1 Environmental Biotechnology Laboratory, Railway Technical Research Institute, Kokubunji, Tokyo 185-8540, Japan
2 Department of Built Environment, Tokyo Institute of Technology, Yokohama 226-8502, Japan
3 Research Institute of Technology, Okayama University of Science, Okayama 703-8232, Japan
Correspondence
Minoru Shimura
mino{at}rtri.or.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AB113649 and AB181508.
A figure showing amino acid sequence comparisons is available as supplementary data with the online version of this paper.
Present address: Genetics and Genomic Biology Program, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M2G 1X8.
Present address: Department of Microbiology and Biochemistry, University of British Columbia, 300-6174 University Blvd, Vancouver, BC, Canada V6T 1Z3.
Present address: Research Institute for Bioresources, Okayama University, 2-20-1 Chuo, Kurashiki, Okayama 710-0046, Japan.
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INTRODUCTION |
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Utilization of a thermophile for the degradation of xenobiotics in a biotechnological process offers several advantages, i.e. increased solubility of the pollutant, reduced risk of mesophile contamination and increased stability exhibited by thermophilic enzymes. Although thermophiles degrading aromatic compounds such as BTEX (benzene, toluene, ethylbenzene, xylene) and phenol/cresol have been isolated, there is limited molecular information about the genes/operons involved (Chen & Taylor 1995; Dong et al., 1992
; Duffner & Müller, 1998
; Miyazawa et al., 2004
; Mutzel et al., 1996
; Natarajan et al., 1994
). We have isolated and characterized a naphthalene- and biphenyl-utilizing Gram-positive thermophilic bacterium, Bacillus sp. JF8, which can degrade PCBs (Shimura et al., 1999
). To study the structure, molecular organization, regulation and phylogeny of the genes/enzymes involved in degradation of biphenyl, we initially cloned and characterized the extradiol dioxygenase of the biphenyl pathway, BphC_JF8 (Hatta et al., 2003
). Unlike any other BphC reported so far, the thermostable BphC_JF8 exhibited manganese dependence. In most reports on extradiol dioxygenases that utilize metals other than Fe(II) (Boldt et al., 1995
; Gibello et al., 1994
; Miyazawa et al., 2004
; Que et al., 1981
), information on the flanking or adjoining genes, which might comprise an operon, is not available. Here we characterize the three genes that flank the gene encoding the Mn(II)-dependent BphC and that are responsible for the degradation of biphenyl in the thermophilic Bacillus sp. JF8.
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METHODS |
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Molecular techniques.
Total DNA from strain JF8 was isolated by a modification of the procedure described by Marmur, while plasmid DNA from JF8 was isolated by the hot alkaline-pH method of Kado & Liu (Johnson, 1994). Standard procedures were used for plasmid DNA preparation and manipulation, and for agarose gel electrophoresis. E. coli MV1184, DH5
and JM109 were transformed by the CaCl2 procedure (Sambrook et al., 1989
). To clone the upstream region of bphC_JF8, initially a 46 kb SacI gene bank of Bacillus sp. JF8 was constructed in Charomid vector 9-42 (Nippon Gene) and was used to transfect E. coli DH5
using the Gigapack II packaging extract kit (Stratagene) following the manufacturer's instructions. Colonies harbouring recombinant plasmids were transferred onto Hybond-N+ membranes (Amersham) and probed with a 1 kb HindIIIKpnI fragment encoding the C-terminal region of bphB and the N-terminal region of bphC. Southern blot hybridization was done with the non-radioactive DIG DNA hybridization kit (Roche Diagnostics) and probes were labelled according to the manufacturer's instructions. SacI inserts from colonies that showed a positive hybridization signal were cloned into pBluescript II SK+ and KS+ vectors (Stratagene). The upstream region of the 4892 bp SacI fragment (pBSc5) was similarly isolated by constructing and screening a 67 kb HindIII gene bank. A Kilosequence deletion kit (Takara Biomedicals) was used to construct a series of deletion derivatives, whose nucleotide sequences were determined by the dideoxy termination method (Sanger et al., 1977
). Sequences were analysed using the program OMIGA 2.0 (Oxford Molecular).
