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Applied and Environmental Microbiology, November 2008, p. 7085-7089, Vol. 74, No. 22
0099-2240/08/$08.00+0     doi:10.1128/AEM.01491-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Cloning and Characterization of {alpha}-Methylacyl Coenzyme A Racemase from Gordonia polyisoprenivorans VH2{triangledown} ,{dagger}

Quyen Arenskötter, Jens Heller, David Dietz, Matthias Arenskötter, and Alexander Steinbüchel*

Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstrasse 3, D-48149 Münster, Germany

Received 2 July 2008/ Accepted 18 September 2008


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ABSTRACT
 
The mcr gene of Gordonia polyisoprenivorans VH2 is not clustered with genes required for rubber degradation. Its disruption by insertion of a kanamycin resistance cassette impaired growth on methyl-branched isoprenoids but not on linear hydrocarbons. Intact mcr from this bacterium or from Nocardia farcinica IFM 10152 complemented the mutant. Reverse transcription analysis showed similar mcrVH2 expression results during cultivation with poly(cis-1,4-isoprene) and propionate. Additional genes coding for a putative cytochrome P450 monooxygenase and a short-chain dehydrogenase/reductase involved in β-oxidation and poly(cis-1,4-isoprene) degradation were also characterized.


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INTRODUCTION
 
{alpha}-Methylacyl coenzyme A (CoA) racemase (Mcr) is a key enzyme in the catabolism by Gordonia polyisoprenivorans VH2 of natural rubber degradation products and of methyl-branched hydrocarbons (2). Only a few bacterial Mcr's have been described so far (2, 21, 22), whereas investigations of mammalian {alpha}-methylacyl CoA racemase have attracted much more interest (19, 24, 25, 27). During catabolism of isoprenoids, Mcr catalyzes inversion of (R)-enantiomers to (S)-enantiomers of various substrates (7, 10, 20), e.g., 2-arylpropionic acids (ibuprofen). Although microbial rubber degradation has been investigated since 1914, only two enzymes involved in the initial step of poly(cis-1,4-isoprene) biodegradation yielding intermediates with aldehyde and keton groups have been identified—RoxA from Xanthomonas sp. (3, 4) and Lcp from Streptomyces sp. strain K30 (18, 28).

This study aimed at the construction of a stable mcr gene disruption mutant of G. polyisoprenivorans VH2 and its characterization with regard to rubber degradation. Therefore, the nucleotide sequence of mcr was identified. Homologous (McrVH2) and heterologous (McrNf) expression of Mcr should exclude a polar effect of disrupted mcr on adjacent genes. Furthermore, other transposon-induced mutants impaired in rubber degradation and the affected genes involved in β-oxidation as well as their organization were characterized. Reverse transcription analysis was done to confirm induced expression of some of these genes in strain VH2 during rubber degradation.


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Identification of transposon insertion loci in mutants D6, C22, and I45.
 
Approximately 33,000 transposon-induced mutants of G. polyisoprenivorans VH2 were generated using the suicide vector pMA5096 as described before (2). Mutants exhibiting diminished growth on mineral salts medium (MSM)-isoprene rubber-sandwich agar plates were subjected to a second screening on MSM agar plates, and only those stable mutants which also exhibited a negative or leaky phenotype in liquid MSM were further analyzed (23).

Two mutants exhibited a leaky phenotype during cultivation in liquid MSM with poly(cis-1,4-isoprene) as the sole carbon and energy source. For these mutant genomes, pMA5096 was mapped in the genes that are most likely to be involved in β-oxidation. Sequence analysis of the genomic DNA was done as described before (2) and by modified directional genome walking (15). The vector had integrated in genes encoding enzymes that catalyze reactions of the β-oxidation or into intergenic regions (Fig. 1). In mutant D6, plasmid pMA5096 was mapped in the intergenic region of two genes coding for an acetyl CoA acetyltransferase (EC 2.3.1.9) and for a short-chain dehydrogenase/reductase (EC 1.1.1). In mutant C22, pMA5096 was mapped in a ferA gene coding for a putative feruloyl CoA synthetase (EC 6.2.1.34). Both the acyl CoA synthetase and feruloyl CoA synthetase enzymes catalyze the transfer of CoA to the carboxyl group of their substrates, thereby forming the corresponding CoA thioesters.


