Sequence variation in dichloromethane dehalogenases ... - CiteSeerX

Microbiology (2001), 147, 611–619

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Sequence variation in dichloromethane dehalogenases/glutathione S-transferases Ste! phane Vuilleumier,1 Nikola Ivos) ,1 Mariangela Dean2 and Thomas Leisinger1 Author for correspondence : Ste! phane Vuilleumier. Tel : j41 1 632 33 57. Fax : j41 1 632 11 48. e-mail : svuilleu!micro.biol.ethz.ch

1

Institut fu$ r Mikrobiologie, ETH Zu$ rich, Schmelzbergstraße 7, CH-8092 Zu$ rich, Switzerland

2

Dipartimento di Biochimica e Biologia Molecolare, Universita' degli Studi, 44100 Ferrara, Italy

Dichloromethane dehalogenase/glutathione S-transferase allows methylotrophic bacteria to grow with dichloromethane (DCM), a predominantly man-made compound. Bacteria growing with DCM by virtue of this enzyme have been readily isolated in the past. So far, the sequence of the dcmA gene encoding DCM dehalogenase has been determined for Methylobacterium dichloromethanicum DM4 and Methylophilus sp. DM11. DCM dehalogenase genes closely related to that of strain DM4 were amplified by PCR and cloned from total DNA from 14 different DCM-degrading strains, enrichment cultures and sludge samples from wastewater treatment plants. In total, eight different sequences encoding seven different protein sequences were obtained. Sequences of different origin were identical in several instances. Sequence variation was limited to base substitutions ; strikingly, 16 of the 19 substitutions in the dcmA gene itself encoded amino acids that were different from those of the DM4 sequence. The kinetic parameters kcat and Km, the pH optimum and the stability of representative DCM dehalogenase variants were investigated, revealing minor differences between the properties of DCM dehalogenases related to that from strain DM4.

Keywords : dichloromethane, dehalogenase, glutathione S-transferase

INTRODUCTION

Dichloromethane (DCM) is a solvent, produced and used industrially in large quantities, which easily escapes into the environment because of its volatile and watersoluble character (Mckay et al., 1993 ; van Agteren et al., 1998). The contamination of groundwater and drinking water supplies by DCM became a source of concern because of evidence of genotoxic effects associated with DCM exposure (reviewed by Green, 1997). This led to the inclusion of DCM in the list of priority pollutants in 1977 (Keith & Telliard, 1979) and provided an incentive for investigating the degradation of DCM by bacteria and its applications in the treatment and prevention of environmental pollution with DCM. Bacteria that rely on a single enzyme, DCM dehalogenase, for growth with DCM as the sole carbon source were readily isolated from environments contaminated .................................................................................................................................................

Abbreviations : CAH, chlorinated aliphatic hydrocarbon ; DCM, dichloromethane ; GST, glutathione S-transferase. The GenBank accession numbers for the sequences determined in this work are AJ271131–38 (see text for details). 0002-4393 # 2001 SGM

with chlorinated aliphatic hydrocarbons (CAHs). More than a dozen strains, isolated from different locations, that possess DCM dehalogenase have been reported (see Vuilleumier, 2001, for an overview). DCM dehalogenase was purified and shown to require glutathione as a cofactor to transform DCM to formaldehyde and two molecules of HCl (Kohler-Staub & Leisinger, 1985). Cloning of the corresponding gene from Methylobacterium sp. strain DM4 (La Roche & Leisinger, 1990), now called Methylobacterium dichloromethanicum DM4 (Doronina et al., 2000), showed that the encoded protein belonged to the glutathione S-transferases (GSTs), a large family of detoxification enzymes in all aerobic organisms, including bacteria (Josephy, 1997 ; Vuilleumier, 1997). With the exception of Methylophilus sp. strain DM11, known methylotrophic DCM-degrading strains were shown to possess a DCM dehalogenase that is very similar, if not identical, to that of Methylobacterium dichloromethanicum DM4, since they showed strong cross-reactivity with antibodies raised against the DM4 enzyme (Kohler-Staub et al., 1986) and hybridized with the dcmA gene of strain DM4 as a probe (Scholtz et al., 611