Homology search and phylogenetic analysis.
A homology search was performed with BLAST 2.0 (gapped BLAST) (Altschul et al., 1997). Amino acid sequences retrieved from the protein database were aligned using CLUSTAL W version 1.9 (Thompson et al., 1994
) and a neighbour-joining tree constructed using the Blosum62 distance matrix, confidence levels being determined by bootstrap analysis. The results were depicted using Tree View version 1.5 (Page, 1996
).
Construction of expression plasmids.
To clone bphA1A2 of strain JF8 into the expression vector pET-21a(+) (Novagen), PCR was used to engineer a SacI restriction site and a ShineDalgarno sequence in front of the ATG start codon of bphA1, and a unique restriction site immediately after the termination codon of bphA2. The HindIII, EcoRV and EcoRI restriction sites in the multicloning site of pBluescript II SK+ were removed using the DNA blunting kit (Takara Biomedicals) to give pSK+HE and the 3052 bp SacIPstI fragment (encoding bphA1A2) from pBSc5 was ligated into pSK+
HE to give pBScP
. A SacI site followed by an efficient ShineDalgarno sequence, AAGGAG (underlined in the primer), was introduced 8 bp upstream of the ATG start site of bphA1 (double underlined in the primer) using the primer 5'-GAGCTCAAGGAGGTATAGCGATGGGAGAAAGAAAATGGCGA-3'. The reverse primer used (giving a 800 bp product) was 5'-ATGCTTTCCGGATACCCCATAAGCTTTGGGGAGGCAACGT-3', which extended up to an internal HindIII site (underlined in the primer) in the bphA1 gene. The PCR product was cloned into the pCR 2.1-TOPO vector using the TOPO TA cloning kit (Invitrogen) to give pCRA1, and the DNA sequence was verified. After digestion with SacI and HindIII the 800 bp product was cloned into the SacI and HindIII site of pBScP
to give pBScP
2. To introduce a XhoI site after the termination codon of bphA2, the PCR primers used were 5'-GACGGATGATATCGTATACAG-3' (forward primer) and 5'-TGCGCTTGCCTCGAGCTAGAGAA-3' (reverse primer). This amplified a 392 bp product, from an internal EcoRV restriction site (underlined in the forward primer) in bphA2 to the TAG stop codon followed by the introduced XhoI site (underlined in the reverse primer). The PCR product was cloned into the pCR 2.1-TOPO vector using the TOPO TA cloning kit to give pCRA2, and the DNA sequence was verified. After digestion with EcoRV and XhoI, the 392 bp product was cloned into the similarly digested pBScP
2 to give pBScP
3. Digesting pBScP
3 with SacI/XhoI gave a 1·9 kb fragment containing the bphA1A2 genes preceded by an efficient ShineDalgarno sequence, which was cloned into pET-21a(+) to give pET21_A1A2. To test if the ORFs were being translated into proteins of the predicted size, the bphA1A2 genes were introduced into E. coli BL21(DE3) after initial transformation into a non-expression host. The culture was grown on Lennox broth at 37 °C to OD550 0·5, then the T7-mediated gene expression was induced by adding IPTG to a final concentration of 1 mM. The cultures were allowed to grow for 23 h and cells were harvested from 1 ml culture. The cell pellet obtained was resuspended in 500 µl 3x reducing SDS sample buffer (New England Biolabs) and heated. Some of the sample (510 µl) was analysed by SDS-PAGE (Laemmli, 1970
) using broad range prestained protein markers (New England Biolabs).
