The phn Genes of Burkholderia sp. Strain RP007 Constitute a ...

JOURNAL OF BACTERIOLOGY, Jan. 1999, p. 531–540 0021-9193/99/$04.0010 Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Vol. 181, No. 2

The phn Genes of Burkholderia sp. Strain RP007 Constitute a Divergent Gene Cluster for Polycyclic Aromatic Hydrocarbon Catabolism ANDREW D. LAURIE1,2

AND

GARETH LLOYD-JONES2*

Department of Biological Sciences, University of Waikato,1 and Landcare Research,2 Hamilton, New Zealand Received 18 May 1998/Accepted 4 November 1998

Cloning and molecular ecological studies have underestimated the diversity of polycyclic aromatic hydrocarbon (PAH) catabolic genes by emphasizing classical nah-like (nah, ndo, pah, and dox) sequences. Here we report the description of a divergent set of PAH catabolic genes, the phn genes, which although isofunctional to the classical nah-like genes, show very low homology. This phn locus, which contains nine open reading frames (ORFs), was isolated on an 11.5-kb HindIII fragment from phenanthrene-degrading Burkholderia sp. strain RP007. The phn genes are significantly different in sequence and gene order from previously characterized genes for PAH degradation. They are transcribed by RP007 when grown at the expense of either naphthalene or phenanthrene, while in Escherichia coli the recombinant phn enzymes have been shown to be capable of oxidizing both naphthalene and phenanthrene to predicted metabolites. The locus encodes iron sulfur protein a and b subunits of a PAH initial dioxygenase but lacks the ferredoxin and reductase components. The dihydrodiol dehydrogenase of the RP007 pathway, PhnB, shows greater similarity to analogous dehydrogenases from described biphenyl pathways than to those characterized from naphthalene/phenanthrene pathways. An unusual extradiol dioxygenase, PhnC, shows no similarity to other extradiol dioxygenases for naphthalene or biphenyl oxidation but is the first member of the recently proposed class III extradiol dioxygenases that is specific for polycyclic arene diols. Upstream of the phn catabolic genes are two putative regulatory genes, phnR and phnS. Sequence homology suggests that phnS is a LysR-type transcriptional activator and that phnR, which is divergently transcribed with respect to phnSFECDAcAdB, is a member of the s54-dependent family of positive transcriptional regulators. Reverse transcriptase PCR experiments suggest that this gene cluster is coordinately expressed and is under regulatory control which may involve PhnR and PhnS. explain genotypically by comparison with nah-like sequences. Employing nah-like probes provides a useful screen for only 45% of strains isolated for a naphthalene-degrading phenotype, yet for strains isolated for their phenanthrene-degrading phenotype (which can also readily degrade naphthalene), the same probes were only effective for 15% of isolates (43). It is evident that as-yet-undescribed PAH genes must play a significant role in PAH degradation. The objective of this study was to isolate PAH catabolic genes that are involved in phenanthrene and naphthalene degradation which are different from the classical nah-like genes. Burkholderia sp. strain RP007 was isolated from a PAHcontaminated site in New Zealand, and its identification is based on biochemical tests, fatty acid analysis, and 16S ribosomal DNA nucleotide sequence (42). RP007 was isolated for its ability to degrade phenanthrene and was subsequently shown to be a versatile degrader of low-molecular-weight PAHs, readily utilizing naphthalene, phenanthrene, and anthracene as sole carbon sources. Naphthalene and phenanthrene are degraded through common routes via salicylic acid and 1-hydroxy-2-naphthoic acid, respectively (42). Here we describe the characterization of a gene cluster from RP007 which specifies the upper pathway for naphthalene and phenanthrene degradation.

The aerobic catabolism of low-molecular-weight polycyclic aromatic hydrocarbons (PAHs) by bacteria has been extensively studied (11). Naphthalene has often been selected as a model compound for the study of PAH degradation because of its high aqueous solubility and the ease of isolation of microbes capable of its degradation. Since the first report of a biochemical pathway for naphthalene oxidation by Pseudomonas species in 1964 (12), extensive studies have rigorously defined the metabolic pathway, genes, and enzymes involved (10, 17, 26). It is now accepted that one highly conserved group of genes cannot reflect the true diversity of microbial genes involved in the catabolism of PAHs such as naphthalene and phenanthrene (27, 85). Several groups of genes, which we shall term nah-like, which show a high degree of homology to the nah genes from Pseudomonas putida G7, have been described. These include the nah (15, 29, 69) and ndo (40) genes encoding the multicomponent naphthalene dioxygenase from two naphthalene-degrading pseudomonads, the pah genes cloned from two phenanthrene-degrading Pseudomonas strains, (37, 76, 77), and the dox genes derived from a Pseudomonas strain which degrades dibenzothiophene (14). Despite originating from diverse geographical locations, the genes carried by these strains show .90% amino acid homology to the P. putida G7 nah genes and have a conserved gene arrangement. Furthermore, a wealth of organisms present in contaminated soils express a PAH-degrading phenotype which we cannot

MATERIALS AND METHODS Bacterial strains and culture conditions. Burkholderia sp. strain RP007 has been deposited in the ICMP culture collection (Landcare Research, Auckland, New Zealand) under strain designation ICMP 13529. The organism was routinely maintained on plate count agar (Difco Laboratories) or on solid mineral salts medium by using a phenanthrene overlay method (7). Minimal medium

* Corresponding author. Mailing address: Landcare Research, Private Bag 3127, Hamilton, New Zealand. Phone: (64) 7 858 3700. Fax: (64) 7 858 4964. E-mail: [email protected]. 531

