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1 Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany
2 Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany
Correspondence
Joachim Klein
joachim.klein{at}lonza.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the sequence determined in this work is U65001.
Present address: Deutsches Ressourcenzentrum für Genomforschung, Heubnerweg 6, 14059 Berlin, Germany.
Present address: Lonza AG, Biotechnology Research and Development, CH-3930 Visp, Switzerland.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Since arylsulfonates are very rarely found in nature, NSAs are referred to as xenobiotics. Naphthalenesulfonates are only slowly degraded in the environment and have been repeatedly isolated from contaminated environments (Wellens, 1990; Alonso et al., 1999; Riediker et al., 2000; Ruckstuhl et al., 2002). Nevertheless, several bacterial strains have been isolated which convert different naphthalenesulfonates (Brilon et al., 1981a, b; Ohe & Watanabe, 1986, 1988; Wittich et al., 1988; Zürrer et al., 1987). The most intensively studied bacterial strain with the ability to degrade naphthalenesulfonates is Sphingomonas xenophaga BN6, which degrades various amino- and hydroxynaphthalenesulfonates to the corresponding amino- or hydroxysalicylic acids (Nörtemann et al., 1986, 1994; Stolz et al., 2000). The catabolism of the naphthalenesulfonates by S. xenophaga BN6 is initiated by an oxygenolytic desulfonation of the substrates in connection with a spontaneous rearomatization to the corresponding (substituted) 1,2-dihydroxynaphthalenes (Fig. 1). The following enzymic steps are identical in the degradative pathways of naphthalene and naphthalenesulfonates. The (substituted) 1,2-dihydroxynaphthalenes are cleaved by a 1,2-dihydroxynaphthalene dioxygenase (NsaC) to 2-hydroxychromene-2-carboxylates, which are converted by a 2-hydroxychromene-2-carboxylate isomerase (NsaD) to trans-2'-hydroxybenzalpyruvates, which are finally converted by 2'-hydroxybenzalpyruvate aldolase/hydratases (NsaEs) to pyruvate and (substituted) salicylaldehydes. Several of these key enzymes of the naphthalenesulfonate degradative pathway have been studied in S. xenophaga (Kuhm et al., 1991a; Stolz, 1999). Three of the relevant enzymes, NsaC, NsaD and NsaE, have been purified and biochemically characterized. Furthermore, the N-terminal amino acid sequences of NsaC and NsaE have been determined (Kuhm et al., 1991a, 1992, 1993).
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, 1997), but sequence and biochemical data show that the two extradiol dioxygenases are not involved in the degradation of NSAs. Recently, the genes for the degradation of NSAs have been localized on a 180 kb megaplasmid in S. xenophaga BN6 (Basta et al., 2004). Therefore, in the present study, the relevant gene cluster was analysed in detail. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. The isolation and characterization of S. xenophaga BN6 has been described previously (Nörtemann et al., 1986; Stolz et al., 2000). The strain has been deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany, as DSM 6383. Strain BN6 was either grown in nutrient broth or a mineral medium (Dorn et al., 1974) supplemented with glucose (MMG, 25 mM), naphthalene-2-sulfonic acid (2NSA, 5 mM), glucose (25 mM) and 2NSA (1 mM), or 6-aminonaphthalene-2-sulfonic acid (5 mM).
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). E. coli strains carrying different plasmids were grown at 37 °C in 2x yeast tryptone (YT) liquid medium or on 2x YT agar plates (Sambrook et al., 1989). The antibiotics ampicillin, kanamycin and tetracycline were used at final concentrations of 100, 50 and 20 µg ml–1, respectively.
DNA manipulation and preparation, cell transformation, and conjugation.
Small-scale plasmids were prepared by the method of Kieser (1984). Genomic DNA was isolated and all manipulations were carried out as described by Sambrook et al. (1989). All enzymes were purchased from Roche Diagnostics and used according to the manufacturer's suggestions. E. coli was transformed with plasmid DNA by the method of Chung et al. (1989). The conjugation experiments were performed as described by Keck et al. (1997).
