and 4-alkylphenol degradation pathway in Pseudomonas ... - CiteSeerX

Microbiology (2003), 149, 3265–3277

DOI 10.1099/mic.0.26628-0

3- and 4-alkylphenol degradation pathway in Pseudomonas sp. strain KL28: genetic organization of the lap gene cluster and substrate specificities of phenol hydroxylase and catechol 2,3-dioxygenase Jae Jun Jeong,13 Ji Hyun Kim,1 Chi-Kyung Kim,2 Ingyu Hwang3 and Kyoung Lee1 Correspondence

1

Department of Microbiology, Changwon National University, Kyongnam 641-773, Korea

Kyoung Lee

2

[email protected]

3

Received 7 July 2003 Revised 7 August 2003 Accepted 26 August 2003

Department of Microbiology, Chungbuk National University, Cheongju 361-736, Korea School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea

The enzymes and genes responsible for the catabolism of higher alkylphenols have not been characterized in aerobic bacteria. Pseudomonas sp. strain KL28 can utilize a wide range of alkylphenols, which include the 4-n-alkylphenols (C1–C5). The genes, designated as lap (for long-chain alkylphenols), encoding enzymes for the catabolic pathway were cloned from chromosomal DNA and sequenced. The lap genes are located in a 13?2 kb region with 14 ORFs in the order lapRBKLMNOPCEHIFG and with the same transcriptional orientation. The lapR gene is transcribed independently and encodes a member of the XylR/DmpR positive transcriptional regulators. lapB, the first gene in the lap operon, encodes catechol 2,3-dioxygenase (C23O). The lapKLMNOP and lapCEHIFG genes encode a multicomponent phenol hydroxylase (mPH) and enzymes that degrade derivatives of 2-hydroxymuconic semialdehyde (HMS) to TCA cycle intermediates, respectively. The PlapB promoter contains motifs at positions ”24(GG) and ”12(GC) which are typically found in s54-dependent promoters. A promoter assay using a PlapB : : gfp transcriptional fusion plasmid showed that lapB promoter activity is inducible and that it responds to a wide range of (alkyl)phenols. The structural genes encoding enzymes required for this catabolism are similar (42–69 %) to those encoded on a catabolic pVI150 plasmid from an archetypal phenol degrader, Pseudomonas sp. CF600. However, the lap locus does not include genes encoding HMS hydrolase and ferredoxin. The latter is known to be functionally associated with C23O for use of 4-alkylcatechols as substrates. The arrangement of the lap catabolic genes is not commonly found in other meta-cleavage operons. Substrate specificity studies show that mPH preferentially oxidizes 3- and 4-alkylphenols to 4-alkylcatechols. C23O preferentially oxidizes 4-alkylcatechols via proximal (2,3) cleavage. This indicates that these two key enzymes have unique substrate preferences and lead to the establishment of the initial steps of the lap pathway in strain KL28.

INTRODUCTION Phenol is produced naturally by the breakdown of plant materials during microbial degradation. In contrast, phenols with bulky alkyl group(s) that are found in the environment originate predominantly from anthropogenic

sources. For instance, nonyl- and octylphenol are pollutants that are commonly found in aquatic environments. They are recalcitrant intermediates that are formed during the microbial breakdown of alkylphenol ethoxylates (Ferguson et al., 2001; Giger et al., 1984). Alkylphenol

3Present address: KOMED Co., Yatap 151, Bundang, Sungnam, Kyunggi, Korea. Abbreviations: C12O, catechol 1,2-dioxygenase; C23O, catechol 2,3-dioxygenase; GFP, green fluorescent protein; HMS, 2-hydroxymuconic semialdehyde; IHF, integration host factor; lap, long-chain alkylphenol; mPH, multicomponent phenol hydroxylase. The GenBank accession numbers for the sequences reported in this study are AY324319 (the partial sequence of 16S rDNA) and AY324644 (orf1lapRBKLMNOPCEHIFG).

0002-6628 G 2003 SGM

Printed in Great Britain

3265

J. J. Jeong and others

ethoxylates are highly effective cleaning agents or surfactants used in the textile, petroleum and paper industries. Alkylphenols, which are used for the synthesis of alkylphenol ethoxylates, are also extensively used as lubricating oil additives, antioxidants, stabilizers for rubbers and plastics, dispersants, emulsifiers and plasticizers for resins. Alkylphenols are environmentally hazardous due to their toxicity, and they have been detected in some aquatic biological systems (Ferrara et al., 2001; Keith et al., 2001). Some alkylphenols are also known as endocrine-disrupting chemicals that mimic oestrogenic compounds, causing adverse effects such as developmental and reproductive toxicity in animals (Sharpe et al., 1995; Tanaka & Grizzle, 2002). Studies on the degradation of higher alkylphenols by micro-organisms are therefore receiving significant attention. 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 microorganisms 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 C1–C5, 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.

METHODS Bacterial strains and culture conditions. Alkylphenol-degrading

strains were initially isolated from soils in an industrial complex in Changwon, Korea. Enrichment was carried out using 4-ethylphenol as a carbon and energy source in minimal salts basal (MSB) medium (Stanier et al., 1966). One strain, designated KL28, which showed the highest growth, was isolated for further study. Strain KL28 was routinely grown in Luria–Bertani (LB) medium (Sambrook & Russell, 2001). Alkylphenols were supplied in the vapour phase in plates or added directly to liquid culture at 0?04 % (v/v) as sole sources of carbon and energy in MSB medium. Other solid chemicals were supplemented at 0?04 % (w/v) and volatile chemicals such as benzene, toluene and styrene were supplied in the 3266

vapour phase. Escherichia coli DH5a (Ausubel et al., 1990) was used as a host strain for the maintenance of plasmids. To culture transformed E. coli or Pseudomonas, ampicillin (Ap), gentamicin (Gm) or tetracycline (Tc) was incorporated into the culture media at the amounts indicated previously (Choi et al., 2003). E. coli and Pseudomonas cells were grown at 37 uC and 28 uC, respectively. In liquid culture, cells were grown in a 50 ml culture volume in 250 ml Erlenmeyer flasks in a shaking incubator at 180 r.p.m. Pseudomonas putida G7.C-1 is an NAH-7 plasmid cured derivative of strain G7 (Dunn & Gunsalus, 1973). Construction of the genomic library and the cloning of alkylphenol catabolic genes. The total DNA of strain KL28 was puri-