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(MM) contained Na2HPO4 (4 g/liter), KH2PO4 (2 g/liter), (NH4)2SO4 (1 g/liter), and Herbert’s salts (2 ml/liter) (58). In liquid culture, phenanthrene was added directly to the medium (0.05 g/liter), while naphthalene was supplemented initially as a vapor and, once growth was observed, added directly to the medium. All incubations were at 28°C. Escherichia coli DH5a was used as the host strain for pUC18 plasmids and derivatives. E. coli JM105 was used as the host strain for recombinants constructed in the expression vector pKK223-3 (Pharmacia). Recombinant constructs in E. coli were routinely grown and maintained on LuriaBertani (LB) solid medium (1.6% agar) or broth amended with ampicillin (100 mg/ml). Molecular techniques. Standard procedures were used for plasmid DNA preparation and manipulation and agarose gel electrophoresis (60). Total bacterial DNA was prepared by the method of Ausubel et al. (4), and plasmid DNA from RP007 was prepared by the method of Wheatcroft and Williams (83). A nested deletion series for nucleotide sequencing was generated by using a double-stranded nested deletion kit (Pharmacia). Plasmid DNA for sequencing was isolated with a Quantum Prep plasmid miniprep kit (Bio-Rad), and cloned sequences were determined by the Waikato DNA sequencing facility with a PRISM Ready Reaction DNA terminator cycle sequencing kit (Perkin-Elmer). The reactions were resolved with an Applied Biosystems Inc. (ABI) model 377 sequencer. Nucleotide sequence data was assembled by using the ABI Fractura and Assembler computer packages and analyzed by using ClustalW (79) and Omiga (version 1.1) (Oxford Molecular Group, Oxford, England). Phylogenetic trees were constructed by using the PHYLIP software package (version 3.57c) (22) with the SEQBOOT, PROTDIST, NEIGHBOR, and CONSENSE programs. Dendrograms were redrawn by using the ClustalW homology data to determine branch lengths. For protein labelling experiments, recombinant pKK223-3 constructs in E. coli JM105 were induced with isopropyl-b-D-thiogalactopyranoside (IPTG) for 2 h and then incubated with [35S]methionine (.1,000 Ci/mmol; NEN Life Science Products) for 1 h. Crude cell extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by the method of Laemmli (41). RT-PCR. Samples (1.5 ml) of RP007 culture, grown to early exponential phase at the expense of either naphthalene, phenanthrene, or acetate, were pelleted, snap frozen in liquid nitrogen, and stored at 270°C until required. RNA was isolated from cells by using an RNeasy total RNA kit (Qiagen). Purified RNA was treated with DNase I (GibcoBRL) and RNase inhibitor (Boehringer Mannheim) to ensure complete removal of DNA and to maintain the integrity of mRNA. Reverse transcriptase PCR (RT-PCR) was carried out with the Titan One Tube RT-PCR system (Boehringer Mannheim) by using primers P6897 (59-GCGATTCCGGTTTATCTCAA-39) and P8420 (59-CTCCACCTTGCCAA TTTCAT-39). Appropriate positive and negative PCR controls were included with each experiment. The cycling conditions (Techne Cyclogene thermal cycler) for the RT-PCR amplification were as follows: 50°C for 30 min; 94°C for 2 min; followed by 10 cycles of 94°C for 30 s, 52°C for 30 s, and 68°C for 1 min; followed by 15 cycles of 94°C for 30 s, 52°C for 30 s, and 68°C for 80 s; followed by 15 cycles of 94°C for 30 s, 52°C for 30 s, and 68 °C for 2 min; followed by 94°C for 30 s, 52 °C for 30 s, and 68°C for 10 min; followed by cooling to 4°C. PCR amplification controls omitted the initial incubation at 50°C. The optimal MgCl2 concentration was 1.5 mM. The identities of the products amplified by RT-PCR were verified by using primers P7525 (59-GTCGTGGAGGATCTTAAGCG-39) and P8197 (59CGCATCACAATCACCTCATC-39) located internal to the P6897 and P8420 primers. Additional primer sets designed to amplify across the phnSF and phnFEC genes and intercistronic regions were, for phnSF, P3076 (59-CAGCGCAT ACAGTTCCTGGT-39)-P3953 (59-GGTCATCGACAACACACCTG-39), and for phnFEC, P4970 (59-CCGAACTAAAGTGGATCACGA-39)-P6510 (59-GAT CAGTGGATCGTGGGG-39) (see Fig. 7A). Assay of recombinant PAH extradiol dioxygenase. The extradiol dioxygenase gene (phnC) was cloned into the vector pKK223-3 (Pharmacia) and expressed in E. coli JM105. Following growth of 100-ml cultures to an absorbance at 600 nm of 2, IPTG was added to 500 mM and the incubation was continued for 2 h to induce expression of phnC. E. coli cells were pelleted and washed in MM and then resuspended in 4 ml of 0.1 M sodium phosphate buffer (pH 7.5) containing 10% (vol/vol) acetone. Cells were disrupted by sonication, and cellular debris was removed by centrifugation to give a crude cell extract. Activity against 1,2dihydroxynaphthalene was determined spectrophotometrically by the method of Kuhm et al. (38). One hundred microliters of crude cell extract was added to 900 ml of 50 mM acetic acid–NaOH buffer (pH 5.5), and the reaction was started by the addition of 0.5 mmol of 1,2-dihydroxynaphthalene (1,2-DHN; Tokyo Chemical Industry [TCI], Tokyo, Japan) in 10 ml of N,N-dimethylformamide. The initial rate of decrease of the absorbance at 331 nm was measured. Three hundred thirty-one nanometers is an isobestic point for the oxidation of 1,2DHN to 1,2-naphthoquinone, which occurs rapidly in aqueous solution but is minimized at pH 5.5 (38). A molar extinction coefficient (e) for 1,2-DHN at 331 nm of 2,600 M21cm21, as calculated by Kuhm et al. (38), was used in the enzyme activity calculations. The PhnC extradiol dioxygenase was also assayed with other arene diols as substrates in 0.1 M sodium phosphate buffer (pH 7.5) containing 10% acetone. Extradiol dioxygenase activities were calculated for catechol by monitoring the increase of absorption at 375 nm, and reaction rates were calculated by using a molar extinction coefficient of 36,000 M21cm21 (51); absorbance maxima and molar extinction coefficients were 388 nm and 15,000 M21cm21, respectively, for 3-methylcatechol and 382 nm and 31,500 M21cm21, respec-