The upstream region of the 4892 bp SacI fragment (pBSc5) was isolated as an overlapping 7 kb HindIII fragment and cloned into the vector pUC118 to give pBUH7. A 4·5 kb PstIHindIII fragment from pBUH7, containing a functional hydrolase gene, was subcloned into pUC118 to give pBUPH2.
PCR primers were used to amplify the bphB gene of strain JF8. The PCR product was cloned into the BamHI/EcoRI site of the vector pBluescript II SK+, and the DNA sequence of the PCR fragment was verified; the plasmid was then introduced into E. coli MV1184. The BamHIEcoRI fragment encoding the bphB gene was cloned into the low copy number vector pSTV29 (Takara Biomedicals) to give pSTV29_B, which is compatible with pET-21a(+), and therefore, the bphB gene could be introduced into E. coli BL21(DE3) along with the bphA1A2 genes.
The plasmids, pUAD1, with a 1·2 kb StuIHindIII fragment encoding bphD, and pUAD2, with a 1·0 kb SacISphI fragment encoding etbD1 of Rhodococcus sp. RHA1 (Yamada et al., 1998), were used in thermostability studies.
Biotransformation experiments with recombinant proteins and identification of metabolites.
Liquid cultures of E. coli BL21(DE3) carrying the bphA1A2 and bphB genes were inoculated into Lennox broth containing 100 µg ampicillin ml1 and 30 µg chloramphenicol ml1 . They were grown at 37 °C to an OD550 0·5 (about 4 h), and IPTG to a final concentration of 1 mM was added. The cultures were incubated for a further 23 h. The cells were collected by centrifugation, washed with ice-cold 50 mM phosphate buffer (pH 7·5) and resuspended in the same buffer after adjusting cell density to OD550 1·0 for resting cell experiments. Biphenyl, naphthalene and phenanthrene dissolved in ethyl acetate were supplied at a final concentration of 500 µM, while benzene was supplied as vapour. PCB congeners dissolved in DMSO were supplied at a concentration of 25 µM. The cells were incubated at 37 °C for 24 h, after which the culture was acidified to pH 2·0 with HCl, and extracted twice with an equivalent amount of ethyl acetate. The extract was evaporated to dryness, resuspended in a minimal amount of ethyl acetate, derivatized with BSTFA (N,O-bis(timethylsilyl)trizfluoroacetamide)+TMCS (trimethylchlorosilane) (Supelco) prior to GC-MS analysis. E. coli BL21(DE3) cells containing the vectors pET-21a(+) and pSTV29 were used as controls in all experiments. To obtain a mass fragmentation pattern of authentic standards, 2,3-dihydroxybiphenyl, 1,2-dihydroxynaphthalene and catechol were added to control samples, which were acidified, extracted with ethyl acetate and then derivatized with BSTFA+TMCS. In the case of PCB congeners, the ethyl acetate extract was directly used for analysis by GC-MS.
Extracted samples were analysed by GC (Hewlett Packard; model 6890) equipped with an HP-5ms capillary column (50 m, 0·2 mm, 0·33 µm; Hewlett Packard) and a mass selective detector (Hewlett Packard; model 5972A), integrated by Hewlett Packard MS Chemstation software. Some of the sample (1 µl) was injected by an auto-injector (Hewlett Packard; model 7673) in the spitless mode. Identification of PCB congeners has been described previously (Shimura et al., 1999). For the identification of the hydroxylated derivatives of benzene, biphenyl, naphthalene and phenanthrene, the initial oven temperature of 60 °C was programmed to increase by 20 °C min1 to 300 °C, where it was held for 5 min. The injector temperature was 250 °C and the transfer line temperature was 280 °C. The carrier gas was helium and its flow rate was kept constant at 1 ml min1. Ions at m/z 188,190; 222,224; and 256,258 were chosen for monitoring PCB congeners, while the electron-ionization mass spectra for the hydroxylated derivatives were obtained using the scanning mode (50650 m/z).
Purification of native and recombinant 6-phenyl HODA hydrolase.
