Novel organization of genes in a phthalate degradation operon of Mycobacterium vanbaalenii PYR-1

Robin L. Stingley1, Barbara Brezna1,2, Ashraf A. Khan1 and Carl E. Cerniglia1

1 Division of Microbiology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR 72079, USA
2 Institute of Molecular Biology, Slovak Academy of Sciences, 845 51 Bratislava, Slovakia

Correspondence
Ashraf A. Khan
Ashraf{at}nctr.fda.gov


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Mycobacterium vanbaalenii PYR-1 is capable of degrading polycyclic aromatic hydrocarbons (PAHs) to ring cleavage metabolites. This study identified and characterized a putative phthalate degradation operon in the M. vanbaalenii PYR-1 genome. A putative regulatory protein (phtR) was encoded divergently with five tandem genes: phthalate dioxygenase large subunit (phtAa), small subunit (phtAb), phthalate dihydrodiol dehydrogenase (phtB), phthalate dioxygenase ferredoxin subunit (phtAc) and phthalate dioxygenase ferredoxin reductase (phtAd). A 6·7 kb EcoRI fragment containing these genes was cloned into Escherichia coli and converted phthalate to 3,4-dihydroxyphthalate. Homologues to the operon region were detected in a number of PAH-degrading Mycobacterium spp. isolated from various geographical locations. The operon differs from those of other Gram-positive bacteria in both the placement and orientation of the regulatory gene. In addition, the M. vanbaalenii PYR-1 pht operon contains no decarboxylase gene and none was identified within a 37 kb region containing the operon. This study is the first report of a phthalate degradation operon in Mycobacterium spp.


Abbreviations: PAH, polycyclic aromatic hydrocarbon; PFGE, pulsed field gel electrophoresis; TMS, trimethylchlorosilane

The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AY365117 (M. vanbaalenii PYR-1 pht operon region), AY372761 and AY372763 (Mycobacterium sp. PAH2.135 phtAa and phtB PCR products, respectively), and AY372762 and AY372764 (M. flavescens PYR-GCK phtAa and phtB PCR products, respectively).


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Phthalates are a class of widely used industrial compounds known technically as dialkyl or alkyl aryl esters of 1,2-benzenedicarboxylic acid. The chemicals are used in the manufacture of many products, including plastics, lubricants, solvents and cosmetics (Graham, 1973; Peakall, 1975). As a result of this extensive use, phthalates are commonly found at low levels in the environment and humans are exposed to phthalates in consumer products, diet and medical treatments (Cadogan et al., 1993; Cobellis et al., 2003; Duty et al., 2003; Tickner et al., 2001). Although phthalates used in fragrances and cosmetic products do not appear to pose significant health risks (Api, 2001), those found in medical devices containing polyvinyl chloride (PVC) may be linked to a number of possible adverse health effects in the liver, reproductive tract, kidneys, lungs and heart (Cobellis et al., 2003; Duty et al., 2003; Tickner et al., 2001).

Because phthalates are widely used, they have undergone extensive testing to determine their environmental fate. Generally they do not persist in the environment and biodegrade rapidly. Phthalate is an intermediate in the biodegradation pathways of some polycyclic aromatic hydrocarbons (PAHs), including pyrene (Heitkamp et al., 1988b), phenanthrene (Barnsley, 1983; Kiyohara & Nagao, 1978; Moody et al., 2001), fluorene (Grifoll et al., 1994) and fluoranthene (Kelley et al., 1993; Sepic et al., 1998). The phthalate degradation pathway of Gram-positive bacteria involves oxygenation to form 3,4-dihydro-3,4-dihydroxyphthalate, dehydrogenation to 3,4-dihydroxyphthalate and finally decarboxylation to form protocatechuate (Eaton, 2001; Habe et al., 2003) (Fig. 1). The pathway in Gram-negative bacteria progresses through oxygenation and dehydrogenation at carbons 4 and 5 to form 4,5-dihydroxyphthalate, with subsequent decarboxylation to form protocatechuate (Chang & Zylstra, 1998; Nomura et al., 1992).



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Fig. 1. Schematic representation of the phthalate degradation pathway in Gram-positive bacteria. Genes identified in M. vanbaalenii PYR-1 that encode enzymes along the pathway are indicated.

 
Mycobacterium vanbaalenii PYR-1 is capable of degrading a number of aromatic hydrocarbons, such as anthracene, phenanthrene, pyrene, biphenyl, benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene (Heitkamp et al., 1988b; Khan et al., 2002; Moody et al., 2001, 2002, 2003, 2004). Based on the degradation of various PAHs by M. vanbaalenii PYR-1, this species is likely to have multiple monooxygenases and dioxygenases to perform the initial steps in degradation pathways, in addition to a number of other enzymes that perform subsequent aromatic ring cleavage steps in the pathways (Heitkamp et al., 1988b; Moody et al., 2003). Two genes were characterized previously, nidA and nidB, which encode large and small subunits, respectively, of a polycyclic aromatic ring dioxygenase in M. vanbaalenii PYR-1 (Khan et al., 2001). The products of these genes share limited sequence homology with known dioxygenases, including PhdA and PhdB of Nocardioides sp. KP7, and have conserved homologues in a number of Mycobacterium species that degrade PAHs (Brezna et al., 2003).