fied as previously described (Ausubel et al., 1990). The DNA was partially digested with Sau3AI. Fragments in the range 25–35 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 l 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(2) (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 XhoI–EcoRI 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 SalI–BamHI 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 PstI–DraI 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 59-CTATGAAGGAGTGACGTCATGAAC-39 and 59-CATCTGCACAATCTCTGCAATAAG-39 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 59-CCGCATCAAGCCAATAATGGAG-39 and 59-GGCTGCATGTCCAGTGACTC-39 from pJJ2 into pEZ-T. PCR was carried out as previously described (Choi et al., 2003) but with a polymerization time of 1 min. RT-PCR. c-DNA from the total RNA of strain KL28, grown on

Microbiology 149

Bacterial aerobic alkylphenol degradation

Fig. 1. Genetic organization of the lap gene cluster in Pseudomonas sp. KL28. The locations and transcriptional directions of the genes encoding the pathway enzymes are indicated by arrows. Plac is a promoter from pLAFR3. Positions of primers used for RT-PCR are indicated at the top. The plasmids used in this study are shown at the bottom.

4-ethylphenol as sole source of carbon and energy, was produced and used for PCR as previously described (Cho et al., 2000), but with a polymerization time of 1?5 min. Positive controls contained the chromosomal DNA of strain KL28 as a template in the PCR reaction. Negative controls were tested by PCR without the reverse transcription step for possible DNA contamination in the sample preparations. The products formed by PCR were confirmed by DNA sequence analysis. The primers used were 790, 59-CCGAGCTAGACAGATAGGTC-39; 2120, 59-CAGCAGCAGCACTGTTATC-39; 2550, 59-CTGCGTCATCGTAAGTCGGAT-39; 3937, 59-GGTCTAGCTGTATGGGAG-39; 4195, 59-AACGTGGACATCGGTCCAA-39; 5197, 59-GTGCCGTAGCCATAGCTACAG-39. 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. http://mic.sgmjournals.org

Biotransformation of (alkyl)phenols by recombinant E. coli DH5a(pJJPMO2) cells expressing mPH. E. coli DH5a(pJJPMO2)

was grown in LB broth with Gm at 28 uC. 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 uC 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 uC. 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 DH5a

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 mg) and incubated 3267

J. J. Jeong and others at 28 uC 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 DH5a(pJJC23O) in the same phosphate buffer by measuring the rate of formation of meta-cleavage products from (substituted) catechols. The wavelengths (absorption coefficients in mM21 cm21) used to monitor the formation of the metacleavage 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 mmol product formed min21 (mg protein)21. 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 2,3dihydroxybiphenyl 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.

RESULTS Identification of strain KL28 and its growth on various alkylphenols The almost complete nucleotide sequence of 16S rDNA (1453 bp) of strain KL28 showed 99 % sequence identity to P. putida strains ATCC 12633 and ATCC 11172 and P. asplenii ATCC 23835T. In addition, biochemical testing with API 20NE showed that strain KL28 is closest to the genus Pseudomonas. In MSB liquid medium, strain KL28 was tested for its ability to degrade a range of alkylphenols as the sole sources of carbon and energy. It grew from an OD600 of 0?02 to an OD600 of 0?4–0?5 with a doubling time of 5–7 h within 2 days in the presence of m-cresol, p-cresol, 3-ethylphenol, 4-ethylphenol or 4-propylphenol. The same level of growth was obtained within 4–5 days in the presence of 4-butylphenol or 4-pentylphenol with doubling times of 15 and 21 h, respectively. However, the strain did not grow in the presence of phenol, o-cresol, 2-ethylphenol, 4-n-alkylphenols (C6–C9), 4-hydroxyacetophenone, 2- or 4-hydroxybiphenyl, 2,3-, 2,6- or 3,5-dimethylphenol, 2-, 3or 4-chlorophenol, benzene, toluene, styrene, biphenyl or naphthalene. The strain also grew in the presence of 4methylcatechol or 4-ethylcatechol, but not in the presence of catechol. During growth with p-cresol and 4-ethylphenol, strain KL28 produced a yellow-coloured diffusible product, presumably a meta-ring cleavage product. 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 3268

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 (C1–C5) 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 DH5a, 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 DH5a, 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 Microbiology 149


purified mPH. The conserved amino acid sequence fingerprints were found at positions 138–142 and 233–237 in LapN, which constitutes a catalytic oxygenase component with LapL and LapO in a putative a2b2c2 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 (33–74 %) 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

Table 1. Properties of the lap genes identified from pJJ2 Gene

Coding region

G+C (mol%)

Protein*

orf1

1–1249

61

Putative methyl-accepting protein

lapR

1362–3041

58

XylR/DmpR-type regulator

lapB

3478–4404

59

C23O

lapK

4417–4707

61

mPH (assembly)

lapL

4758–5747

61

mPH (b subunit)

lapM

5760–6026

56

mPH (activator)

lapN

6045–7562

58

mPH (a subunit)

lapO

7620–7973

60

mPH (c subunit)

lapP

7987–9042

62

mPH (reductase)

lapC

9258–10715

63

HMS dehydrogenase

lapE

10726–11520

61

2-Hydroxypent-2,4-dienoate hydratase

lapH

11520–12320

59

4-Oxalocrotonate decarboxylase

lapI

12347–12556

58

4-Oxalocrotonate isomerase

lapF

12556–13467

62

Acetaldehyde dehydrogenase (acylating)

lapG

13470–14471

63

4-Hydroxy-2-oxovalerate aldolase

No. of amino acid residues and size (kDa)

(truncated) 417 43?6 540 60 309 35?1 97 11?2 362 41?1 89 10 506 58?9 118 13?2 379 41?5 486 52?9 265 28?3 267 28?9 70 7?7 304 32?7 (truncated) 334 35?5

Equivalent to Dmp/AphD Identity (%)d

GenBank accession no.