E. coli MV1184 harbouring pBUPH2 (BphD_JF8) was grown overnight in 1·5 litres of Lenox broth containing 100 µg ampicillin ml1 and 1 mM IPTG. The cells were collected by centrifugation at 4000 g for 10 min. For native 6-phenyl HODA hydrolase, cells of strain JF8 were grown overnight in Lennox broth with biphenyl. The cells were washed twice with 25 mM phosphate buffer (pH 7·5) and resuspended in the same buffer. The cells were disrupted by a French pressure cell (Aminco) and centrifuged at 12 000 g for 30 min and then at 105 000 g for 60 min. The supernatant was recovered and used as crude extract.
For enzyme purification, all manipulations were carried out at 10 °C in 25 mM potassium phosphate buffer (pH 7·5) containing 1 mM -mercaptoethanol (buffer A) unless otherwise stated. The enzyme activities of the eluted fractions were assayed against 6-phenyl HODA (as described below). For DEAE-Toyopearl chromatography, the crude extract was loaded onto a DEAE-Toyopearl column (2·6x15 cm) previously equilibrated with buffer A. Proteins were eluted with a linear gradient of KCl from 0 to 0·5 M, in a total volume of 800 ml buffer A. Active fractions, eluted around 0·2 M KCl, were collected. For phenyl sepharose column chromatography, the collected fractions were dialysed against buffer A containing 1·0 M ammonium sulfate. The resulting protein solution was loaded onto a phenyl sepharose HP 26/10 column (Amersham Biosciences) equilibrated with buffer A containing 1·0 M ammonium sulfate. The enzyme was eluted with 600 ml of a gradient of 1·00·0 M ammonium sulfate. The enzyme was eluted at around 0·1 M ammonium sulfate. For MonoQ column chromatography, the active fractions eluted from the phenyl sepharose column were pooled and dialysed against buffer A. The resulting solution was applied to a MonoQ HR 16/10 column (Amersham Biosciences) equilibrated with buffer A. After the column was washed with 60 ml buffer A, the enzyme was eluted with 400 ml of a linear gradient of KCl from 0·0 to 0·25 M. The enzyme eluted at around 0·15 M KCl. Protein concentration was estimated by the method of Bradford (1976)
using BSA as a standard. The purity and size of the enzyme proteins were estimated by SDS-PAGE according to the method of Laemmli (1970)
. Protein staining of the gel was performed with Coomassie brilliant blue R-250.
BphD assay and kinetic measurements.
The hydrolase activity was assayed by monitoring the decrease in A434 for 6-phenyl-HODA, 13 200 cm1 M1, A388 for HOHDA,
32 000 cm1 M1 and A375 for 2-hydroxymuconic semialdehyde (HMSA),
36 000 cm1 M1 (Asturias & Timmis, 1993). 6-Phenyl HODA, HOHDA and HMSA were prepared from 2,3-dihydroxybiphenyl, 3-methylcatechol and catechol by incubating with BphC_JF8. The assay was performed at 25 or 60 °C in 50 mM phosphate buffer (pH 7·5). The reaction was initiated by the addition of 5 µl enzyme solution to the reaction mixture. One unit of activity was defined as the amount of enzyme required to degrade 1 µmole of meta-cleavage compound min1.
The substrate concentration used was 0·42·5 µM for 6-phenyl HODA, and 1300 µM for HOHDA and HMSA. The activation energy (Ea) was estimated using the Arrhenius equation for the temperature range of 2075 °C. The value of Ea was determined from the slope of the straight line that resulted when the logarithm of the reaction constant, k, was plotted against 1/T. To determine thermal stability, a temperature range of 3080 °C was utilized. In this case crude extract of recombinant BphD_JF8, BphD_RHA1 and EtbD_RHA1 were used.
N-terminal sequence analysis.
Purified native BphD_JF8 was subjected to N-terminal amino acid sequencing by the Edman degradation process using a model 477A protein sequencer (Applied Biosystems) in accordance with the manufacturer's instructions.
