1 Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
2 Department of Chemistry, Faculty of Sciences, Science University of Tokyo, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan
3 Department of Biotechnology, Faculty of Bioresource Sciences, Akita Prefectural University, 241-7 Kaidobata-nishi, Shimoshinjo-nakano, Akita 010-0195, Japan
4 Geo and Water Environmental Engineering Department, Obayashi Corporation, 4-640 Shimokiyoto, Kiyose-shi, Tokyo 204-0011, Japan
Correspondence
Hiroshi Habe
uhhabe{at}mail.ecc.u-tokyo.ac.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AP008980.
A figure showing RT-PCR and expression analysis of pca genes, and a table of annotated ORFs are available as supplementary material with the online version of this paper.
Present address: Department of Industrial Chemistry, Faculty of Engineering, Shibaura Institute of Technology, Minato-ku, Tokyo 108-8548, Japan.
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INTRODUCTION |
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Strain DBF63 was originally isolated as a bacterium capable of utilizing both dibenzofuran (DF) and FN (Monna et al., 1993). Initially, dbfA1 and dbfA2 were isolated from strain DBF63 as genes encoding novel terminal oxygenase components of the angular dioxygenase for DF degradation (Kasuga et al., 2001
), and later, the genes were revealed to be located within an FN-catabolic gene cluster (Habe et al., 2004
). The FN-catabolic genes, designated dbf-fln (involved with the degradation of FN to phthalate) and pht (involved with the degradation of phthalate to protocatechuate), were located on linear plasmids pDBF1 and pDBF2 (approx. 160 and 190 kb, respectively), and pDBF2 is thought to be derived from pDBF1 (Nojiri et al., 2002a
). The presence of protocatechuate catabolic genes in strain DBF63, which are required for the complete degradation of FN to TCA cycle intermediates, is supported by the fact that strain DBF63 can grow with protocatechuate as its sole carbon and energy source.
Two major catabolic pathways for protocatechuate have been proposed. The first is the meta-cleavage pathway initiated by protocatechuate 4,5-dioxygenase, and the second is the -ketoadipate pathway initiated by protocatechuate 3,4-dioxygenase (Dagley et al., 1960
; Fig. 1
). The
-ketoadipate pathway, in particular, is considered to be a good model system for studying mechanisms of evolution in an ecologically important catabolic pathway (Buchan et al., 2004
; Parke et al., 2000
). Following initial ortho-cleavage by protocatechuate 3,4-dioxygenase (PcaHG), five additional enzymes, i.e.
-carboxy-cis,cis-muconate cycloisomerase (PcaB),
-carboxymuconolactone decarboxylase (PcaC),
-ketoadipate enol-lactone hydrolase (PcaD),
-ketoadipate succinyl CoA transferase (PcaIJ) and
-ketoadipyl CoA thiolase (PcaF), convert the ring cleavage product to TCA cycle intermediates (Fig. 1
). In the case of the proteobacteria, this pathway is biochemically conserved among phylogenetically different strains, but operon organization, regulatory proteins, coinducer molecules and transport proteins for the pathway are remarkably diverse, most likely reflecting subtle aspects of niche adaptation (Buchan et al., 2004
; Parke, 1997
; Parke et al., 2000
). In contrast, there is still little information available concerning protocatechuate catabolic gene clusters from actinobacteria (Eaton, 2001
; Eulberg et al., 1998
; Iwagami et al., 2000
). The protocatechuate branch of the
-ketoadipate pathway (involving pca gene products) has only been functionally analysed in two systems, i.e. Rhodococcus sp. (Eulberg et al., 1998
; Patrauchan et al., 2005
) and Streptomyces sp. (Iwagami et al., 2000
). Analysis of the pca genes in Terrabacter may, therefore, provide a better perspective on the evolution of the
-ketoadipate pathway in actinobacteria, if this strain has such genes.
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METHODS |
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Sequencing and annotation of the 70 kb DNA region of the linear plasmid.
Shotgun sequencing of (i) two previously isolated cosmid clones (designated pCC4 and pCC19) that contain the phtA1 gene (Kasuga et al., 2001), and (ii) one cosmid clone (designated pCC31) that contains the flanking region of the pCC4 insert (this study) was performed by Dragon Genomics. Fragmented, blunt-ended DNA was ligated into the SmaI site of pUC118. Determination of the nucleotide sequence was carried out by the chain-termination method. To identify ORFs, the nucleotide sequence was analysed with DNASIS-Mac software (version 3.7; Hitachi Software Engineering). Homology searches were carried out using the SWISS-PROT protein sequence database or the DDBJ, EMBL and GenBank nucleotide sequence databases with BLAST programs (Altschul et al., 1997
). Results from the automated ORF prediction and functional assignment were manually controlled for the entire DNA contiguous sequence (70 kb). The nucleotide sequence was deposited at the DDBJ, EMBL and GenBank nucleotide sequence databases under the accession number AP008980.
