Characterization of a gene cluster encoding the maleylacetate reductase from Ralstonia eutropha 335T, an enzyme recruited for growth with 4-fluorobenzoate

Volker Seibert1,{dagger}, Monika Thiel2, Isabelle-S. Hinner1 and Michael Schlömann1,2

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
A gene cluster containing a gene for maleylacetate reductase (EC 1.3.1.32) was cloned from Ralstonia eutropha 335T (DSM 531T), which is able to utilize 4-fluorobenzoate as sole carbon source. Sequencing of this gene cluster showed that the R. eutropha 335T maleylacetate reductase gene, macA, is part of a novel gene cluster, which is not related to the known maleylacetate-reductase-encoding gene clusters. It otherwise comprises a gene for a hypothetical membrane transport protein, macB, possibly co-transcribed with macA, and a presumed regulatory gene, macR, which is divergently transcribed from macBA. MacA was found to be most closely related to TftE, the maleylacetate reductase from Burkholderia cepacia AC1100 (62 % identical positions) and to a presumed maleylacetate reductase from a dinitrotoluene catabolic gene cluster from B. cepacia R34 (61 % identical positions). By expressing macA in Escherichia coli, it was confirmed that macA encodes a functional maleylacetate reductase. Purification of maleylacetate reductase from 4-fluorobenzoate-grown R. eutropha 335T cells allowed determination of the N-terminal sequence of the purified protein, which was shown to be identical to that predicted from the cloned macA gene, thus proving that the gene is, in fact, recruited for growth of R. eutropha 335T with this substrate.


The GenBank accession number for the sequence reported in this article is AF130250.

{dagger}Present address: Europroteome, Neuendorfstr. 24 b, 16761 Henningsdorf, Germany.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Maleylacetate reductases (EC 1.3.1.32) play a crucial role in the aerobic microbial degradation of aromatic compounds. They catalyse the NADH- or NADPH-dependent reduction of maleylacetate or 2-chloromaleylacetate to 3-oxoadipate (Fig. 1) or of substituted maleylacetates to substituted 3-oxoadipates. In fungi, maleylacetate reductases contribute to the catabolism of very common substrates, such as tyrosine, gentisate, benzoate, 4-hydroxybenzoate, protocatechuate, vanillate, resorcinol and phenol (Karasevich & Ivoilov, 1977; Buswell & Eriksson, 1979; Gaal & Neujahr, 1979; Sparnins et al., 1979; Anderson & Dagley, 1980; Jones et al., 1995). For bacteria, it has been shown that maleylacetate reductases are involved in the degradation of resorcinol and 2,4-dihydroxybenzoate via hydroxyquinol (Larway & Evans, 1965; Chapman & Ribbons, 1976; Stolz & Knackmuss, 1993). Other substrates whose catabolic routes comprise this activity have a more complex structure, such as 2-hydroxydibenzo-p-dioxin and 3-hydroxydibenzofuran (Armengaud et al., 1999), or carry unusual substituents, such as nitro or sulfo groups (Spain & Gibson, 1991; Feigel & Knackmuss, 1993; Jain et al., 1994; Rani & Lalithakumari, 1994), fluorine or chlorine atoms (Duxbury et al., 1970; Schlömann et al., 1990a; Latus et al., 1995; Zaborina et al., 1995; Daubaras et al., 1996; Miyauchi et al., 1999).



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Fig. 1. Reactions catalysed by maleylacetate reductase.

 
The maleylacetate reductase genes characterized so far tend to belong to specialized gene clusters for the degradation of aromatic compounds. Thus, 3-hydroxydibenzofuran and 2-hydroxydibenzo-p-dioxin degradation via hydroxyquinol appears to be encoded by a specialized gene cluster comprising most of the genes for the pathway including a maleylacetate reductase gene (Armengaud et al., 1999). However, the dinitrotoluene degradative gene cluster of Burkholderia cepacia R34 contains a maleylacetate reductase gene which does not appear to play a role in the degradation (Johnson et al., 2002).

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).