Protein

–

–

–

42 48 51 42 43 41 53 52 54 56 61 59 44 46 60 64 69 70 60 60 55 51 43 33 55 74 55 67

DmpR AphR DmpB AphB DmpK AphK DmpL AphL DmpM AphM DmpN AphN DmpO AphO DmpP AphP DmpC AphC DmpE AphE DmpH AphH DmpI AphI DmpF AphF DmpG AphG

X68033 AB006480 M33263 AB006479 M60276 AB006479 M60276 AB006479 M60276 AB006479 M60276 AB006479 M60276 AB006479 M60276 AB006479 X52805 AB029044 X60835 AB029044 X60835 AB029044 X60835 AB029044 X60835 AB029044 X60835 AB029044

–, No corresponding gene was found in strains CF600 and TA441. *Deduced from the corresponding genes of Pseudomonas sp. CF600 and C. testosteroni TA441 (see text for references). DThe arrangement and composition of the catabolic genes are not equal in the three (alkyl)phenol degradation operons (see Discussion). dResults of NCIB BLAST 2 sequences. http://mic.sgmjournals.org

3269


encodes 4-hydroxy-2-oxovalerate aldolase and is located at the end of the cloned DNA fragment. However, it lacks 16–18 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. RT-PCR analysis of the transcription of lap genes DmpR in the dmp operon belongs to the XylR/DmpR subfamily of the NtrC family of positive transcriptional activators (Inouye et al., 1988; Shingler et al., 1993). The putative transcriptional regulatory protein LapR is homologous to DmpR (Table 1) and also shares 43 % sequence identity with XylR, which controls the upper pathway operon of the TOL plasmid pWW0 at the Pu promoter (Abril et al., 1991), suggesting that LapR also belongs to the same family of transcriptional activators. The lapR gene is transcribed in the same direction as the flanking genes (Fig. 1). In order to determine if the lapR gene is cotranscribed with the adjacent genes, RT-PCR was carried out as described in Methods. Although positive and negative controls gave the expected PCR results, only PCR primers of set 3 in Fig. 1 generated the expected RT-PCR product (data not shown). This result indicates that the lapR gene is independently transcribed from adjacent genes, and that the gene encoding C23O is co-transcribed with genes encoding mPH. The possibility of the presence of a promoter upstream of the lapR gene was investigated with a GFP reporter, as constructed in pJJR2 (Fig. 1). The level of the specific GFP expression of Pseudomonas sp. KL28(pJJR2) was 6?7±1, whereas the control level of the specific GFP expression by Pseudomonas sp. KL28 (pPROBE-GT) was 1?8±0?3. This result further indicates the presence of a promoter in front of the lapR gene.

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 s54-dependent 224/ 212-type promoter [59-TGGCACCATCTCTGCA-39 of consensus sequence 59-TGGC-N8-TGCA-39, where N represents any nucleotide (Thony & Hennecke, 1989)], the putative IHF-binding region [59-GATCAATGCTTTA-39 of consensus sequence 59-WATCAAN4TTR-39 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.

Fig. 2. Regulatory region of the lap operon. The ATG initiation codon of lapB is indicated in bold. Putative ribosome-binding (RBS) and IHF-binding (IHFBS) regions are indicated by the thin and double underlines, respectively. The ”12 and ”24 sequences of the lapB promoter, PlapB, are boxed and labelled accordingly. Arrows in both directions indicate an inverted repeat. 3270

Microbiology 149


Table 2. Expression of the lap operon based on PlapB in response to different alkylphenols Pseudomonas sp. KL28(pJJR1) was used as a reporter strain. Other experimental conditions are described in Methods. Chemical

Specific GFP expression* at 24 h

None Phenol o-Cresol m-Cresol p-Cresol 2-Ethylphenol 3-Ethylphenol 4-Ethylphenol 2-Propylphenol 4-Propylphenol 4-Butylphenol 4-Pentylphenol 4-Hexylphenol 4-Heptylphenol 4-Octylphenol 4-Nonylphenol

1?6 7?8 2?5 26?3 5?7 1?7 21?2 20?2 5?7 11?2 23?2 19?3 2?1 2?3 2?2 2?3

48 h

72 h

3?1 14?6 4?2 36?2 20?8 2?3 21?4 15?2 11?3 11?3 18?0 24?5 1?5 1?8 1?5 2?0

3?2 10?3 2?9 27?7 24?3 2?5 16?9 15?4 7?1 12?6 18?2 23?1 4?8 4?0 2?5 2?1

*Units are arbitrary and represent the mean of duplicate determinations in two independent experiments.

Substrate preference of mPHKL28 While the products of the lap genes are compatible with those in the dmp and aph operons, the first two steps of the alkylphenol degradation, which involve the hydroxylation

of alkylphenols and the ring cleavage of catechols, can show variations in regioselectivity and in the mode of ring cleavage, respectively. First, to determine the substrate preference and regioselectivity of mPHKL28, biotransformations were carried out as described in Methods with E. coli DH5a(pJJPMO2). The products formed were identified by TLC and GC-MS. mPH catalysed the formation of catechols from a broad-range of (alkyl)phenols with a preference for 3- and 4-alkylphenols over 2-alkylphenols (Table 3). In the case of 3-alkylphenols, hydroxylation occurred at the carbon at the distal position from the alkyl substitution, yielding 4-alkylcatechols. Products from 2-ethylphenol and 4-alkylphenols with an alkyl group greater than C4 were not detected under the given experimental conditions. This result may be due either to the inability of substrate and/or product to be transported across the E. coli membrane or to the diminished activity of mPH with higher alkylphenols. In contrast to whole-cell transformations, which were carried out in 48 h, the mPH activity of cell extract was too low to be distinguished from the basal activity in an oxygen consumption assay (data not shown). Substrate preference and the mode of ring cleavage by C23OKL28, LapB Extradiol dioxygenases catalyse the incorporation of molecular oxygen into a carbon–carbon bond adjacent to the hydroxyl group of catechols. In terms of the metacleavage of 4-alkylcatechols by extradiol dioxygenases, molecular oxygen can be incorporated at a position near to the alkyl substitution or at a position distant from the substitution, which are called (2,3) proximal and (1,6) distal cleavages, respectively (Nozaki et al., 1970). The site of ring cleavage of the C23O of strain KL28 (LapB) was determined

