1 Department of Microbiology, Changwon National University, Kyongnam 641-773, Korea
2 Department of Microbiology, Chungbuk National University, Cheongju 361-736, Korea
3 School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea
Correspondence
Kyoung Lee
kyounglee{at}changwon.ac.kr
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ABSTRACT |
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The GenBank accession numbers for the sequences reported in this study are AY324319 (the partial sequence of 16S rDNA) and AY324644 (orf1lapRBKLMNOPCEHIFG).
Present address: KOMED Co., Yatap 151, Bundang, Sungnam, Kyunggi, Korea.
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INTRODUCTION |
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Numerous studies on the microbial catabolism of phenol and the methylphenols (e.g. cresols) have been carried out, and have led to a deeper understanding of the microbial degradation of aromatic compounds, in terms of enzymology, genetics, microbial diversity, and the use of micro-organisms for the removal of toxic chemicals. Studies on the microbial degradation of higher alkylphenols will provide important information to supplement results obtained from simple phenols. To date, the microbial degradation of higher alkylphenols has been studied in pure cultures, which show that microbial degradation is initiated at the phenolic moiety, rather than at the alkyl chain (Ajithkumar et al., 2003; Fujii et al., 2000
; Soares et al., 2003
; Tanghe et al., 1999
, 2000
). However, no information is available on the degradation pathway and the bacterial catabolic genes for higher alkylphenols.
We have isolated a Pseudomonas strain, designated KL28, which can degrade a broad range of 4-n-alkylphenols with an alkyl chain length of C1C5, and identified the gene cluster, designated as lap (for long-chain alkylphenols), responsible for their complete degradation. The results obtained by comparing the deduced amino acid sequences of the Lap proteins with those of previously reported proteins and of studies on the substrate specificities of multicomponent phenol hydroxylase (mPH; EC 1.14.13.7) and catechol 2,3-dioxygenase (C23O; EC 1.13.11.2) allowed us to determine the degradation pathway of higher alkylphenols (the lap pathway) in strain KL28.
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METHODS |
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Construction of the genomic library and the cloning of alkylphenol catabolic genes.
The total DNA of strain KL28 was purified as previously described (Ausubel et al., 1990). The DNA was partially digested with Sau3AI. Fragments in the range 2535 kb were isolated by ultracentrifugation over a sucrose density gradient and ligated into cosmid pLAFR3 (Staskawicz et al., 1987
) that had been treated with BamHI and calf intestine dephosphatase. After in vitro packaging into
bacteriophage, using a Gigapack III Packaging extract (Stratagene), the E. coli VCS257 (Stratagene) host strain was transfected with the phage. Cosmid clones in pLAFR3 were mobilized from the E. coli strain to P. putida G7.C-1, by triparental mating in the presence of E. coli HB101(pRK2013) (Figurski & Helinski, 1979
). Recombinant P. putida G7.C-1 cells containing the alkylphenol catabolic genes were positively selected in Tc-containing MSB medium with 4-ethylphenol supplied in the vapour phase. One recombinant plasmid, designated pJJ2, conferred upon P. putida G7.C-1 the ability to grow on higher alkylphenols and was selected for further study. Other molecular genetic techniques were performed using standard procedures (Sambrook & Russell, 2001
) and as recommended by the reagent suppliers.
DNA sequence analysis.
The insert in pJJ2 was digested with various restriction enzymes and subcloned in pBluescript SK(-) (Stratagene). Subclones were used as templates for DNA sequencing. Nucleotide sequences were determined by Genotech Co. (Taejeon, Korea) using an automated sequencing unit (ABI PRISM 377, PE Biosystems) with M13 and sequence-based primers. Searches for specific nucleotide or amino acid sequences were carried out using the BLAST program (Altschul et al., 1997) provided by DDBJ/GenBank/EMBL and the ExPASy Interface to EMBnet-CH/SIB/CSCS provided by the Swiss Institute of Bioinformatics (SIB), as available on the internet. The nucleotide sequence of the partial 16S rDNA gene of strain KL28 was determined by direct sequencing of the PCR product amplified using 27F and 1522R primers (Johnson, 1994
) with Ex-Taq DNA polymerase (TaKaRa, Japan).