S. V U I L L E U M I E R a n d O T H E R S

1988). In contrast, the sequence of the DCM dehalogenase from Methylophilus sp. strain DM11 (266 residues) deduced from the gene sequence is only 56 % identical to that of the enzyme from strain DM4 (287 residues) (Bader & Leisinger, 1994). The kinetic parameters of the DM4 and DM11 enzymes differ quite significantly, the DM11 enzyme displaying a kcat value about sixfold higher, and Km value sixfold higher, than that of the DM4 enzyme (Vuilleumier & Leisinger, 1996). The kinetic properties of these enzymes also determine the growth properties of DCM-degraders using DCM as the sole carbon source. Hence, a bacterium expressing the DM11 enzyme was superior to an otherwise identical strain expressing the DM4 enzyme in batch culture, but the reverse applied during growth under limiting conditions of DCM in the chemostat (Gisi et al., 1998). Nevertheless, only DCMdegraders with a DCM dehalogenase gene similar to that of strain DM4 were detected by hybridization analysis in sludge from wastewater treatment plants and in enrichment batch cultures (growing with DCM) that were derived from them (Gisi et al., 1998). Hybridization analysis also revealed that the DNA loci of DCM-degrading bacteria with a DCM dehalogenase very similar to that of strain DM4 displayed significant variation (Schmid-Appert et al., 1997). Strains were classified into different groups on the basis of the number, type and pattern of overlapping insertion sequence elements flanking a common conserved 4n2 kb DNA fragment containing the dcmA gene. This suggested that horizontal gene transfer may have been involved in the spread of the DCM-degrading phenotype in environments contaminated with CAHs. However, it also raised specific questions regarding the evolution of DCM dehalogenase genes. Did dcmA genes of different origins display sequence variation, and, if so, what were the consequences of this variation at the enzyme level ? We report here on the PCR amplification, the cloning and the sequence analysis of dcmA genes from DCMdegrading strains and from other sources, and on the functional characterization of some of the corresponding enzymes. METHODS Materials. All chemicals were reagent grade or better and were purchased from Fluka, unless otherwise indicated. Bacterial strains and growth conditions. DCM-degrading strains (Table 1) were grown in liquid cultures at 30 mC in phosphate minimal medium with DCM as the carbon source, as described previously (Scholtz et al., 1988). Enrichment cultures of DCM-degrading strains were obtained in the same medium by incubating sewage sludge (1 %, w\v, pelleted and washed inoculum) in the presence of 100 µg cycloheximide ml−" ; this was followed by periodic serial transfer (1 : 1000, v\v, inoculum) into fresh medium. Escherichia coli XL-1 Blue (Stratagene) was used in conjunction with plasmid pBLS KS II(j) (Stratagene) for cloning and grown in Luria–Bertani medium supplemented with ampicillin (100 µg ml−") when appropriate. DNA techniques. Cloning procedures followed standard methods (Ausubel et al., 1987–2000). Restriction and DNA-

612

modifying enzymes were purchased from Fermentas. Oligonucleotides were purchased from Microsynth. DNA isolation. Sequencing-quality plasmid DNA from E. coli

was obtained with Jetstar ion-exchange columns (Genomed). Total DNA from enrichment cultures and methylotrophic DCM-degrading strains was isolated by using the cetyltrimethylammonium bromide method (Ausubel et al., 1987–2000). Total DNA from sludge was isolated as follows. Sewage sludge (160 ml) was collected by centrifugation (10 min, 9000 r.p.m.), resuspended in 50 ml cold extraction buffer [100 mM Tris\HCl, pH 7n6 ; 10 mM EDTA ; 5 mM thiourea ; 10 mM DTT ; 1n5 % w\v SDS ; 1 %, w\v, deoxycholate ; 1 %, w\v, Nonidet P-40 (Sigma)] with 5 g glass beads (0n11 mm diameter), treated for 1 min in a cooled MiniBeadbeater (Biospec Products), and immediately put on ice. The resulting emulsion was centrifuged (10 min, 12 000 r.p.m., 4 mC) and the resulting supernatant was extracted once with phenol and twice with chloroform. After the addition of 0n1 vol. 3 M sodium acetate (pH 5n4), total DNA was precipitated with 2 vols cold ethanol, resuspended in 40 µl HPLC-grade water, incubated briefly with 0n5 ml polyvinylpolypyrrolidone (Aldrich) suspension (7n5 %, w\v ; Holben et al., 1988), and recovered by centrifugation through a spin column (Bio101) (Berthelet et al., 1996). PCR amplification. Amplifications were performed with Taq polymerase for the detection of dcmA genes, and with either a 10 : 1 mix of Taq polymerase with Vent polymerase exo− (New England Biolabs), TaqExpand (Roche Diagnostics) or Pfu polymerase (Stratagene) for the cloning of dcmA genes from genomic DNA. Amplifications were performed with 100–300 ng total DNA in a 50 µl volume for 30 cycles of denaturation (95 mC), annealing (50 mC, unless otherwise indicated) and extension (72 mC) of 1 min each, using 0n2 mM dNTP and 50 pmol of each primer (see Fig. 1). Degenerate primers Cfor (ATSATCYKGCRTCMCAGC) and Crev (TMAGCMAGTAWTYCTA), corresponding to conserved DNA and amino acid sequence segments in DM4 and DM11 DCM dehalogenases, were used to detect dcmA dehalogenases from all DCM-degrading methylotrophic bacteria containing a dcmA gene (a 450 bp product for DM4-like dcmA genes and a 441 bp product for the DM11 dcmA gene). A 1225 bp PCR product encompassing the dcmA gene in strains closely related to strain DM4 was amplified (annealing temperature 55 mC) using primers DMfor (AAAAAAAACATCTAGAGAATGACAACCGTGCGC, an intergenic sequence at position k193 to k169 upstream of the dcmA gene start codon) and DMrev (AAAAAAAAAAGGATCCGGTCATCGAAGGAATGC ; position 125–149 relative to the dcmA gene stop codon, in the orf353 gene of unknown function downstream of dcmA). These sequences permitted the introduction of XbaI and BamHI restriction sites (underlined) to facilitate the cloning of PCR products. For direct amplification of the strongly conserved dcmA gene region from total DNA from sludge samples, an initial PCR reaction (annealing temperature 65 mC) was performed with primers DMforout (CCTCCCGTTACGCCTTCCTCCCC ; position k376 to k354 relative to the dcmA gene start codon in the upstream intergenic region) and DMrevout (TCCCGATGTTCCATCACCGCCC ; position 302–323 relative to the dcmA gene stop codon in orf353 downstream of dcmA). The primers were chosen as a suitable pair for amplification using the program 3 (Rozen & Skaletsky, 1998 : http :\\www.genome. wi.mit.edu\cgi-bin\primer\primer3Iwww.cgi). The resulting 1566 bp product (1 µl of the 50 µl reaction) was then used in a second PCR reaction with primers DMfor and DMrev as described above.