Table 3. Identification of products formed from (alkyl)phenols by E. coli DH5a(pJJPMO2) expressing mPH Conditions for biotransformation and GC/MS analysis are described in Methods. –, No products were detected. Substrate

Product GC/MS data

TLC RF Rt (min) Phenol o-Cresol m-Cresol p-Cresol 2-Ethylphenol 3-Ethylphenol 4-Ethylphenol 4-Propylphenol 4-Butylphenol 4-Pentylphenol

0?17 0?23 0?18 0?18 – 0?19 0?19 0?19 – –

15?165 16?117 16?424 16?424 – 17?664 17?652 18?835 – –

m/z of fragment ions (% relative intensity) (M+, (M+, (M+, (M+,

92 (10?9), 81 (13?8), 64 (36?2), 53 (11?6), 39 (7?2) 106 (21?7), 78 (75?4), 51 (18?8), 44 (21?7) 107 (13), 78 (59?4), 51 (18?8), 39 (15?9) 107 (13), 78 (58), 51 (18?8), 39 (15?9) – 138 (M+, 42), 123 (100), 91 (8), 77 (13) 138 (M+, 36?2), 123 (100), 91 (7?2), 77(13) 152 (M+, 21?7), 123 (100), 77 (11?6), 44 (24?6) – –

110 124 124 124

100), 100), 100), 100),

Name Yield (%)* 72?1 4?6 85?7 44?1 – 29?4 19?6 3?6 – –

Catechol 3-Methylcatechol 4-Methylcatechol 4-Methylcatechol – 4-Ethylcatechol 4-Ethylcatechol 4-PropylcatecholD – –

*Yield was obtained from the mean of duplicate determinations in two independent experiments. DTentatively identified. http://mic.sgmjournals.org

3271


by comparing the visible spectra of the ring fission products with those formed by TodE and XylE. The latter two enzymes are found in the degradation pathways of toluene and xylenes and are known to catalyse the dioxygenation of 3- and 4-methylcatechol by (2,3) proximal cleavage (Cerdan et al., 1994; Cho et al., 2000; Klecka & Gibson, 1981; Ramos et al., 1987). In addition, XylE and TodE are known to catalyse the dioxygenation of 4-ethylcatechol and 2,3dihydroxybiphenyl, respectively, via the same mode of ring cleavage (Cerdan et al., 1994; Cho et al., 2000; Ramos et al., 1987). TodE and/or XylE produced maximum absorbance values (lmax) from 3- and 4-methylcatechol, 2,3-dihydroxybiphenyl and 4-ethylcatechol, which were the same as the lmax values previously determined (Table 4). In addition, LapB yielded the same absorbance spectra with different levels of absorbance yielding the same lmax values as those formed by TodE and XylE from the same substrates, indicating a (2,3) proximal cleavage mode for LapB. LapB preferentially oxidized 4-methylcatechol and 4-ethylcatechol, with relatively weak activity upon 3-methylcatechol, catechol and 2,3-dihydroxybiphenyl. The meta-ring cleavage product of 4-ethylcatechol was not degraded in the absence of NAD+ or NADP+ by the cell extract of KL28 cells grown on 4-ethylphenol, indicating a lack of HMS hydrolase in the lap pathway.

DISCUSSION Pseudomonas sp. strain KL28 is an alkylphenol degrader, which can assimilate 4-n-alkylphenols with a side chain length of C1–C5. The range of compounds that are degraded by strain KL28 is similar to that found for an activated sludge isolate of Pseudomonas veronii INA06 (Ajithkumar et al., 2003), which can assimilate phenol, p-cresol and other 4-n-alkylphenols (C3–C6). However, because strains KL28 and INA06 cannot use octyl- or nonylphenol as a carbon source, they are distinguished from other strains which catabolize these chemicals and have been identified mainly as Sphingomonas (Fujii et al., 2000; Tanghe et al., 1999, 2000) and as cold-adapted Pseudomonas and Stenotrophomonas (Soares et al., 2003). Despite the fact that the side chain length is different, all these strains preferentially degrade

alkylphenols with a para substitution and start the degradation at the phenolic moiety, suggesting a similar degradation pathway. Because the degradation pathway of higher alkylphenols has not been elucidated, we have characterized in this study the enzymes and genes for the alkylphenol degradation pathway present in strain KL28. 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, 3,4dimethylphenol 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 woodfeeding 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 33–74 % 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

Table 4. Activity of C23Os in cell extracts of cloned E. coli Experimental conditions are described in Methods. –, No detectable activity;

not applicable.

lmax (nm) of product/absorbance by

Substrate TodE Catechol 3-Methylcatechol 4-Methylcatechol 4-Ethylcatechol 2,3-Dihydroxybiphenyl

NA,

376/0?75 389/0?60 382/0?38 – 434/0?49

XylE

LapB

376/0?86 389/0?58 382/0?82 381/0?21 434/0?57

376/0?97 389/0?41 382/0?81 381/0?82 434/0?58

Cleavage mode of LapB

Activity of LapB (mU)

NA

20±2 68±21 593±45 320±23 10±1

P* P P P

*(2,3) Proximal cleavage. 3272

Microbiology 149


Fig. 3. Comparison of the lap gene cluster of strain KL28 with the (methyl)phenol degradative genes of Pseudomonas sp. CF600 (dmp) and of C. testosteroni TA441 (aph). Gene designations are described in Table 1 with the following additions: dmpQ and aphQ, ferredoxin genes; dmpD, HMS hydrolase gene; aphS and aphT, transcriptional regulator genes; aphX, aphY and aphJ, unknown genes.