M. vanbaalenii PYR-1 nidA and nidB are clustered on the genome with an aldehyde dehydrogenase, nidD (Khan et al., 2001). Genes encoding enzymes involved in PAH degradation are clustered in a similar manner in other bacterial species, including Arthrobacter keyseri 12B, Terrabacter sp. DBF63 and Nocardioides sp. KP7 (Eaton, 2001; Habe et al., 2003; Nojiri et al., 2002; Saito et al., 1999, 2000). The present study focuses on molecular cloning, sequencing and characterization of additional genes in the nidDBA region of the M. vanbaalenii PYR-1 genome. Approximately 9 kb upstream of the nidDBA BamHI fragment, we have found a putative operon containing genes that encode enzymes involved in the degradation of phthalate. Since phthalate has been isolated as an intermediate metabolite in the degradation of pyrene, phenanthrene and fluoranthene in M. vanbaalenii PYR-1 (Heitkamp et al., 1988b; Kelley et al., 1993; Moody et al., 2001, 2002, 2003, 2004), we wanted to characterize the genes involved in phthalate degradation. This is the first description of the operon involved in the phthalate degradation pathway from Mycobacterium spp. The operon is conserved in several PAH-degrading Mycobacterium spp. isolated from various geographical locations (Table 1).


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Table 1. Bacterial strains

 

   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and genomic DNA isolation.
Bacterial strains are listed in Table 1. Mycobacterium vanbaalenii PYR-1 was grown on Middlebrook 7H10 agar at 25 °C for 4 days. Cells were scraped from the plates into 1 ml H2O and pelleted by centrifugation for 10 min at 5900 g. Genomic DNA was isolated according to the protocol for Gram-positive bacteria with the Qiagen DNeasy Kit. Additional strains (Table 1) were cultivated on mineral basal salt (MBS) medium supplemented with sorbitol and phenanthrene as described previously (Brezna et al., 2003).

Cloning, sequencing and sequence analyses.
A genomic library was previously prepared in pCC1FOS (Epicentre) and probed for nidA (Stingley et al., 2004). One clone, pFOS608, contained a putative phthalate degradation operon approximately 9 kb upstream of nidA. A 6·7 kb EcoRI fragment from pFOS608 containing the pht operon region, including the putative regulatory protein, was cloned into pGEM-11z f(+) (pPHT) and introduced into E. coli JM109.

DNA sequencing was performed on an Applied Biosystems model 377 DNA sequencer at the University of Arkansas for Medical Sciences. Sequences were compiled, translated and analysed using Lasergene software (DNASTAR) and compared to similar genes and proteins using BLAST and Conserved Domain Database searches (Marchler-Bauer et al., 2003). Phylogenetic analyses were performed with putative amino acid sequences by using PHYLIP, version 3.572c. The percentage confidence was estimated by a bootstrap analysis with 1000 replications.

Screening Mycobacterium spp. and Rhodococcus spp. for pht genes.
PCR was performed using genomic DNA as template with Taq DNA polymerase and supplied PCR solutions according to the manufacturer's instructions (Qiagen). The PCR regime consisted of a 3 min preincubation at 95 °C, 30 cycles of 30 s denaturation at 94 °C, 30 s annealing at 55 °C and 1 min extension at 72 °C, followed by a final hold at 72 °C for 7 min. All primers used are listed in Table 2. Primers phtAa-2 and phtAa-3 were used to detect phtAa, primers phtB-1 and phtB-3 to detect phtB, and primers phtAa-1 and phtAd-1 to detect a 3·1 kb region of the pht operon, which includes part of phtAa, spans through phtAb, phtB, phtAc and ends inside phtAd.


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Table 2. PCR primers and oligonucleotide probes

 
Hybridizations and confirmation of PCR screening.
M. vanbaalenii PYR-1 fosmid DNA BamHI restriction digests were separated on agarose gels stained with ethidium bromide for visualization, then transferred and cross-linked to positively charged nylon membranes (Roche). The resulting blots were incubated at 65 °C for at least 30 min in DIG Easy Hyb (Roche) prior to addition of digoxigenin (DIG)-labelled DNA probes (denatured at 95 °C for 10 min) specific for nidA. Hybridization was carried out at 65 °C for 16–18 h. DIG-labelled probe was detected with alkaline phosphate-conjugated anti-DIG antibody (Roche) and the chemiluminescent substrate disodium 3-(4-methoxyspiro(1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1]decan)-4-yl)phenyl phosphate (CSPD; Roche).