Dichloromethane dehalogenase sequence variation Table 1. Origin of cloned DCM dehalogenase sequences and summary of observed sequence variation Cloned PCR amplicon

Origin

Amino acid (base) differences

Methylobacterium dichloremethanicum DM4* Methylophilus sp. DM11 Hyphomicrobium sp. DM2‡ Methylorhabdus multivorans DM13 Hyphomicrobium sp. GJ21

CAH-contaminated soil

Reference sequence

Heavily DCM-contaminated groundwater CAH-contaminated soil CAH-contaminated groundwater

115 (332)† 1 (1) 6 (7)

CAH-contaminated sludge and waterways

9 (15)

LZ

Trickling filter bioreactor for DCM removal from waste air Sewage sludge, industrial wastewater treatment plant Sewage sludge enrichment culture, municipal wastewater treatment plant Sewage sludge enrichment culture, municipal wastewater treatment plant

4 (7)

S1§ S2-E1 S3-E1

Reference

La Roche & Leisinger (1990) Bader & Leisinger (1994) Stucki et al. (1981) Doronina et al. (1995)

5 (7)

Ottengraf et al. (1986) ; Janssen et al. (1991) Zuber (1995) ; Zuber et al. (1997) ; this work This work

7 (11)

This work

0 (2)

This work

* Amplicons from strain DM1 (which contains two copies of the dcmA gene ; Schmid-Appert et al., 1997), from strain DM5 and from the DCM-grown enrichment culture S1-E1 obtained from sludge S1 gave sequences identical to that of strain DM4. † Minimal estimate of base changes accounting for the observed amino acid differences between aligned sequences, as calculated by the  program (Lewontin, 1989). The DM11 protein sequence contains 266 residues, as opposed to the 287 residues of all DM4-like protein sequences. ‡ Amplicons from enrichment culture S3-E2 gave a sequence identical to that of strain DM2. § Amplicons from enrichment culture S2-E2 gave a sequence identical to that of sewage sludge S1.

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Fig. 1. Strategy for the PCR amplification of DCM dehalogenase genes, and an overview of observed sequence variability in the dcmA region, taking the dcmA gene of Methylobacterium dichloromethanicum DM4 (La Roche & Leisinger, 1990) as a reference. The aligned, 56 % identical dcmA gene from Methylophilus sp. strain DM11 (Bader & Leisinger, 1994) is indicated by a dashed arrow. The primers used in PCR detection and cloning of the dcmA genes (see Methods) are shown as triangles labelled with their names. The transcriptional start (‘ j1 ’) and the putative k35/k10 region upstream of the dcmA gene (small boxes) from strain DM4 (La Roche & Leisinger, 1990) are shown, along with two prominent inverted repeats detected upstream and downstream of dcmA. The locations of sequence variation in cloned PCR amplicons are indicated by circles (open circles, silent nucleotide changes ; solid circles, nucleotide changes leading to an altered protein sequence relative to the DCM dehalogenase of strain DM4 ; see Table 2).