(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 (65–98 %) 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 (61– 87 %) 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. One of the intriguing features in the lap gene cluster is that it lacks a gene encoding ferredoxin, which is generally located adjacent to the gene for C23O, as shown in the above examples of the dmp, ahp, phl, phh, pox, phc, phe and phk operons for phenol catabolism. The role of ferredoxin (XylT) from the TOL plasmid has been studied in detail. XylT contains a plant-type [2Fe–2S] cluster, and is known to reactivate C23O (XylE), when XylE is inactivated by the oxidation of the ferrous iron at the active site during catalysis (Hugo et al., 1998). The inactivation of XylE by 4-methylcatechol was dramatic and hence it was shown that such a ferredoxin gene is necessary for http://mic.sgmjournals.org

degradation pathways using 4-methylcatechol as an intermediate. This property enables XylE to degrade 4-methylcatechol in addition to 3-methylcatechol and thus to extend the catabolic capacity of the degradation pathway (Polissi & Harayama, 1993). It was unexpected that a ferredoxin gene was not found adjacent to the gene for C23O in the lap gene cluster, because the lap pathway contains enzymes that metabolize alkylphenols that form 4-methylcatechol and other 4-alkylcatechol intermediates. However, LapB maintained high activity with 4-methylcatechol and 4-ethylcatechol with the cell extract obtained from recombinant E. coli (Table 4), suggesting the enzyme is active even in the absence of XylT-type ferredoxin. The requirement for ferredoxin might be absolute for the catalysis of 4-methylcatechol by C23Os that are found in degradation pathways utilizing both 3- and 4-methylcatechols. However, enzymes encoded by the lap operon constitute a degradation pathway utilizing exclusively 4-alkylcatechols; thus it is assumed that LapB might have been optimally evolved to catalyse 4-alkylcatecols without the aid of ferredoxin. In the available databases, more than 250 extradioltype dioxygenases were found. The amino acid sequence of LapB is closest to Avin0687 from an A. vinelandii genome and the partial sequence of BupB from strain MT4, with identities of 84 and 65 %, respectively. These three enzymes might have arisen from the same origin because the gene organizations in their operons are similar, as mentioned above. LapB is next closest to the C23Os from P. putida UCC22(pTDN1) (GenBank accession no. BAB62050), P. stutzeri OX1 (GenBank accession no. CAD43168) and P. putida plasmid pDK1 (GenBank accession no. A42733), with identities of 62, 52 and 52 %, respectively. These genes participate in the degradation of aniline, o-xylene and toluene/xylene, respectively (Benjamin et al., 1991; Bertoni et al., 1996; Saint & Venables, 1990) and have XylT-type ferredoxin associated with the genes for C23O. In terms of the divergence in amino acid sequence and catalytic properties, LapB, probably with Avin0687 and BupB, appears to belong to its own subfamily of extradiol dioxygenases, which were categorized by Eltis & Bolin (1996). 3273


Fig. 4. The 3- and 4-n-alkylphenol degradation (lap) pathway in Pseudomonas sp. KL28. HMS derivatives undergo the 4-oxalocrotonate branch that is initiated by LapC but do not follow the hydrolytic branch because of the lack of HMS hydrolase.

The first step in lap degradation is the conversion of 3and 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). 3274

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). On the basis of the substrate specificities of mPH and C23O in the lap operon, and of the amino acid sequence similarities between the lap gene products and known enzymes, the 3- and 4-alkylphenol degradation pathway present in strain KL28 can be depicted as shown in Fig. 4. This pathway contrasts with the degradation pathways for p-cresol by P. putida NCIMB 9869 and NCIMB 9866 (Kim et al., 1994) and 4-ethylphenol by Pseudomonas sp. JD1 (Reeve et al., 1989), which start degradation from the side chain, but is compatible with the p-cresol degradation pathways present in P. putida U (Bayly et al., 1966), Alcaligenes eutrophus 345 (Hughes et al., 1984) and Pseudomonas sp. CF600 (Shingler, 1996), and part of the degradation pathway for alkylbenzoates in P. putida containing the TOL plasmid (Ramos et al., 1987). The results obtained from assays of GFP-based promoter activity and the substrate specificities of the first two enzymes (mPH and C23O) revealed that the lap pathway present in strain KL28 is best suited for the catabolism of m- and p-cresol and 3-and 4-ethylphenol, and that it may be expanded further to accommodate larger side chains due to the relaxed specificities of catabolic enzymes, as well as a regulatory protein.

ACKNOWLEDGEMENTS This work was supported by a Korea Research Foundation Grant (KRF 200-005-D20020). We would like to thank Professor David T. Gibson (University of Iowa) for providing plasmid pDTG617 and strains P. putida G7.C-1 and P. putida mt-2 for this study, and Dr Daniel J. Lessner (University of Iowa) for critical reading and suggestions. Microbiology 149


REFERENCES Abril, M. A., Buck, M. & Ramos, J. L. (1991). Activation of the

Pseudomonas TOL plasmid upper pathway operon. Identification of binding sites for the positive regulator XylR and for integration host factor protein. J Biol Chem 266, 15832–15838. Ajithkumar, B., Ajithkumar, V. P. & Iriye, R. (2003). Degradation of 4-amylphenol and 4-hexylphenol by a new activated sludge isolate of Pseudomonas veronii and proposal for a new subspecies status. Res Microbiol 154, 17–23. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a

Choi, E. N., Cho, M. C., Kim, Y., Kim, C. K. & Lee, K. (2003).

Expansion of growth substrate range in Pseudomonas putida F1 by mutations in both cymR and todS, which recruit a ring-fission hydrolase CmtE and induce the tod catabolic operon, respectively. Microbiology 149, 795–805. de Lorenzo, V., Herrero, M., Metzke, M. & Timmis, K. N. (1991). An

upstream XylR- and IHF-induced nucleoprotein complex regulates the sigma 54-dependent Pu promoter of TOL plasmid. EMBO J 10, 1159–1167. Duggleby, C. J. & Williams, P. A. (1986). Purification and some

new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402.

properties of the 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase (2hydroxymuconic semialdehyde hydrolase) encoded by the TOL plasmid pWW0 from Pseudomonas putida mt-2. J Gen Microbiol 132, 717–726.