Figure 1
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  		FIG. 1. Localization of transposon insertions in mutants of G. polyisoprenivorans VH2 and localization of the mcr gene locus in G. polyisoprenivorans VH2. Three regions of the genome of G. polyisoprenivorans VH2 that were sequenced in this study and in which transposon insertions were mapped are shown. (a) orf1 (310 aa), encoding methionyl-tRNA formyltransferase (M. vanbaalenii PYR-1; accession no. YP_953500.1; aa identity = 57%); orf2 (394 aa), encoding acetyl CoA acetyltransferase (Rhodococcus sp. strain RHA1; accession no. YP_708690.1; aa identity = 30%); orf3 (275 aa), encoding a short-chain dehydrogenase/reductase (Frankia sp. strain EAN1pec; accession no. ZP_00570976.1; aa identity = 43%); orf4 (385 aa), encoding acyl CoA synthetases (AMP forming)/AMP-acid II ligases (Brevibacterium linens BL2; accession no. ZP_00377854.1; aa identity = 36%). (b) orf1 (509 aa), encoding hypothetical protein RHA1_ro08129 (Rhodococcus sp. strain RHA1; accession no. YP_707334.1; aa identity = 34%); orf2 (316 aa), encoding oxidoreductase, short-chain dehydrogenase/reductase family (Arthrobacter aurescens TC1; accession no. YP_946751.1; aa identity = 70%); orf3 (146 aa), encoding a transcriptional regulator (Rhodococcus sp. strain RHA1; accession no. YP_705064.1; aa identity = 40%); ferA (511 aa), encoding a putative feruloyl CoA synthetase (R. aetherivorans; accession no. AAY98502.1; aa identity = 52%); orf4 (261 aa), encoding enoyl CoA hydratase/isomerase (Mycobacterium sp. strain MCS; accession no. YP_637483.1; aa identity = 73%); orf5 (500 aa), encoding gp34 (Mycobacterium sp. phage 244; accession no. YP_654789.1; aa identity = 43%); orf6 (363 aa), encoding a transposase (Rhodococcus sp. strain RHA1; accession no. YP_707596.1; aa identity = 63%) (c) orf1 (387 aa), encoding acetyl CoA acetyltransferase (N. farcinica IFM 10152; accession no. YP_116726.1; aa identity = 74%); orf2 (410 aa), encoding cytochrome P450 [M. vanbaalenii PYR-1; accession no. YP_955928.1; aa identity = 65%); orf3 (139 aa), encoding hypothetical protein nfa5190 (N. farcinica IFM 10152; accession no. YP_116728.1; aa identity = 59%). (d) orf1 (251 aa), encoding a hypothetical creatininase (M. gilvum PYR-GCK; accession no. YP_001136312.1; aa identity = 76%); mcr, encoding a putative methylacyl CoA racemase (Rhodococcus sp. strain RHA1; accession no. YP_705495.1; aa identity = 67%); orf2 (427 aa), encoding a probable aspartate aminotransferase (M. abscessus; accession no. YP_001705021.1; aa identity = 56%); orf3 (285 aa), encoding a short-chain dehydrogenase/reductase (M. vanbaalenii PYR-1; accession no. YP_952126.1; aa identity = 60%). The nucleotide sequence accession numbers were obtained from the GenBank database (http://www.ncbi.nlm.nih.gov/).

One mutant (I45) exhibited a leaky growth phenotype on MSM-isoprene rubber-sandwich agar plates but a wild-type phenotype in liquid MSM with isoprene rubber. The plasmid had integrated between orf2, which codes for a putative cytochrome P450 monooxygenase, and orf3, whose function is unknown. The amino acid (aa) sequence of this cytochrome P450 monooxygenase shared high levels of homology (56% aa identity) with a linalool 8-monooxygenase from Mycobacterium smegmatis strain mc2 155, which catalyzes {omega}-oxidation of linalool. Genes coding for the putative acyl CoA thiolase and cytochrome P450 monooxygenase as well as for a hypothetical protein (Fig. 1c) are organized in a order similar to that seen in Nocardia farcinica IFM 10152 (nfa5170, acyl CoA thiolase; nfa5180, cytochrome P450 monooxygenase; nfa5190, hypothetical protein).

In mutants D6 and I45, plasmid pMA5096 had not inserted into genes, but its insertions into the genome exerted polar effects on the expression of adjacent genes (Fig. 1). Degradation of poly(cis-1,4-isoprene) in G. polyisoprenivorans VH2 was expected to be associated with increased expression of β-oxidation pathway enzymes because of the transposon mapped in genes probably encoding enzymes catalyzing reactions of the β-oxidation or adjacent regions (Fig. 1) in the mutant genomes, which caused a reduced ability to degrade poly(cis-1,4-isoprene) in all three mutants.