PCR amplification of the 1,2-dihydroxynaphthalene dioxygenase (DHNDO) gene fragment.
The PCR reaction mixtures contained, in a volume of 40 µl, 10–100 ng genomic DNA, 0.5 µM of each forward and reverse primer (MWG-Biotech), 10 mM Tris/HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs (Pharmacia Biotech) and 2.5 U Taq DNA polymerase (Pharmacia Biotech). The amino acid sequences GYLGM and WRAYAA found at the N terminus of DHNDO (Kuhm et al., 1991a) were used to design the primers 5'-GGY-TAY-CTS-GGY-ATG-3' (DHNDO1, coding strand) and 5'-GC-SGC-RTA-SGC-SCG-CCA-3' (DHNDO2, non-coding strand). The Pseudomonas codon usage was used (Wada et al., 1992). The mixtures were placed in a thermal cycler (PTC-200; MJ Research). The first step consisted of denaturation for 1 min at 94 °C, followed by 30 cycles of denaturation for 1 min at 92 °C, annealing of primers for 1 min at 35 °C and extension for 2 min at 72 °C, with extension for 5 min during the last cycle. The PCR fragments were separated by electrophoresis through 1 % agarose gels at 10 V cm–1 and stained by ethidium bromide.
Nucleotide sequence analysis.
The DNA sequence was determined by the dideoxy chain-termination method in an automated DNA sequencer (ALF-Sequencer; Pharmacia Biotech) by use of reverse primers, sequencing primers or primers which were derived from known sequences. Database searches were done online with the programs BLASTX, BLASTP and BLASTN provided by the BLAST E-mail server (Altschul et al., 1990; Gish & States, 1993). CLUSTALW (Thomson et al., 1994) was used to align the amino acid sequences. All parameters were set at their default values. The sequence alignments were edited and analysed by using the multiple sequence alignment editor and shading utility GeneDoc (version 1.1.004) (Nicholas & Nicholas, 1996).
Isolation of the genomic DNA region.
The 1.3 kb PCR fragment was treated with the Klenow fragment of DNA polymerase I (Boehringer Mannheim) and inserted into EcoRV-cut pJOE890 (Altenbuchner et al., 1992) to yield pJKS240. A 
RES vector, with a built-in Tn1721-encoded excision system (Altenbuchner, 1993), was used to construct a genomic library of the wild-type S. xenophaga BN6. The PCR fragment was DIG-labelled to identify the phage carrying the relevant genomic region. One of the identified phages was converted into the RTS1-derived pAKE3-5 (Altenbuchner, 1993).
Hybridization of DNA.
Total genomic DNA was isolated and digested with restriction endonucleases. After electrophoresis, the DNA was transferred onto a positively charged nylon membrane (Roche Diagnostics). Probe DNA was DIG-labelled by use of a DNA labelling and detection kit (Roche Diagnostics). Hybridization was carried out at 68 °C in hybridization buffer, as described by the manufacturer. The Southern blots were developed using chemiluminescent detection, according to the protocol of the DNA labelling and detection kit.
Inverse PCR.
Inverse PCR was performed, as described by Ochman et al. (1988), to isolate the 5' and 3' flanking sequences of the genomic sequence fragment of pAKE3-5. To isolate the 5' flanking sequence, genomic DNA of strain BN6 was cut with HindIII and hybridized against the chromosomal fragment of pAKE3-5. The hybridization signal corresponded to a fragment of 3.5 kb for the additional 5' sequence. The DNA of strain BN6 was cut with HindIII, ligated and used for PCR studies (forward primer S1659, 5'-GAC-GAT-GAG-CAG-GTA-GAA-CT-3'; reverse primer S1660, 5'-ACC-TTC-ATC-ATG-TCG-TCT-TC-3'). To isolate the 3' flanking sequence, genomic DNA of S. xenophaga BN6 was cut with PstI and hybridized against the 12 kb chromosomal insert of pAKE3-5. The hybridization signal corresponded to a fragment of 
1.5 kb for the additional 3' sequence. Thus, genomic DNA of strain BN6 was cut with PstI and ligated in different concentrations (100 ng, 1 µg). The ligated DNA was used for PCR with the primer pair S2124 (5'-GCA-AAT-CGT-CGT-CAG-GAT-TA-3') and S2125 (5'-GCG-CCG-ACT-GTT-ATC-TTC-TT-3'). The 5' (3.5 kb) and 3' (1.5 kb) flanking fragments of the genomic DNA region were cloned into EcoRV-cut pUC18 and sequenced.