Arai, H., Akahira, S., Ohishi, T., Maeda, M. & Kudo, T. (1998).

Dunn, N. W. & Gunsalus, I. C. (1973). Transmissible plasmid coding

Adaptation of Comamonas testosteroni TA441 to utilize phenol: organization and regulation of the genes involved in phenol degradation. Microbiology 144, 2895–2903. Arai, H., Akahira, S., Ohishi, T. & Kudo, T. (1999). Adaptation of Comamonas testosteroni TA441 to utilization of phenol by spontaneous mutation of the gene for a trans-acting factor. Mol Microbiol 33, 1132–1140. Arai, H., Ohishi, T., Chang, M. Y. & Kudo, T. (2000). Arrangement and regulation of the genes for meta-pathway enzymes required for degradation of phenol in Comamonas testosteroni TA441. Microbiology 146, 1707–1715.

early enzymes of naphthalene oxidation in Pseudomonas putida. J Bacteriol 114, 974–979.

Arenghi, F. L., Berlanda, D., Galli, E., Sello, G. & Barbieri, P. (2001).

Organization and regulation of meta cleavage pathway genes for toluene and o-xylene derivative degradation in Pseudomonas stutzeri OX1. Appl Environ Microbiol 67, 3304–3308. Ausubel, F. M. R. B., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (1990). Current Protocols in Molecular

Biology. New York: Wiley. Ballou, P. D. (1982). Flavoprotein monooxygenases. In Flavins and Flavoproteins, pp. 301–310. Edited by V. Massey & C. H. Williams. Amsterdam: Elsevier Science Publishing. Bartilson, M. & Shingler, V. (1989). Nucleotide sequence and

expression of the catechol 2,3-dioxygenase-encoding gene of phenolcatabolizing Pseudomonas CF600. Gene 85, 233–238. Bayly, R. C., Dagley, S. & Gibson, D. T. (1966). The metabolism of cresols by species of Pseudomonas. Biochem J 101, 293–301. Benjamin, R. C., Voss, J. A. & Kunz, D. A. (1991). Nucleotide sequence of xylE from the TOL pDK1 plasmid and structural comparison with isofunctional catechol-2,3-dioxygenase genes from TOL, pWW0 and NAH7. J Bacteriol 173, 2724–2728. Bertoni, G., Bolognese, F., Galli, E. & Barbieri, P. (1996). Cloning of the genes for and characterization of the early stages of toluene and o-xylene catabolism in Pseudomonas stutzeri OX1. Appl Environ Microbiol 62, 3704–3711. Buswell, J. A. (1975). Metabolism of phenol and cresols by Bacillus stearothermophilus. J Bacteriol 124, 1077–1083. Cadieux, E., Vrajmasu, V., Achim, C., Powlowski, J. & Mu¨nck, E. (2002). Biochemical, Mo¨ssbauer, and EPR studies of the diiron

cluster of phenol hydroxylase from Pseudomonas sp. strain CF 600. Biochemistry 41, 10680–10691. Cerdan, P., Wasserfallen, A., Rekik, M., Timmis, K. N. & Harayama, S. (1994). Substrate specificity of catechol 2,3-dioxygenase encoded by

TOL plasmid pWW0 of Pseudomonas putida and its relationship to cell growth. J Bacteriol 176, 6074–6081. Cho, M. C., Kang, D.-O., Yoon, B. D. & Lee, K. (2000). Toluene degradation pathway from Pseudomonas putida F1: substrate specificity and gene induction by 1-substituted benzenes. J Ind Microbiol Biotechnol 25, 163–170. http://mic.sgmjournals.org

Ehrt, S., Schirmer, F. & Hillen, W. (1995). Genetic organization, nucleotide sequence and regulation of expression of genes encoding phenol hydroxylase and catechol 1,2-dioxygenase in Acinetobacter calcoaceticus NCIB8250. Mol Microbiol 18, 13–20. Elango, N., Radhakrishnan, R., Froland, W. A., Wallar, B. J., Earhart, C. A., Lipscomb, J. D. & Ohlendorf, D. H. (1997). Crystal structure

of the hydroxylase component of methane monooxygenase from Methylosinus trichosporium OB3b. Protein Sci 6, 556–568. Eltis, L. D. & Bolin, J. T. (1996). Evolutionary relationships among

extradiol dioxygenases. J Bacteriol 178, 5930–5937. Ensley, B. D., Ratzkin, B. J., Osslund, T. D., Simon, M. J., Wackett, L. P. & Gibson, D. T. (1983). Expression of naphthalene oxidation

genes in Escherichia coli results in the biosynthesis of indigo. Science 222, 167–169. Ferguson, P. L., Iden, C. R. & Brownawell, B. J. (2001). Distribution

and fate of neutral alkylphenol ethoxylate metabolites in a sewageimpacted urban estuary. Environ Sci Technol 35, 2428–2435. Ferrara, F., Fabietti, F., Delise, M., Bocca, A. P. & Funari, E. (2001).

Alkylphenolic compounds in edible molluscs of the Adriatic Sea (Italy). Environ Sci Technol 35, 3109–3112. Figurski, D. H. & Helinski, D. R. (1979). Replication of an origin-

containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A 76, 1648–1652. Fox, B. G., Shanklin, J., Somerville, C. & Mu¨nck, E. (1993). Stearoylacyl carrier protein D9 desaturase from Ricinus communis is a diiron-

oxo protein. Proc Natl Acad Sci U S A 90, 2486–2490. Friedman, D. I. (1988). Integration host factor: a protein for all

reasons. Cell 55, 545–554. Fujii, K., Urano, N., Ushio, H., Satomi, M., Iida, H., Ushio-Sata, N. & Kimura, S. (2000). Profile of a nonylphenol-degrading microflora

and its potential for bioremedial applications. J Biochem 128, 909–916. Giger, W., Brunner, P. H. & Schaffner, C. (1984). 4-Nonylphenol in

sewage sludge: accumulation of toxic metabolites from nonionic surfactants. Science 225, 623–625. Harayama, S., Mermod, N., Rekik, M., Lehrbach, P. R. & Timmis, K. N. (1987). Roles of the divergent branches of the meta-cleavage

pathway in the degradation of benzoate and substituted benzoates. J Bacteriol 169, 558–564. Herrmann, H., Muller, C., Schmidt, I., Mahnke, J., Petruschka, L. & Hahnke, K. (1995). Localization and organization of phenol

degradation genes of Pseudomonas putida strain H. Mol Gen Genet 247, 240–246. Hino, S., Watanabe, K. & Takahashi, N. (1998). Phenol hydroxylase

cloned from Ralstonia eutropha strain E2 exhibits novel kinetic properties. Microbiology 144, 1765–1772. 3275