RT-PCR analysis.
Strain DBF63 was grown to mid-exponential phase in CFMM supplemented with FN, DF or protocatechuate solutions to a final substrate concentration of 0·1 % (w/v). Both FN and DF were dissolved in dimethylformamide (DMF; 100 mg ml1), and protocatechuate was dissolved in ethanol (100 mg ml1). These solutions were sterilized by filtration with a 0·2 mm pore-size membrane filter, and 50 µl each solution was added to 5 ml CFMM. Two millilitres of each culture was then centrifuged, and total RNA was extracted from the harvested cells using a NucleoSpin RNA II purification kit (Macherey-Nagel), combined with RQ1 RNase-free DNase (Promega), according to the manufacturer's instructions. The gene-specific reverse primers used to synthesize cDNA were RT-Pr3R for pcaHG, RT-Pr5R for pcaDC, RT-Pr7Rb for pcaFJI and RT-Pr1R for pcaR (Table 2). cDNA of each gene was synthesized from 10 ng total RNA using SuperScript III reverse transcriptase (Invitrogen), according to the manufacturer's instructions. The primers used are listed in Table 2
. After the cDNA samples were properly diluted, PCR was performed as follows: (i) for pcaHG amplification, 95 °C for 10 min and 5 s, 60 °C for 10 s, and 72 °C for 30 s, for 40 cycles; (ii) for pcaDC amplification, 95 °C for 10 min and 30 s, 50 °C for 30 s, and 72 °C for 90 s, for 40 cycles; (iii) for pcaFJI amplification, 95 °C for 10 min and 5 s, 55 °C for 30 s, and 72 °C for 30 s, for 40 cycles; (iv) for pcaR amplification, 95 °C for 10 min and 30 s, 55 °C for 30 s, and 72 °C for 90 s, for 40 cycles.
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Construction of plasmids for pcaD, pcaC and catBC expression.
The 820 bp and 415 bp DNA fragments containing pcaD and pcaC, respectively, were prepared by PCR using pCC12 (Kasuga et al., 2001) as the template. The primer sets used are listed in Table 2
. PCR was carried out using LA Taq with GC buffer (Takara Shuzo) and PCR Thermal Cycler Dice (Takara Shuzo). The cycling conditions were as follows: 96 °C for 1 min, followed by 30 cycles of 96 °C for 30 s, 55 °C for 30 s, and 72 °C for 2 min, followed by 72 °C for 6 min. The PCR products were cloned using the pT7Blue (R) vector (Novagene), and the nucleotide sequences of the PCR products were confirmed by sequencing. The clones were digested with both HindIII and EcoRI (sites derived from the primer, Table 2
), and then the fragments were cloned between the HindIII and EcoRI sites of pSTV29 to give pDFS604 (carrying pcaD) and pDFS603 (carrying pcaC), respectively. To construct the expression plasmid for catBC genes, the 1·9 kb XhoIEcoRI fragment of pUCA811 (Nojiri et al., 2002b
) was cloned between the corresponding sites of pBluescript II KS() to give pUCA832 (carrying catBC).
Resting cell reactions and analysis of the products.
Transformation of E. coli cells with the constructed plasmids was performed as described by Hanahan (1983). An appropriate E. coli transformant was grown in 100 ml LB broth supplemented with ampicillin, chloramphenicol and IPTG, as appropriate, in a 500 ml flat-bottom flask at 30 °C with reciprocal shaking at 120 strokes min1. The culture broth was centrifuged (4000 g), and the pelleted cells were washed twice with 100 ml buffer (containing 2·2 g Na2HPO4, 0·8 g KH2PO4, and 3·0 g NH4NO3 per litre of distilled water; pH 7·0), and resuspended in the same buffer to an OD600 of approximately 10. We added cis,cis-muconate (0·1 % w/v) to 5 ml resting cell suspension in test tubes and incubated the tubes at 30 °C for 2 h. The reaction mixtures were acidified with 1 M HCl and extracted with 5 ml ethyl acetate. The extracts were analysed by GC-MS after derivatization with N-methyl-N-trimethylsilyltrifluoroacetamide. GC-MS analyses were performed with a JMS-Automass 150 GC-MS system (JEOL) fitted with a fused-silica chemically bonded capillary column (DB-5; 0·25 mm inside diameter by 15 m, 0·25 µm film thickness; J&W Scientific). Each sample was injected into the column at 80 °C in the splitless mode. After 2 min at 80 °C, the column temperature was increased at 16 °C min1 to 280 °C. The head pressure of the helium carrier gas was 65 kPa.