PCR products were analysed by ethidium-bromide-stained agarose gels and transferred to positively charged nylon membranes (Roche), then subjected to Southern hybridization with the following internal DIG-labelled probes: oligoprobe phtAa-4 (Table 2) for phtAa and oligoprobe phtB-2 (Table 2) for phtB and phtAaAbBAcAd PCR products. Oligoprobes were prepared with the DIG oligonucleotide 3'-end labelling kit (Roche). Hybridization was at 30 °C and washes with 0·5x SSC, 0·1 % SDS were at 48 °C for phtAa and 55 °C for phtB and phtAaAbBAcAd. The phtAa and phtB PCR products from Mycobacterium sp. strain PAH2.135 were confirmed by sequencing.

To screen bacterial strains for phtAa, total genomic DNA was embedded in agarose plugs, digested by XbaI and separated by pulsed field gel electrophoresis (PFGE) as described previously (Brezna et al., 2003). Restriction fragments were separated at 14 °C in a 1 % agarose gel in 0·5x TBE buffer using a contour-clamped homogeneous electric field (CHEF) apparatus (CHEF-DR II, Bio-Rad). Electrophoresis was performed at 6 V cm–1 with a 1·2–8·5 s linear ramp time for 30 h.

DNA was analysed by Southern hybridization with a phtAa-specific DIG-labelled DNA probe, which was prepared using the PCR DIG probe synthesis kit (Roche) with primers phtAa-2 and phtAa-3. Hybridization was performed at 41 °C and washes with 0·5x SSC, 0·1 % SDS were performed at 61 °C.

Degradation of phthalate.
E. coli JM109 cells containing either pPHT, pRS14 or pRS42 were grown in 50 ml LB broth supplemented with 100 µg ampicillin ml–1 at 30 °C with shaking. The cells were pelleted by centrifugation at 4000 g, washed twice with 50 ml 50 mM phosphate buffer (pH 6·8) and suspended in 20 ml buffer to an OD600 of 10. IPTG (Invitrogen) and phthalic acid (dipotassium salt; Aldrich) were added to each at a final concentration of 0·1 % and the cultures were incubated at 30 °C for 16–18 h. The cells were pelleted by centrifugation at 4000 g and the resultant supernatants were extracted three times with an equal volume of ethyl acetate. The samples were derivatized with 1 % trimethylchlorosilane (TMS; Regis Technologies) for GC-MS analyses. The samples were dissolved in 250 µl acetonitrile. Dissolved sample (100 µl) was mixed with 150 µl silylation reagent and allowed to react for 1 h at 60 °C. GC-MS analysis was performed on the Thermo Finnigan TSQ 700 triple quadrupole mass spectrometer operated in EI mode. Separation was achieved in a J&W DB5-ms capillary column (30 mx0·25 mm i.d.x0·25 µm). GC-MS analyses were performed with a column temperature rate of 10 °C min–1 and a total analysis time of 25 min. The amount of degradation was calculated from the peak area as compared to control.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Nucleotide sequencing and operon organization
An M. vanbaalenii PYR-1 genomic library was constructed previously in a fosmid vector and screened for nidA (Stingley et al., 2004). A single nidA-positive fosmid clone, pFOS608, was chosen for initial sequencing studies. The clone was digested with BamHI and the resultant fragments were subcloned into pGEM-11z and sequenced. Initial sequences were obtained from T7 and Sp6 primers located on either end of the insert site in pGEM-11z and subsequent sequencing used walking primers in both directions. Subclone junctions were amplified by high-fidelity PCR using pFOS608 as template and primers that were insert-specific. The resulting PCR products were sequenced to confirm the relative organization of the BamHI fragments in the fosmid insert (Fig. 2a).



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Fig. 2. Schematic representation of the putative M. vanbaalenii PYR-1 pht operon region. (a) The pFOS608 insert, approximately 39 kb in length, is represented in the top schematic, with all BamHI (B) and two EcoRI (E) restriction sites indicated above. The pht operon and its regulatory gene are completely contained on a 6·7 kb EcoRI fragment from pFOS608, which was cloned into pGEM-11z (pPHT). Boxes represent the location and direction of genes within the EcoRI fragment. A partial restriction map and scale of pPHT are included. BamHI subclones pRS14 and pRS42 are also indicated. (b) Operons containing phthalate-degrading genes from M. vanbaalenii PYR-1, Terrabacter sp. DBF63, A. keyseri 12B, B. cepacia DBO1 and P. putida NMH102-2 are compared. Light grey boxes represent the dioxygenase large subunit (phtAa/phtA1), small subunit (phtAb/phtA2), ferredoxin subunit (phtAc/phtA3) and ferredoxin reductase (phtAd/phtA4). Medium grey boxes represent the dihydrodiol dehydrogenase (phtB), dark grey boxes represent the transcriptional regulator (phtR) and solid white boxes represent the decarboxylase (phtC). The cross-hatched boxes represent the putative phthalate transporter gene phtI, orfI and ophD. Immediately downstream of the dioxygenase small subunit gene, each species contains a putative gene with unknown function, which is not represented.