J. J. Jeong and others Hughes, E. J., Bayly, R. C. & Skurray, R. A. (1984). Evidence for

isofunctional enzymes in the degradation of phenol, m- and p-toluate, and p-cresol via catechol meta-cleavage pathways in Alcaligenes eutrophus. J Bacteriol 158, 79–83. Hugo, N., Armengaud, J., Gaillard, J., Timmis, K. N. & Jouanneau, Y. (1998). A novel [2Fe-2S] ferredoxin from Pseudomonas putida mt2

promotes the reductive reactivation of catechol 2,3-dioxygenase. J Biol Chem 273, 9622–9629. Inouye, S., Nakazawa, A. & Nakazawa, T. (1988). Nucleotide

sequence of the regulatory gene xylR of the TOL plasmid from Pseudomonas putida. Gene 66, 301–306. Johnson, J. L. (1994). Similarity analyses of rRNAs. In Methods

for General and Molecular Bacteriology, pp. 683–700. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology. Johnson, G. R. & Olsen, R. H. (1997). Multiple pathways for toluene

degradation in Burkholderia sp. strain JS150. Appl Environ Microbiol 63, 4047–4052. Keith, T. L., Snyder, S. A., Naylor, C. G., Staples, C. A., Summer, C., Kannan, K. & Giesy, J. P. (2001). Identification and quantitation

of nonylphenol ethoxylates and nonylphenol in fish tissues from Michigan. Environ Sci Technol 35, 10–13. Kim, J., Fuller, J. H., Cecchini, G. & McIntire, W. S. (1994). Cloning,

sequencing, and expression of the structural genes for the cytochrome and flavoprotein subunits of p-cresol methylhydroxylase from two strains of Pseudomonas putida. J Bacteriol 176, 6349–6361. Klecka, G. M. & Gibson, D. T. (1981). Inhibition of catechol 2,3-

dioxygenase from Pseudomonas putida by 3-chlorocatechol. Appl Environ Microbiol 41, 1159–1165. Kovach, M. E., Elzer, P. H., Hill, D. S., Robertson, G. T., Farris, M. A., Roop, R. M., 2nd & Peterson, K. M. (1995). Four new derivatives of

the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166, 175–176. Kukor, J. J. & Olsen, R. H. (1992). Complete nucleotide sequence of

tbuD, the gene encoding phenol/cresol hydroxylase from Pseudomonas pickettii PKO1, and functional analysis of the encoded enzyme. J Bacteriol 174, 6518–6526. Lee, K., Resnick, S. M. & Gibson, D. T. (1997). Stereospecific

oxidation of (R)- and (S)-1-indanol by naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4. Appl Environ Microbiol 63, 2067–2070. Miller, W. G., Leveau, J. H. & Lindow, S. E. (2000). Improved gfp and

inaZ broad-host-range promoter-probe vectors. Mol Plant Microbe Interact 13, 1243–1250. Murray, K., Sala-Trepat, J. M. & Williams, P. A. (1972). The divergent

meta-cleavage pathway for the metabolism of benzoate, 3-methylbenzoate and 4-methylbenzoate by Pseudomonas arvilla mt-2. Biochem J 128, 89P–90P. Nakamura, K., Ishida, H. & Iizumi, T. (2000). Constitutive

trichloroethylene degradation led by tac promoter chromosomally integrated upstream of phenol hydroxylase genes of Ralstonia sp. KN1 and its nucleotide sequence analysis. J Biosci Bioeng 89, 47–54. Ng, L. C., Shingler, V., Sze, C. C. & Poh, C. L. (1994). Cloning and

phenol hydroxylase from Pseudomonas sp. strain CF600. J Bacteriol 172, 6826–6833. Nozaki, M., Kotani, S., Ono, K. & Seno, S. (1970). Metapyro-

catechase. III. Substrate specificity and mode of ring fission. Biochim Biophys Acta 220, 213–223. Polissi, A. & Harayama, S. (1993). In vivo reactivation of catechol 2,3-dioxygenase mediated by a chloroplast-type ferredoxin: a bacterial strategy to expand the substrate specificity of aromatic degradative pathways. EMBO J 12, 3339–3347. Powlowski, J. & Shingler, V. (1990). In vitro analysis of polypeptide

requirements of multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600. J Bacteriol 172, 6834–6840. Powlowski, J. & Shingler, V. (1994). Genetics and biochemistry of phenol degradation by Pseudomonas sp. CF600. Biodegradation 5, 219–236. Ramos, J. L., Wasserfallen, A., Rose, K. & Timmis, K. N. (1987).