Chemicals.
-Ketoadipic acid (3-oxoadipic acid) was purchased from Sigma-Aldrich. cis,cis-Muconic acid was synthesized by Mitsubishi Chemical. All other chemicals used in this study were of the highest purity commercially available (98100 %; Merck, Sigma-Aldrich, Kanto Chemical, Wako Pure Chemical, Nacalai Tesque).
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RESULTS |
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(ii) Insertion sequences (ISs).
We found several ORFs homologous to transposase genes or IS-like elements around the pca and dbf-fln gene clusters (Fig. 2). The deduced amino acid sequence of ORF58 located downstream of pcaR exhibited 100 % similarity to that of the transposase-encoding gene in ISTesp2, found just upstream of the pht genes in strain DBF63 (Habe et al., 2003
). We also determined that the incomplete ORF78 and its flanking region were homologous to ISTesp2 (Fig. 2
).
The DNA sequence revealed that ORF68 and its flanking region (nucleotides 61 17762 584; Fig. 2) were identical to ORF70 and its flanking region (nucleotides 64 30665 713), and that the full-length of these repetitive elements was 1408 bp. The deduced amino acid sequences of ORF42, ORF68 and ORF70 showed 82, 84 and 84 % overall lengthwise similarity, respectively, to the transposase of IS1601-A from Mycobacterium avium (see the Supplementary Table), a predicted member of the IS256 family (Eckstein et al., 2000
). The similarity between the ORF42 product and ORF68 (ORF70) product was 85 %. At both ends of ORF42 and ORF68 (ORF70), we found 20 and 23 bp imprecise inverted repeats (IRs), respectively. However, the formation of direct repeats, as duplicated target sequences at both ends, was not observed in the immediate vicinity of these IRs.
The polypeptide encoded by ORF67 and ORF66 showed 6668 % and 5269 % overall lengthwise similarity to the transposase subunits encoded by MC34 and MC35, respectively, from the plasmid pSD10 of Micrococcus sp. strain 28 (AY034092.1), and by PDB2.181 and PDB2.182, respectively, from plasmid pBD2 of Rhodococcus erythropolis strain BD2 (Stecker et al., 2003). This IS is potentially a member of the IS3 family. The 24 bp imperfect IR was found around the two ORFs, but a direct repeat, duplicated target site, was not observed in the immediate vicinity of the IR.
Transcriptional analysis of pca genes by RT-PCR
We used RT-PCR analysis to investigate whether pca genes were transcribed in strain DBF63 during its growth with FN. Total RNA was prepared from DBF63 cells grown with FN, DF or protocatechuate. As the pcaHGBDCFIJ gene cluster has been shown, by RT-PCR, to be transcribed as an operon (data not shown), we only present here the results with the three primers specific for amplifying the pcaHG, pcaDC and pcaFIJ genes within this cluster (Table 2; Supplementary Fig. 1a
with the online journal). Clear amplification of the appropriate portion of the respective pca genes (1·2 kb for pcaHG, 0·85 kb for pcaDC and 1·4 kb for pcaFIJ) was detected when RNA prepared from both FN- and protocatechuate-grown cells were used, whereas PCR products were not detected when RNA from DF-grown cells was used. No amplified products were detected in the negative control lacking reverse transcriptase (data not shown). These results indicate that pcaHGBDCFIJ genes were transcribed in DBF63 cells grown with FN, as well as protocatechuate, suggesting that the genes are probably required for FN degradation in this strain. As strain DBF63 degrades DF via gentisate (Monna et al., 1993
), it is reasonable that PCR products are not detected from cells grown on DF. In contrast, the pcaR gene was not specifically expressed during the degradation of FN or protocatechuate, and the same levels of PCR products were detected from RNA prepared from DBF63 cells grown on all three substrates (see Supplementary Fig. 1a
). This result suggests that the pcaR gene may be constitutively expressed in this strain. RT-PCR with primers specific for amplifying the intergenic region between pcaR and pcaH (Table 2
) produced no PCR products of the expected size (310 bp; data not shown).
Quantitative RT-PCR
To analyse the expression pattern of pca genes, the pcaD mRNA levels in protocatechuate-induced or non-induced DBF63 cells, within 1 h of induction, were determined by quantitative RT-PCR with SYBR Green. Transcriptional levels were normalized with 16S rRNA as an internal control. As shown in Supplementary Fig. 1(b) (left panel), the pcaD mRNA in cells with protocatechuate was about 21-fold more abundant than in cells with ethanol (control). Similarly, the pcaD mRNA levels in FN-induced or non-induced DBF63 cells were examined (see Supplementary Fig. 1b, right panel), and expression levels 1·7-fold higher were observed in cells with FN. The differences in water solubility of these inducer substrates, and the solvent types, may have had some effect on induction levels. In addition, incubation times longer than 1 h would produce higher induction levels in FN-induced cells. Nevertheless, the patterns of sharp and gradual increase in the pcaD mRNA levels in response to protocatechuate and FN, respectively, indicate that protocatechuate itself or its metabolite, e.g. -ketoadipate, is the inducer of Pca enzymes.