Cloning of PCR products. PCR products were purified from agarose gels by using the QIAquick gel extraction kit (Qiagen), digested with XbaI and BamHI and then ligated to the pBLS KS II(j) vector (Stratagene) cut with the same enzymes.

DNA sequencing. Complete (1159 bp) DNA sequences of cloned PCR products were obtained for both strands by cycle sequencing, with DIG-labelled primers and using a GATC 1500 direct blotting electrophoresis system as described (Pohl

613

S. V U I L L E U M I E R a n d O T H E R S

& Maier, 1995), or with the ABI Prism dye terminator ready reaction mix and an ABI Prism 310 genetic analyser (PerkinElmer). Sequences were obtained from two clones each from at least two independent PCR amplification reactions. The different 1159 nt sequences obtained and analysed here were submitted to the GenBank\EMBL database.The GenBank accession numbers are as follows : AJ271131 (strains DM1 and DM5, identical to Methylobacterium dichloromethanicum DM4), AJ271132 (Hyphomicrobium sp. DM2), AJ271133 (Methylorhabdus multivorans DM13), AJ271134 (Hyphomicrobium sp. GJ21) ; AJ271136 (LZ), AJ271135 (S2-E1), AJ271138 (S3-E1) and AJ271137 (S1). Sequence analysis. Sequence alignments were generated using

the programs  and  from the GCG package (version 10.0). Phylogenetic analysis was performed using the  package of programs (version 3.5 ; Felsenstein, 1993 : http :\\evolution.genetics.washington.edu\phylip.html). Distance measurements were performed using the Kimura default option and the programs  or . Trees were constructed with ,  and , using the global optimization and randomized input options (100 jumbles). Bootstrapping analysis was performed with the same programs by first generating 1000 bootstrapped datasets from the original datasets, using , and then calculating the consensus tree with . Trees were visualized using  (Perrie' re & Gouy, 1996 : http :\\ pbil.univ-lyon1.fr\software\njplot.html).

DCM dehalogenase purification. DCM dehalogenases were purified from cell-free extracts of methylotrophic bacteria by ammonium sulfate fractionation and anion-exchange chromatography as described previously (Vuilleumier & Leisinger, 1996). An additional hydrophobic-interaction chromatography step (Resource Iso ; Pharmacia – 50 % saturated ammonium sulfate in 100 mM phosphate pH 8 buffer to 100 mM phosphate pH 8 buffer) was performed when required. The protein concentration was determined using a commercial Bradford reagent (Bio-Rad) with bovine serum albumin as the standard. Enzyme assays. DCM dehalogenase activity was determined by detecting the dihydropyridine derivate of formaldehyde at 412 nm, or with the formaldehyde dehydrogenase coupled assay by monitoring NADH formation at 340 nm as described previously (Vuilleumier & Leisinger, 1996). The specific activity of DCM dehalogenases was determined at 30 mC in 100 mM phosphate buffer (pH 8), using 1 mM DCM or dibromomethane and 2 mM glutathione. The kinetic parameters Km and kcat for DCM and glutathione were obtained as described previously (Vuilleumier & Leisinger, 1996) under the same conditions, by fitting data directly to the Michaelis– Menten equation by non-linear least-square minimization using  (Abelbeck Software). Standard errors were observed to be less than 20 % of the Km values and less than 10 % of the kcat values, respectively. The pH optimum of DCM dehalogenases was obtained with the formaldehyde derivatization assay by measuring specific activity in MES buffer (pH 5n3–6n5), phosphate (pH 6n5–7n7), Bicine (pH 7n4–8n8) and glycine (pH 8n9–9n9) buffers of constant (200 mM) buffer strength. DCM dehalogenase inactivation. The residual activity of

DCM dehalogenases in the presence of different concentrations of urea (electrophoresis grade ; Bio-Rad) was investigated with the formaldehyde derivatization assay, by measuring their specific activity (V) in 200 mM Bicine buffer 614

(pH 8n3) with 2 mM glutathione and 1 mM DCM at T l 25 mC. The urea concentration at half-maximal enzyme activity, [U] / , was determined. "#