J. BACTERIOL. tively, for 4-methylcatechol. Extradiol cleavage of 2,3-dihydroxybiphenyl was assayed with an initial substrate concentration of 0.1 mM 2,3-dihydroxybiphenyl, the increase of absorption at 434 nm was measured, and reaction rates were calculated by using a molar extinction coefficient of 22,000 M21cm21 (74). Protein was estimated by the method of Lowry et al. (45). Transformation of naphthalene and phenanthrene by recombinant phn genes. E. coli DH5a(pB1) or E. coli DH5a (pB9R) was grown in 100 ml of M9 medium to an absorbance at 600 nm of 2. Cells were washed and resuspended in 20 ml of fresh M9 medium to give a high-density cell suspension. Naphthalene or phenanthrene was added to 1 mg/ml, and the culture was incubated at 28°C for 16 h. The culture supernatant was acidified to pH 3.0 with HCl and extracted three times with an equal volume of ethyl acetate. The extract was evaporated to dryness and resuspended in a minimal volume of methanol and analyzed by thin-layer chromatography (TLC) or gas chromatography-mass spectrometry (GC-MS). For TLC (Silica Gel 260; Merck), we used a benzene-acetate-water (125:74:1, vol/ vol/vol) solvent system designed to resolve polar and acidic metabolites (47) which were visualized at 302 nm, and metabolites were recovered from the TLC plate in methanol for spectrophotometric analysis. GC-MS analysis was conducted with a Hewlett-Packard 5890 GC, fitted with an HP-1 column (25 m by 0.2 mm) and 0.33-mm film, and a Hewlett-Packard 5970 mass selective detector. For 14C experiments, [U-14C]naphthalene (49.8 mCi/mmol; Sigma) or [9-14C]phenanthrene (13.3 mCi/mmol; Sigma) was mixed with unlabelled PAH and added to cell suspensions to give a concentration of 50 mg/ml of the unlabelled substrate. After extraction and TLC analysis as described above, colored metabolites were recovered from the TLC plate in methanol for measurement of disintegrations per minute associated with these metabolites. All experiments used E. coli DH5a as a control run alongside the E. coli strains carrying recombinant genes. Nucleotide sequence accession number. The nucleotide sequence of 11,451 bp of the pB1 fragment is available in GenBank under accession no. AF061751.

RESULTS Cloning and sequencing of PAH catabolic genes from RP007. Southern hybridization and PCR experiments using probes and primers based on the pah genes of P. putida OUS82 (77) and the nah genes of P. putida G7 (69) revealed that RP007 did not possess genes that were homologous to these genes for PAH degradation (data not shown). To clone the genes responsible for the early stages of PAH degradation, genomic DNA from RP007 was digested with common restriction enzymes and ligated into appropriately digested pUC18 to generate a genomic library. The library was screened for the ability to oxidize indole, leading to the formation of the blue product indigo, which is indicative of the presence of aromatic oxygenase genes (20). A single clone, designated pB1, which gave a positive indole reaction was obtained. Restriction mapping of pB1 revealed that it contained an 11.5-kb HindIII fragment ligated into the HindIII site of the pUC18 multiplecloning site. A 1.7-kb EcoRI fragment of pB1 hybridized to a corresponding fragment of a large (.100-kb) plasmid present in RP007. pB1 did not hybridize to either P. putida G7 genomic DNA or to the pahAB genes of P. putida OUS82 (37) (data not shown). Analysis of the pB1 nucleotide sequence. The complete nucleotide sequence (cds 1 to 11451) of the pB1 HindIII fragment of RP007 was determined, and analysis revealed the presence of nine complete open reading frames (ORFs) (Fig. 1). Each was initiated by the canonical ATG start codon and preceded by a putative ribosomal binding site. To verify that the hypothetical ORFs encoded peptides of the predicted size, complete ORFs were cloned into the expression vector pKK223-3 (Pharmacia). Five recombinant peptides were successfully expressed in E. coli JM105 and visualized by SDS-PAGE analysis (data not shown). The sizes as estimated by SDS-PAGE agree well with the predicted sizes, although the experimentally determined molecular size for PhnB, 35 kDa, was larger than the predicted size of 28.4 kDa (Table 1). The function of individual ORFs was determined based on nucleotide homology to previously described genes and their corresponding amino acid sequences (Table 1). ORFs 3 to 9 have functions analogous to genes from previously described

phn GENES FOR PAH CATABOLISM IN BURKHOLDERIA

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FIG. 1. Physical map of pB1 showing the phn gene cluster of Burkholderia sp. strain RP007. The insert fragment is denoted by the bold line, and open arrows indicate the positions and orientations of the ORFs identified. The gene designations of each ORF are shown, as are the sites of common restriction enzymes. The subclone, pB9R, contains the phnDAcAdB genes under the control of the lac promoter of pUC18. The phenotype expressed by pB9R is PhnAcAdB since the phnC gene coding for extradiol dioxygenase, the next pathway enzyme, is not present on this fragment.

