1 Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany
2 Interdisziplinäres Ökologisches Zentrum, Technische Universität Bergakademie Freiberg, Leipziger Str. 29, D-09599 Freiberg, Germany
Correspondence
Michael Schlömann
Michael.Schloemann{at}ioez.tu-freiberg.de
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ABSTRACT |
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Present address: Europroteome, Neuendorfstr. 24 b, 16761 Henningsdorf, Germany.
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INTRODUCTION |
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In particular, the occurrence of maleylacetate reductase genes as parts of specialized gene clusters has been found in the degradation of chloroaromatic compounds. It has been reported for the 2,4,5-trichlorophenoxyacetate conversion via 5-chloro-2-hydroxyquinol and hydroxyquinol by B. cepacia AC1100 (Daubaras et al., 1995, 1996
; Zaborina et al., 1998
) as well as for pentachlorophenol degradation via 2,6-dichloroquinol by Sphingomonas chlorophenolica ATCC 39723 (Cai & Xun, 2002
). It has also been shown for pathways that use chlorocatechols as their central intermediates. Although the maleylacetate reductase gene, tfdF, of Ralstonia eutropha JMP134(pJP4) was initially misinterpreted as encoding a chlorodienelactone isomerase (Don et al., 1985
), the chlorocatechol gene clusters of pJP4, pAC27, Pseudomonas sp. B13, and pP51 by DNA sequencing, by correspondence of experimentally determined N-terminal sequences to sequences predicted from DNA and by expression of cloned genes, were shown to contain maleylacetate reductase genes in addition to genes for chlorocatechol 1,2-dioxygenases, chloromuconate cycloisomerases and dienelactone hydrolases (Frantz & Chakrabarty, 1987
; Perkins et al., 1990
; Laemmli et al., 2000
; van der Meer et al., 1991
; Schell et al., 1994
; Kasberg et al., 1995
, 1997
; Seibert et al., 1993
; Plumeier et al., 2002
). Sequence similarities suggest that this is true also for the chlorocatechol gene clusters of various other plasmids or strains. While these results provide clear-cut proof that many chlorocatechol operons do contain maleylacetate reductase genes, some recently characterized chlorocatechol gene clusters do not appear to contain such genes (Eulberg et al., 1998
; Moiseeva et al., 2002
).
The dioxygenase, cycloisomerase and lactone hydrolase of chlorocatechol catabolism have functional analogues or even evolutionary homologues among the enzymes of catechol catabolism (for summary, see Schlömann, 1994), while maleylacetate reductases have no such equivalent. This raises the question as to where the maleylacetate reductase gene was recruited from during the evolution of the ancestral chlorocatechol pathway. A maleylacetate reductase gene (macA) not belonging to a chlorocatechol operon was cloned and characterized from the Gram-positive chlorophenol degrader Rhodococcus opacus 1CP (Seibert et al., 1998
). However, the natural function of this gene could not be inferred from the neighbouring region, and the Rhodococcus macA gene has not been shown to play a role in halocatechol catabolism. Various genome-sequencing projects, including that of Ralstonia metallidurans (GenBank accession no. NZ_AAAI01000075), have recently revealed hypothetical maleylacetate reductase genes of which the exact function has remained unclear.
During growth with 4-fluorobenzoate, R. eutropha 335T and R. eutropha JMP222, a pJP4-free derivative of the 2,4-dichlorophenoxyacetate-utilizing strain JMP134, induce a maleylacetate reductase, but not the other enzymes typical for chlorocatechol catabolism (Schlömann et al., 1990a). Thus, these strains contain a presumably chromosomal maleylacetate reductase gene of still unknown metabolic affiliation. The work described in the present study was concerned with characterizing the maleylacetate reductase genes of these R. eutropha strains. We report on the cloning of the macA gene from strain 335T. Sequencing of the gene itself and of the surrounding region revealed that macA is located in a region completely dissimilar to those regions neighbouring other maleylacetate reductase genes. The macA gene was shown to be expressed during growth with 4-fluorobenzoate, thus proving that it is recruited to allow growth of R. eutropha with this unusual substrate. Some of the results given here have previously been presented in a preliminary form (Seibert & Schlömann, 1996
).