RESULTS Detection of DCM dehalogenase genes by PCR

We developed a PCR-based strategy for the detection and isolation of dcmA genes in DNA from purified strains, sludge and other samples (Fig. 1). Adequate combinations of primers useful for detecting both the DM4 (La Roche & Leisinger, 1990) and DM11 (Bader & Leisinger, 1994) types of genes were difficult to design, because of the large differences between DM4 and DM11 gene sequences (56 % sequence pairwise identity). Using the 32-\16-fold degenerate primer pair Cfor\Crev, which leads to the amplification of an internal dcmA gene fragment with both DM4 and DM11 strains, DM4like dcmA genes were readily detected in sludge from wastewater treatment plants, as well as in enrichment cultures derived from them, but DM11-like genes were not observed (data not shown). Although some dcmA genes may have remained undetected with this approach, these results confirm findings obtained previously by hybridization analysis using DM4- and DM11-specific dcmA probes (Gisi et al., 1998). Following these observations, other sets of primers were used for further characterization of dcmA genes closely similar to that of strain DM4. It was possible to amplify a 1n2 kb PCR product from DNA of DCM-degrading DM strains DM1 to DM14, except Methylophilus sp. strain DM11, using the non-degenerate primers DMfor and DMrev (corresponding to flanking sequences of the dcmA gene from strain Methylobacterium dichloromethanicum DM4) (Fig. 1). This confirmed the extremely conserved character of the DNA region encompassing the dcmA gene of DCM-degrading strains with a DCM dehalogenase closely related to that in strain DM4 hinted at by previous hybridization studies (Scholtz et al., 1988 ; Schmid-Appert et al., 1997). In sludge, however, DM4-like dcmA genes were present at very low levels, and the resulting PCR products were detected only upon subsequent hybridization with a dcmA probe (data not shown). In such cases, dcmA genes were amplified and cloned using a nested PCR strategy, by prior amplification with primers DMforout and DMrevout flanking the dcmA gene sequence from strain DM4 and subsequent amplification of the resulting 1n5 kb product with DMfor and DMrev. Sequence analysis of cloned PCR products

DCM-degrading bacteria with a DM4-like dcmA gene were previously classified into different groups based on the arrangement of insertion-sequence elements flanking the conserved 4n2 kb BamHI DNA fragment with the dcmA gene (Scholtz et al., 1988 ; Schmid-Appert et al., 1997). PCR products were obtained with primers DMfor and DMrev from total DNA of representative strains of each subgroup, as well as from enrichment cultures and

Dichloromethane dehalogenase sequence variation Table 2. Summary of observed variation in cloned DCM dehalogenase sequences Position

Codon

T(k137)C C6T A48T G61T C62G G63C G82A C83T G127T T139A C152A G196A C458T A645G A708G T721G A746G C818T T829A A833T T(j25)A

T C* AGC AGT CTA CTT GCG TGC GCG KGS GCG KGC GCA ACA GCA GTA GTC TTC TCC ACC ACC AAC GTT ATT ACC ATC AAA AAG ATA ATG TTA GTA AAG AGG GCA GTA TTC ATC AAG ATG T A*

Protein – S1w† L15w† A20C A20G A20C, A20G A27T A27V V42F S46T T50N V65I T152I K214w† I235M L240V K248R A272V F276I K277M –

DM4 T C A G C G G C G T C G C A A T A C T A T

DM2 DM13 GJ21

T T G

LZ

C T

C T

G C

T G C

T

T

A A

A

S1

S2-E1 C T

T G C

S3-E1 C T

G C

A T A A

T A

T G

A A A

T G G

A

G T A T A

A T

A T

* Sequence flanking the dcmA gene. † w, Synonymous change at the third, wobble position of the codon.

1). Identical changes relative to the DM4 sequence were observed in sequences from samples and strains isolated at different locations and at different times (Table 2). In total, eight different gene sequences encoding seven different protein sequences (287 amino acids) were obtained. The sequence displaying most sequence differences relative to strain DM4 was that of strain GJ21, isolated in the Netherlands (Ottengraf et al., 1986 ; Janssen et al., 1991), having 15 base differences encoding nine amino acid differences (Fig. 1, Tables 1 and 2). The observed nucleotide differences showed a striking bias for changes that encoded a different amino acid (Tables 1 and 2). Of the 19 base differences observed relative to the dcmA gene from strain DM4, 16 encoded differences in the protein sequence of the DCM dehalogenase. Upstream of the dcmA gene, differences in the nucleotide sequence were observed, altbeit repeatedly, at one position only (Fig. 1, Table 2). Downstream of dcmA, a single difference was noted in the case of the sequence from strain GJ21 (Fig. 1, Table 2).