Nucleotide sequence accession number.
The nucleotide sequence reported here has been submitted to the DDBJ and GenBank nucleotide sequence databases under accession nos AB113649 and AB181508.
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RESULTS |
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The deduced amino acid sequence of the two ORFs upstream of the BphB_JF8-encoding gene showed similarity to the large () and small (
) subunits of terminal oxygenases from multicomponent primary dioxygenases, and the gene products were designated BphA1 and BphA2, respectively. The consensus sequence Cys-X1-His-X16-Cys-X2-His of a Rieske-type [2Fe-2S] cluster binding site can be identified in BphA1_JF8. The motif Glu-X34-Asp-X2-His-X35-His, separated from the Rieske type site by 90100 aa, is the potential mononuclear non-haem iron coordination site (Kauppi et al., 1998
). In BphA1_JF8, the initial Glu of the above-mentioned motif is replaced with Asp (also observed in some naphthalene- and phenanthrene-degrading proteins, and in TdnA1_UCC and AtdA3_YAA, Fig. 7
), while other amino acids of the motif are conserved.
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There is an ORF (420 aa, 53 mol% G+C) 260 bp upstream of the putative bphR exhibiting homology (4344 % identity) to the transposase for insertion sequence element IS256 in transposon Tn4001 of Staphylococcus aureus (Ito et al., 2003) and the transposase for insertion sequence ISRM5 of Sinorhizobium meliloti strain 1021 (Capela et al., 2001
).
Expression and characterization of the bphA1A2 genes
A plasmid pET21_A1A2 was constructed and introduced into E. coli BL21(DE3) for the expression of the bphA1A2 genes. SDS-PAGE analysis indicated that the expressed BphA1A2 proteins were of the expected size (results not shown). The bphB gene, on a plasmid (pSTV29_B) compatible with pET-21a(+), was introduced into the E. coli containing the plasmid pET21_A1A2. Resting cells of E. coli BL21(DE3) expressing recombinant BphA1A2 and BphB proteins exhibited biphenyl-transforming activity indicating that non-specific ferredoxin and ferredoxin reductase supplied by the host cell complemented the recombinant proteins. GC-MS analysis of the extracted supernatant revealed the presence of a molecular ion with the molecular mass (m/z 330) and signature (fragment ion of m/z 315, 242, 227, 212 and 165) observed in authentic samples of trimethylsilanized 2,3-dihydroxybiphenyl (results not shown). Benzene, naphthalene and phenanthrene were also used as substrates for resting cell assays. Transformation products catechol and 1,2-dihydroxynaphthalene were detected by GC-MS analysis in the case of benzene & naphthalene, respectively, and the molecular masses and signature patterns of the extracted compounds were consistent with authentic standards (results not shown). Bacillus sp. JF8 does not utilize benzene as a carbon source, nevertheless, benzene was a substrate for BphA1A2B. Although 1,2-dihydroxynaphthalene is known to be unstable in aqueous solutions (Eaton & Chapman, 1992), we could identify the derivatized compound, albeit at very low concentrations. No transformation products were identified when phenanthrene was added. To check for an indole-positive phenotype, E. coli BL21(DE3) expressing recombinant BphA1A2 was grown on LB agar plates in the presence of IPTG and indole vapour. However, indigo was not produced.
Bacillus sp. JF8 can degrade some congeners of PCB (Shimura et al., 1999). To verify that the cloned bphA1A2 gene products were responsible for the degradation specificity observed in Bacillus sp. JF8, the congener-transforming activity of E. coli BL21(DE3) expressing recombinant BphA1A2 and BphB proteins was analysed in a defined mixture of di- and tri-chlorinated PCB. When supplied with a combination of 2,2'-, 4,4'- and 3,3'-dichlorobiphenyl, the recombinant protein completely transformed 3,3'- and 4,4'-dichlorobiphenyl, while 2,2'-diclorobiphenyl was hardly transformed. In a mixture of 2,5,2'-, 2,4,4'-, 2,3,3'- and 2,4,3'-trichlorobiphenyl, 2,3,3'-trichlorobiphenyl was completely transformed, 2,5,2'-trichlorobiphenyl did not show any transformation while the other two congeners were partly transformed. Table 2
compares transformation of the above-mentioned congeners by the recombinant BphA1A2 with that of Bacillus sp. JF8.