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and culture conditions.
R. eutropha 335T (DSM 531T), and JMP222 and JMP289, 2,4-dichlorophenoxyacetate-negative derivatives of the 2,4-dichlorophenoxyacetate-degrading strain R. eutropha JMP134, cured of pJP4 (Don & Pemberton, 1981), were shown to grow with 4-fluorobenzoate (Schlömann et al., 1990a). Unless mentioned otherwise, all strains were cultivated as described by those authors. Escherichia coli DH5{alpha} was obtained from Gibco-BRL and E. coli BL21(DE3)(pLysS) was obtained from Novagen. Usually, E. coli was grown aerobically with constant shaking (120 r.p.m.) at 37 °C in baffled Erlenmeyer flasks with dYT medium (1·6 % tryptone, 1 % yeast extract, 0·5 % NaCl). When appropriate for selection, ampicillin was added to a final concentration of 100 µg ml-1. The plasmids used in this study are listed in Table 1.


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Table 1. Plasmids used in this study

 
DNA manipulation, cloning procedures and DNA sequence analysis.
Genomic DNA from R. eutropha 335T was available from a previous purification (Hinner, 1998). General methods of DNA manipulation as well as the overall cloning strategy and hybridization conditions used have been described previously (Eulberg et al., 1997; Seibert et al., 1998).

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{alpha}, 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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Table 2. Enrichment of the R. eutropha 335T maleylacetate reductase

 
The maleylacetate reductase enriched from 4-fluorobenzoate-grown R. eutropha 335T, after separation from contaminating proteins by SDS-PAGE, was Western blotted onto a Millipore Immobilon-P membrane by using the procedure described in the Immobilon Tech Protocol (staining with Coomassie brilliant blue R250). The protein band was excised and subsequently N-terminally sequenced in an Applied Biosystems model 473A protein sequencer.


   RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Experiments for cloning of the maleylacetate reductase genes from R. eutropha 335T and R. eutropha JMP222 or JMP289
Primers NH2-3 and HOM, derived from similar regions of tfdF of pJP4 and tcbF of pP51 and from the N terminus of TfdFII of pJP4, allowed the amplification of a fragment with the expected size of approximately 980 bp from R. eutropha 335T DNA. The fragment was cloned into pBluescript II SK(+), to yield pREMARP1. Use of a 600 bp HindIII fragment of pREMARP1 as a probe resulted in the cloning of several fragments of genomic DNA from strain 335T, thus yielding, among others, plasmid pREMAR1 with an approximately 20 kb EcoRI insert (Table 1). By using the same probe, an approximately 5·9 kb EcoRV fragment was subcloned from pREMAR1 to give pREMAR5 (Table 1). Further analysis showed that pREMAR5 encodes a functional maleylacetate reductase (see below).

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 DNA–DNA 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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Fig. 2. Map of pREMAR5 carrying 5861 bp of R. eutropha 335T DNA in the EcoRV site of pBluescript IISK(+). ORFs are represented by arrows. The position of a presumed hairpin loop is indicated by a vertical arrow.

 
The predicted amino acid sequence of the R. eutropha 335T maleylacetate reductase turned out to be most similar to TftE, the maleylacetate reductase of the 2,4,5-trichlorophenoxyacetate-degrading strain B. cepacia AC1100 (Daubaras et al., 1995, 1996), and to the putative maleylacetate reductase of the 2,4-dinitrotoluene-degrading strain B. cepacia R34 (Johnson et al., 2002), with which it shares 62 and 61 %, respectively, of the positions in the alignment of Fig. 3. The relatively close relationship between these enzymes is also evident in the dendrogram shown in Fig. 4. The similarity of R. eutropha 335T MacA to enzymes of chlorocatechol catabolic pathways was somewhat lower – between 51 and 57 % identical positions (in the alignment of Fig. 3) with the maleylacetate reductases of pP51, pJP4 and Pseudomonas sp. B13. Among known and confirmed maleylacetate reductases, the lowest similarity for R. eutropha 335T MacA was found with MacA from Rhodococcus opacus 1CP (44 % identical positions).