-Ketoadipate enol-lactone hydrolase (PcaD) activity
We constructed the plasmids pUCA832 (carrying catBC) and pDFS604 (carrying pcaD; Table 1), and performed a biotransformation experiment with cis,cis-muconate using E. coli cells harbouring both pUCA832 and pDFS604. E. coli JM109 cells carrying both pUCA832 and pSTV29 were used as a control. GC-MS analysis of products from cis,cis-muconate after treatment with N-methyl-N-trimethylsilyltrifluoroacetamide, gave a mass spectrum that exhibited fragment ions at m/z 378 (M+, 6), 361 (27), 317 (6), 286 (16), 259 (6), 231 (21), 169 (59), 147 (39), 125 (8) and 73 (100), where the numbers in parantheses represent relative intensity, detected with a retention time of 8·4 min. This fragmentation pattern of the trimethylsilyl derivative of the metabolite was identical to that of authentic
-ketoadipate (data not shown). In contrast,
-ketoadipate was not detected in the control sample (data not shown). Instead, the trimethylsilyl derivative of
-ketoadipate enol-lactone was tentatively identified because the mass spectrum (m/z) 286 (M+, 14), 271 (5), 169 (100), 147 (19) and 73 (100) and the retention time (6·3 min) were consistent with those of the product when we performed biotransformation experiments with protocatechuate using E. coli cells harbouring both pARO523 (carrying pcaHGB; Parke, 1995
) and pDFS603 (carrying pcaC; see Fig. 1
). The above result indicates that the pcaD gene encodes
-ketoadipate enol-lactone hydrolase.
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DISCUSSION |
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Eulberg et al. (1998) first found a pcaL gene that encoded a merged enzyme with
-ketoadipate enol-lactone hydrolase and
-carboxymuconolactone decarboxylase in the rhodococcal pca gene cluster, although these two enzymes are encoded by separate genes, pcaD and pcaC, respectively, in most proteobacteria (Buchan et al., 2004
). In addition, Iwagami et al. (2000)
identified the pcaL gene homologue in the streptomycete pca gene cluster (strain 2065). Recent genome sequence analyses also show that S. coelicolor strain A3(2) (SCO939128), Streptomyces avermitilis MA-4680 (AP005027.1) and Corynebacterium glutamicum strain ATCC13032 (BX927155) possess the pcaL gene. This fusion of the two proteins into one is predicted to be beneficial to the stabilization of the enzyme itself, and to the efficient delivery of substrates and products by minimizing the distance between the active sites of each protein (Eulberg et al., 1998
). Furthermore, it is hypothesized that the separate pcaD and pcaC gene arrangement of proteobacteria may be more ancient, and that the presence of a fused pcaL gene is a Gram-positive trait (Eulberg et al., 1998
; Iwagami et al., 2000
). However, in this study we found that pDBF1 carried the separate pcaD and pcaC genes that had not been found in the pca gene cluster of actinobacteria (Fig. 3
). The pcaD gene was expressed in both FN- and protocatechuate-induced DBF63 cells (Supplementary Fig. 1), and the PcaD enzyme exhibited its activity as a single protein, indicating that PcaD functions during FN or protocatechuate degradation by strain DBF63. Therefore, the presence of the pcaL gene is not quite a Gram-positive trait. Considering that the merged pcaL gene also exists in Ralstonia metallidurans (a
-proteobacteria) and Caulobacter crescentus (an
-proteobacteria) within the pca gene cluster (Jimenez et al., 2002
), whether the Pca enzyme arrangement of actinobacteria is more evolved than that of proteobacteria may be irrelevant.
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In conclusion, this report is believed to be the first to (i) elucidate all catabolic pathway genes for the conversion of FN to TCA cycle intermediates, (ii) find the genes encoding the protocatechuate branch of the -ketoadipate pathway on a linear plasmid, and (iii) find separate pcaD and pcaC genes, but not the merged pcaL gene, within the pca gene cluster of an actinobacteria species. The study of further examples of actinobacterial pca gene clusters, and comparisons of both the organization and regulatory systems of these pca operons may illuminate an evolutionary trait in actinobacteria that could be a consequence of the distinctive selection pressures faced by organisms maintaining the
-ketoadipate pathway.
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ACKNOWLEDGEMENTS |
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Received 24 May 2005;
revised 28 July 2005;
accepted 11 August 2005.
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