 
BLAST searches with the resulting sequence data indicated the presence of a putative phthalate-degrading (pht) operon (Fig. 2, Table 3). A putative regulatory protein gene (phtR), transcribed from the opposite strand, is upstream of the putative operon. The operon consists of large (phtAa) and small (phtAb) dioxygenase subunits, an unknown ORF (phtU) (not shown in Fig. 2), a dihydrodiol dehydrogenase (phtB), a dioxygenase ferredoxin subunit (phtAc) and a dioxygenase ferredoxin reductase (phtAd). The stop sites of phtAa, phtAb and phtU overlap with the start sites of phtAb, phtU and phtB, respectively, and the stop site of phtAc overlaps the start site of phtAd. In each instance, the stop/start overlap consists of four bases, ATGA. The operon gene products share 53–78 % identity and 66–88 % similarity with their counterparts in Terrabacter sp. DBF63 and A. keyseri 12B (Table 3). However, the M. vanbaalenii PYR-1 operon differs from operons in these species in the placement and orientation of the regulatory gene, which is encoded upstream of the operon and transcribed divergently, rather than encoded in tandem with the operon (Fig. 2b). In contrast to the pht operons in Terrabacter sp. DBF63, A. keyseri 12B, Burkholderia cepacia DBO1 and Pseudomonas putida NMH102-2, the M. vanbaalenii PYR-1 pht operon did not contain a decarboxylase gene (Fig. 2b) and none was located within approximately 2·5–3 kb of either end of the operon region.


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Table 3. Putative pht operon gene products

 
M. vanbaalenii PYR-1 phthalate dioxygenase (PhtAaAbAcAd)
Sequence alignments with proteins from BLASTX searches revealed that the large subunit of the phthalate dioxygenase (PhtAa) was closely related to aromatic ring hydroxylation dioxygenase E (gene E) of Rhodococcus sp. RHA1 (78 % identical and 88 % similar residues), PhtAa of A. keyseri 12B (75 % identical and 87 % similar residues) and PhtA1 of Terrabacter sp. DBF63 (71 % identical and 82 % similar residues) (Table 3). The putative protein sequence contained a number of conserved domains related to those of large subunit ring-hydroxylating dioxygenases (Table 4). M. vanbaalenii PYR-1 PhtAa and the product of Rhodococcus sp. RHA1 gene E branch together in an unrooted phylogenetic tree (Fig. 3a). These proteins share membership in a clade with PhtAa and PhtA1 of A. keyseri 12B and Terrabacter sp. DBF63, respectively. PhtAa was more distantly related to the previously characterized large subunit dioxygenase, NidA, of M. vanbaalenii PYR-1, which is involved in pyrene degradation (Khan et al., 2001) (Fig. 3a). These two enzymes share only 41 % identity and 56 % similarity over 437 residues. Alignment of the phthalate dioxygenase large subunit from M. vanbaalenii PYR-1 and 13 of the most closely related Nocardioform spp. proteins from a BLASTX search indicates significant homology in the Rieske domain and a consensus Fe–S-binding motif, especially with A. keyseri 12B PhtAa, Terrabacter sp. DBF63 PhtA1 and the Rhodococcus spp. large subunit dioxygenases. Alignment of two Gram-negative phthalate dioxygenases from B. cepacia DBO1 and P. putida NMH102-2 showed less than 24 % similarity with PYR-1 phthalate dioxygenase.


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Table 4. Conserved domains in putative pht operon proteins

 