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Cloning of mcr of G. polyisoprenivorans VH2.
 
Sequence analysis mapped the transposon insertion in mutant D21 (2) in a gene whose translational product showed high aa sequence identity to the {alpha}-methylacyl CoA racemase of Rhodococcus sp. strain RHA1. The missing 5' and 3' regions were obtained by modified nested PCR (15) using the nonbiotinylated primers Mcr_1016_fw and Mcr_924_rv, respectively, and the walker primers Walker1 to Walker4, respectively, in the first amplification step (see Table S2 in the supplemental material). After purification of the PCR products, a second amplification step was performed to obtain the nucleotide sequences in the 3' and 5' regions, using the PCR products as templates and the primers Mcr_1042_nsd_fw and Mcr_33_nsd_rv in, respectively, combination with the primer Nested (see Table S2 in the supplemental material). The mcr gene was amplified from total genomic DNA of G. polyisoprenivorans VH2 by employing oligonucleotides 5'VH2_mcr and 3'StoppVH2_mcr (see Table S2 in the supplemental material) and yielded the entire 1,113-bp mcr gene. The aa sequence of McrVH2 (370 aa) shares highest homologies with the {alpha}-methylacyl CoA racemase of Rhodococcus sp. strain RHA1 (GenBank accession no. YP_705495.1; 67% identical aa) and the L-carnitine dehydratase/bile acid-inducible F protein of Mycobacterium vanbaalenii PYR-1 (GenBank accession no. YP_955359.1; 67% identical aa). In addition, it showed high levels of homology to the {alpha}-methylacyl CoA racemases of mice (GenBank accession no. EDL03288.1; 50% identical aa) and humans (GenBank accession no. AF158378_1; 46% identical aa).


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Genetic localization of mcr.
 
Directional genome walking was carried out to identify genes adjacent to mcrVH2. In G. polyisoprenivorans VH2, the mcr gene seems not to be clustered with genes coding for other enzymes that could be involved in the degradation of polyisoprenoids such as the mcr gene cluster involved in isoprene catabolism in Rhodococcus sp. strain AD45 (26). In silico analysis gave no indications of the presence of adjacent genes coding for enzymes involved in the β-oxidation pathway. The mcr gene is flanked by two open reading frames coding for a putative creatinase (EC 3.5.3.3) and a putative aspartate aminotransferase (EC 2.6.1.1), respectively (Fig. 1d).


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Disruption of mcr.
 
To confirm an essential function of mcr for utilization of methyl-branched isoprenoids and poly(cis-1,4-isoprene) in strain VH2, a disruption mutant was generated. For this purpose, mcrVH2 was amplified with primers 5'VH2_mcr and 3'StoppVH2_mcr and cloned into pGEMTeasy, yielding pGEMTeasy::mcrVH2. To disrupt mcr, an {Omega}Km cassette was cloned into the single KpnI site of pGEMTeasy::mcrVH2 at position 651 of mcr. The {Omega}Km cassette was then isolated from pSKsym{Omega}Km (17) by digestion with SmaI and ligated with KpnI-linearized pGEMTeasy::mcrVH2 DNA, which was blunted by T4 DNA polymerase treatment (Fermentas, Germany), yielding pGEMTeasy::mcrVH2{Omega}Km. Gene disruption mutants generated by homologous recombination via a double-crossover event in G. polyisoprenivorans were obtained by transfer of this mcr{Omega}Km DNA fragment. Among 500 transformants, which were transferred onto MSM agar plates with geranylacetone as the sole carbon source, five mutants were impaired in growth. To confirm correct integration of the mcr{Omega}Km fragment at the DNA level, Southern analysis of genomic DNA was performed with total DNA prepared from putative mcr disruption mutants and from the wild type, employing a digoxigenin-labeled internal 600-bp mcr fragment: whereas genomic DNA of the wild type digested with EcoRI gave a 2.4-kbp signal, all five disruption mutants gave a 3.4-kbp signal (data not shown), thereby indicating integration of the 1.0-kbp {Omega}Km cassette into mcr by homologous recombination. Inactivation of mcr resulted in a total loss of the ability to metabolize methyl-branched isoprenoids but had no effect on the degradation of long-chain alkanes or water-soluble substrates such as acetate and propionate. Obviously, no genes are present in the G. polyisoprenivorans VH2 genome encoding functionally active isoenzymes, because disruption of mcr yielded a fully negative phenotpye.


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Complemention of the mcr disruption mutant G. polyisoprenivorans VH2 2-22 by intact mcr genes.
 