sludge samples (Table 1). These products were cloned by taking advantage of restriction sites introduced into the primers used for PCR amplification (Fig. 1). In all cases, two clones from two independent amplifications were sequenced in order to avoid PCR artefacts (Table 2). For strain DM1, which belongs to the group of strains possessing two copies of the dcmA gene on distinct DNA fragments (Schmid-Appert et al., 1997), amplicons from both copies of the gene were obtained from sized fractions of PstI-digested total DNA containing either the 7 kb or the 10n5 kb PstI fragment with a copy of the dcmA gene (Schmid-Appert et al., 1997). Amplicons were also characterized from total DNA of a prototype bioreactor for DCM removal from waste air (Zuber, 1995 ; Zuber et al., 1997 ; Table 1), and from enrichment liquid cultures, derived from sludge from three different wastewater treatment plants (Table 1), growing with DCM as the sole carbon source. PCR products were obtained and cloned directly from sludge from an industrial wastewater treatment plant, but not from two municipal plants that were investigated (Table 1).

Phylogenetic analysis of DCM dehalogenase genes

In all cases, the sequenced DNA fragment was 1159 bp in length, comprising the 867 bp of the complete dcmA gene and 292 bp in the upstream and downstream regions. This fragment was identical to that of strain DM4 in both dcmA gene copies of strain DM1, in strain DM5 and in the enrichment culture S1-E1 derived from industrial sludge. Nucleotide differences were found in nine other amplicons that were sequenced (Fig. 1, Table

Distance-based and parsimony methods yielded the same tree topology describing the relationships between DCM dehalogenases using both DNA (Fig. 2) and protein (not shown) sequence information. The nodes in the preferred tree topology were the highest-ranking clusters obtained by bootstrapping analysis, with the exception of the position of the LZ sequence, which, at the protein level, clustered with either the DM2\DM4 or 615

S. V U I L L E U M I E R a n d O T H E R S

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Fig. 2. Unrooted phylogenetic tree of DCM dehalogenase DNA sequences from cloned PCR products (1159 bp) (see Table 1) obtained with the DNADIST, FITCH and DNAPARS programs from the PHYLIP package (Felsenstein, 1993). Isolated DCM-degrading strains are indicated in bold. Numbers above and below branches indicate the percentages at which the branches were obtained, as calculated by bootstrap analysis (SEQBOOT, 1000 replicates) using distance-based (FITCH) and parsimony (DNAPARS, italics) methods, respectively.

the GJ21\S2-E1 group (data not shown). The low bootstrapping values obtained (Fig. 2) can be explained by the limited information present in the sequences (13 and nine phylogenetically informative positions at the DNA and protein levels, respectively). A reliable rooting of the tree using the DM11 sequence as an outgroup was not possible, because of the extensive differences beween DM4-like sequences and the DM11 sequence. Indeed, at least 332 substitutions separate the DM4 sequence from the DM11 sequence (Table 1) (Lewontin, 1989) ; this compares with just 15 substitutions from the most divergent DM4-like sequence found (that of strain GJ21). Functional consequences of sequence variation in DCM dehalogenases

The consequences of sequence variation at the enzyme level were evaluated in the case of the DCM dehalogenases from strains DM2, DM4, DM13 and GJ21 (Table 3, Fig. 3), which were purified from their natural

host. Purification factors (five- to sixfold) similar to those obtained previously for strain DM2 (Kohler-Staub & Leisinger, 1985) rather than to that of strain DM11 (13-fold ; Scholtz et al., 1988) were noted for these enzymes. For DCM, the kinetic parameters kcat and Km of DM4-type enzymes very closely resembled those of the DM4 strain (Vuilleumier et al., 1997). However, the kcat for dibromomethane and the Km for glutathione of the enzymes from Hyphomicrobium sp. GJ21 and Methylorhabdus multivorans DM13 appeared more similar to those of the DM11 enzyme (Table 3). Notably, the enzymes from DM13 and GJ21 display the same residue at position 27 as the DM4 enzyme. The single difference between DM2 and DM4 enzymes at position 27 was previously shown to be associated with a minor difference in the glutathione affinity of the enzyme (Vuilleumier et al., 1997 ; Table 3), and variants of the DM11 enzyme with differences at the corresponding position obtained by protein engineering also displayed an altered affinity for glutathione (Vuilleumier et al., 1997). Therefore, the residue at position 27 of DM4-like DCM dehalogenases is not unique in modulating the affinity of DCM dehalogenases for the glutathione cofactor. The pH optimum and resistance towards inactivation of DM4-like DCM dehalogenases was determined (Fig. 3). The pH optima were very similar. However, the DM13 and GJ21 enzymes were more active at the low and high ends of the pH range (Fig. 3a). What was striking (and previously unremarked upon) was the qualitative difference in pH profiles between DM4-like and DM11 enzymes, the latter showing a plateau extending up to pH 10 rather than a pH optimum around pH 8n5 (Fig. 3a). Sequence variation may result in increased structural stability, rather than in increased catalytic proficiency. Systematic protein-engineering studies in many systems have demonstrated that single amino acid changes can significantly affect the stability of an enzyme (Fersht, 1999). The retention of DCM dehalogenase activity upon urea denaturation was remarkably similar (Fig. 3b, Table 3) among the enzymes, although those of