Subcloning of ORF5, ORF7, ORF12 and ORF13 into pJOE2702.
The primers which were used for the subcloning experiments are summarized in Table 2. The forward primer S1817 and the reverse primer S1818 were used to amplify a 1519 bp fragment containing the ORF5 gene. The PCR was performed as described for the DHNDO gene fragment. The primer pairs S1252/S1352, S1821/S1822 and S1823/S1824 were used to generate PCR fragments of ORF7 (916 bp), ORF12 (607 bp) and ORF13 (1006 bp), respectively. To facilitate cloning of the PCR products, NdeI and BamHI restriction sites were added to the primers. The appropriate PCR products were cut with NdeI and BamHI and inserted into NdeI/BamHI-cleaved pJOE2702 (Volff et al., 1996) to give pAKE16 (ORF5), pJKS325 (ORF7), pAKE18 (ORF12) and pAKE17 (ORF13). E. coli strains JM109(pAKE16), JM109(pJKS325), JM109(pAKE18) and JM109(pAKE17) were each grown to the early exponential growth phase (OD600 0.3) and induced for 4 h with 0.2 % (w/v) rhamnose. The cell extracts were analysed by 12 % (w/v) SDS-PAGE.
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). The ORFs were deleted and replaced by the kanamycin-resistance gene (nptII) of Tn5. The chromosomal regions for homologous recombination of the 5' and 3' flanking regions of the inactivated gene, which were ligated to the nptII gene, were set to 1 kb. This resulted in pAKE36 (for ORF6), pAKE37 (for ORF8), pAKE39 (for ORF9) and pAKE41 (for ORF16). The plasmids were transferred separately from E. coli S17-1 (Simon et al., 1983) to S. xenophaga BN6 by conjugation. Since pAKE35.1 (Tcr) does not replicate in strain BN6, integration mutants were selected on MMG agar plates supplemented with tetracycline. To isolate integration mutants which had lost the vector fragment by homologous recombination, the strains were selected on MMG supplemented with sucrose (4 %) and kanamycin. Genomic DNA was isolated, digested with different restriction enzymes and hybridized against the deleted gene, the nptII gene and the 5' and 3' flanking fragments of the deleted gene, to verify the integration of the nptII gene into the corresponding gene. The primers used are listed in Table 2, and the procedure was performed as described previously (Keck et al., 2002).
Preparation of cell extracts.
Cells were harvested by centrifugation at 6000 g, washed with 5 ml and resuspended in 1 ml 50 mM sodium/potassium phosphate buffer (pH 7.3). The cells were disrupted by sonication (Ultrasonics Sonicator W-385; Heat Systems-Ultrasonics; Microtip, 2x30 s, duty cycle 50 % s–1). Cell debris was removed by centrifugation for 30 min at 100 000 g. Protein concentrations were determined according to the method of Bradford (1976), by use of the Bio-Rad protein assay.
Enzyme assays.
The naphthalenesulfonate dioxygenase (NsaA) activity was quantified, as described by Nörtemann et al. (1986), using resting cells of strain BN6 incubated with 2NSA (1 mM), and is given as nanomoles of 2NSA transformed per minute per milligram of protein present in the crude extract (Nörtemann et al., 1994). The NSA degradation pathway was induced by the addition of 0.5 mM salicylate to the exponentially growing cells and the culture was further incubated at 30 °C for 3 h.
NsaC and salicylaldehyde dehydrogenase (NsaF) activity were measured as described by Kuhm et al. (1991a, b). NsaE and NsaD were assayed spectrophotometrically, as described by Kuhm et al. (1992, 1993).