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Fig. 3. Multiple sequence alignment of selected confirmed and putative maleylacetate reductases (MARs). Positions in which at least 16 of the 20 sequences are identical are highlighted black. Designations in quotation marks indicate putative maleylacetate reductases from genome-sequencing projects. Numbers above the sequences refer to positions in the alignment, not in a single sequence. Open triangles below the sequences indicate two patterns from the PROSITE database typical for iron-containing alcohol dehydrogenases, ‘[STALIV]-[LIVF]-x-[DE]-x(6,7)-P-x(4)-[ALIV]-x-[GST]-x(2)-D-[TAIVM]-[LIVMF]-x(4)-E’ and ‘[GSW]-x-[LIVTSACD]-[GH]-x(2)-[GSAE]-[GSHYQ]-x-[LIVTP]-[GAST]-[GAS] x(3)-[LIVMT]-x-[HNS]-[GA]-x-[GTAC]’ (PROSITE entry PDOC00059). Solid triangles indicate positions in which at least one of the maleylacetate reductases shows a deviation from the published motif. The alignment was created using CLUSTAL_X 1.81 (Thompson et al., 1997) with the outgroups used in Fig. 4. The outgroups were removed afterwards and resulting column gaps were eliminated. References for the published sequences (order as in the alignment): AF130250 (this study); U19883 (Daubaras et al., 1995); AF169302 (Johnson et al., 2002); NZ_AAAI01000075, Ralstonia metallidurans Reut_75 genome shotgun sequence; AF512952 (Cai & Xun, 2002); NC_003304, positions 2 501 013–2 502 068, Agrobacterium tumefaciens C58 chromosome (Wood et al., 2001); NC_004463, positions 2 938 247–2 939 326, Bradyrhizobium japonicum USDA 110 genome (Kaneko et al., 2002); AJ536297 (Fritsche, 1998); X72850 (Armengaud et al., 1999); U32188 (Vedler et al., 2000); AF030176 (Seibert et al., 1998); M57629 (van der Meer et al., 1991); AF019038 (Kasberg et al., 1997); M35097 (Perkins et al., 1990); AB050198 (Liu et al., 2001); P94135 (Laemmli et al., 2000); NC_004369, positions 3 077 929–3 079 059, Corynebacterium efficiens YS-314T genome; NC_003450, positions 1 214 871–1 215 941 and 3 257 401–3 258 492, Corynebacterium glutamicum ATCC 13032T genome; NC_004463, positions 1 096 165–1 097 388, Bradyrhizobium japonicum USDA 110 genome (Kaneko et al., 2002).

 


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Fig. 4. Dendrogram showing the relatedness of selected maleylacetate reductases. The dendrogram was constructed using the programs PROTDIST and FITCH from the PHYLIP 3.5c package based on an alignment created using CLUSTAL_X 1.81 (Thompson et al., 1997). For construction of the dendrogram, only amino acids corresponding to positions 28–408 in the alignment of Fig. 3 were used. Numbers on the branches indicate those bootstrap values that were higher than 50. For references of maleylacetate reductases see legend to Fig. 3. The following enzymes were used as outgroups: 1,3-propanediol dehydrogenases (DhaT) from Citrobacter freundii (U09771), Klebsiella pneumoniae (U30903) and Clostridium pasteurianum (AF006034); alcohol dehydrogenases from Zymomonas mobilis (M15394) and Rhodospirillum rubrum (AB023641); methanol dehydrogenase (Mdh) from Bacillus sp. C1 (M65004); and hydroxybutyrate dehydrogenase (Gbd) from Ralstonia eutropha (L36817).

 
Corresponding to the finding of van der Meer et al. (1992) that TfdF of pJP4 and TcbF of pP51 are homologous to iron-containing (type III) alcohol dehydrogenases, signature sequences of this protein family could be located also in the sequence of the Ralstonia eutropha 335T maleylacetate reductase (Fig. 3). However, several deviations from the signature sequences occurred, and the sequence similarity between R. eutropha MacA and those members of the type III alcohol dehydrogenases which are not maleylacetate reductases was considerably lower than that to other maleylacetate reductases (e.g. 22–30 % identical positions for comparisons of MacA to the outgroup sequences used for the dendrogram in Fig. 4). Possibly related to the deviations from the signature sequences is the fact that, so far, iron has not been identified as a prosthetic group of maleylacetate reductases.

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 4349–3144, 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.


   ACKNOWLEDGEMENTS
 
We are indebted to H.-J. Knackmuss for stimulating discussions and for providing excellent facilities at Universität Stuttgart. For amino acid sequencing, we thank H. Weber, Fraunhofer-Institut für Grenzflächen und Bioverfahrenstechnik, Stuttgart, and R. Getzlaff, Gesellschaft für Biotechnologische Forschung, Braunschweig, Germany. We thank R. Schmid and H. Atomi, Institut für Technische Biochemie, Universität Stuttgart, for providing the facility for automated sequencing, and S. Bürger for performing the sequencing.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Received 27 June 2003; revised 15 September 2003; accepted 17 September 2003.



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