loci for the catabolism of naphthalene and phenanthrene (Fig. 2), and the designations of individual genes are consistent with those given for these loci. The RP007 genes were designated the phn genes (for phenanthrene degradation) to indicate their divergence from the nah/ndo/pah/dox group. Our analysis frequently compares the phn genes of RP007 and their products to the analogous nah-like genes or Nah-like peptides, respectively. Since previously described loci for naphthalene/phenanthrene catabolic pathways (nah [15, 29, 69], ndo, [40], pah [76, 77], and dox [14]) usually show .90% nucleotide homology between individual genes, the phn genes are compared with this highly similar group as a whole. We apply the generic term PAH to describe the phn gene products to account for relaxed substrate specificities which allow for the degradation of the PAHs naphthalene and phenanthrene by enzymes encoded by the phn gene cluster. ORFs 7 and 8 are the phnAc and phnAd genes that encode the iron sulfur protein (ISP) large (a) and small (b) subunits of a PAH initial dioxygenase. The ISPa subunit has been implicated in defining the substrate specificity properties of 2-nitrotoluene dioxygenase (53). The two cysteine and histidine residues, which in ISPs form a conserved motif involved in coordination of a Rieske-type [2Fe-2S] center, are found at the expected positions in PhnAc. Figure 3 presents a dendrogram of ISPa subunits from aromatic dioxygenases and shows that PhnAc is located on the same branch as the NahAc-like ISPa

subunits. The 56% homology of PhnAc to this group is significantly lower than that between members of this group (at .80%), which also includes the divergent branch comprising NtdAc of Pseudomonas sp. strain JS42 (52), DntAc of Burkholderia sp. strain DNT (72), and NagAc of Pseudomonas sp. strain U2 (23). The phylogeny of the PhnAd ISPb subunit shows a similar arrangement, but similarity to the NahAd group is significantly less at 32 to 36%. ORF 9 is the phnB gene which encodes a PAH dihydrodiol dehydrogenase. The predicted amino acid sequence of the phnB product shows most similarity to analogous dehydrogenases from biphenyl catabolic pathways. As shown in Fig. 4, the NahB-like dehydrogenases are a tightly clustered group which form a separate branch with ,35% amino acid similarity to the BphB branch, which includes PhnB. ORF 6 is identified as the phnD gene which encodes a putative isomerase in the RP007 PAH catabolic pathway. Previously described 2-hydroxychromene-2-carboxylate isomerases from naphthalene pathways (PahD [76], PahD [77], NahD [15], and DoxJ [14]) are .80% similar at the amino acid level although only 47% similar to the PhnD sequence. ORF 5 is a gene coding for an extradiol dioxygenase which we designated phnC. The predicted amino acid sequence of the phnC product shows no homology to that of NahAc-like 1,2dihydroxynaphthalene dioxygenases or to extradiol dioxygenases from biphenyl catabolic pathways. PhnC shows greatest

TABLE 1. Properties of the phn genes identified on the pB1 fragment of RP007 ORF

1 2 3 4 5 6 7 8 9 a

Gene

phnR phnS phnF phnE phnC phnD phnAc phnAd phnB

Nucleotide positions

No. of encoded aaa

155–1843 2377–3519 3529–5022 5073–6065 6480–7307 7339–7929 7992–9344 9406–9993 10076–10894

562 380 497 330 275 196 450 195 272

aa, amino acids. GenBank sequence showing greatest homology. ND, overexpressed protein not detected in E. coli. d ID, PAH initial dioxygenase. b c

Molecular mass (kDa) Predicted

Experimental

62.2 41.7 52.6 36.5 30.0 21.9 50.5 23.7 28.4

67 NDc ND 39 32 ND 54 ND 35

Closest relative (reference)b

% aa identity

Protein feature

phhR (49) nodD (70) pahF (77) nahE (15) carBb (61) pahD (77) pahAc (76) dntAd (72) bphB (46)

44 22 65 73 26 48 56 36 55

Regulatory Regulatory Aldehyde dehydrogenase Hydratase-aldolase Extradiol dioxygenase Isomerase ISPa (large) subunit of IDd ISPb (small) subunit of ID Dihydrodiol dehydrogenase

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class III enzymes, particularly among the members of the second family, three histidines and four aspartates which could possibly comprise the iron coordination sphere (18) are highly conserved and are found in PhnC. Also conserved are DHG and GXSH motifs (where X is any residue) which may function as iron-coordinating ligands (18). The type II or class III extradiol dioxygenase group has also been referred to as the protocatechuate 4,5-dioxygenase family (50), and most members of this group attack substituted singlering catechol compounds. To confirm that PhnC is indeed an extradiol dioxygenase of the RP007 naphthalene/phenanthrene pathway, we have assayed the cleavage of 1,2-DHN and demonstrated the preference of the PhnC enzyme for arene diols containing fused aromatic ring structures. phnC was expressed in E. coli JM105, and the activity in crude cell extracts against arene diol substrates was measured. The relative activ-

FIG. 2. Proposed functions of enzymes encoded by the phn genes of Burkholderia sp. strain RP007. The dashed arrow indicates a spontaneous chemical reaction, and numerals indicate the following metabolites of the recombinant phn genes which were identified: I, 3,4-dihydro-3,4-dihydroxyphenanthrene; II, 1,2-naphthoquinone; III, phenanthrene-3,4-quinone; IV, salicylic acid.

similarity to members of a recently described but expanding group of extradiol dioxygenases which show no homology to the bulk of extradiol dioxygenases so far characterized. This group has been termed either type II (18) or class III (71) extradiol dioxygenases. The amino acid alignment of members of this class (Fig. 5) shows the two families recognized by Eltis and Bolin (18): in the first group, HppB, EdoD, MhpB, and MpcI have 43 to 58% amino acid homology; the second group, which includes PhnC, is more disparate, with 10 to 26% homology, and characteristically differs from the MpcI group by having a deletion of 36 to 44 residues in the central part of the enzyme. Despite the low overall homology of these type II or