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METHODS |
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An approximately 980 bp fragment of the maleylacetate reductase gene of R. eutropha 335T was amplified from genomic DNA as template by using the same primers as for the cloning of macA of Rhodococcus opacus 1CP (Seibert et al., 1998): NH2-3 [5'-(A/C)G(G/C)GT(G/C)GT(G/C)TTCGG(G/C)GC(G/C)GG-3'] and HOM [5'-GG(A/T)CG(G/C)GG(A/G)TT(G/C)GG(A/G)TA(C/T)TG-3'], which had been designed corresponding to the N-terminal sequence of the maleylacetate reductase purified from Ralstonia eutropha JMP134 (Seibert et al., 1993
), later identified as TfdFII (Laemmli et al., 2000
), and to regions similar in tfdF of pJP4 and tcbF of pP51, respectively. PCR amplification was performed under a layer of light mineral oil in a 50 µl reaction mixture which contained 50 pmol each primer, 0·1 µg genomic template DNA, 100 µM dNTPs, 0·5 U Goldstar DNA polymerase (Eurogentech), the corresponding buffer, 1·5 mM MgCl2, and 5 % formamide as denaturing agent. The temperature programme was as follows: denaturation at 94 °C initially for 3 min (addition of the polymerase after 2 min) and during the cycles for 40 s, annealing for 90 s with the temperature decreasing from 55 to 42 °C in 42 cycles (0·3 °C per cycle) and staying constant at 42 °C for 10 more cycles, elongation at 72 °C for 60 s during the cycles and at the end for 15 min.
PCR products that appeared to have the expected size of approximately 980 bp were ligated into the T-tailed EcoRV site of pBluescript II SK(+), yielding pREMARP1, and checked by sequencing. A 600 bp HindIII fragment of pREMARP1 was labelled and then used to detect the corresponding fragment on a Southern blot of 5 µg EcoRI-, XhoI-, PstI- and BamHI-digested R. eutropha 335T DNA. From a second gel, the area containing DNA of approximately the right size [20 kb (EcoRI) or 3 kb (XhoI, PstI and BamHI)] was excised. DNA was eluted from the gel slice and ligated into pBluescript II SK(+). After transformation of the ligation mixture into E. coli DH5
, the labelled HindIII fragment of pREMARP1 was used again to identify the correct clones by colony hybridization.
From subcloned inserts and inserts carrying nested deletions, the nucleotide sequences of both strands were determined with an ABI 373A automated DNA sequencer (Applied Biosystems) with a cycle sequencing protocol. Sequences were assembled by using the PC/GENE program package (intelligenetics) and compared to database entries by using BLASTX (Altschul et al., 1997). Multiple sequence alignments were created using CLUSTAL_X 1.81 (Thompson et al., 1997
). Dendrograms were constructed by using the PROTDIST and FITCH algorithms of the PHYLIP program package version 3.5c (Felsenstein, 1993
) and visualized by using TREEVIEW (Page, 1996
). Bootstrap values were calculated using SEQBOOT, of the PHYLIP package, with 100 replicates. The protein family database (Pfam) at the Sanger Institute (http://www.sanger.ac.uk/Software/Pfam/) was used to assign MacB to a protein family.
Overproduction of maleylacetate reductase.
For overexpression of maleylacetate reductase by use of the T7lac expression system (Studier et al., 1990), the macA gene was first amplified from pREMAR5 as template. One primer (335NDEI; 5'-GATCATATGGTTCCTTTCGCCTACCAG-3') contained an NdeI restriction endonuclease site (underlined) and 21 bases of the start of the macA gene (bold); the second primer (335KPNI; 5'-GTGGTACCTCAGGCCGACGGCGCTGTT-3') carried a KpnI restriction endonuclease site (underlined) and 19 bases complementary to the end of the macA gene (bold). The PCR mixture corresponded to the one given above with the exception that only 25 pmol of the primers were used, and 100 ng of pREMAR5 served as template. The temperature programme comprised an initial denaturation at 94 °C for 4 min (addition of polymerase after 3 min), 30 cycles of annealing at 58 °C for 45 s, elongation at 72 °C for 60 s, and denaturation at 94 °C for 30 s, and finally annealing at 50 °C for 45 s and elongation at 72 °C for 15 min. The resulting PCR product was digested with NdeI and KpnI and inserted into pET11a* digested with the same enzymes, to yield plasmid pREMARE1. E. coli BL21(DE3)(pLysS) was used as the host strain for inducible overexpression of maleylacetate reductase. The strain containing pREMARE1 was grown at 37 °C in Erlenmeyer flasks containing dYT medium with 100 µg ampicillin ml-1. For induction of macA in E. coli, 0·6 mM IPTG was added to the cultures when they reached OD600 1. After induction, the cells were incubated at 30 °C for 7 h and then harvested by centrifugation. They were washed with 50 mM Tris/HCl (pH 7·5), 1 mM DTT, and stored at -20 °C until required. Cell extracts of BL21(DE3)(pLysS)(pREMARE1) were prepared in the same buffer by a French pressure cell procedure (Seibert et al., 1993
) followed by centrifugation (4 °C, 30 min, 130 000 g) and filtration of the supernatant through a 0·22 µm pore-size filter.