Table 3. Properties of purified DCM dehalogenases from isolated DCM-degraders Strain Methylobacterium dichloromethanicum DM4* Methylophilus sp. DM11 Hyphomicrobium sp. DM2 Methylorhabdus multivorans DM13 Hyphomicrobium sp. GJ21

KGSH m ‡ (µM)

pH optimum

[U]1/2 (M)

9*

0n9*

403*

8n0–8n5

0n81p0n14

59† 11* 6 8

1n8† 1n0* 1n7 1n7

66† 174* 74 44

8n0–10n0 8n0–8n5 8n0–9n0 7n5–9n0

1n59p0n20 0n66p0n10 1n05p0n17 1n29p0n19

KDCM (µM) m

0n6* 3n3† 0n6* 0n5 0n8

* Data from Vuilleumier et al. (1997). † Data from Vuilleumier & Leisinger (1996). ‡ DBM, dibromomethane ; GSH, glutathione. 616

DCM kDBM cat /kcat ‡

kDCM (s−1) cat

Dichloromethane dehalogenase sequence variation

Specific activity (%)

100

100

(a)

80

80

60

60

40

40

20

20

0 5

(b)

0 6

7

8

9

pH

10

0

0·4 0·8 1·2 1·6 2·0 2·4 2·8 Urea (M)

.................................................................................................................................................................................................................................................................................................................

Fig. 3. Dependence of DCM dehalogenase activity from isolated DCM-degraders on (a) pH and (b) urea concentration. Methylobacterium dichloromethanicum DM4 ($, solid line), Methylophilus sp. DM11 (#, dotted line), Hyphomicrobium sp. GJ21 ( , broken line), and Methylorhabdus multivorans DM13 ( , broken/dotted line). The data for Hyphomicrobium sp. DM2 were omitted, for clarity, since they did not differ significantly from those of Methylobacterium dichloromethanicum DM4.

DM13 and GJ21 were slightly more resistant to inactivation than those of DM2 and DM4. DISCUSSION

Strains and environmental samples containing DM4like dcmA genes showed, at most, 15 nucleotide differences with respect to the DM4 sequence in a fragment of 1159 nt encompassing the entire dcmA gene. This low sequence variability may reflect constraints in the recovery of a functional dcmA gene when DCM is the sole growth substrate, but perhaps there is also a bias arising from the conditions used for cultivation. Indeed, DCM-degraders with dcmA genes closely related to the DM4 sequence were predominantly obtained. In particular, only the DM4 sequence was recovered from enrichment culture S1-E1 derived from industrial wastewater sludge, whereas in this sludge itself, a sequence with seven nucleotide differences relative to the DM4 sequence (S1) had been detected (Table 1). Sequences of dcmA genes from strains isolated by other workers showed more variation relative to the DM4 sequence ; these included the sequences from DM13 (Doronina et al., 1995) and GJ21 (Ottengraf et al., 1986 ; Janssen et al., 1991), as well as the sequence LZ obtained from a bioreactor initially inoculated with strain DM4 but operated under different conditions (Zuber, 1995). Notably, the few differences in DCM dehalogenase sequences recovered in this analysis were exclusively single nucleotide exchanges. Triazine dehalogenase gene sequences from different bacteria were also observed to be very strongly conserved (De Souza et al., 1998), and haloalkane dehalogenase (dhlA) genes from several different strains showed a sequence identical to that originally published for Xanthobacter autotrophicus GJ10 (van den Wijngaard et al., 1992). In contrast, however, haloalkane dehalogenase variants selected by growth on an alternative substrate of the enzyme displayed quite extensive differences, including several