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Fig. 3. Phylogenetic analyses of phthalate dioxygenase proteins. Protein sequences with the greatest homology to M. vanbaalenii PYR-1 phthalate enzymes, based on BLASTX results, were used in the analyses. The multiple-alignment analysis was performed using the PHYLIP software package and phylogenetic unrooted trees were drawn using TREEVIEW. The numbers on some branches refer to the percentage confidence, estimated by a bootstrap analysis with 1000 replications. The scale bars indicate percentage divergence for each tree. Large subunit dioxygenases (a), small subunit dioxygenases (b), dioxygenase ferredoxin subunits (c), dioxygenase ferredoxin reductases (d), dihydrodiol dehydrogenases (e) and transcriptional regulators (f) were analysed. GenBank accession numbers are as follows. (a) PhtAa of M. vanbaalenii PYR-1, AY365117; Protein E of Rhodococcus sp. RHA1, BAB62289; PhtA1 of Terrabacter sp. DBF63, BAC54156; PhtAa of A. keyseri 12B, AAK16534; NidA of Rhodococcus sp. I24, AAD25395; NarAa of Rhodococcus sp. NCIMB12038, AAD28100; NarA of Rhodococcus sp. 1BN, CAC14063; PhdA of Nocardioides sp. KP7, BAA84712; NidA of M. vanbaalenii PYR-1, AAF75991; NidA of M. gilvum BB1, AAN78316; NidA of M. flavescens PYR-GCK, AAN78312; PdoA1 of Mycobacterium sp. 6PY1, CAD38647; NidA of M. frederiksbergense FAn9, AAN78314; PdoA2 of Mycobacterium sp. 6PY1, CAD38643. (b) PhtAb of M. vanbaalenii PYR-1, AY365117; PhtAb of A. keyseri 12B, AAK16535; PhtA2 of Terrabacter sp. DBF63, BAC54157; RnoA4 of Rhodococcus sp. CIR2, BAA76339; NidB of M. frederiksbergense FAn9, AAN78315; NidB of Rhodococcus sp. I24, AAD25396; PhdB of Nocardioides sp. KP7, BAA84713; NidB of M. vanbaalenii PYR-1, AAF75992; NidB of M. gilvum BB1, AAN78317; NidB of M. flavescens PYR-GCK, AAN78313. (c) PhtAc of M. vanbaalenii PYR-1, AY365117; PhtAc of A. keyseri 12B, AAK16536; PhtA3 of Terrabacter sp. DBF63, BAC54160; ferredoxin-related protein of M. tuberculosis CDC1551, NP_335215; hypothetical protein of M. tuberculosis H37Rv, NP_215277; Orf7 protein of Rhodococcus sp. YK2, BAC00808; Msi331 of Mesorhizobium loti R7A, CAD31363; Yp015 of Rhizobium etli CFN42, NP_659824; NysM of Streptomyces noursei ATCC 11455, AAF71770. (d) PhtAd of M. vanbaalenii PYR-1, AY365117; PhtAd of A. keyseri 12B, AAK16537; PhtA4 of Terrabacter sp. DBF63, BAC54161; Orf8 protein of Terrabacter sp. DBF63, BAB55881; FprC of Streptomyces avermitilis MA-4680, NP_828132; Rv0688 of M. tuberculosis H37Rv, NP_215202; MT0716 of M. tuberculosis CDC1551, NP_335128; BphA4 of Novosphingobium aromaticivorans F199, NP_049182; FprA of S. avermitilis MA-4680, NP_821758; FprF of S. avermitilis MA-4680, NP_828132. (e) PhtB of M. vanbaalenii PYR-1, AY365117; PhtB of Terrabacter sp. DBF63, BAC54159; NarB of Rhodococcus sp. NCIMB12038, AAD30203; BphB of Bacillus sp. JF8, BAC79228; NidC of Rhodococcus sp. I24, AAD25397; PhdE of Nocardioides sp. KP7, BAA94705; ThnB of Sphingopyxis macrogoltabida TFA, AAN26445; IpbB of Rhodococcus sp. I1, CAA06877; RnoB of Rhodococcus sp. CIR2, BAA76340; BphB of Rhodococcus sp. RHA1, BAA06873; PhtB of A. keyseri 12B, AAK16533. (f) PhtR of M. vanbaalenii PYR-1, AY365117; PhtR of Terrabacter sp. DBF63, BAB55883; PhtR of A. keyseri 12B, AAK16539; YagI of E. coli K-12, NP_414806; regulatory protein 1 of S. avermitilis MA-4680, NP_828130; regulatory protein 2 of S. avermitilis MA-4680, NP_826918; KdgR of Bacillus halodurans C-125, NP_244592.

 
The small subunit phthalate dioxygenase (PhtAb) was most closely related by sequence alignment to PhtAb of A. keyseri 12B (68 % identical and 80 % similar residues) and PhtA2 of Terrabacter sp. DBF63 (67 % identical and 79 % similar residues) (Table 3). The putative protein sequence contained domains related to small subunit dioxygenases (Table 4). In addition, M. vanbaalenii PYR-1 PhtAb is a member of the same clade of an unrooted phylogenetic tree as PhtAb and PhtA2 of A. keyseri 12B and Terrabacter sp. DBF63, respectively (Fig. 3b). PhtAb shares only 45 % identity and 61 % similarity with NidB, a previously described dioxygenase small subunit involved in pyrene metabolism in M. vanbaalenii PYR-1 (Khan et al., 2001).

The putative protein sequence of the phthalate dioxygenase ferredoxin subunit (PhtAc) contains a ferredoxin domain (Table 4). Upon sequence alignment, PhtAc shared the greatest homology with PhtAc of A. keyseri 12B (69 % identical and 77 % similar residues) and PhtA3 of Terrabacter sp. DBF63 (55 % identical and 66 % similar residues) (Table 3). M. vanbaalenii PYR-1 PhtAc shares membership in a clade with these proteins; however, it appears to be more phylogenetically related to NysM of Streptomyces noursei ATCC 11455 and the product of orf7 of Rhodococcus sp. YK2 (Fig. 3c). NysM is a ferredoxin that participates in electron transfer in P450 monooxygenase systems (Brautaset et al., 2000) and the product of orf7 is a putative ferredoxin.