Enzyme assay and estimation of protein concentration.
Maleylacetate reductase activities were determined by following the maleylacetate-dependent oxidation of NADH at 340 nm (Schlömann et al., 1990a). Maleylacetate was prepared by alkaline hydrolysis from cis-dienelactone, which was kindly provided by S. Kaschabek and W. Reineke. Protein concentrations were determined by the method of Bradford (1976)
, with BSA as the standard.
Enrichment and N-terminal sequencing of maleylacetate reductase from 4-fluorobenzoate-grown cells of R. eutropha 335T.
Maleylacetate reductase was partially purified from R. eutropha 335T cells grown with 4-fluorobenzoate in a simplified minimal medium consisting of 25 g KH2PO4, 9·5 g Ca(NO3)2.4H2O, 1·9 g FeIII(NH4)citrate, 285 g (NH4)2SO4 and 38 g MgSO4.7H2O in tap water, with the volume increasing by repeated doses of 4-fluorobenzoate from 125 to 167 litre (Hinner, 1998). The purification procedure during the first steps was aimed mainly at the purification of dienelactone hydrolase (Hinner, 1998
) and thus was not optimized for maleylacetate reductase. Frozen cells were resuspended in 25 mM Bistris/HCl (pH 7·0), 4 mM MnSO4, 0·1 mM DTT, and disintegrated as described above (Hinner, 1998
). As an initial step, the cell extract was heated for 20 min at 55 °C, a step helpful for the purification of dienelactone hydrolase, but not favourable for the stability of maleylacetate reductase (Table 2
). The chromatographic steps were performed at room temperature by using the FPLC system as well as columns and chromatography media from Pharmacia. The anion-exchange chromatography of the cell extract was performed on a Q-Sepharose HP HR 16/10 column (bed volume, 20 ml; 5 parallel runs) with 25 mM Bistris/HCl (pH 7·0), 4 mM MnSO4, 0·1 mM DTT and a linear NaCl gradient from 0 to 0·6 M over 400 ml and from 0·6 to 2 M over 65 ml for elution of the proteins. The maleylacetate reductase and the dienelactone hydrolase both eluted at about 0·3 M NaCl. The most active fractions were combined and (NH4)2SO4 was added to a final concentration of 1·5 M. The pooled sample was chromatographed on a Phenyl-Sepharose FF HR 16/10 column (bed volume, 20 ml) with 25 mM Bistris/HCl (pH 7·0), 1 mM DTT, 1 mM MnSO4, and a decreasing (NH4)2SO4 gradient from 1·5 to 1·125 M (NH4)2SO4 over 10 ml and from 1·125 to 0 M (NH4)2SO4 over 200 ml. The maleylacetate reductase eluted at 0·5 M (NH4)2SO4 separated from dienelactone hydrolase. The pooled maleylacetate reductase sample was desalted by dialysis against 25 mM Tris/HCl (pH 7·5), 1 mM DTT, and then applied to a Blue-Sepharose CL-6B HR 26/10 column (bed volume, 50 ml) equilibrated with 25 mM Tris/HCl (pH 7·5), 1 mM DTT. A linear NaCl gradient from 0 to 2 M over 25 ml was used for elution of the proteins (elution of maleylacetate reductase at about 1 M NaCl). The pooled maleylacetate reductase sample was again desalted by dialysis against 25 mM Tris/HCl (pH 7·5), 1 mM DTT, and then further purified on a Resource Q column (bed volume, 1 ml) using 25 mM Tris/HCl (pH 7·5), 1 mM DTT, and a linear NaCl gradient from 0 to 1 M NaCl over 50 ml (elution of maleylacetate reductase at about 0·3 M NaCl). Finally, SDS-PAGE was performed as described previously (Seibert et al., 1993
), but with Coomassie blue staining.
