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
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ABSTRACT |
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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).
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INTRODUCTION |
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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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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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METHODS |
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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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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 cm1 with a 1·28·5 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 ml1 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 1618 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 min1 and a total analysis time of 25 min. The amount of degradation was calculated from the peak area as compared to control.
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RESULTS |
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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 helixturnhelix 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 1618 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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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 3397 kb range. No hybridization signal was detected in Mycobacterium sp. PAH2.135.
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DISCUSSION |
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Based upon sequencing data and metabolite studies, a phthalate-degrading operon is positioned approximately 1219 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.
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ACKNOWLEDGEMENTS |
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Received 20 April 2004;
revised 25 June 2004;
accepted 12 August 2004.
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