Sequence alignment of the phthalate dioxygenase ferredoxin reductase (PhtAd) indicated that it was most similar to PhtAd of A. keyseri 12B (58 % identical and 73 % similar residues) and PhtA4 of Terrabacter sp. DBF63 (57 % identical and 71 % similar residues) (Table 3). The putative protein sequence contained several domains related to ferredoxin reductases (Table 4). Upon phylogenetic analysis, these proteins cluster together, along with the product of orf8 of Terrabacter sp. DBF63 (a ferredoxin reductase) to form a clade, with M. vanbaalenii PYR-1 PhtAd branching most closely to PhtAd of A. keyseri 12B (Fig. 3d).

Other proteins from the M. vanbaalenii PYR-1 pht operon region
Sequence alignments indicate that the phthalate dihydrodiol dehydrogenase (PhtB) is most closely related to PhtB of Terrabacter sp. DBF63 (65 % identical and 76 % similar residues) and NarB of Rhodococcus sp. NCIMB12038 (49 % identical and 66 % similar residues) (Table 3). The alignments suggest that M. vanbaalenii PYR-1 PhtB is not significantly similar to that of A. keyseri 12B. Phylogenetic analysis, however, indicates that M. vanbaalenii PYR-1 PhtB is more closely related to that of A. keyseri 12B than to Rhodococcus sp. NCIMB12038 NarB (Fig. 3e). In addition, the putative protein sequence of PhtB contained a number of dehydrogenase domains (Table 4).

In sequence alignments the M. vanbaalenii PYR-1 pht regulatory protein (PhtR) was most similar to PhtR of Terrabacter sp. DBF63 (55 % identical and 69 % similar residues) and PhtR of A. keyseri 12B (53 % identical and 71 % similar residues) (Table 3). Phylogenetic analysis supports the relatedness of these regulatory proteins, as they are co-members of a clade in an unrooted tree (Fig. 3f). The M. vanbaalenii PYR-1 PhtR contains a helix–turn–helix motif near the N terminus and a conserved region near the C terminus that is typical of the isocitrate lyase regulation (IclR) family (Table 4) (Donald et al., 2001). Members of this family have been implicated in both repression (Yamamoto & Ishihama, 2003) and activation (Torres et al., 2003; Trautwein & Gerischer, 2001) of bacterial transcription. A number of aromatic metabolic pathways are regulated by members of the IclR family (Contzen & Stolz, 2000; Eulberg & Schlomann, 1998; Martin & Mohn, 2000; Torres et al., 2003; Trautwein & Gerischer, 2001).

A putative protein of unknown function is encoded by phtU and contains no conserved domains. This protein is similar to hypothetical proteins of unknown function in the Terrabacter sp. DBF63 pht operon (57 % identical and 71 % similar residues) and in a region of the Mycobacterium sp. 6PY1 genome that contains PAH-degradation enzymes (44 % identical and 63 % similar residues) (Krivobok et al., 2003) (Table 3). In addition, the A. keyseri 12B pht operon contains a putative ORF that encodes a similar protein and is immediately downstream of the phtAb ORF (analysis of GenBank accession no. AF331043).

Degradation of phthalate
To determine whether enzymes from the M. vanbaalenii PYR-1 pht operon were capable of phthalate degradation, the pht region, which is completely contained on a 6·7 kb EcoRI fragment from pFOS608 (Fig. 2a), was cloned into the pGEM-11z vector (pPHT). M. vanbaalenii PYR-1, clone pFOS608, and subclones pRS42 and pRS14 (negative control) were incubated with 0·1 % phthalate in phosphate buffer (pH 6·8) at 30 °C for 16–18 h and the resulting supernatant was extracted with ethyl acetate for GC-MS analysis. M. vanbaalenii PYR-1 and clone pFOS608 produced 3,4-dihydroxyphthalate which eluted at 16·2 min and gave major ions at m/z (percentage intensity, proposed composition) 486 (8, M+), 471 (57, [M-CH3]+), 349 (30), 309 (20, [M-CH3-OTMS-TMS]+), 259 (38), 220 (45), 206 (100), 192 (49), 103 (40) and 91 (21). M. vanbaalenii PYR-1 was able to degrade 95 % of phthalate and clone pFOS608 degraded 75 % of phthalate under similar conditions. Subclones pRS14 (containing the 3' end of phtAa and the 5' end of phtAb) and pRS42 (containing unrelated putative genes for membrane proteins) (Fig. 2a) were used in control experiments. 3,4-Dihydroxyphthalate was not formed by control strains.