Construction of plasmids.
pJJPMO2 (Gmr) was constructed by cloning the 5·8 kb XhoIEcoRI fragment carrying the mPH genes from pJJ2 into the corresponding restriction sites of pBBR1MCS-5 (Kovach et al., 1995) (see Fig. 1
). pJJR1 (Gmr) is a PlapB-gfp vector and was constructed by cloning a 1·3 kb SalIBamHI fragment of pJJ2 into the same restriction sites of pPROBE-GT (Miller et al., 2000
) (see Fig. 1
). pJJR2 (Gmr) is a PlapR-gfp vector and was constructed by cloning the 1·0 kb PstIDraI fragment into pPROBE-GT digested with SmaI and PstI (see Fig. 1
). pJJXYLE (Apr) was constructed by cloning the 1·0 kb PCR fragment of the xylE gene amplified with the primers 5'-CTATGAAGGAGTGACGTCATGAAC-3' and 5'-CATCTGCACAATCTCTGCAATAAG-3' from the total DNA of P. putida mt-2 (Murray et al., 1972
) into a PCR product cloning vector, pEZ-T (RNA Co.). pJJC23O (Apr) was made by cloning a 1·0 kb PCR fragment carrying the lapB gene amplified with 5'-CCGCATCAAGCCAATAATGGAG-3' and 5'-GGCTGCATGTCCAGTGACTC-3' from pJJ2 into pEZ-T. PCR was carried out as previously described (Choi et al., 2003
) but with a polymerization time of 1 min.
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Induction studies with green fluorescent protein (GFP) as the reporter.
Pseudomonas sp. strain KL28(pJJR1) was grown in MSB liquid medium containing pyruvate (10 mM) and Gm. After 24 h, alkylphenols (final concentration 1 mM from 1 M stock in methanol) were added to the culture media. Cells were further grown and 2 ml aliquots were harvested every 24 h by centrifugation, washed twice with saline, and resuspended in saline at an OD600 of around 0·2. The expression level of GFP was measured using a spectrofluorophotometer (model RF-5391PC, Shimadza Co.) as previously described (Choi et al., 2003). Pseudomonas sp. strain KL28(pJJR2) and its control strain Pseudomonas sp. strain KL28(pPROBE-GT) were grown in LB liquid medium with Gm. The expression level of GFP was measured 24 h after inoculation.
Biotransformation of (alkyl)phenols by recombinant E. coli DH5(pJJPMO2) cells expressing mPH.
E. coli DH5(pJJPMO2) was grown in LB broth with Gm at 28 °C. Expression of the cloned genes was induced by adding IPTG (final concentration 0·25 mM), as described previously (Lee et al., 1997
). Cells harvested by centrifugation were suspended in 50 mM sodium phosphate buffer (pH 7·0) with 20 mM glucose to an OD600 of 1. Phenol or alkylphenol was then added to each flask to a concentration of 0·05 % (v/v) in a culture volume of 50 ml. Biotransformation was carried out at 28 °C with shaking at 180 r.p.m. for 48 h. The culture supernatants were extracted with ethyl acetate and the extracts were concentrated by rotary evaporation under vacuum at 45 °C. The concentrated extracts were analysed by TLC using silica gel 60 F254 (2 mm thickness; E. Merck) with chloroform/acetone (95 : 5, v/v). GC-MS analysis and oxygen consumption assays with cell extracts were carried out as previously described (Cho et al., 2000
).
Measurement of substrate preference of C23O.
E. coli DH5 harbouring pDTG617 (Zylstra & Gibson, 1991
), pJJXYLE and pJJC23O are recombinant cells expressing catechol dioxygenase encoded by todE, xylE and lapB, respectively. Cell extracts were obtained as previously described (Cho et al., 2000
) and used to determine if the three C23Os could form the same product from a given catechol substrate. Assay mixtures (total 1 ml) contained potassium phosphate buffer (0·1 M, pH 7·5), catechol substrate (final concentration 0·1 mM) and cell extract (75 µg) and incubated at 28 °C with gentle agitation for 30 min. Product formation was monitored by scanning with a spectrophotometer (model 2130, Scinco, Korea). The specific activity of LapB was determined using a cell extract of E. coli DH5
(pJJC23O) in the same phosphate buffer by measuring the rate of formation of meta-cleavage products from (substituted) catechols. The wavelengths (absorption coefficients in mM-1 cm-1) used to monitor the formation of the meta-cleavage products of catechol, 3-methylcatechol, 4-methylcatechol, 4-ethylcatechol and 2,3-dihydroxybiphenyl were 376 (40), 389 (11·9), 382 (24·5), 381 (36·0) and 434 (19·8) nm, respectively (Bayly et al., 1966
; Duggleby & Williams, 1986
; Ramos et al., 1987
; Seah et al., 1998
). Protein concentrations were determined using the BCA protein assay (Pierce) with BSA as the standard. Specific enzyme activities are reported as µmol product formed min-1 (mg protein)-1. Activity assays were conducted in triplicate, and the initial rates of the assays were determined and used to calculate mean and standard deviations.
