JOURNAL OF BACTERIOLOGY, June 2008, p. 3817–3823 Vol. 190, No. 110021-9193/08/$08.00�0 doi:10.1128/JB.00180-08Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Characterization of a Novel Methanol Dehydrogenase in Representatives ofBurkholderiales: Implications for Environmental Detection of

Methylotrophy and Evidence for Convergent Evolution�

Marina G. Kalyuzhnaya,1 Krassimira R. Hristova,3 Mary E. Lidstrom,1,2 and Ludmila Chistoserdova2*Departments of Microbiology1 and Chemical Engineering,2 University of Washington, Seattle, Washington 98195, and

Department of Land, Air, and Water Resources, University of California, Davis, California 956163

Received 5 February 2008/Accepted 24 March 2008

Some members of Burkholderiales are able to grow on methanol but lack the genes (mxaFI) responsible forthe well-characterized two-subunit pyrroloquinoline quinone-dependent quinoprotein methanol dehydroge-nase that is widespread in methylotrophic Proteobacteria. Here, we characterized novel, mono-subunit enzymesresponsible for methanol oxidation in four strains, Methyloversatilis universalis FAM5, Methylibium petroleiphi-lum PM1, and unclassified Burkholderiales strains RZ18-153 and FAM1. The enzyme from M. universalis FAM5was partially purified and subjected to matrix-assisted laser desorption ionization–time of fight peptide massfingerprinting. The resulting peptide spectrum was used to identify a gene candidate in the genome of M.petroleiphilum PM1 (mdh2) predicted to encode a type I alcohol dehydrogenase related to the characterizedmethanol dehydrogenase large subunits but at less than 35% amino acid identity. Homologs of mdh2 wereamplified from M. universalis FAM5 and strains RZ18-153 and FAM1, and mutants lacking mdh2 weregenerated in three of the organisms. These mutants lost their ability to grow on methanol and ethanol, demon-strating that mdh2 is responsible for oxidation of both substrates. Our findings have implications for environmentaldetection of methylotrophy and indicate that this ability is widespread beyond populations possessing mxaF, thegene traditionally used as a genetic marker for environmental detection of methanol-oxidizing capability. Ourfindings also have implications for understanding the evolution of methanol oxidation, suggesting a convergencetoward the enzymatic function for methanol oxidation in MxaF and Mdh2-type proteins.

Methanol dehydrogenase (MDH) is a key enzyme in utili-zation of methane and methanol by methylotrophic proteobac-teria (1, 2). This is a pyrroloquinoline quinone (PQQ)-depen-dent quinoprotein that acts in the periplasm. Like otherquinoproteins, MDH is assayed in vitro in a dye-linked systemusing artificial electron acceptors such as phenazine methosul-phate and dichlorophenolindophenol. It is typically measuredat high pH (9–11) in the presence of ammonia as an essentialactivator (1–3). MDH oxidizes a wide range of primary alco-hols but has especially high affinity for methanol (Km value ofabout 20 �M). MDH and its prosthetic group were first de-scribed more than 40 years ago, making it one of the mostthoroughly studied quinoprotein dehydrogenases (9, 12, 32, 44,46). Gene clusters encoding the structural subunits of MDH(mxaFI), the specific electron acceptor cytochrome cL (mxaG),and a number of accessory proteins (mxaJRSACKLD) havebeen found well conserved in a variety of methylotroph ge-nomes (5, 6, 25, 42), and the gene encoding the large subunit,mxaF, has been used as a marker for methylotrophy in envi-ronmental studies (27, 28). However, a few methylotrophicisolates have been described that appear to lack mxa genes. Insome cases this is evidenced by the analysis of complete ge-nomes (10, 20) and in others by the inability to PCR amplifythe mxaF gene (18, 37) or detect the protein via Western blot

hybridization (31). Dye-linked quinoprotein ethanol dehydro-genases have been implicated in enabling methylotrophy instrains lacking mxaFI (33). However, the identity of the gene(s)involved has not been revealed.