To exclude the possibility that the negative phenotypes observed in mcr knockout mutants were caused by a polar effect, we performed genetic complementation of the mcr disruption mutant G. polyisoprenivorans VH2 2-22, which was one of the knockout mutants previously verified by Southern hybridization (see above). Therefore, the mcr of N. farcinica IFM 10152 was amplified from total DNA by the use of primers 5'NF_mcr and 3'NFStopp_mcr (see Table S2 in the supplemental material). The PCR product was cloned into pGEMTeasy, yielding pGEMTeasy::mcrNf. Inserts of pGEMTeasy::mcrVH2 and pGEMTeasy::mcrNf, were afterwards cloned as XbaI-BamHI fragments into the shuttle vector pDBMCS-5 (5), yielding pDBMCS-5::mcrVH2 and pDBMCS-5::mcrNf, respectively.

The plasmids were transferred to competent cells of this mutant (1), and transformants were selected on MSM agar plates containing kanamycin (50 µg/ml) and gentamicin (50 µg/ml), with geranylacetone (35 µl/plate) as the carbon source (Fig. 2). All randomly chosen clones harbored pDBMCS5::mcrVH2 or pDBMCS5::mcrNf. The negative phenotype of the mcr knockout mutant in degradation of poly(cis-1,4-isoprene) and methyl-branched isoprenoids was complemented by homologous expression of McrVH2 and also by heterologous expression of a fatty acid CoA racemase from N. farcinica IFM 10152 (McrNf; GenBank accession no. YP_121581) exhibiting 62% aa identity with McrVH2 (Fig. 2a and b), indicating the same function of Mcr as that seen in N. farcinica. McrMtub harbors four highly conserved amino acid residues (Arg91, His126, Asp156, and Glu241), which are also found in McrNf (Arg56, His134, Asp164, and Glu249) and McrVH2 (Arg105, His140, Asp170, and Glu255) (22). Amino acid sequence comparisons showed that both racemases—McrVH2 and McrNf—belong to the family III CoA transferases, which are divided into groups of racemases and CoA transferases (13). The conserved Glu residue occurs only in racemase enzymes (22) and confirms McrVH2 and McrNf to be racemases also on the basis of comparisons of aa sequences.


Figure 2
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  		FIG. 2. Comparison of growth of G. polyisoprenivorans VH2 wild type and mutants. (a) Cells were incubated on MSM agar plates containing geranylacetone (35 µl/plate) as the single carbon and energy source for 7 days at 30°C. Section 1, knockout mutant of G. polyisoprenivorans VH2 mcr{Omega}Km; section 2, G. polyisoprenivorans VH2 wild type; section 3, mutant G. polyisoprenivorans VH2 mcr{Omega}Km complemented with pDBMCS5::mcrVH2 6-33; section 4, mutant G. polyisoprenivorans VH2 mcr{Omega}Km complemented with pDBMCS5::mcrVH2 1-23; section 5, mutant G. polyisoprenivorans VH2 mcr{Omega}Km complemented with pDBMCS5::mcrNf 2-2; section 6, mutant G. polyisoprenivorans VH2 mcr{Omega}Km complemented with pDBMCS5::mcrNf 16. (b) Comparison of levels of growth of G. polyisoprenivorans VH2 wild type and mutants in liquid MSM with 0.2% (wt/vol) poly(cis-1,4-isoprene) as the sole carbon and energy source. Cultures were cultivated in Erlenmeyer flasks and incubated on a horizontal rotary shaker at 130 rpm and 30°C. Kanamycin (50 µg/ml) and gentamicin (50 µg/ml) were added when needed. The optical densities of the wild type and the mcr disruption mutant at 600 nm (OD600nm) were measured twice. {square}, G. polyisoprenivorans VH2 wild type; {blacksquare}, knockout mutant of G. polyisoprenivorans VH2 mcr{Omega}Km; {blacktriangleup}, mutant G. polyisoprenivorans VH2 mcr{Omega}Km complemented with pDBMCS5::mcrVH2 1-23; {Delta}, mutant G. polyisoprenivorans VH2 mcr{Omega}Km complemented with pDBMCS5::mcrVH2 6-33; {blacklozenge}, mutant G. polyisoprenivorans VH2 mcr{Omega}Km complemented with pDBMCS5::mcrNf 2-2; {diamond}, mutant G. polyisoprenivorans VH2 mcr{Omega}Km complemented with pDBMCS5::mcrNf 16.


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Transcription analysis of mcr, ferA, the P450 gene, and a gene coding for a short-chain dehydrogenase in strain VH2.
 