SDS-PAGE.
The proteins were separated on a 12 % (w/v) denaturing polyacrylamide gel by use of the discontinuous buffer system described by Laemmli (1970) and stained with Coomassie blue R250.
HPLC analysis.
The degradation of NSA was monitored by HPLC (Lichrograph L-6200 gradient pump equipped with a L-4200 UV-VIS detector; Merck). A reversed-phase column (250x4 mm internal diameter; GROM), filled with 5 µm diameter particles of Lichrospher RP18 (Merck), was used to identify individual compounds which were detected spectrophotometrically at 210 nm. The mobile phase consisted of 50 % (v/v) methanol, 49.75 % (v/v) H2O and 0.25 % (v/v) H3PO4, and the flow rate was 1 ml min–1.
Chemicals.
NSAs were obtained from Bayer. 2'-Hydroxybenzalpyruvate was kindly provided by A. Kuhm, Institut für Mikrobiologie, Stuttgart. 2-Hydroxychromene-2-carboxylate was prepared enzymically with whole cells of strain BN6, as described by Kuhm et al. (1993). All other chemicals were obtained from Sigma-Aldrich or Merck. Biochemicals were from Boehringer Mannheim.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The primers DHNDO1 and DHNDO2 (Table 2) were derived from the N-terminal amino acid sequence and used for the attempted amplification of the corresponding part of the gene (60 bp) encoding NsaC. Surprisingly, amplification of a 1.3 kb DNA fragment was achieved from the genomic DNA of strain BN6 with primer DHNDO1 alone, using a low annealing temperature (35 °C). The 1.3 kb PCR fragment was cloned into pUC18 and sequenced. Database searches demonstrated a significant homology of the encoded peptide to different bacterial 2,3-dihydroxybiphenyl-1,2-dioxygenases and NsaCs. Furthermore, these sequence comparisons demonstrated that the encoded protein was different from two other extradiol dioxygenases (BphC1 and BphC2) which were previously cloned from strain BN6 (Heiss et al., 1995, 1997).
Isolation of the DNA region surrounding the gene encoding the NsaC
A genomic library of the wild-type S. xenophaga BN6 was constructed using a RES vector (Altenbuchner, 1993). The PCR fragment encoding the NsaC was labelled and used to screen the library. pAKE3-5 was identified, which carries the genomic context of the nsaC gene. Using inverse PCR, 3.5 kb upstream and 1.5 kb downstream of the genomic insert of pAKE35.1 were identified and sequenced. This resulted in a total continuous DNA sequence of 16 915 bp. The G+C content of this DNA sequence was 57.7 %, a value which is lower than the total G+C content of the genomic DNA of S. xenophaga BN6 (62.1±0.2 %) (Stolz et al., 2000). Computer analysis revealed 15 complete (ORF2–ORF13, ORF15–ORF17), one partial (ORF1) and one disrupted (ORF14) ORFs. ORF10 and ORF11 were homologous to the N- and C-terminal parts, respectively, of the large component of an oxygenase. This indicated that ORF10 and ORF11 are inactive fragments generated by an amber mutation (Q
stop) from the gene encoding the large component of a ring-hydroxylating aromatic oxygenase (Fig. 2). The deduced protein sizes of all identified proteins are summarized in Table 3. The identified genes are presumably organized in at least six different clusters. Two of the putative transcriptional units are separated by a putative rho-independent transcription terminator (http://www.bioinfo.rpi.edu/applications/mfold; Fig. 2).