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RESULTS AND DISCUSSION |
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However, when the 600 bp HindIII fragment of pREMAR1 was employed as a probe in Southern hybridization experiments with DNA from strains R. eutropha JMP222 or JMP289, to clone the corresponding maleylacetate reductase genes, no significant hybridization could be detected. Likewise, the use of primers NH2-3 and HOM under various PCR conditions did not allow amplification of a band with the expected size from the DNA of strains JMP222 and JMP289. These observations indicated considerable sequence divergence between the maleylacetate reductase gene of R. eutropha 335T and those of strains JMP222 and JMP289, and are in concordance with the report of Jenni et al. (1988), who by DNADNA hybridization experiments found that strain JMP134, and consequently its derivatives JMP222 and JMP289, are too dissimilar to the type strain 335T to be included in the species R. eutropha (formerly Alcaligenes eutrophus).
Identification and characterization of the maleylacetate reductase gene of R. eutropha 335T
The nucleotide sequence of the 5861 bp EcoRV fragment of pREMAR5 was determined and found to comprise six open reading frames (ORFs) (Fig. 2). The maleylacetate reductase gene macA was detected between sequence coordinates 2001 and 3083; it comprises 1083 nt and thus encodes a protein of 360 aa, with a calculated molecular mass of 37 460 Da. The level of macA expression in E. coli BL21(DE3)(pLysS) from vector pET11a* (plasmid pREMARE1) increased in response to IPTG by a factor of 15 [specific activity at the time of induction was 1·2 U (mg protein)-1; after 4 h induction it was 18 U (mg protein)-1]. This result proved that a functional maleylacetate reductase gene had been cloned.
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Preliminary characterization of ORFs neighbouring the maleylacetate reductase gene
About 50 bp upstream of macA an ORF was detected which is probably transcribed in the same orientation as macA; therefore, this ORF was tentatively designated macB. Of the two possible translation initiation sites found for macB at positions 824 and 926, the latter appears to be the more likely initiation site based on comparison to homologous genes (see below) and on the occurrence of potential ribosome binding sequences. The macB gene extends to position 1948 and thus translation initiation at position 926 would yield a 35 655 Da protein (340 aa). Database searches revealed that the deduced amino acid sequence of MacB shows significant similarity to various hypothetical proteins of R. metallidurans and to numerous putative exported proteins or putative lipoproteins of Bordetella strains. Similarity was also found between MacB and the predicted products of ORFs of several chlorocatechol gene clusters including the products of ORF3 from the clc and tcb operons of plasmids pAC27 [Frantz & Chakrabarty, 1987; GenBank accession no. M16964] and pP51 [van der Meer et al., 1991
; GenBank accession no. M57629], respectively. These ORF3 products, in turn, have previously been found to be similar to the terephthalate and isophthalate transporter proteins TphC and IphC of Comamonas testosteroni YZW-D (G. J. Zylstra, personal communication; Wang et al., 1995
). In a multiple sequence alignment of these five proteins, MacB shared 35, 35, 37 and 32 % identical positions with the tcb-ORF3, the clc-ORF3, IphC and TphC, respectively. The sequence similarities to the transporter proteins TphC and IphC suggest that MacB may play a similar role. This conclusion is supported by the assignment in a Pfam database search of a Bug (Bordetella uptake gene) domain (Pfam accession no. PF03401) to MacB. The occurrence of homologous maleylacetate reductase genes and homologous presumed transporter genes in both the mac gene cluster of R. eutropha and some chlorocatechol gene clusters raises the question as to whether this is indicative of an evolutionary scenario in which a maleylacetate reductase gene and a transporter gene were recruited for a new function together, i.e. as constituents of the same genetic module. The considerably lower degree of sequence similarity among the presumed transporters as compared to the similarity among the corresponding maleylacetate reductases, however, suggests that such a co-evolution is very unlikely to have happened.
Divergently transcribed from macB, an ORF was detected which started at position 747 of the sequence and was not complete on the insert of pREMAR5. The predicted product showed significant sequence similarity to the LysR family of transcriptional regulators (Henikoff et al., 1988). In all comparisons to database entries, the similarity of the predicted protein to deposited protein sequences was lower than 40 % identical positions. Because of its presumed regulatory role and because the arrangement of the genes suggested a functional association with macA and macB, the incomplete ORF was tentatively designated macR'.
One base pair downstream of macA an inverted repeat was detected which should form a hairpin loop of high free energy (-15 kcal). This could function as a transcriptional terminator and might indicate the end of the gene cluster functioning in maleylacetate conversion.