Burkholderiales have recently emerged as an order withinBetaproteobacteria containing methylotrophic representatives.These so far belong to two new genera, Methylibium (20, 29)and Methyloversatilis (18). Phenotypically and metabolicallymethylotrophic Burkholderiales differ from the well-character-ized betaproteobacterial methylotrophs of the family Methylo-philaceae (1, 24) by being able to grow on a variety of multi-carbon compounds in addition to C1 compounds (methanol,methylamine, and formaldehyde but not methane) and em-ploying the serine cycle for formaldehyde assimilation insteadof the ribulose monophosphate cycle (18, 20). These organismshave also been shown to lack the traditional MDH encoded bymxaFI, suggesting that they use an alternative enzyme (18, 20).Knowledge about this enzyme and the respective genes wouldbe important for accurate estimates of numbers of potentiallymethylotrophic Burkholderiales in the environment. In addi-tion, revealing the identity of the genes encoding alternativeMDH enzymes would contribute to a better understanding ofthe evolution of methylotrophy as a metabolic capability. Inthis work, we explored properties of MDH enzymes in methylo-trophic Burkholderiales, identified the respective genes, and con-firmed their role in methylotrophy via mutation.

MATERIALS AND METHODS

Isolation and phylotyping of two novel methylotrophic Burkholderiales. StrainRZ18-153 was isolated from Lake Washington sediment from an enrichment

* Corresponding author. Mailing address: Department of ChemicalEngineering, University of Washington, Box 355014, Seattle, WA98195. Phone: (206) 616-1913. Fax: (206) 616-5721. E-mail: milachis@u.washington.edu.

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supplemented by 5 mM formaldehyde (made up of a 37% formaldehyde stocksolution stabilized with 10 to 15% methanol; Fisher Scientific), essentially aspreviously described for Methyloversatilis universalis FAM5 (18). Strain FAM1was isolated via a dilution-to-extinction technique essentially as previously de-scribed for M. universalis 500 (18) except that 1 mM formaldehyde made up byautoclaving paraformaldehyde (Sigma-Aldrich) was used as a substrate. Bothstrains were found to be able to grow on methanol as a single carbon and energysource (data not shown). 16S rRNA genes were PCR amplified and sequencedas described before (18), revealing that both strains belonged to Burkholderialesand were closely related to M. universalis FAM5 (94 and 96% similarity, respec-tively).

Strains and growth condition. M. universalis FAM5 (18), Methylibium petro-leiphilum PM1 (28) and strains RZ18-153 and FAM1 were grown in 0.3� Hyphomedium (15) supplemented with 2 ml/liter of filter-sterilized vitamin stock solu-tion containing the following (mg/ml): 0.25 vitamin B12, 0.05 thiamine, 0.025 folicacid, 0.075 ascorbic acid, 0.05 riboflavin, 0.05 niacin, and 0.05 vitamin B5. Meth-anol (60 mM), methylamine (70 mM), ethanol (40 mM), or succinate (90 mM)was used as a growth substrate. Nutrient agar or triple soy agar (BD) was usedfor matings. Methylobacterium extorquens AM1 and Xanthobacter autotrophicusH4-14 were grown in Hypho medium (15) in the presence of 125 mM methanol.Escherichia coli TOP10 (Invitrogen), JM109, and S17-1 were grown on Luria-Bertani medium (BD). Filter-sterilized antibiotics were added as follows: 100�g/ml ampicillin, 100 �g/ml kanamycin, and tetracycline at 0.5 �g/ml for themethylotrophic strains and 10 �g/ml for E. coli strains.