Screening Mycobacterium and Rhodococcus strains for pht genes
PCR amplification of specific regions within the pht operon was used to screen additional Mycobacterium and Rhodococcus strains. PCR products of expected sizes, 1·36 kb for phtAa and 0·45 kb for phtB, were observed in the PAH-degrading strains Mycobacterium sp. PAH2.135 (RJGII-135), M. flavescens PYR-GCK (ATCC 700033), M. gilvum BB1 (DSM 9487), M. frederiksbergense FAn9 (DSM 44346), Rhodococcus sp. (Dean-Ross et al., 2001), M. austroafricanum GTI-23 and in the positive control strain M. vanbaalenii PYR-1 (Fig. 4). The 3·1 kb band that spans the phtAaAbBAcAd region was present in all of the strains, except Mycobacterium sp. PAH2.135. Southern hybridization using internal probes designed from the M. vanbaalenii PYR-1 sequence confirmed the presence of the regions in these strains, except putative phtAa and phtB bands from Mycobacterium sp. PAH2.135 (Fig. 4).



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Fig. 4. PCR screening of Mycobacterium spp. and Rhodococcus spp. for genes phtAa, phtB and for locus phtAaAbBAcAd. Corresponding Southern hybridization results are shown below each gel photograph. Lanes: 1, Mycobacterium sp. PAH2.135 (RJGII-135); 2, M. flavescens PYR-GCK; 3, M. gilvum BB1; 4, Rhodococcus sp. R-22; 5, M. aurum (ATCC 23366); 6, M. austroafricanum (ATCC 33464); 7, M. vanbaalenii PYR-1; 8, M. vaccae JOB-5; 9, Mycobacterium sp. 7E1B1W; 10, Rhodococcus rhodochrous 7E1C; 11, M. chlorophenolicum PCP-1; 12, M. frederiksbergense FAn9; 13, ‘M. petroleophilum’; 14, M. austroafricanum GTI-23; 15, M. gilvum (ATCC 43909); 16, Rhodococcus sp. (Dean-Ross et al., 2001); 17, M. smegmatis mc2155.

 
The phtAa PCR product from Mycobacterium sp. PAH2.135 was sequenced and shared 90 % identity with that of M. vanbaalenii PYR-1 between bases 64 and 947 of phtAa. The phtB PCR product from Mycobacterium sp. PAH2.135 was also sequenced and shared 87 % identity between bases 61 and 465 of phtB in M. vanbaalenii PYR-1. Three of the 19 bases in each of the two oligoprobes, phtAa-4 and phtB-2, were different between the two species. The phtAa and phtB PCR products from M. flavescens PYR-GCK were obtained as well and share 96 and 99 % identity, respectively, with the same regions in M. vanbaalenii PYR-1. Of the 19 bases in oligoprobe sequences for phtAa-4 and phtB-2, the M. flavescens PYR-GCK sequence contained 18 and 19 identical bases, respectively.

Strains that were positive in PCR screening for pht operon components were further investigated by Southern analysis. Genomic DNA was subjected to XbaI restriction digestion, PFGE separation and subsequent hybridization with a phtAa-specific probe (Fig. 5). No XbaI site is present in the sequence of M. vanbaalenii PYR-1 phtAa. M. vanbaalenii PYR-1 was used as a positive control and M. austroafricanum (ATCC 33464) and M. gilvum (ATCC 43909) were used as negative controls. Two bands were detected between 82 and 112 kb in M. vanbaalenii PYR-1. Single bands between 15 and 48·5 kb were detected in M. frederiksbergense FAn9 and Rhodococcus sp. (Dean-Ross et al., 2001). Three bands between 82 and 112 kb were detected in M. austroafricanum GTI-23. Three bands in M. flavescens PYR-GCK and two bands in M. gilvum BB1 were detected, all within a 33–97 kb range. No hybridization signal was detected in Mycobacterium sp. PAH2.135.



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Fig. 5. Screening Mycobacterium spp. and Rhodococcus sp. for phtAa. Bacterial genomic DNA was digested with XbaI, separated by PFGE and hybridized with a phtAa-specific probe. Lanes: 1, M. vanbaalenii PYR-1; 2, Mycobacterium sp. PAH2.135 (RJGII-135); 3, M. gilvum BB1; 4, M. flavescens PYR-GCK; 5, M. frederiksbergense FAn9; 6, M. austroafricanum GTI-23; 7, Rhodococcus sp. (Dean-Ross et al., 2001); 8, M. austroafricanum (ATCC 33464); 9, M. gilvum (ATCC 43909).

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In an effort to identify additional M. vanbaalenii PYR-1 genes involved in aromatic hydrocarbon degradation, a genomic library was constructed and used to examine a region of the genome near previously characterized dioxygenase genes (Khan et al., 2001; Stingley et al., 2004). Sequence from one fosmid clone of the region, pFOS608, indicated that multiple PAH-degrading genes are clustered together on the genome.