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; Kuhm et al., 1991a). Thus, the initial desulfonating, ring-hydroxylating dioxygenase is of central importance in this pathway. Analysis of the 17 kb genomic sequence revealed several ORFs which shared significant homologies with different proteins of multicomponent dioxygenases. Thus, ORF6, ORF8, ORF9 and ORF16 demonstrated sequence similarities with the 
, ferredoxin, small and reductase components of ring-hydroxylating dioxygenases (Table 3). To analyse if these ORFs encode (parts of) the initial naphthalenesulfonate dioxygenase or different multicomponent dioxygenases, the corresponding genes were each deleted by site-specific integration mutagenesis and replaced by the kanamycin-resistance gene (nptII) of Tn5. The integration cassettes were constructed via gene splicing by overlap extension (Horton et al., 1989). The strains which were further analysed were designated AKE2 (
ORF6), AKE3 (ORF9), AKE4 (ORF8) and AKE5 (ORF16). The integration of the nptII gene into each ORF was verified by Southern blot analysis, as described previously (Keck et al., 2002).
Biochemical analysis of the deletion mutants in the genes presumably encoding NsaA
The conversion of 2NSA by cell suspensions of the integration mutants AKE2 (ORF6 : : nptII), AKE3 (ORF9 : : nptII), AKE4 (ORF8 : : nptII) and AKE5 (ORF16 : : nptII) was tested. Resting cells of these strains were analysed for NsaA activity and the corresponding cell extracts were analysed for NsaC, NsaE and NsaF activity. The addition of salicylate induced NsaC, NsaE and NsaF activities in the mutant and wild-type strains (Table 4). NsaA activity was only detected with resting cells of strains AKE2, AKE3 and the wild-type strain. Strains AKE4 and AKE5 did not show any measurable NsaA activity, irrespective of the presence of salicylate. These results strongly support the view that ORF8 and ORF16 encode the ferredoxin (nsaA3) and reductase (nsaA4) components of NsaA, whereas ORF6 and ORF9 might encode the oxygenase component of a not-yet-identified ring-hydroxylating dioxygenase.
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, 1992). This allowed the identification of ORF7 and ORF13 as the genes encoding the NsaC and NsaE of the naphthalenesulfonate pathway, because the N-terminal 29 or 26 aa of the predicted protein sequences were identical to the data obtained experimentally by protein sequencing. Furthermore, the predicted molecular masses of the proteins encoded by ORF7 (33 301 Da) and ORF13 (35 459 Da) were in good agreement with the values of 33 000 and 38 500 Da determined experimentally for the purified NsaC and NsaE (Kuhm et al., 1991a, 1992).
The amino acid sequence of ORF12 shared significant homologies to different NsaDs (Table 3), such as NahD from S. aromaticivorans F199 and Sphingomonas sp. CHY-1 (76–77 % identical residues). ORF5 shared the highest degree of homology with different presumed benzaldehyde or salicylaldehyde dehydrogenases from different sphingomonads (Table 3). Thus, it appeared that all enzymes necessary for the conversion of (substituted) 1,2-dihydroxynaphthalene(s) to (substituted) salicylate(s) were encoded on the analysed DNA fragment.
Experimental identification of the genes encoding the NsaC, NsaD, NsaE and NsaF involved in the degradation of naphthalenesulfonates
The genes encoding the putative NsaC, NsaD, NsaE and NsaF were amplified from genomic DNA of strain BN6 via PCR (Table 2). The genes were integrated into the expression plasmid pJOE2702 (Volff et al., 1996) to give pAKE16 (ORF5), pJKS325 (ORF7), pAKE18 (ORF12) and pAKE17 (ORF13) (Table 5). The corresponding strains E. coli JM109(pAKE16), JM109(pJKS325), JM109(pAKE18) and JM109(pAKE17) were each grown to the early exponential growth phase and induced by the addition of rhamnose (see Methods). The cell extracts were analysed via SDS-PAGE. Additional proteins of the predicted sizes were only present in cell extracts of the rhamnose-induced strains (Fig. 3). The corresponding enzyme activity was analysed in comparison to cell extracts of the uninduced strain E. coli JM109(pJOE2702). As summarized in Table 5, the putative enzyme functions were confirmed for ORF7 (NsaC), ORF12 (NsaD), ORF13 (NsaE) and ORF5 (NsaF). The cell extracts of the uninduced strains and the control strain JM109(pJOE2702) did not show any NsaC, NsaD, NsaE or NsaF activity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and phenanthrene by Sphingomonas sp. P2 (Pinyakong et al., 2003a), clearly demonstrate a unique conserved organization of the genes involved in the degradation of bicyclic and polycyclic aromatic compounds by sphingomonads. Thus, in