Chemicals.
The aromatic compounds used in this study were obtained from Aldrich, except for the following: 4-n-butylphenol and 4-n-pentylphenol from Lancaster Synthesis, Morecambe, UK; 4-n-hexylphenol from Kanto Chemical, Japan; 3-ethylphenol, 2-hydroxy- and 4-hydroxybiphenyl from Fluka Chemica; and 2,3-dihydroxybiphenyl from Wako Pure Chemicals. Enzymes and reagents used for DNA manipulation were purchased from Takara, KOSCO, Promega and Pharmacia. The commercial phenotype identification API 20NE kit was obtained from API Analytab Products.
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RESULTS |
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Cloning of alkylphenol catabolic genes from strain KL28
Various efforts to detect a plasmid in strain KL28 failed, indicating that the genes for alkylphenol degradation are encoded on its chromosome. In order to identify the genes responsible for the degradation of alkylphenols in strain KL28, a genomic library was made using pLAFR3, as described in Methods. pLAFR3 is a cosmid vector (Staskawicz et al., 1987). It can be mobilized by conjugation and can replicate in various Gram-negative bacteria. The genomic library in E. coli cells was transferred via conjugation to P. putida G7.C-1, which cannot grow on alkylphenols. The recombinant strains obtained were screened for their ability to grow on 4-ethylphenol as a carbon and energy source. Eight different transconjugants were isolated and found to contain recombinant plasmids, all of which were apparently identical in size and orientation in the insert. One of the recombinant strains, containing a recombinant plasmid named pJJ2, was selected for further studies. P. putida G7.C-1(pJJ2) could grow on 4-n-alkylphenols (C1C5) as the sole sources of carbon and energy at growth rates similar to those by KL28 (data not shown). The transconjugant strain also grew on 4-n-hexylphenol as the sole source of carbon and energy. When plasmid pJJ2 was introduced into E. coli DH5
, the recombinant strain obtained did not degrade alkylphenols and did not produce indigo from indole. The latter biochemical reaction is caused by E. coli cells that express mPHKL28 (see below). These results showed that the alkylphenol catabolic genes cloned from strain KL28 are not expressed in E. coli, indicating that cis or/and trans elements necessary for the transcription of the cloned genes in E. coli and Pseudomonas may be different.
Analyses of the nucleotide and deduced amino acid sequences of the lap genes
Subcloning and restriction analysis of pJJ2 showed that the insert was approximately 25·5 kb in size. When a subcloned plasmid (pJJPMO2) (Fig. 1) was introduced into E. coli DH5
, the recombinant E. coli strain was able to produce indigo on LB agar even in the absence of IPTG. This result indicated that the DNA fragment contains genes encoding an oxygenase with expression under control of the lac promoter from the vector. The production of indigo is due to indole formation by tryptophanase in E. coli and indoxyl formation by the oxygenase from the plasmid, as was previously shown by E. coli cells expressing naphthalene dioxygenase (Ensley et al., 1983
). Sequence analysis of the fragment showed that it contains six complete genes (designated lapKLMNOP) for mPH, which was first identified in Pseudomonas sp. CF600 by Nordlund et al. (1990)
. Comparison of the amino acid sequence of the oxygenase component with those of other non-haem iron oxygenases revealed that the active site of the oxygenase component contains a dinuclear iron centre that is liganded by a pair of a conserved motif (Asp/Glu)-Glu-Xaa-Arg-His (Fox et al., 1993
). The role of the amino acid sequence motif was further confirmed in methane monooxygenase crystal structures (Elango et al., 1997
; Rosenzweig et al., 1997
). Recently, Cadieux et al. (2002)
reported experimental evidence for the presence of the binuclear iron centre in a purified mPH. The conserved amino acid sequence fingerprints were found at positions 138142 and 233237 in LapN, which constitutes a catalytic oxygenase component with LapL and LapO in a putative
2
2
2 hexamer (Cadieux et al., 2002
). In most cases, the phenol catabolic genes are clustered. Thus, we further subcloned and sequenced in both directions from the oxygenase genes including the AvrII site to the downstream end (Fig. 1
). The sequenced region consisted of 14 471 bp with a G+C content of 59·8 mol%. It contained 13 complete and 2 incomplete ORFs (Table 1
). The gene products showed some degree of sequence identity (3374 %) with their counterparts in the (methyl)phenol catabolic dmp operon in Pseudomonas sp. CF600 (Bartilson & Shingler, 1989
; Nordlund et al., 1990
; Shingler et al., 1992
, 1993
) and the phenol catabolic aph operon from Comamonas testosteroni TA441 (Arai et al., 1998
, 1999
, 2000
) (Table 1
). The complete nucleotide sequences containing mPH genes are only known for the dmp and aph operons. Thus, the functions of the regulatory gene product (LapR) and of the catabolic gene products (LapBKLMNOPCEHIFG) can be readily inferred as indicated in Table 1
. The identified genes constitute a catabolic pathway for the degradation of alkylphenols to TCA cycle intermediates. The lapG gene encodes 4-hydroxy-2-oxovalerate aldolase and is located at the end of the cloned DNA fragment. However, it lacks 1618 amino acids at the carboxyl terminal compared to known LapG homologues, but may be functionally active because its insert in pJJ2 supported the growth of strain G7.C-1 on alkylphenols. Interestingly, the lap gene cluster does not contain genes encoding ferredoxin, which are generally located adjacent to a gene encoding C23O, and 2-hydroxymuconic semialdehyde (HMS) hydrolase for the hydrolytic branch of the meta pathway. Furthermore, the order of the lap genes is not found in other meta-cleavage operons.