MDH assay. Methanol-grown cells (optical density at 600 nm of 0.7 to 0.9)were harvested by centrifugation at 4,500 � g using the Sorvall RC-5B centrifugeat 4°C for 25 min. Cells were resuspended in 50 mM potassium phosphate buffer(pH 7.5) and passed two times through a French pressure cell at 1.2 � 108 Pa.Centrifugation was performed at 21,000 � g for 30 min at 4°C to remove celldebris. MDH activity was measured using a modification of the method ofAnthony and Zatman (3) based on following the phenazine methosulfate-medi-ated reduction of DCPIP (2,6-dichlorophenol-indophenol). The modificationincluded the addition of CaCl2 (10 mM) to the reaction mixture for proteinstabilization/activation (41), increasing the molarity of the buffer system (0.3 MTris-HCl, pH 8.8), and decreasing the concentration of methanol to 2.5 mM. Thereaction was initiated by the addition of essential activator NH4Cl (45 mM).Assays were performed routinely at room temperature in plastic 1.5-ml cuvettes(Bio-Rad) in a total volume of 1 ml. DCPIP reduction was monitored spectro-photometrically at 600 nm. For the calculations, a ε600 absorbance coefficient of21.9 mM�1 cm�1 was used (4).

Partial enzyme purification and enzyme kinetics. Enzymes from M. universalisFAM5, M. petroleiphilum PM1, and strain RZ18-153 were partially purified asfollows. Crude cell extracts obtained as above were treated with (NH4)2SO4

(75% saturation; supplied as powder) for 30 min at 4°C with stirring, followed bycentrifugation at 4,500 � g for 10 min at 4°C. To the resultant supernatants,(NH4)2SO4 was then added to 85% saturation, and samples were treated asabove. The precipitated proteins were resuspended in 25 mM HEPES (pH 7.5),0.5 M NaCl, and 2 mM dithiothreitol buffer and desalted and concentrated in 25mM HEPES buffer using Amicon Ultra-4 centrifugal filter devices (Millipore).The following alcohols were tested as substrates: methanol, ethanol, 1-propanol,and 1-butanol. Km values were deduced from double reciprocal plots of the initialreaction rates versus concentrations of the alcohols.

Isoelectrofocusing and in-gel activity staining. Crude extracts were isoelectro-focused in a pH range of 3 to 9 using a PhastSystem instrument, as described bythe manufacturer (Pharmacia Biotech). The running conditions were pro-grammed as advised by the manufacturer. A total of 45 to 60 �g of protein wasloaded per well. Gels were neutralized in 100 mM Tris-HCl buffer, pH 7.5, for 10min at room temperature. In-gel activity staining was performed as described byChistoserdova and Lidstrom (7). Gels were incubated for 40 min at 35°C in thedark for activity bands to develop.

Protein purification and mass spectrometric analysis. Crude extracts of meth-anol-grown cells of M. universalis FAM5 and M. petroleiphilum PM1 (opticaldensity at 600 nm of 0.7 to 0.9; prepared as above) were subjected to preparativeprotein separation in 1% agarose gel prepared in 1� Tris-glycine buffer (23).In-gel activity staining was performed as above. Activity-positive bands wereexcised from gels and subjected to denaturing (12% sodium dodecyl sulfate)polyacrylamide gel electrophoresis as described previously (23). Proteins werevisualized by staining with Coomassie brilliant blue R250 (Amersham Bio-sciences). The major polypeptide band of M. universalis FAM5 with a molecularmass of approximately 65 kDa was excised from the gel and submitted to Al-phalyse (http://www.alphalyse.com/) for matrix-assisted laser desorption ioniza-tion–time of fight (MALDI-TOF) peptide mass fingerprinting after trypsin di-gestion. The Mascot (version 1.9.03) search program was used to match the

MALDI-TOF mass spectrum against the 11 PQQ-dependent dehydrogenasespredicted from the genome sequence of M. petroleiphilum PM1 (20). We verifiedthese predictions by carrying out BLAST analyses using known PQQ-dependentquinoprotein dehydrogenase sequences (MDH, type I and type II alcohol dehy-drogenase [ADH], glucose dehydrogenase, etc.) (see Fig. 3). A BLAST searchwith each query produced the same 11 protein candidates (Table 1).