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Recombinant 6-phenyl HODA hydrolase was purified 42-fold with an overall yield of 54 %. (Table 3). The molecular mass of the enzyme was determined to be 33 kDa by SDS-PAGE (Fig. 6
), which is in agreement with the value calculated from the deduced amino acid sequence of BphD_JF8. Recombinant BphD_JF8 exhibited a high affinity for 6-phenyl-HODA as evidenced by a Km of 0·71±0·07 µM at the physiological growth temperature of 60 °C, while at 25 °C, the Km was 0·85±0·15 µM. At 60 °C, the specific activity of BphD_JF8 for 6-phenyl-HODA was 1·34 U mg1, Vmax was 2·1 U mg1, while Kcat was calculated to be 1·11 s1. BphD_JF8 exhibited faint activity towards HOHDA and HMSA. The optimum temperature of the enzyme was 85 °C. Fig. 7(a)
compares thermal stability of BphD_JF8 with a mesophilic HOHDA hydrolase (EtbD1_RHA1) and 6-phenyl HODA hydrolase (BphD_RHA1) after 30 min incubation at temperatures ranging from 3080 °C. The Arrhenius plot was discontinuous with an inflection temperature of 47 °C (Fig. 7b
). The activation energy for hydrolysis of 6-phenyl-HODA by BphD_JF8 was determined to be 7·5 kcal mol1 (31·4 kJ mol1) for the temperature range of 2046 °C, while it was 2·9 kcal mol1 (12·1 kJ mol1) at the higher range (5075 °C).
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DISCUSSION |
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The genetic organization of the bph locus in JF8 is uncommon with the meta-cleavage product hydrolase gene (bphD), which carries out the fourth step in the degradation of biphenyl, directly linked to the ring-hydroxylating dioxygenase, upstream of the bphA1A2 cluster, forming a single transcriptional unit, while bphB, which encodes the second enzyme (dihydrodiol dehydrogenase) in the pathway, is transcribed as a separate unit with bphC. The genes encoding the ferredoxin (bphA3) and ferredoxin reductase (bphA4) components of the ring-hydroxylating dioxygenases were not present in the bph locus. Sequencing regions 3 kb upstream and downstream of the bph locus failed to locate bphA3A4. It is possible that they are located elsewhere on the plasmid or they may be supplied by the cellular housekeeping genes. The interchangeability of electron-transfer components between evolutionary related primary dioxygenases has been reported previously (Furukawa et al., 1993; Romine et al., 1998
), and a tolerance between redox and oxygenase partners has been proposed to function as an evolutionary process for multicomponent oxygenases (Harayama et al., 1992
).
Indels (insertions or deletions) are signature sequences in proteins that can be used as phylogenetic markers to determine the course of evolutionary history (Gupta, 1998). We compared the amino acid sequences in the regions of the Rieske-type [2Fe-2S] cluster and the non-haem iron-coordination site in the 35 ring-hydroxylating dioxygenases shown in Fig. 4
to check for the presence of indels (see the Supplementary Figure available with the online journal). While the amino acids around the Rieske-type [2Fe-2S] cluster binding site are highly conserved, the environment of the Fe(II) ligands revealed differences, and indels flanked by regions conserved in all sequences could be used to categorize the dioxygenases into four groups (group I to IV), which appears to reflect the clustering on the phylogenetic tree (Fig. 4
). Probably the geometry of the ligands of the active site where oxygen activation is thought to occur differs depending on the range of substrates that can be oxidized.