Redesigning metabolic routes: manipulation of TOL plasmid pathway for catabolism of alkylbenzoates. Science 235, 593–596. Reeve, C. D., Carver, M. A. & Hopper, D. J. (1989). The purifica-

tion and characterization of 4-ethylphenol methylenehydroxylase, a flavocytochrome from Pseudomonas putida JD1. Biochem J 263, 431–437. Rosenzweig, A. C., Brandstetter, H., Whittington, D. A., Nordlund, P., Lippard, S. J. & Frederick, C. A. (1997). Crystal structures of the

methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath): implications for substrate gating and component interactions. Proteins 29, 141–152. Saint, C. P. & Venables, W. A. (1990). Loss of Tdn catabolic genes by deletion from and curing of plasmid pTDN1 in Pseudomonas putida: rate and mode of loss are substrate and pH dependent. J Gen Microbiol 136, 627–636. Sala-Trepat, J. M., Murray, K. & Williams, P. A. (1972). The metabolic

divergence in the meta cleavage of catechols by Pseudomonas putida NCIB 10015. Physiological significance and evolutionary implications. Eur J Biochem 28, 347–356. Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. Sarand, I., Skarfstad, E., Forsman, M., Romantschuk, M. & Shingler, V. (2001). Role of the DmpR-mediated regulatory circuit

in bacterial biodegradation properties in methylphenol-amended soils. Appl Environ Microbiol 67, 162–171. Schirmer, F., Ehrt, S. & Hillen, W. (1997). Expression, inducer

spectrum, domain structure, and function of MopR, the regulator of phenol degradation in Acinetobacter calcoaceticus NCIB8250. J Bacteriol 179, 1329–1336. Seah, S. Y., Terracina, G., Bolin, J. T., Riebel, P., Snieckus, V. & Eltis, L. D. (1998). Purification and preliminary characterization of a serine

hydrolase involved in the microbial degradation of polychlorinated biphenyls. J Biol Chem 273, 22943–22949. Sharpe, R. M., Fisher, J. S., Millar, M. M., Jobling, S. & Sumpter, J. P. (1995). Gestational and lactational exposure of rats to xenoestrogens

sequences of the first eight genes of the chromosomally encoded (methyl) phenol degradation pathway from Pseudomonas putida P35X. Gene 151, 29–36.

results in reduced testicular size and sperm production. Environ Health Perspect 103, 1136–1143.

Ng, L. C., Poh, C. L. & Shingler, V. (1995). Aromatic effector

phenol degradation by Pseudomonas sp. strain CF600. In Pseudomonas: Molecular Biology and Biotechnology, pp. 153–164. Edited by T. Nakazawa. Washington, DC: American Society for Microbioloy.

activation of the NtrC-like transcriptional regulator PhhR limits the catabolic potential of the (methyl)phenol degradative pathway it controls. J Bacteriol 177, 1485–1490. Nordlund, I., Powlowski, J. & Shingler, V. (1990). Complete

nucleotide sequence and polypeptide analysis of multicomponent 3276

Shingler, V. (1996). Metabolic and regulatory check points in

Shingler, V. & Moore, T. (1994). Sensing of aromatic compounds by the DmpR transcriptional activator of phenol-catabolizing Pseudomonas sp. strain CF600. J Bacteriol 176, 1555–1560.

Microbiology 149


Shingler, V., Franklin, F. C., Tsuda, M., Holroyd, D. & Bagdasarian, M. (1989). Molecular analysis of a plasmid-encoded phenol hydroxylase

Tanaka, J. N. & Grizzle, J. M. (2002). Effects of nonylphenol on the

from Pseudomonas CF600. J Gen Microbiol 135, 1083–1092.

gonadal differentiation of the hermaphroditic fish, Rivulus marmoratus. Aquat Toxicol 57, 117–125.

Shingler, V., Powlowski, J. & Marklund, U. (1992). Nucleotide

Tanghe, T., Dhooge, W. & Verstraete, W. (1999). Isolation of a

sequence and functional analysis of the complete phenol/3, 4dimethylphenol catabolic pathway of Pseudomonas sp. strain CF600. J Bacteriol 174, 711–724.

bacterial strain able to degrade branched nonylphenol. Appl Environ Microbiol 65, 746–751.

Shingler, V., Bartilson, M. & Moore, T. (1993). Cloning and

nucleotide sequence of the gene encoding the positive regulator (DmpR) of the phenol catabolic pathway encoded by pVI150 and identification of DmpR as a member of the NtrC family of transcriptional activators. J Bacteriol 175, 1596–1604. Soares, A., Guieysse, B., Delgado, O. & Mattiasson, B. (2003).

Aerobic biodegradation of nonylphenol by cold-adapted bacteria. Biotechnol Lett 25, 731–738.

Tanghe, T., Dhooge, W. & Verstraete, W. (2000). Formation of the

metabolic intermediate 2, 4, 4-trimethyl-2-pentanol during incubation of a Sphingomonas sp. strain with the xeno-estrogenic octylphenol. Biodegradation 11, 11–19. Teramoto, M., Futamata, H., Harayama, S. & Watanabe, K. (1999).

Characterization of a high-affinity phenol hydroxylase from Comamonas testosteroni R5 by gene cloning, and expression in Pseudomonas aeruginosa PAO1c. Mol Gen Genet 262, 552–558. Thony, B. & Hennecke, H. (1989). The 224/212 promoter comes of

Stanier, R. Y., Palleroni, N. J. & Doudoroff, M. (1966). The

age. FEMS Microbiol Rev 5, 341–357.

aerobic pseudomonads: a taxomonic study. J Gen Microbiol 43, 159–271.

Wigmore, G. J., Bayly, R. C. & Di Berardino, D. (1974). Pseudomonas

Staskawicz, B., Dahlbeck, D., Keen, N. & Napoli, C. (1987).

Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea. J Bacteriol 169, 5789–5794. Sze, C. C., Laurie, A. D. & Shingler, V. (2001). In vivo and in vitro effects of integration host factor at the DmpR-regulated s54-

dependent Po promoter. J Bacteriol 183, 2842–2851.

http://mic.sgmjournals.org

putida mutants defective in the metabolism of the products of meta fission of catechol and its methyl analogues. J Bacteriol 120, 31–37. Xu, Y., Chen, M., Zhang, W. & Lin, M. (2003). Genetic organization of genes encoding phenol hydroxylase, benzoate 1,2-dioxygenase alpha subunit and its regulatory proteins in Acinetobacter calcoaceticus PHEA-2. Curr Microbiol 46, 235–240. Zylstra, G. J. & Gibson, D. T. (1991). Aromatic hydrocarbon

degradation: a molecular approach. Genet Eng 13, 183–203.

3277