DNA manipulations. DNA was isolated using a QIAamp DNA minikit (Qia-gen). Plasmid DNA was purified using a GeneJet plasmid miniprep kit (Fermen-tas). E. coli transformation, restriction enzyme digestion, and ligation reactionswere carried out as described by Sambrook et al. (36). PCR amplification reac-tions were performed using Taq polymerase (Qiagen) in accordance with themanufacturer’s instructions.

PCR amplification and primer design. Partial mxaF and xoxF fragments (0.56kb) were amplified using previously described primer sets (27, 38). Degenerateprimers for PCR amplification of nearly complete (1.6 kb) mdh2 genes (PQQDH-215F, 5�-CAGCGCTACAGCCCGCTCAAG; PQQDH-1805R, 5�-GTACTGCTCGCCGTCCTGCTCCC) were designed based on alignments of DNA sequencesencoding ADHs from M. petroleophilum PM1 (MpeA0476) (20), Pseudomonasbutanovora (41) (NCBI accession number AF326086), Azoarcus sp. BH72 (22)(NCBI accession number NC_008702.1, gene ID 4607717), and Pseudomonas aerugi-nosa (21) (NZ_AAKW01000014.1, locus tag Paer2_01001089). Vector NTI Ad-vance 10 AlignX software (Invitrogen) was used for alignment. Degenerate primersfor PCR amplification of nearly complete (1.3 kb) xoxF genes (XoxF-f, 5�-CGGCGTGCTGCGCGGCCACG; XoxF-r, 5�-GCCCAGCCGCCRATRCCCGAG) weredesigned in a similar fashion based on the alignment of homologous gene sequencesfrom M. petroleiphilum PM1 (MpeA3393), Methylobacillus flagellatus KT (Mfla2314)(6), Methylobacterium extorquens AM1 (NCBI accession number U72662) (7), andParacoccus denitrificans (NCBI accession number U34346) (33). Homologous prim-ers for PCR amplification of nearly complete (1.6 kb) mxaF from RZ18-530 (RZ18-236F, 5�-CCTGACCCAGATCAACCG, and mxaF-RZ18-1850R, 5�-CATCATGCCGCCACCCATC) were designed after aligning mxaF sequences of M. flagellatus(Mfla2044) (6) and Verminephrobacter eiseniae EF01-2 (NCBI accession CP00542).PCR amplification reactions were performed using Taq polymerase (Qiagen) inaccordance with the manufacturer’s instructions. The resulting PCR products werepurified, cloned into pCR2.1 (Invitrogen), and sequenced as previously described(18). After verification of the nucleotide sequences of the inserts, the respectiveplasmids were used as templates for amplification of the corresponding 5� and 3�gene fragments for subsequent cloning into the allelic exchange vector pCM184 (26)in order to create deletion/insertion mutant constructs, essentially as describedpreviously (26). The resulting plasmids were transformed into E. coli S17-1, andstrains harboring appropriate plasmids were used as donors in biparental matings.The kanamycin-resistant recombinants were selected on succinate plates andchecked for resistance to tetracycline. Tetracycline-sensitive recombinants were cho-sen as potential double-crossover recombinants. The identity of the double-crossover

TABLE 1. Sequence comparisons between putative quinoproteinsencoded in the genome of M. petroleiphilum PM1 andrepresentatives of MDHs, type I ADHs, and enzymes

of unknown function (XoxF)

Polypeptideidentifier

% Amino acid sequence identitya

M.methylotrophus

MxaF

P.butanovora

BDH

P.aeruginosa

ADH

P.denitrificans

XoxF

MpeA0341 33 34 37 34MpeA0363 32 33 35 33MpeA0473 34 50 54 33MpeA0476 32 80 69 34MpeA0877 33 36 37 35MpeA0905 30 32 34 30MpeA1591 30 35 38 36MpeA1594 24 22 22 23MpeA1595 31 33 33 32MpeA3393 49 34 35 66MpeA3660 32 30 32 31

a Sequences of MxaF from M. methylotrophus (46), butanol dehydrogenasefrom P. butanovora (40), ethanol dehydrogenase from P. aeruginosa (21), andXoxF of P. denitrificans (34) were used as queries against the genome of M.petroleiphilum PM1 (20). Matches producing identity scores above 60% arehighlighted in boldface. Sequence accession numbers are as shown in Fig. 3.