Based upon sequencing data and metabolite studies, a phthalate-degrading operon is positioned approximately 12–19 kb upstream of the dioxygenase large subunit gene, nidA. Putative products from genes in the operon share homology with those characterized in other species, especially Terrabacter sp. DBF63 (Habe et al., 2003) and A. keyseri 12B (Eaton, 2001). Although BLAST searches indicate little homology at the DNA level, searches of the protein database suggest substantial homology to proteins from the pht operons in these species. Phthalate dioxygenase from two Gram-negative strains, P. putida NMH102-2 and B. cepacia DBO1, showed different genetic organization as compared to nocardiforms and showed less than 25 % sequence homology. The M. vanbaalenii PYR-1 phthalate operon genes were therefore named in the manner of those of A. keyseri 12B (Eaton, 2001).

Phylogenetic analyses of the dioxygenase proteins (PhtAa, PhtAb, PhtAc and PhtAd) from the M. vanbaalenii PYR-1 pht operon confirm a significant relationship with counterpart genes from the Terrabacter sp. DBF63 (Habe et al., 2003) and A. keyseri 12B (Eaton, 2001) pht operons. Sequence alignments indicated that PhtB showed considerable divergence from A. keyseri 12B PhtB (Eaton, 2001), yet was related to Terrabacter sp. DBF63 PhtB (Habe et al., 2003) and other dihydrodiol dehydrogenases (Larkin et al., 1999; Masai et al., 1995; Saito et al., 2000; Treadway et al., 1999). Phylogenetic analysis of PhtB suggests, however, that the protein is closely related to PhtB of both Terrabacter sp. DBF63 and A. keyseri 12B. The M. vanbaalenii PYR-1 regulatory protein, PhtR, and a putative protein of unknown function are also related to similar proteins encoded by Terrabacter sp. DBF63 and A. keyseri 12B genes.

A clone containing the M. vanbaalenii PYR-1 pht operon in E. coli exhibited the ability to degrade phthalate to the expected dihydroxyphthalate. The fragmentation pattern of the TMS derivative of the metabolite obtained was similar to those previously reported for 3,4-dihydroxyphthalate (Chang & Zylstra, 1998; Eaton, 2001; Habe et al., 2003). Based on the sequence homologies among M. vanbaalenii PYR-1 pht operon proteins and those of Terrabacter sp. DBF63 and A. keyseri 12B, the product of phthalate degradation by enzymes from this operon is most likely 3,4-dihydroxyphthalate, rather than 4,5-dihydroxyphthalate, which is more commonly produced by Gram-negative species (Chang & Zylstra, 1998; Eaton, 2001; Habe et al., 2003; Nomura et al., 1992).

The pht operon appears to be conserved within PAH-degrading Mycobacterium spp. Based on PCR and Southern hybridization analyses, all of the PAH-degrading strains examined in this study, except strain PAH2.135, appear to have homologues to phtAa, phtB and the phtAaAbBAcAd region. Although phtAa and phtB probes did not bind to PCR products or genomic digests of strain PAH2.135, the sequences of the PCR products share significant homology with the related portions of strain PYR-1 genes. Among the strains analysed in this study, a correlation between the PAH degradative phenotype and the presence of nidA, nidB and pht genes was observed (Brezna et al., 2003; Khan et al., 2001).

This report is the first to identify and characterize a functional operon involved in biodegradation of phthalate in Mycobacterium spp. Although putative proteins from this operon share significant homology with those from similar operons in Terrabacter sp. DBF63 (Habe et al., 2003) and A. keyseri 12B (Eaton, 2001), the M. vanbaalenii PYR-1 pht operon is distinct both in its overall organization and in its nucleotide sequence. The placement and orientation of the gene encoding the putative regulatory protein are distinctive in M. vanbaalenii PYR-1 (Fig. 2b). In addition, in contrast to the composition of pht operons in Terrabacter sp. DBF63 (Habe et al., 2003) and A. keyseri 12B (Eaton, 2001), no decarboxylase gene is present in the M. vanbaalenii PYR-1 pht operon and none has been identified within that region of the genome. Therefore, the M. vanbaalenii PYR-1 pht operon is distinctly different from related operons in other bacterial genera.


   ACKNOWLEDGEMENTS
 
This work was supported in part by an appointment to the Postgraduate Research Program at the National Center for Toxicological Research administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the US Department of Energy and the US Food and Drug Administration. The authors would like to thank Mr Allen Gies at the University of Arkansas for Medical Sciences for sequencing, Dr James P. Freeman for GC-MS analyses, Ms Joanna Moody and Ms Lisa Mullis for technical support, and Dr Mark Hart and Dr Chris Elkins for critical review of the manuscript.


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Received 20 April 2004; revised 25 June 2004; accepted 12 August 2004.



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