a typical leader peptide structure (amino acids 1–20), the calculated mass of the mature azu gene product was 14,158 and corresponded well with the molecular masses reported for other azurins. At 72 bp upstream of ehyA, an ORF (eugR) of 1,881 bp was identified, whose putative translational product exhibited high similarity to transcriptional activators of the NtrC family.

Fig. 5 Expression of Pseudomonas sp. strain OPS1 ehyAB genes in Escherichia coli XL1blue. Cytoplasmic fractions obtained from Pseudomonas sp. cells grown on gluconate plus eugenol or E. coli cells grown for 12 h in Luria-Bertani medium in the presence of tetracycline (12.5 µg/ml) or ampicillin (100 µg/ml) were separated in a 7.4% (w/v) polyacrylamide gel and were stained for eugenol hydroxylase activity as described in Materials and methods. Lane 1 E. coli XL1-blue (pBluescript SK–), lane 2 E. coli XL1-blue (pSKehyOPS1), lane 3 Pseudomonas sp. strain OPS1

Heterologous expression of ehyA and ehyB from Pseudomonas sp. strain OPS1 in E. coli The genes ehyA and ehyB were amplified together with ORF2 in a PCR reaction. Since the upstream primer exhibited a SacI site and the downstream primer exhibited a NotI site, the PCR product was cloned in pBluescript SK– with the genes ehyA and ehyB colinear to and downstream of the lacZ promoter. The resulting hybrid plasmid pSKehyOPS1 conferred eugenol hydroxylase activity to E. coli XL1-Blue (Fig. 5). After growth of the re-

729 Table 2 Expression of ehyA and ehyB of Pseudomonas sp. strain OPS1 in Escherichia coli. Cells of P. eugenolovorans were grown at 30 °C in mineral salts medium, containing eugenol as a carbon source, to the late exponential phase and harvested. Cells of recombinant strains of E. coli were grown for 12 h at 37 °C in LuriaBertani medium. To obtain plasmids inducing expression from the lacZ promoter, the recombinant strains of E. coli harboring pBluescript SK– derivatives were cultivated in the presence of 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The eugenol hydroxylase activities were determined in soluble extracts at 30 °C, by the photometric assay as described in Materials and methods Bacterial strain

Plasmid (inductor)

Pseudomonas sp. strain OPS1 E. coli XL1-blue E. coli XL1-blue E. coli XL1-blue

– pBluescript SK– (+IPTG) pSKehyOPS1 (–IPTG) pSKehyOPS1 (+IPTG)

Specific activity of eugenol hydroxylase (U/mg protein) 0.024 <0.001 0.003 0.014

combinant strain in the presence of the inductor isopropyl-β-D-thiogalactopyranoside, an eugenol hydroxylase activity of 0.014 U/mg protein was obtained in cytoplasmic fractions of the cells (Table 2).

Discussion Eugenol was recently discovered as cheap substrate for a biotransformation process to produce natural vanillin (Rabenhorst and Hopp 1990; Steinbüchel et al. 1998). However, the bioconversion of eugenol is problematic, since it can only be applied in low concentrations, due to its antibacterial properties. Thus, there are only a few reports on microorganisms able to degrade this aromatic compound. The first reports on Corynebacterium sp. and Pseudomonas sp. postulated the formation of the epoxide eugenol oxide (Tadasa 1977; Tadasa and Kayanara 1983), implying an initial reaction catalyzed by a monooxygenase. However, the molecular characterization of the eugenol hydroxylase genes ehyA and ehyB of Pseudomonas sp. strain HR199, which revealed strong sequence similarities to p-cresol methylhydroxylases, suggested a different mechanism via a p-quinone propenide intermediate (Priefert et al. 1999). A similar mechanism was shown for the same reaction catalyzed by eugenol dehydrogenase of P. fluorescens E118 (Furukawa et al. 1998) and vanillyl alcohol oxidase of Penicillium simplicissimum (Fraaije and van Berkel 1997). Interestingly, vanillyl alcohol oxidase differs considerably from the eugenol hydroxylases, because it lacks a cytochrome c subunit, although its primary structure (Benen et al. 1998) shares extensive regions of homology with the amino acid sequence of the flavoprotein subunit of the eugenol hydroxylase (Fig. 3; Priefert et al. 1999). In the present study, we isolated a new eugenoldegrading bacterium from soil. Pseudomonas sp. strain OPS1 was identified as an effective eugenol-degrading bacterium and the genes ehyA and ehyB encoding eugenol hydroxylase were characterized.

As in Pseudomonas sp. strain HR199, the ehyA gene product exhibited a signal sequence for export into the periplasmic space. This sequence was much less conserved (32% identity) than the amino acid sequence of the mature cytochrome c subunit (70% identity). Upstream and downstream of the ehyAB gene cluster of Pseudomonas sp. strain OPS1, eugR and azu were identified, respectively. The eugR gene product exhibited significant similarities to positive transcriptional regulators of the NtrC family (up to 50% identity) and to the product of an homologous gene, which was identified in the direct neighborhood of the eugenol catabolism genes in Pseudomonas sp. strain HR199 (64% identity; Priefert, unpublished data). The azu gene product exhibited significant similarities to azurins from other bacteria (Fig. 4). This is remarkable, since azurins are believed to transfer electrons from the cytochrome c subunit of p-cresol methylhydroxylase to the cytochrome oxidase of the respiratory chain (Causer et al. 1984). Thus, it seems likely that the azu gene product may represent the natural electron-acceptor of the eugenol hydroxylase. The involvement of this azurin in electron transfer from eugenol to the respiratory chain will be investigated in the future, as will be the function of eugR on the regulation of the expression of these genes. In this regard, the present study also provides molecular data for the generation of chimeric and site-specifically mutated forms of eugenol hydroxylases. Acknowledgements H.P. and A.S. are indebted to Haarmann & Reimer GmbH for providing a collaborative research grant.

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