The occurrence of transposon insertions in the ferA gene (mutant C22), in the P450 gene (mutant I45), and in the gene coding for a short-chain dehydrogenase (mutant D6) raised the issue of whether expression of these genes is specifically induced during cultivation of cells in MSM containing poly(cis-1,4-isoprene) as the carbon source. Therefore, transcription of these three genes and additionally of the mcr gene was investigated by one-step reverse transcription-PCR (RT-PCR). RNA isolation from cells cultivated with 0.2% (wt/vol) sodium propionate (2 days at 30°C) or 0.2% (wt/vol) isoprene rubber (4 weeks at 30°C) and PCR were done using an RNeasy RNA purification kit and a Qiagen OneStep RT-PCR kit (both Qiagen, Hilden, Germany). Control experiments for DNA contamination that were set up by adding the RNA template after the reverse transcriptase step and before activating Taq polymerase did not yield PCR products. Cells cultivated in liquid MSM in the presence of sodium propionate or poly(cis-1,4-isoprene) exhibited constitutive expression of all gene transcripts (Fig. 3). RT-PCR analysis clearly showed that mcr and ferA are constitutively expressed in G. polyisoprenivorans VH2. Although the genes coding for the short-chain dehydrogenase/reductase and the P450 monooxygenase were also expressed on nonisoprenoid substrates in VH2, induction occurred during cultivation of cells in media containing polyisoprenoids (Fig. 3). Degradation products occurring during growth of G. polyisoprenivorans on rubber must induce expression of both genes. In N. farcinica, cytochrome P450 monoxygenase is putatively involved in fatty acid metabolism (12). P450 cytochromes belong to a superfamily of heme-containing enzymes, which occur in prokaryotes as well as in all eukaryotes (16) and catalyze the oxidation of a wide range of organic molecules. Since {omega}-oxidation of fatty acid catalyzed by P450 cytochromes yielding dicarboxylic acids is widespread in nature (for a review, see reference 6), it is possible that intermediates of poly(cis-1,4-isoprene) undergo {omega}-oxidation.


Figure 3
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  		FIG. 3. Transcription analysis of mcr, ferA, the P450 gene, and a gene coding for a short-chain dehydrogenase identified in mutant D6. Expression of the genes was analyzed by RT-PCR using samples derived from cells cultivated in the presence of 0.2% (wt/vol) sodium propionate or 0.2% (wt/vol) poly(cis-1,4-isoprene) as the carbon source. M, 100-bp leader; K, poly(cis-1,4-isoprene); P, sodium propionate.


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Detection of biotinylated proteins.
 
Biotinylated carboxylases are key enzymes in degradation of acyclic isoprenoids in some pseudomonads (8, 9, 11). Therefore, the proteome of G. polyisoprenivorans VH2 was investigated for the presence of biotinylated proteins specially expressed during poly(cis-1,4-isoprene) catabolism (14). However, no additionally biotinylated proteins were detectable during rubber degradation (data not shown). These findings were not surprising, since in Pseudomonas species geranyl CoA carboxylase and methylcrotonyl CoA carboxylase catalyze the carboxylation of branched methyl groups, which would inhibit β-oxidation (8, 9, 11). In G. polyisoprenivorans VH2, no specifically expressed carboxylase is necessary, because Mcr is the key enzyme in the β-oxidation of methyl-branched hydrocarbons. This indicates that for acyclic monoterpenes like citronellol, another degradation pathway exists in VH2, as described for some Pseudomonas species.


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Conclusions.
 
Our studies have demonstrated the crucial role of Mcr in the catabolism of poly(cis-1,4-isoprene) and of other methyl-branched hydrocarbons in G. polyisoprenivorans. Further studies will focus on other enzymes in addition to Mcr that are involved in degradation of poly(cis-1,4-isoprene) cleavage products such as the cytochrome P450 monoxygenase and the short-chain dehydrogenase to unravel the role of these enzymes in poly(cis-1,4-isoprene) degradation.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Correnstrasse 3, D-48149 Münster, Germany. Phone: 49-251-8339821. Fax: 49-251-8338388. E-mail: steinbu{at}uni-muenster.de Back

{triangledown} Published ahead of print on 26 September 2008. Back

{dagger} Supplemental material for this article may be found at http://aem.asm.org/. Back


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Applied and Environmental Microbiology, November 2008, p. 7085-7089, Vol. 74, No. 22
0099-2240/08/$08.00+0     doi:10.1128/AEM.01491-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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