FIG. 3. Dendrogram showing the levels of homology between predicted amino acid sequences of different ISPa subunits of aromatic compound dioxygenases. This is an unrooted tree rerooted by using CarAa of Pseudomonas sp. strain CA10 (62) as an outgroup. Bootstrap values of the major branch points are shown; these represent the numbers of times the group consisting of the species which are to the right of that branch occurred, out of 100 trees. The sequences are for the following proteins: PhnAc, Burkholderia sp. strain RP007 (this study); DntAc, Burkholderia sp. strain DNT (72); NagAc, Pseudomonas sp. strain U2 (23); NtdAc, Pseudomonas sp. strain JS42 (52); NahAc, P. putida NCIB 9816-4 (69); DoxB, Pseudomonas sp. strain C18 (14); NdoB, P. putida NCIB 9816 (40); PahAc, P. putida OUS82 (77); NahAc, P. putida G7 (69); PahA3, Pseudomonas aeruginosa PaK1 (76); CarA1, Sphingomonas sp. strain CB3 (66); BphA1, Rhodococcus sp. strain RHA1 (46); IpbA1, Rhodococcus erythroposis BD2 (35); BphA1, Rhodococcus globerulus P6 (2); BpdC1, Rhodococcus sp. strain M5 (81); BedC1, P. putida ML2 (78); BnzA, P. putida (33); TodC1, P. putida F1 (84); TecA1, Burkholderia sp. strain PS12 (6); TcbAa, Pseudomonas sp. strain P51 (80); BphA1, Pseudomonas pseudoalcaligenes KF707 (75); BphA1, Burkholderia sp. strain LB400 (21); CumA1, Pseudomonas fluorescens IP01 (1); IbpA1, Pseudomonas sp. strain JR1 (55); BphA1, Pseudomonas sp. strain KKS102 (24); BphA, Comamonas testosteroni B-356 (73); XylC1, Cycloclasticus oligotrophus RB1 (82); CmtAb, P. putida F1 (16); TdnA1, P. putida UCC22 (25); BenA, Acinetobacter calcoaceticus ADP1 (48); XylX, P. putida mt-2 (30).

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FIG. 4. Dendrogram showing the levels of homology between predicted amino acid sequences of different aromatic dihydrodiol dehydrogenases. This is an unrooted tree rerooted by using CmtB of P. putida F1 (16) as an outgroup. Bootstrap values of the major branch points are shown. The sequences are for the following proteins: PahB, P. putida OUS82 (77); DoxE, Pseudomonas sp. strain C18 (14); PahB, Pseudomonas aeruginosa PaK1 (76); CarB, Sphingomonas sp. strain CB3 (66); PhnB, Burkholderia sp. strain RP007 (this study); BphB, Rhodococcus sp. strain RHA1 (46); BphB, Burkholderia sp. strain LB400 (21); BphB, Pseudomonas pseudoalcaligenes KF707 (75); IpbB, Pseudomonas sp. strain RJ1 (55); CumB, Pseudomonas fluorescens IP01 (1); BphB, Comamonas testosteroni B-356 (73); BphB, Pseudomonas sp. strain KKS102 (24); BphB, Rhodococcus globerulus P6 (3); BpdD, Rhodococcus sp. strain M5 (81); TcbC, Pseudomonas sp. strain P51 (80); TodD, P. putida F1 (84); XylL, P. putida mt-2 (30); BenD, Acinetobacter calcoaceticus ADP1 (48).

ities of the phnC PAH extradiol dioxygenase against arene diol substrates were: 1,2-DHN, 100% (564 6 158 mmol/min/mg of protein); 3,4-dihydroxybiphenyl, 20.2%; 4-methylcatechol, 7.3%; 3-methylcatechol, 5.5%; catechol, 1.2%. 3,4-Dihydroxyphenanthrene is not commercially available; however, these results demonstrate the preference of PhnC for arene diols containing fused aromatic ring structures (1,2-DHN), as compared to the single and unfused ring structures of catechol, methylcatechols, and 2,3-dihydroxybiphenyl, and confirms that PhnC is a PAH extradiol dioxygenase. ORF 4 is a putative hydratase-aldolase gene designated phnE. The predicted amino acid sequence of the phnE product is 73% similar to the sequences of four highly homologous NahE-like trans-o-hydroxy-benzylidenepyruvate hydratase-aldolase peptides (PahE [76], PahE [77], NahE [15], and DoxI [14]), which have .94% amino acid homology. The putative aldehyde dehydrogenase of the RP007 PAH pathway is encoded by ORF 3, which we designated phnF. The predicted amino acid sequence of the phnF product is 65% similar to the sequences of NahF-like salicylaldehyde dehydrogenases (14, 15, 76, 77), which are .91% similar. The LELG GKSP sequence, which is highly conserved in the aldehyde dehydrogenase superfamily (31), is conserved in the PhnF sequence, with the exception of serine which is replaced by alanine. The predicted amino acid sequence of the ORF 2 product is similar to that of a number of LysR-type transcriptional regulators; ORF 2 is designated phnS, and it encodes a putative transcriptional regulator involved in the regulation of the RP007 PAH catabolic genes. PhnS shows low homology to LysR-type regulators involved in transcription of nodulation genes in rhizosphere bacteria (70) and is 21% similar to NahR,