Consistent with the occurrence of BphD_JF8 in a pathway responsible for the catabolism of biphenyl, the enzyme exhibited a narrow substrate preference and hydrolysed 6-phenyl HODA. The narrow substrate specificity could be the key determining factor governing the selectivity of the pathway with respect to the aromatic compound degraded. BphD_JF8 bears the characteristic catalytic residues of nucleophile-acid-histidine of the /
hydrolase family (Nardini & Dijkstra, 1999
). Of the polar residues (Asn-46, Asn-109, Gln-266) in the substrate-binding pocket of BphD_RHA1, determined to be sites for potential electrostatic interaction (Nandhagopal et al., 2001
), the analogous Asn residues (Asn-43, Asn-104) are conserved in BphD_JF8, while Gln-266 is replaced by a non-polar methionine residue, and this is also true for DxnB_RW1 (Armengaud et al., 1998
) and CarC_CA10 (Sato et al., 1997
), which cluster with BphD_JF8 on the phylogenetic tree.
As BphD_JF8 is from a thermophile with an optimum growth temperature of 60 °C, it was expected that the optimum temperature for the enzyme would be higher than that of hydrolases from mesophiles. The optimum temperature was 85 °C for BphD_JF8 compared to 48 °C for CumD_IP01 (Saku et al., 2002), and 65 °C for ThnD_TFA (Hernaez et al., 2000
) and BphD_RHA1 (Hatta et al., 1998
). Temperature stability of mesophilic HODA hydrolases have been rarely reported. XylF_TOL, the HMSA hydrolase retained 50 % activity after 15 min at 42 °C (Diaz & Timmis, 1995
), while CumD_IP01 retained 80 % activity at 50 °C for 30 min (Saku et al., 2002
). Compared to XylF_TOL, CumD_IP01, BphD_RHA1 and EtbD1_RHA1, the temperature stability of BphD_JF8 is higher. Surprisingly, the stability of BphD_JF8 was found to be lower than ThnD_TFA from the mesophilic tetralin degrader, Sphingomonas macrogoltabidas, which retained 78 % residual activity at 70 °C after 2 h (Hernaez et al., 2000
), while BphD_JF8 retained 41 % residual activity at 70 °C after 30 min.
Molecular analysis of catabolic pathways in bacteria degrading xenobiotic compounds indicates that bacteria might have adapted to the appearance of these compounds by expressing new functions to counteract potential toxic effects, or by using the available compounds as alternative sources of carbon or nitrogen (Copley, 2000; Top & Sringael, 2003
). Adaptation to the presence of xenobiotic compounds could involve horizontal gene transfer, mutations and gene rearrangements (van der Meer et al., 1992
), and the construction of pathways by assembling pre-existing genes or gene modules (referred to as a patchwork assembly or pathway assembly) has been observed (Copley, 2000
; Springael & Top, 2004
; van der Meer, 1997
). To account for the divergent phylogenetic clustering of the bph genes from Bacillus sp. JF8 and the variance in gene order, we hypothesize that the locus was created by the recruitment of genes from a variety of catabolic pathways. While BphC and BphD appear to be specific for the products of the biphenyl pathway (although their genes do not cluster with the bph genes on a phylogenetic tree), biotransformation experiments indicate that BphA and BphB have relaxed substrate specificity and they cluster with proteins that attack polyaromatic hydrocarbons. The recruitment and fusion of a meta-cleavage enzyme and hydrolase specific for 2,3-dihydroxybiphenyl and 6-phenyl-HODA, respectively, with a ring-hydroxylating dioxygenase and dehydrogenase with broad substrate specificity could have given rise to the bph operon of JF8. The presence of an ORF with homology to the transposase genes of insertion sequences upstream of the bph genes implies the likely involvement of transposition in the evolution of the operon.
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Received 12 August 2005;
revised 2 September 2005;
accepted 2 September 2005.
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