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mutants was further verified by diagnostic PCR tests using PCR primers specific tothe insertion sites.

Phylogenetic analysis. Amino acid sequences of complete or nearly completeproteins were aligned using the CLUSTAL W program (39). For phylogeneticanalyses, the PHYLIP package (12) was used. Maximum likelihood, distance,and parsimony methods were employed; 1,000 bootstrap analyses were per-formed. Tree-branching patterns were similar for the three analyses.

Nucleotide sequence accession numbers. The nucleotide sequences obtainedin this study have been deposited in the GenBank under the following accessionnumbers (in respective order): EU548062 and EU548068 for the mdh2 and xoxFgene fragments from M. universalis FAM5; EU548066, EU548065, andEU548067 for the mdh2, xoxF, and mxaF gene fragments from strain RZ18-153;and EU548063 for the mdh2 gene fragment from strain FAM1.

RESULTS

MDH activity in methylotrophic Burkholderiales. MDH ac-tivity was tested in four strains of methylotrophic Burkholderia-les. These included M. petroleiphilum PM1, which lacks in itsgenome the typical MDH gene cluster previously characterizedin model alpha-, beta-, and gammaproteobacterial methylo-trophs (20); M. universalis FAM5, which is negative for MDHactivity as measured in standard assay conditions and for thepresence of mxaF as judged by PCR amplification-based tests(18); and two other recently isolated representatives of Burk-

holderiales referred to here as strains RZ18-153 and FAM1.While closely related to M. universalis, these strains were pos-itive for PCR amplification of mxaF using standard PCR prim-ers (data not shown). Sequencing of the mxaF gene fragmentfrom strain RZ18-153 revealed close relatedness to the se-quence present in the genome of V. eiseniae EF01-2 (86%identity at the DNA level), another member of Burkholderiales(http://genome.jgi-psf.org/finished_microbes/verei/verei.home.html).

As we previously encountered difficulties measuring MDHactivity in M. universalis, we performed plus-minus qualitativetests on crude extracts from cells grown on methanol, ethanol,methylamine, and succinate by activity staining after isoelec-trofocusing in native polyacrylamide gels. Methylobacterium ex-torquens AM1 (5), and X. autotrophicus H4-14 (43), methanolutilizers possessing the classical MDH, were used as controls.We observed positive activity bands for all the strains grown onmethanol or ethanol. Activity bands were faint or absent inextracts of cells grown on methylamine or succinate (Fig. 1 anddata not shown), suggesting that the enzyme responsible foractivity staining was inducible by both methanol and ethanol.The position in gels of the activity bands corresponding to theBurkholderiales MDH enzymes was distinctly different from thepositions of the bands corresponding to MDH enzymes fromMethylobacterium extorquens and X. autotrophicus, suggesting asignificantly more basic pI for these “novel” enzymes.

We obtained further indication of the novel nature of Burk-holderiales MDH enzymes by testing for optimal MDH assayconditions. The enzymes in question were shown to be morelabile than MDH enzymes encoded by mxaFI (8), losing all oftheir activity in cells that were frozen and thawed. They re-quired a high-molarity buffer system (0.3 M Tris-HCl) formaximum activity (approximately fivefold higher than at 0.1 MTris-HCl) and lower methanol concentration than the standardprotocol (3) (see Materials and Methods), and they had aslightly more acidic pH optimum than known MDH enzymes(pH 8.5 versus pH 9 to 11). However, like MDH as well as typeI ADH enzymes, the novel enzymes required ammonia foractivation. Using the optimized assay, we measured MDH ac-tivity in cell extracts of all the Burkholderiales strains involvedin this study and obtained activities from 0.10 � 0.03 to 0.15 �0.05 �mol/min/mg of protein (from three biological replicates)(Table 2). Km values were determined for partially purifiedenzymes from M. universalis FAM5, M. petroleiphilum PM1,