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which is the regulator of the nah genes on the NAH7 plasmid of P. putida G7 (64). LysR-type regulators are characterized by an N-terminal helix-turn-helix motif involved in DNA binding, and seven highly conserved residues [Ala27, Thr(Ser)33, Gln34, Pro35, Ser(Thr)38, Leu44, and Glu45, numbered in accordance with the NahR sequence (63)] within this region are conserved in PhnS, apart from a Pro-to-Ser mutation at position 35. The C-terminal region, which is also important for DNA interactions and transcriptional activation (NahR residues 227 to 253), is also highly conserved in PhnS, with 13 residues identical to those in NahR. ORF 1 encodes a large peptide of 562 amino acids and is divergently transcribed with respect to the other eight phn genes. The predicted amino acid sequence of the product of the ORF 1 gene, designated phnR, has high homology to the sequences of a group of positive transcriptional regulators which form the NtrC family, so called because of their similarity to the Klebsiella pneumoniae NtrC and NifA proteins, which are transcriptional regulators for the ntr and nif genes for nitrogen metabolism (8). Members of the NtrC family of transcriptional regulators control a variety of physiological processes in response to environmental signals and are characterized by their dependence on RNA polymerase that utilizes the alternative s54 cofactor; they are therefore referred to as s54-dependent regulators (67). PhnR shows .40% amino acid similarity to a number of s54-dependent regulators involved in regulation of aromatic degradative pathways which are able to recognize specific effector molecules to induce transcription (13a). These include PhhR and DmpR regulators of (methyl)phenol catabolism from P. putida P35X (49) and Pseudomonas sp. strain CF600 (68) and the XylR regulator of xylene catabolism from P. putida mt-2 (32). s54-dependent regulators are modular proteins consisting of four functional domains. The central C domain possesses a nucleoside triphosphate binding site which mediates the ATPase activity of this domain. This region is thought to be involved in the association with the promoter-bound s54-RNA polymerase holoenzyme complex and is highly conserved among regulators of this class. Across this region, PhnR shows high similarity to other s54dependent regulators and the motifs (GXXGXK and QXXL LRVL) implicated in ATP binding and hydrolysis (67) are present. The C-terminal D domain of s54-dependent regulators contains a helix-turn-helix motif (AX10AAXXLG) involved in DNA binding which is also conserved in the PhnR sequence. Detection of phn transcripts by RT-PCR. An in vivo assay of naphthalene dioxygenase indicated that the enzymes required for PAH catabolism are induced during growth of RP007 at the expense of naphthalene and phenanthrene. Naphthalene dioxygenase activity of RP007 cells was assayed by the method of Shamsuzzaman and Barnsley (65). Cell suspensions grown on naphthalene and phenanthrene had a naphthalene dioxygenase activity, removing naphthalene at 8 6 0.1 mmol/min/mg of protein, compared with no observable activity for acetategrown cells. To analyze expression of the phn genes further, we isolated DNA-free RNA from RP007 grown at the expense of naphthalene, phenanthrene, and acetate. These extracts were used as the target for RT-PCR amplification of partial gene sequences and intercistronic regions corresponding to a 1,524-bp fragment of phnC/phnD/phnAc. RNA extracts derived from phenanthrene- and naphthalene-grown cells both showed amplified products of the anticipated size, while RNA from acetate-grown cells consistently failed to give a product (Fig. 6). All positive and negative PCR controls gave expected results for each experiment. The identity of the RT-PCR products was confirmed by applying the P7525-P8197 internal primer pair to

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FIG. 5. Amino acid alignment of members of the type II (18) or class III (71) family of extradiol dioxygenases. The first five sequences (including that of PhnC) comprise the LigB subfamily recognized by Eltis and Bolin (18), and each has a 36- to 44-residue deletion beginning at a position corresponding to residue 206 in the LigB enzyme. The remaining four enzymes form the MpcI subfamily (18). The conserved histidine and aspartate residues and the DHG and GXSH motifs which may form the iron coordination sphere are shown. Shading represents the degree of conservation among aligned residues, with black indicating 90% and grey indicating 60%. The sequences are for the following enzymes: PhnC, Burkholderia sp. strain RP007 (this study); CarBb, Pseudomonas sp. strain CA10 (61); LigB, Sphingomonas paucimobilis SYK6 (50); HpcB, E. coli C (57); AmnA, Pseudomonas pseudoalcaligenes JS45 (13); MpcI, Alcaligenes eutrophus JMP222 (34); MhpB, E. coli K-12 (71); HppB, Rhodococcus globerulus PWD1 (5) EdoD, Rhodococcus sp. strain I1 (39).

amplify the expected 673-bp product. From these results, we can conclude that phnCDAc is induced by phenanthrene and naphthalene but not acetate, and these genes are therefore regulated at the transcriptional level. This experiment also demonstrated that the phnCDAc genes are cotranscribed, which was expected due to the close arrangement of these genes. To determine whether the other phn genes, excluding phnR, are also transcribed on the same fragment, we designed primers for RT-PCR that would amplify across the phnSF genes and across the phnFEC genes, including the 414-bp intercistronic space between phnE and phnC (Fig. 7A). Both the P3076-P3953 (phnSF) and P4970-P6510 (phnFEC) primer sets amplified products of the expected size from a DNA-free RNA extract of RP007 cells grown on phenanthrene (Fig. 7B). These results show that phnSFECDAc genes are transcribed on a single transcript of at least 7.2 kb. The regions amplified by these primers did not include phnAd and phnB, but since both phnAc and phnAd encode subunits of the same enzyme (ISP), we assume they must be cotranscribed. The intergenic space between phnAd and phnB is 82 bp, and since phnB appears to be the most downstream gene of this operon, it is probable that it is also transcribed on the same fragment as phnSFECDAc. These results suggest that since phnSFECDAcAdB genes are cotranscribed, they may constitute an operon. We expect that

the transcription initiation site of this operon is located upstream of phnS, but we were unable to identify this site by primer extension analysis. RNA was extracted from RP007 cells growing at the expense of naphthalene or phenanthrene

FIG. 6. RT-PCR amplification of a 1,524-bp fragment from total RNA of Burkholderia sp. strain RP007 using the P6897-P8420 primer pair. Lanes 6 to 8 show the RT-PCR experiment for acetate-, naphthalene-, and phenanthrenegrown RP007 cells, respectively; a product is only observed in lanes 7 and 8. Other lanes show PCR controls with the following templates: RP007 genomic DNA (lane 1), no template DNA (lane 2), total RNA from acetate-grown cells (lane 3), total RNA from naphthalene-grown cells (lane 4), and total RNA from phenanthrene-grown cells (lane 5).