all three strains, three conserved gene clusters were identified, which shared significant nucleotide sequence identities ranging from 60 to 90 %. One of these gene clusters has also recently been identified in the chrysene-degrading strain Sphingomonas sp. CHY-1 (Demanèche et al., 2004), and a similar genetic organization also seems to be present in the biphenyl-degrading strain Sphingomonas yanoikuyae B1 (Kim & Zylstra, 1999), although unfortunately the relevant gene sequence has not yet been deposited in a public database. Furthermore, some recent PCR experiments, using primers derived from conserved sequences within these gene clusters, suggest that these gene clusters are also present in other aromatic-degrading sphingomonads (although in different arrangements and at different distances from each other) (Basta et al., 2005). The conservation of these gene clusters among different sphingomonads is also reflected by the results of the amino acid sequence comparisons of the proteins of the S. xenophaga BN6 aromatic degradation pathway and the sequences deposited at the NCBI database. These alignments always resulted in the identification of genes from other sphingomonads as the most closely related sequences. This clearly demonstrates that, although the relevant genes are encoded on plasmids in S. aromaticivorans F199 and S. xenophaga BN6 (Romine et al., 1999; Basta et al., 2004), some restrictions seem to occur which prevent a broad dissemination of the relevant genetic information among bacterial strains which do not belong to the sphingomonads. This was also indicated by our failure to transfer Sphingomonas plasmids labelled with antibiotic resistance markers to bacterial strains not belonging to the Sphingomonadaceae (Basta et al., 2004). Thus, the organization of the genes and the sequence comparisons of the individual genes both suggest some unique lines of evolution for the degradation of aromatics among the sphingomonads. The previous work with S. aromaticivorans F199 and Sphingomonas sp. P2 was mainly concerned with the sequencing of the relevant parts of the bacterial genomes, and the putative functions of the identified genes were almost exclusively deduced from their homologies with genes of known function from the databases. In contrast to this situation, for S. xenophaga BN6, much more biochemical data were available from previous studies. This, together with the extensive generation of mutants performed in the course of the present study, allowed an unequivocal identification of the nsaC and nsaE genes participating in the degradation of the naphthalenesulfonates. This furnished the final experimental proof that these two genes are indeed not part of the same operon in various sphingomonads.
The phenotypic analysis of the mutants clearly suggested that ORF8 and ORF16 encode the ferredoxin (nsaA3) and ferredoxin reductase (nsaA4), respectively, of the initial desulfonating NsaA, because these mutants no longer converted 2NSA. This demonstrated that the genes encoding the ferredoxin and ferredoxin reductase also belong to different gene clusters. In contrast, the mutations in ORF6 and ORF9 (which presumably encode the large and small components, respectively, of a hydroxylating oxygenase) clearly demonstrated that these ORFs either do not encode the initial desulfonating activity or can be replaced by some other (redundant) activity. A similar observation was made when the putative oxygenase gene of a ring-hydroxylating dioxygenase from S. yanoikuyae B1 was deleted, but no influence on the ability of the strain to oxidize naphthalene or biphenyl was found. The analysis of the relevant gene sequence from S. yanoikuyae B1 demonstrated the presence of at least six oxygenase components of ring-hydroxylating dioxygenases (XylXY, BphA1a2a, BphA1b2b, BphA1c, BphA1d and BphA1e). On the other hand, there is only one copy of the genes specifying the ferredoxin (bphA3) and ferredoxin reductase (bphA4) (Zylstra & Kim, 1997). Thus, analysis of the relevant genomic regions of S. aromaticivorans F199 (and also of Sphingomonas sp. P2) suggests that various terminal oxygenases can be synthesized by these strains (Romine et al., 1999; Pinyakong et al., 2003a). These findings indicate that it is a general concept in the metabolism of aromatic compounds by sphingomonads to couple a unique electron transport system (consisting of a ferredoxin and a ferredoxin reductase) with multiple oxygenase components (presumably with different substrate specificities). Thus, it is probable that the genes for the large and small components of the NsaA are located in S. xenophaga BN6 at a different position.