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Nucleotide sequence features in PlapB and the induction of the lap catabolic operon by (alkyl)phenols
The nucleotide sequence upstream of lapB has the following sequence features: a putative 54-dependent -24/-12-type promoter [5'-TGGCACCATCTCTGCA-3' of consensus sequence 5'-TGGC-N8-TGCA-3', where N represents any nucleotide (Thony & Hennecke, 1989
)], the putative IHF-binding region [5'-GATCAATGCTTTA-3' of consensus sequence 5'-WATCAAN4TTR-3' where W=A or T; R=A or G (Friedman, 1988
)], and two palindromic elements (Fig. 2
). These sequence features, except for the second inverted repeat sequence, are well characterized in the Po and Pu promoter regions, which are controlled by the aromatic effector-responsive XylR and DmpR transcriptional activators, respectively (Abril et al., 1991
; de Lorenzo et al., 1991
; Sze et al., 2001
). The promoter sequence and the amino acid sequence similarity of LapR to DmpR and XylR suggest that the expression of PlapB is controlled by the regulatory protein LapR. In order to determine the range of effectors of the lap operon, the transcriptional activity of the lapB promoter (PlapB) in response to various chemicals was assessed by monitoring the expression of GFP from the gfp-fusion plasmid, pJJR1 (Fig. 1
). The expression of GFP was determined from Pseudomonas sp. KL28(pJJR1) as described in Methods. Expression of GFP was induced by phenol and by a broad range of (alkyl)phenols, which served as carbon and energy sources for strain KL28 (Table 2
). GFP expression was high in the presence of 3-ethylphenol, 4-ethylphenol, m-cresol and phenol, with a preference for 3- and 4-alkylphenols over 2-alkylphenols. However, GFP was not expressed in the presence of aromatic hydrocarbons such as benzene, p-xylene, toluene, biphenyl and naphthalene (data not shown). This induction pattern differs from those of DmpR, PhhR and MopR (Ng et al., 1995
; Schirmer et al., 1997
; Shingler & Moore, 1994
), all of which are found in phenol degraders. These regulatory proteins show no preference for 3- and 4-alkylphenols and are more responsive to simple phenols.
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DISCUSSION |
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The chromosomally encoded lap catabolic gene cluster of strain KL28 can be compared to the dmp and aph phenol catabolic gene clusters, where all the operonic genes including those for mPH have been cloned and sequenced. The dmp gene cluster located in the pVI150 catabolic plasmid of Pseudomonas sp. strain CF600 allows strain CF600 to grow on phenol, o-, m- or p-cresol, 3,4-dimethylphenol or 2-ethylphenol as sole sources of carbon and energy (Shingler et al., 1989). Regulatory mutants of strain CF600 also have the ability to grow on 4-ethylphenol as a carbon and energy source (Sarand et al., 2001
). However, the wild-type and mutant strains have not been reported to grow on higher alkylphenols. C. testosteroni TA441, an isolate from the gut of the wood-feeding termite Reticulitermes speratus, does not grow on phenol as a sole carbon source initially, but it is able to utilize phenol by derepression of the aph gene cluster after adaptation in a medium containing phenol as a sole source of carbon and energy (Arai et al., 1998
).