The product of the fourth ORF (ORF4), positions 43493144, showed moderate sequence similarity to various FAD-dependent monooxygenases, including the 2,6-dihydroxypyridine hydroxylase of Arthrobacter nicotinovorans (Baitsch et al., 2001; GenBank accession no. AF373840) with 32 % identical positions, and various salicylate hydroxylases, for example, one from Pseudomonas putida strain PpG7 [You et al., 1991
; GenBank accession no. M60055) with 21 % identical positions. While the similarities of the ORF4 product to specific enzymes are too low to infer a precise function, it is not unreasonable to assume that the protein encoded by ORF4 might also be an FAD-dependent monooxygenase. For the next ORF (ORF5), database searches revealed similarities to various hypothetical proteins without indicating any function. ORF6, presumably transcribed in the opposite direction to ORF5, according to database searches appears to encode another LysR-type transcriptional regulator.
The characterization of the region neighbouring the maleylacetate reductase gene macA in R. eutropha 335T revealed that it is completely dissimilar to all other known gene clusters containing maleylacetate reductase genes. With the exception of the presumed transporter gene (see above), none of the other genes generally constituting the chlorocatechol operons (Schlömann, 1994) occurred in the vicinity of R. eutropha 335T macA. Likewise, besides the maleylacetate reductase gene, no homologue to any of the tft genes occurring in the B. cepacia AC1100 gene cluster for 2,4,5-trichlorophenoxyacetate degradation could be detected. Specifically, in the macA region there was no similarity to hydroxyquinol-1,2-dioxygenase genes, which also in the 2-hydroxydibenzo-p-dioxin and 3-hydroxydibenzofuran catabolic gene cluster of Sphingomonas sp. RW1 (Armengaud et al., 1999
) as well as in the complete sequences of Corynebacterium glutamicum ATCC 13032T (GenBank accession no. NC_003450), Bradyrhizobium japonicum USDA 110 (GenBank accession no. NC_004463), Agrobacterium tumefaciens C58 (GenBank accession no. NC_003304) and R. metallidurans Reut_75 (GenBank accession no. NZ_AAAI01000075) were found next to or within a few kilobases of hypothetical maleylacetate reductase genes, suggesting the respective enzymes to serve a common function in some cases. Finally, in the R. eutropha 335T macA region there was no gene for a short-chain dehydrogenase/reductase, but there is one in the macA region of Rhodococcus opacus 1CP (Seibert et al., 1998
). Obviously, homologous maleylacetate reductase genes appear in quite different genetic contexts. In Ralstonia eutropha 335T, in contrast, the presumed operon to which the macA gene might belong is small and does not encode any other catalytic function, but only a possible transporter.
Recruitment of macA for growth of R. eutropha 335T with 4-fluorobenzoate
Previously, R. eutropha 335T has been shown to induce a maleylacetate reductase during growth with 4-fluorobenzoate, but not during growth with benzoate, 4-hydroxybenzoate or succinate (Schlömann et al., 1990a). Despite a considerable instability, the maleylacetate reductase could now be partially purified from 4-fluorobenzoate-grown cells (Table 2
), giving a major band of the expected size among other bands on an SDS-PAGE gel. From the Western-blotted band of the SDS-PAGE gel, the N-terminal sequence of the maleylacetate reductase was determined to be MVPFAYQTRPQRVVFGPGSLAR. This sequence corresponds exactly to the N terminus predicted for MacA (Fig. 3
), proving that MacA is in fact induced during growth with 4-fluorobenzoate. Thus, MacA represents the maleylacetate reductase which funnels the maleylacetate formed by hydrolysis of 4-fluoromuconolactone (Schlömann et al., 1990b
) into the lower part of the 3-oxoadipate pathway.
The pattern of MacA induction (high activity during growth with 4-fluorobenzoate, but not during growth with benzoate; Schlömann et al., 1990a) indicates that the product of maleylacetate reduction, 3-oxoadipate, or any compound derived from it, cannot be the inducer. Likewise, all the metabolites between 4-fluorobenzoate and 4-fluoromuconolactone are very unlikely to be responsible for the specific induction due to their high structural similarity to the metabolites of benzoate degradation. Thus, it is tempting to speculate that maleylacetate itself is the inducer of the macA gene. If so, the small mac gene cluster of R. eutropha 335T might serve as a bridge between maleylacetate-forming pathways and the 3-oxoadipate pathway in a more general way than just for degradation of the presumably non-natural compound 4-fluorobenzoate.
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ACKNOWLEDGEMENTS |
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Received 27 June 2003;
revised 15 September 2003;
accepted 17 September 2003.
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