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FIG. 7. (A) Diagram showing the locations of the primers used for RT-PCR experiments and the regions amplified. The numbering of the primers reflects the position in the 11,451-bp pB1 fragment. (B) RT-PCR amplification of regions of the phn locus of Burkholderia sp. strain RP007 showing that the phnSFECDAc genes are expressed on a single mRNA transcript. Lanes 3, 6, and 9 show RT-PCR products from total RNA from phenanthrene-grown RP007 cells amplified by using the P3076-P3953, P4970-P6510, and P6897-P8420 primer sets, respectively. Other lanes are PCR controls with the following primers and templates: primer set P3076-P3953, RP007 genomic DNA (lane 1), primer set P3076-P3953, total RNA from phenanthrene grown RP007 (lane 2), primer set P4970-P6510, RP007 genomic DNA (lane 4), primer set P4970-P6510, total RNA from phenanthrene-grown RP007 (lane 5), primer set P6897-P8420, RP007 genomic DNA (lane 7), primer set P6897-P8420, total RNA from phenanthrene-grown RP007 (lane 8).

by using either the RNeasy total RNA kit (Qiagen) or the Trizol reagent method (GibcoBRL), but we were repeatedly unable to detect an extension product from two different primers located near the start codon of phnS. We have therefore refrained from describing the phn gene cluster as an operon. No potential ORFs were detected in the 557-bp downstream of the phnB stop codon to the end of the sequenced region, suggesting that phnB is the most downstream gene of this putative operon. This may be supported by the presence of an inverted repeat 164 bp downstream from the stop codon of phnB which is capable of forming a stem-loop structure and which may serve as a transcription terminator. Expression of the phn genes. We have provided two lines of evidence to support our notion that the phn genes are PAH (naphthalene and phenanthrene) catabolic genes: (i) by demonstrating that the phn genes are transcribed in the wild-type strain during growth at the expense of both naphthalene and phenanthrene as sole carbon and energy sources, but not during growth on acetate (see above); and (ii) by demonstrating that E. coli DH5a expressing the recombinant phn genes was able to oxidize both naphthalene and phenanthrene to more polar compounds (highlighted in Fig. 2). Thus, both PAHs independently induce transcription of the phn genes, and both PAHs are oxidized by the same phn enzymes. Recombinant phnAcAdB genes expressed by E. coli DH5a (pB9R) (Fig. 1) catalyzed the oxidation of [U-14C]naphthalene and [9-14C]phenanthrene. The 14C-labelled polar metabolites formed were resolved by TLC to reveal that distinctly colored metabolites were formed from both PAHs. A yellow metabolite, with an Rf value of 0.44, was formed from [U-14C]naphthalene. This metabolite represents a commonly detected oxidation product derived from naphthalene (28) which arises due to the formation of 1,2-naphthoquinone (9) from the me-

tabolite 1,2-DHN (54). A darker red-brown 14C-labelled metabolite, with an Rf value of 0.3, was formed from [9-14C] phenanthrene. This metabolite bears similarity to previously described oxidation products formed from phenanthrene during whole-cell studies (28, 47) and arises due to the formation of phenanthrene-3,4-quinone (59) from the metabolite 3,4dihydroxyphenanthrene. Furthermore, brown nonpolar 14C-labelled metabolites, which are insoluble in the TLC mobile phase, remained at the origin and would appear to be similar to brown metabolites accumulated during incubation of a doxG mutant with naphthalene and phenanthrene (14). These colored metabolites may have arisen due to the conjugation of quinones formed from the 14C-labelled substrates with other unsaturated molecules during cell incubation and extraction. Additional experiments with unlabelled substrates have also allowed us to identify intermediates of naphthalene and phenanthrene degradation. E. coli DH5a(pB1), which contains the genes required for the first six steps of the PAH catabolic pathway, produced a fluorescent polar metabolite when incubated in the presence of naphthalene. This metabolite was isolated from acidified supernatant and has an identical Rf value (0.95) and UV spectrum to those of authentic salicylic acid. This observation suggests that the recombinant phnFECDAcAdB genes were able to transform naphthalene through to salicylate, although flux through the pathway was poor. In a similar experiment, E. coli DH5a(pB9R), which contains the phnAcAdB genes, was able to oxidize phenanthrene. GC-MS analysis of the extracted supernatant revealed the presence of a molecular ion (m/z 212) with a molecular weight and signature (fragment ions of m/z 194, 181, and 165) consistent with those of 3,4-dihydro-3,4-dihydroxyphenanthrene, the product of the initial dioxygenase. Detection of additional products may have been obscured due to the instability of 3,4-dihydroxy-