The analysed DNA fragment from S. xenophaga BN6 encoded, in addition to ORF6 and ORF9, which were analysed by deletion mutagenesis, two more ORFs (ORF10 and ORF11) which demonstrated homology to large components of ring-hydroxylating oxygenases. The sequence comparisons suggested that the proteins encoded by ORF10 and ORF11 are significantly truncated in comparison to classical, functional, large components of ring-hydroxylating oxygenases. Thus, ORF10 may encode only a protein of 132 aa and ORF11 a protein of 285 aa. The sequence alignments suggested that ORF10 and ORF11 are homologous to the N- and C-terminal parts, respectively, of the large component of an oxygenase. This indicated that ORF10 and ORF11 are inactive fragments generated by an amber mutation (Qstop) from the gene encoding the large component of a ring-hydroxylating aromatic oxygenase. The BLAST searches indicated that both ORF10 and ORF11 show the highest degree of sequence identity with AhdA1c from Sphingomonas sp. P2. Furthermore, we found that ORF9 from strain BN6 showed the highest degree of homology to the small component (AhdA2c) of the same oxygenase from Sphingomonas sp. P2. This indicated that S. xenophaga BN6 only possesses an inactivated derivative of the enzyme from Sphingomonas sp. P2. Interestingly, it has recently been shown that AhdA1c and AhdA2c encode a monooxygenase in Sphingomonas sp. P2 which converts (substituted) salicylate(s) to (substituted) catechol(s) (Pinyakong et al., 2003b). Thus, the amber mutation between ORF10 and ORF11 might be responsible for the inability of S. xenophaga BN6 to oxidize (substituted) salicylate(s) which are formed from (substituted) naphthalenesulfonate(s) (Nörtemann et al., 1986, 1994). This is of central importance for the ability of this strain to establish stable mixed bacterial cultures with salicylate-degrading bacteria and, presumably, is also responsible for the ability of strain BN6 to convert a wide range of substituted naphthalenesulfonates.
It was previously demonstrated that the genes responsible for the degradation of naphthalenesulfonates are encoded on a 180 kb plasmid (pBN6) in S. xenophaga BN6 (Basta et al., 2004). The sequence analysis performed during the present work suggested that the genes encoding the degradation of naphthalenesulfonates are flanked by genes (ORF1, ORF2, ORF3 and ORF17) specifying proteins with homologies to proteins encoded by transposons or insertion elements (Table 4). The presence of such genes suggests that the identified nsa genes may be part of functional or non-functional mobile genetic elements. Similar genetic arrangements might also be responsible for the previous observations that the genes encoding the degradative pathway(s) for naphthalene (and biphenyl) are either found on the chromosome or on large plasmids of certain sphingomonads (Kim et al., 1996). The pronounced rearrangements which have been observed after the transfer of plasmids among sphingomonads might be due to these special structural features and homologies (Feng & Ogram, 1997; Basta et al., 2004).
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Received 19 December 2005;
revised 13 March 2006;
accepted 14 March 2006.
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subunits of a multicomponent dioxygenase are shaded in grey. ORF14, which is disrupted by ORF15, is indicated by a dotted outline. ORF10 and ORF11 are truncated genes of a dioxygenase and are shown as a single, non-functional ORF. The genes encoding proteins which have been shown to be involved in the degradation of naphthalenesulfonates (nsa genes) are displayed in italic type. Relevant restriction sites are shown. A rho-independent terminator-like structure (T) is indicated between ORF12 and ORF13. (b) In comparison to the genes of the BN6 locus, homologous proteins which are encoded on three different, non-contiguous loci of pNL1 from S. aromaticivorans F199 are displayed. Homologous genes from strain F199 which encode proteins with functions similar to those of the nsa genes are shaded.