While the deduced amino acid sequences of the lap genes are 3374 % identical to the corresponding proteins in the dmp and aph operons or show identities similar to isofunctional proteins from other phenol catabolic operons, the arrangement of the lap catabolic genes differs from those of known phenol catabolic genes that include mPH genes. In most cases, mPH genes are followed by genes encoding a XylT-type ferredoxin (Hugo et al., 1998) and C23O, which are equivalent to dmpQB and aphQB in strains CF600 and TA441, respectively (Fig. 3
). Other examples include the phh operon from P. putida P35X (Ng et al., 1994
), the phl operon from P. putida H (Herrmann et al., 1995
), the pox operon R. eutropha E2 (Hino et al., 1998
), the phc operon from C. testosteroni R5 (Teramoto et al., 1999
), the phe operon from Ralstonia sp. KN1 (Nakamura et al., 2000
) and the phk operon from Burkholderia kururiensis (unpublished, GenBank accession no. AB063252). In the case of the mop operon from Acinetobacter calcoaceticus NCIB 8250, the genes encoding mPH are followed by a catechol 1,2-dioxygenase (C12O) gene (Ehrt et al., 1995
). Furthermore, in the mph operon from A. calcoaceticus PHEA-2, the genes encoding mPH and C12O are separated by six ORFs that are related to benzoate degradation (Xu et al., 2003
). In contrast, in the lap genetic organization, the gene encoding C23O precedes the genes encoding mPH, which are followed by genes encoding enzymes that degrade HMS derivatives to TCA cycle intermediates. In addition, the order of the rest of the genes for the meta pathway operon of strain KL28 differs from those of Pseudomonas sp. CF600 and C. testosteroni TA441 (Fig. 3
). The deduced gene products from a DNA fragment encoding bupBA1A2A3A4A5A6 from the butylphenol degrader P. putida MT4 (GenBank accession no. AB107791) showed highest sequence identity (6598 %) to lapBKLMNOP with the same gene order, indicating that its gene organization may be similar to that of the lap operon. The lap gene products also showed high sequence identities (6187 %) with unassigned gene products found in the Azotobacter vinelandii genome (GenBank accession number NZ_AAAU02000027). In the latter case, the order of the gene cluster is the same as the lap catabolic operon, but a group of eight unrelated ORFs is inserted between the genes corresponding to lapP and lapC. These results indicate that the genetic organization of the lap operon is an emerging module for meta-cleavage operons.
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The first step in lap degradation is the conversion of 3- and 4-n-alkylphenols to 4-alkylcatechols, which is catalysed by mPH (LapKLMNOP) (Table 1). Phenol is also oxidized to catechol by flavin-containing single-component phenol hydroxylases (Ballou, 1982
). It is important to note that the mPH in strain KL28 forms only 4-alkylcatechols from hydroxylation of 3-alkylphenols (Table 3
). Many single and multicomponent phenol hydroxylases characterized to date catalyse m-cresol to 3-methylcatechol or a mixture of 3- and 4-methylcatechol (Arenghi et al., 2001
; Johnson & Olsen, 1997
; Kukor & Olsen, 1992
; Shingler, 1996
). An exception is C23O from the thermophilic Bacillus stearothermophilus, which catalyses the conversion of m-cresol to 4-methylcatechol (Buswell, 1975
). In addition, the aforementioned phenol hydroxylases oxidize 2-alkylphenols to 3-alkylcatechols with high activity, whereas the mPH of KL28 shows weak or no activity toward 2-alkylphenols (Table 3
), indicating that mPH in the lap pathway possesses a unique specificity. It is further noted that the ability of mPHKL28 to oxidize indole to indigo is dissimilar from that of mPHCF600, which has been reported to produce a red pigment, not indigo (Powlowski & Shingler, 1990
). Understanding of the reaction mechanism and the regiospecificity of mPH will be greatly aided by elucidation of its structure.
The ring fission products (variably substituted HMS) formed by C23O can be further degraded by dehydrogenation (oxalocrotonate branch) or hydrolysis (hydrolytic branch), where the former needs an electron acceptor such as NAD+ (Fig. 4). It was first observed in P. putida U that the ring fission products formed from 3-methylcatechol and (4-methyl)catechol follow the hydrolytic and oxalocrotonate branches, respectively (Sala-Trepat et al., 1972
; Wigmore et al., 1974
). This was further confirmed in Pseudomonas sp. CF600 and P. putida containing the TOL plasmid pWWO, with mutants defective in the specific genes (Harayama et al., 1987
; Powlowski & Shingler, 1994
). The lack of formation of 3-alkylcatechol intermediates in the lap pathway may have led evolutionarily to a deficiency of the hydrolytic branch enzyme (equivalent to dmpD in strain CF600). The latter enzyme is also not found in the aph pathway in C. testosteroni TA441, which degrades phenol but not alkylphenols (Arai et al., 2000
) (Fig. 3
).
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ACKNOWLEDGEMENTS |
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Received 7 July 2003;
revised 7 August 2003;
accepted 26 August 2003.
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