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phenanthrene, which readily oxidizes to phenanthrene-3,4-quinone (59); this oxidation product is itself unstable and readily forms brown dead-end products, such as during the incubation and extraction, which were not resolved by gas-liquid chromatography. DISCUSSION The phn genes of Burkholderia sp. strain RP007 constitute a plasmid-borne locus which encodes an upper pathway for PAH catabolism that has a different gene order and that contains isofunctional genes of low homology, in comparison to that of previously described PAH catabolic loci. The phn locus bears some similarity to previously described nah-like degradative pathways, but a number of features make this locus novel. Phylogenetic analysis of predicted amino acid sequences of the phnAc, phnAd, phnD, phnE, and phnF gene products reveals that they fall into the same group as the corresponding nah-like genes. However, in each case the phn gene is consistently placed at an outlying position relative to the tightly clustered nah-like genes, to which the corresponding amino acid similarity is 36 to 73%. phnB differs from these genes in that it is more closely related to genes coding for dehydrogenases from biphenyl catabolic pathways than to the nahB-like genes. phnC encodes the PAH extradiol dioxygenase of the phn pathway, which shows a phylogeny not seen before among extradiol dioxygenases from any PAH or biphenyl catabolic pathway. To our knowledge, this is the first observation of a class III extradiol dioxygenase (71) which is not part of a two-component enzyme and which contributes to a catabolic pathway for polycyclic aromatic substrates. It was not possible to compare extradiol dioxygenase activities of the cloned phnC gene with that of the wild-type RP007 since concurrent expression of more than one extradiol dioxygenase confounds analysis (36, 44). Two catechol 2,3-dioxygenase genes, implicated in lower pathways for aromatic degradation, have been cloned and characterized from RP007 (42). The AaAb genes, which encode the ferredoxin and reductase components of the multicomponent PAH initial dioxygenase, are not present on the phn locus. It is expected that the PhnA initial dioxygenase requires these components since there was no evidence of electron transport functions apparent in the predicted amino acid sequence of the phnAcAd product. It is possible the AaAb genes lie downstream of phnB, as shown for the catabolic genes of Comamonas testosteroni GZ39 (27), but no homology to ferredoxin or reductase genes could be found within the 557 bp from the phnB stop codon to the end of the pB1 fragment. The ferredoxin and reductase genes for electron transport functions for PhnA may be located elsewhere in the RP007 genome or may be supplied by cellular housekeeping genes. That the PhnA dioxygenase was fully functional in E. coli was demonstrated by the indole-positive phenotype of pB1 and the ability of the pB9R clone carrying the phnAcAdB genes to oxidize naphthalene and phenanthrene. Ensley et al. (19) found that the cloned nahAcAd gene products (ISPa and ISPb) of P. putida G7 could oxidize indole to indigo, and Simon et al. (69) suggest that E. coli contains a nonspecific reductase which provides the electron transport functions for recombinant dioxygenases. Our results imply that E. coli DH5a must also contain a nonspecific ferredoxin component which complements PhnA ISPa and ISPb. Predicted amino acid sequence data suggest that phnR and phnS encode a s54-dependent regulator and a LysR-type transcriptional regulator. Both classes of regulators include positive transcriptional activators which, in the presence of specific

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coinducers, activate transcription from specific promoters (63, 67). The location and direction of transcription of phnR and the large intercistronic region between phnR and the other phn genes represent an arrangement similar to that of other aromatic catabolic operons regulated by a s54-dependent regulator (49). We have shown that phnS is cotranscribed with the phn catabolic genes as phnSFECDAc, and despite our inability to determine the transcription start site, we believe that the phnSFECDAcAdB genes are coordinately expressed and therefore constitute an operon. Although we have no supplementary data to confirm the role of phnR and phnS, a possible model for the regulatory functions of these genes can be formulated. As an example, by analogy to the XylR/XylS regulatory model of the pWW0 TOL plasmid (56), we could hypothesize that PhnR is expressed constitutively and, in the presence of an inducing substrate, stimulates transcription of the entire phn operon at a 224/212 type of promoter situated upstream of phnS. Thus, PhnS is coordinately expressed with the other phn gene products and in the presence of another inducing substrate acts to induce the lower-pathway genes located distal to the upper-pathway phn genes. The order of the catabolic genes of the phn locus differs from the highly conserved order found in other nah-like operons. The phn genes are arranged FECDAcAdB, as compared with the conserved order for nah-like loci of AaAbAcAdBFCQED (85). A possible scenario to account for this difference, and the divergent phylogeny patterns of the phn genes, is that the phn locus was compiled by the recruitment of individual genes from a variety of catabolic pathways, possibly including nah and bph loci, and subsequently evolved to specifically catabolize PAHs. Similarities to nah and bph genes therefore reflect the origin of these genes, but the gene arrangement and composition are novel. Characterization of the phn genes of Burkholderia sp. strain RP007 expands the current knowledge of the genetics of bacterial PAH degradation. We have described a novel locus encoding a complete upper pathway for PAH degradation which comprises genes analogous to those of nah-like pathways, but significant differences suggest that the phn operon is not closely related to nah-like determinants. The presentation of this data will allow investigation of the prevalence and distribution of this new group of PAH catabolic genes which will broaden the current understanding of the genetic basis for PAH catabolism. Work to assess the contribution of the phn genotype relative to nah in PAH-degrading microbial communities is currently under way. ACKNOWLEDGMENTS Financial support provided by the New Zealand Foundation for Research, Science and Technology (CO9615) and by the New Zealand Lottery Board Commission is acknowledged. The pDI1 plasmid carrying the pahAB genes of P. putida OUS82 was a gift from N. Takizawa. REFERENCES 1. Aoki, H., T. Kimura, H. Habe, H. Yamane, T. Kodama, and T. Omori. 1996. Cloning, nucleotide sequence, and characterization of the genes encoding enzymes involved in the degradation of cumene to 2-hydroxy-6-oxo-7-methylocta-2,4-dienoic acid in Pseudomonas fluorescens IP01. J. Ferment. Bioeng. 81:187–196. 2. Asturias, J. A., E. Diaz, and K. N. Timmis. 1995. The evolutionary relationship of biphenyl dioxygenase from gram-positive Rhodococcus globerulus P6 to multicomponent dioxygenases from gram-negative bacteria. Gene 156:11– 18. 3. Asturias, J. A., L. D. Eltis, M. Prucha, and K. N. Timmis. 1994. Analysis of three 2,3-dihydroxybiphenyl 1,2-dioxygenases found in Rhodococcus globerulus P6. Identification of a new family of extradiol dioxygenases. J. Biol. Chem. 269:7807–7815.

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