FIG. 1. In-gel activity staining of MDH after isoelectrofocusing inpolyacrylamide gels (Pharmacia Biotech). A total of 45 to 60 mg ofprotein was loaded per lane. Running conditions were as recom-mended by the manufacturer (Pharmacia Biotech). Gels were neutral-ized in Tris-HCl buffer, pH 7.5, and stained as described previously (7)for 40 min at 35°C in the dark. Arrows show bands corresponding toMxaFI and Mdh2. Lane 1, M. extorquens AM1 grown on methanol;lane 2, X. autotrophicus H4-14 grown on methanol; lanes 3 to 5, M.petroleiphilum PM1 grown, respectively, on methanol and ethanol andgrown on succinate and then induced with methanol as describedpreviously (7); lane 6, mutant of M. petroleiphilum PM1 defective inxoxF grown on succinate and induced with methanol; lane 7, mutant ofM. petroleiphilum PM1 defective in mdh2 grown on succinate andinduced with methanol; lanes 8 to 10, M. universalis FAM5 grown,respectively, on methanol, methylamine, and succinate; lanes 11 and12, strains FAM1 and RZ18-153, respectively, grown on methanol.

TABLE 2. Reaction velocity and substrate affinity of enzymes partially purified in this work compared to typical methanol andethanol dehydrogenases

Substrate

Kinetics of the indicated straina

M. universalis FAM5 Strain RZ18-153 M. petroleiphilum PM1 M. methylotrophus W3A1 P. aeruginosa

Km(mM) % of Vmax Km (mM) % of Vmax

Km(mM) % of Vmax

Km(mM) % of Vmax

Km(mM) % of Vmax

Methanol 0.01 100 (0.59) 0.02 100 (0.43) 0.29 100 (0.23) 0.02 100 (6.45) 94 100 (5.63)Ethanol 0.05 93 0.04 93 0.10 109 0.03 76 0.01 1601-Propanol 0.50 22 0.19 47 0.09 111 0.28 94 0.02 2221-Butanol 0.50 21 0.20 47 0.10 100 0.29 89 ND 104

a Data for a classic MDH (from M. methylotrophus �12�) and a classic ethanol dehydrogenase (from P. aeruginosa �13�) were used for comparisons. Purity of theenzymes investigated in this work was estimated based on scanning the images of gels after sodium dodecyl sulfate-polyacrylamide gel electrophoresis using GelianceGeneTools software (Perkin-Elmer). Enzyme purity was estimated at 32, 38, and 46% for M. universalis FAM5, strain RZ18-153, and M. petroleiphilum PM1,respectively. Values in parentheses represent Vmax in U/mg of protein. ND, not determined.

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and strain RZ18-153l and compared to the parameters previ-ously observed for a typical mxaFI-encoded MDH (fromMethylophilus methylotrophus) and a typical ethanol dehydro-genase (from P. aeruginosa) (Table 2). Strain FAM1, due to itsclose relatedness to strain RZ18-153, was excluded from theseanalyses. The enzymes from M. universalis FAM5 and strainRZ18-153 revealed kinetic properties very similar to eachother and to those of the classic MDH from M. methylotrophus,having the highest affinity toward methanol and exhibiting thehighest reaction velocity with methanol. The enzyme from M.petroleiphilum PM1 revealed a much lower affinity for metha-nol than the enzymes from M. universalis FAM5 and strainRZ18-153, but this affinity was more than two orders of mag-nitude higher than the affinity of a typical ethanol dehydroge-nase (Table 2).