insertions and deletions, relative to the wild-type enzyme (Pries et al., 1994). In addition, representatives of another class of haloalkane dehalogenases encoded by the dhaA gene and characterized in Rhodococcus and Mycobacterium species (see Poelarends et al., 2000) display enzymic and structural properties that are related to, but distinct from, dhlA-type dehalogenases (see Newman et al., 1999), but which show little similarity (approx. 30 % identity) at the protein-sequence level. In the case of bacterial DCM dehalogenases, the most distant sequence is that of Methylophilus sp. DM11 (56 % identity), which encodes an enzyme with lower affinity, but higher turnover with DCM and dibromomethane, than the enzyme from strain DM4 (Table 3) (see Vuilleumier & Leisinger, 1996 ; Gisi et al., 1998). Unlike haloalkane dehalogenases, however, which have a relatively broad substrate range, bacterial DCM dehalogenases are strongly specific for dihalomethanes as substrates. Therefore, the observed 1n7-fold higher activity of DCM dehalogenases from strains DM13 and GJ21 with dibromomethane compared to DCM (Table 3) may represent a significant property of these enzymes – one which confers a selective advantage upon the corresponding strains in the presence of this compound. Growth of methylotrophic bacteria with dibromomethane has not yet been thoroughly investigated, however, because of the higher toxicity, to bacteria, of dibromomethane compared to DCM (Scholtz et al., 1988 ; Goodwin et al., 1998). The possibility that selection for altered enzymic functional properties of the enzyme has been operating in different environments contaminated with DCM warrants further consideration. Indeed, the large proportion of nucleotide differences in DM4-like DCM dehalogenases that lead to differences in the protein sequence (Fig. 1, Table 1) is quite striking. Despite the relatively minor differences detected in the enzymic properties of DCM dehalogenases (Table 3, Fig. 3), the possibility that some of these variations confer a 617

S. V U I L L E U M I E R a n d O T H E R S

selective advantage under certain conditions in vivo, or that they affect properties of the enzyme that are not manifested in the in vitro assays (Gillespie, 1991), cannot be ruled out. Certainly, much of the observed sequence variation appears to be clustered in certain parts of the protein sequence, suggesting that the corresponding residues contribute to the definition of the active site of the enzyme. In the absence of structural information on DCM dehalogenases, however, this remains entirely speculative. The rare occurrence of base changes leading to synonymous codons rather than amino acid changes (Fig. 1) might also reflect strongly constrained codon usage (Akashi & Eyre-Walker, 1998) in the highly expressed dcmA gene (5–20 % of the soluble cell protein in shaken batch cultures ; Gisi et al., 1998). This would imply either that codon usage is the same in the different DCM-degraders studied or that the dcmA genes recovered in our analysis were acquired by their host too recently for codon optimization to have already taken place. Sequence-variation data such as those obtained here may be used to infer pathways of enzyme evolution (see Eulberg et al., 1998 ; Hill et al., 1999 ; Poelarends et al., 2000, and references cited therein). However, the observed differences with respect to the canonical sequence of strain DM4 appear to be at odds with the notion of a single evolutionary pathway connecting different DCM dehalogenases. For example, pairwise comparisons between DM13, GJ21 and LZ DCM dehalogenases show that DM13 and LZ sequences display the same residue at position 20, that the GJ21 and LZ sequences display the same residues at positions 27 and 277, and that the GJ21 and DM13 sequences display the same residues at positions 46 and 276, respectively (Table 2). In addition, the patterns of insertion sequences flanking the dcmA gene in different strains (Schmid-Appert et al., 1997) do not always correlate with differences in the corresponding dcmA gene. For example, the same dcmA sequence as that in strain DM4 was found in strains DM1 and DM5 (Table 1), which, however, display a different arrangement of insertion sequences. Furthermore, the pattern of insertion sequences associated with one copy of the dcmA gene in strain DM1 was also found in strain Hyphomicrobium sp. GJ21, but the latter strain features a dcmA gene with nucleotide differences at 15 positions relative to that of strain DM4 (Table 1). Thus, the succession of events that led to the observed patterns of sequence variation in strains capable of mineralizing DCM, both at the level of dcmA and at the level of the genes flanking it, remains elusive. To conclude, we note that the sequence and catalytic properties of DCM dehalogenases are strongly conserved among aerobic DCM-degrading methylotrophic bacteria. However, gene-sequence variations with more pronounced effects would be expected to be selected under conditions in which increased enzyme efficiency is an advantage. Thus, it will be interesting to investigate whether any of the sequence differences observed in the 618

present study are selected when gene evolution in the laboratory (see e.g. Myazaki & Arnold, 1999 ; Sutherland, 2000) is performed with the dcmA gene of strain DM4 as a starting point. ACKNOWLEDGEMENTS We thank the following students involved in the early stages of this work : Karin Wu$ thrich, Kurt Zoller, Hubert Traber, Helga Sorribas, Michael Stumpp and Philipp Krummenacher. We are also grateful to Dietmar Stax for advice on isolation of DNA from sludge. This work was supported by grant 5002037905 from the Biotechnology Priority Programme of the Swiss National Science Foundation.

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