Protein identification. To obtain further insights into the iden-tity of the enzyme responsible for MDH activity in Burkholderialesstrains, we performed preparative native agarose gel electro-phoresis using crude cell extracts of M. universalis FAM5 and M.petroleiphilum PM1. The activity bands were excised from gelsand subjected to denaturing polyacrylamide gel electrophoresis.In each case, one major polypeptide band was observed with amolecular mass of approximately 65 kDa, and no light band cor-responding to a typical MxaI was detected (data not shown). TheM. universalis FAM5 band was excised from the gel and submittedto Alphalyse for MALDI-TOF peptide mass fingerprinting. Theresulting MALDI-TOF spectra were screened against an in-house protein database created of 11 predicted quinoprotein de-hydrogenases translated from the genome of M. petroleiphilumPM1 (Table 1). Of the 11 polypeptides tested, only one waspositively matched with the MALDI-TOF spectrum:MpeA0476 (YP_001019673; two polypeptide matches; 3%coverage). Based on its primary sequence, this polypeptideis closely related to type I periplasmic quinoprotein ADHs

(up to 80% amino acid identity), a number of which havebeen purified and characterized (13, 40). The same polypep-tide has been previously implicated in having a role as amajor ethanol dehydrogenase in this organism, based on itsup-regulation during growth on ethanol as judged by tran-scriptional microarray analysis (17). Type I quinoproteinADHs typically exhibit broad substrate specificity towardvarious primary, secondary, and aromatic alcohols but re-veal very poor affinity for methanol (13, 40). MpeA0476shared less than 35% identity with MxaF polypeptides ofknown methylotrophs. Alignment of the sequence of MpeA0476 with the sequences of MxaF and a typical type I quino-protein ADH is shown in Fig. 2.

The predicted polypeptide most closely related to MxaF wasMpeA3393 (approximately 50% amino acid identity). How-ever, this polypeptide was even more closely related to XoxFpolypeptides found in a variety of proteobacterial species in-cluding both methylotrophs and nonmethylotrophs (6, 7, 16,38, 45). xoxF genes have been previously mutated in twomethylotrophs, M. extorquens and Paracoccus denitrificans,without resulting in any visible effect on methanol-oxidizingcapability (7, 16). However, more recently a function was sug-gested for XoxF in photosynthesis-dependent methanol utili-zation by Rhodobacter sphaeroides based on mutant pheno-types (45).

To test whether predicted proteins similar to MpeA0476were encoded in other Burkholderiales methylotrophs, we de-signed primers for PCR amplification, as described in the Ma-terials and Methods section. Gene fragments were amplifiedfrom M. universalis FAM5 and strains RZ18-153 and FAM1,followed by sequencing. These gene fragments were highlysimilar to each other (90 to 96% identical at the DNA level)and to the gene encoding MpeA0476 (84% identical). Phylo-genetic analyses demonstrated that predicted polypeptide se-

FIG. 2. Alignment of the sequence of Mdh2 from M. petroleiphilum with the sequences of MxaF from M. methylotrophus and type I ADH (Edh)from P. aeruginosa. Amino acid residues conserved in all three proteins are shaded in black, and residues conserved in two out of three proteinsare shaded in gray. Accession numbers are as shown in Fig. 3.

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quences translated from these genes clustered together withtype I ADH sequences and formed a branch clearly separatedfrom the branch containing the MxaF predicted polypeptides(Fig. 3).

Mutant generation and phenotypic characterization. In or-der to confirm the involvement of MpeA0476-like genes (ten-tatively named mdh2) in methanol oxidation in methylotrophicBurkholderiales, these genes were inactivated in M. petroleiphi-lum PM1, M. universalis FAM5, and strain RZ18-153 using apreviously described insertion/deletion mutagenesis technique(26). We also constructed mutants of M. petroleiphilum PM1and M. universalis FAM5 lacking functional xoxF homologs.While the only MDH activity band in strain RZ18-153 corre-sponded to Mdh2, the organism was positive for the presenceof an mxaF gene homolog; so we mutated this gene as well andinvestigated the phenotypes of all the mutants. All of the mu-tants lacking functional mdh2 have lost the ability to grow onboth methanol and ethanol, and they were also negative forMDH activity (Fig. 1 and data not shown). However, the mu-tation of the mxaF gene homolog had no effect on methylotro-phic growth of the strain RZ18-153. Mutants of M. universalisFAM1 and M. petroleiphilum PM1 with lesions in xoxF ho-mologs as well as the mutant of strain RZ18-153 with a lesionin the mxaF gene homolog retained wild-type growth charac-teristics and remained positive for in-gel MDH activity stain-ing. These results strongly point toward Mdh2 as the majormethanol oxidation enzyme in methylotrophic Burkholderiales.

DISCUSSION

Until recently, the ability to oxidize methanol by gram-neg-ative bacteria has been attributed almost exclusively to theMDH enzyme encoded by mxaFI (14, 24). The mxa genes arewell conserved among different classes of proteobacteria(alpha, beta, and gamma) in terms of both gene clustering andprotein sequence identity (5, 6, 25, 42), suggesting a monophy-letic origin for the mxa (mox)-encoded methanol oxidationmachinery. Based on its conservation, mxaF has served as agenetic marker for environmental detection of methylotrophy(27, 28). However, recent studies involving discovery and anal-ysis of novel methylotrophs, both within and outside of Pro-teobacteria, demonstrated the lack of mxaFI gene homologs(10, 18, 31, 37), implicating the existence of alternative en-zymes responsible for methanol oxidation. In this work, weinvestigated the nature of methanol-oxidizing enzymes inmethylotrophs belonging to the order of Burkholderiales.Methylotrophs of this group have already been shown to relyon noncanonical biochemical schemes for methylotrophy asthey are the first examples of betaproteobacterial methylo-trophs to employ the serine cycle instead of the traditionalribulose monophosphate cycle for formaldehyde assimilation(18, 20). Based on the experiments presented here, these or-ganisms also employ a noncanonical MDH for methanol oxi-dation, named here Mdh2. In terms of the primary sequence aswell as subunit composition, Mdh2 enzymes are more similarto type I ADHs that typically exhibit low affinity for metha-

FIG. 3. Consensus parsimony phylogenetic tree of quinoprotein ADHs. Numbers correspond to bootstrap values for 1,000 analyses (parsi-mony). GenBank protein accession numbers are shown. Proteins investigated in this study are shown in bold. GDH, glucose dehydrogenase.

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nol than to the mxaFI-encoded MDH enzymes (13, 40).Akin to traditional MDH enzymes, Mdh2 appears to beresponsible for the oxidation of both methanol and ethanol.While a function in methanol oxidation has been recentlysuggested for the ubiquitous Xox system (45), analyses pre-sented here demonstrate that in Burkholderiales, as in thepreviously tested nonphotosynthetic alphaproteobacterialmethylotrophs (7, 16), a function for xoxF in methylotrophycould not be demonstrated.

The discovery of the novel MDH has major implications for (i)understanding the evolution of enzymes and pathways that enablemethylotrophy and (ii) environmental detection of methanol-ox-idizing capabilities. Previous analyses of methylotrophy pathwaysin betaproteobacterial methylotrophs suggested that representa-tives of Methylophilales and Burkholderiales must have acquiredgenes and pathways enabling methylotrophy independently,based on pathway distribution in these organisms and phylogenyof the proteins involved in the shared pathways (6, 19). This workdemonstrated that enzymes enabling methanol oxidation are alsodifferent in Methylophilales and Burkholderiales. The novel MDHenzymes characterized here are closely related to ADHs typicallyexhibiting very low affinity for methanol. The most likely evolu-tionary scenario to explain the emergence of Mdh2 would be asingle or a few point mutations in the catalytic center of a type IADH, resulting in increased affinity for methanol rather thandivergence from MxaF under selective pressure. The existence ofsuch selective pressure is also unlikely, as Methylophilales andBurkholderiales methylotrophs coinhabit the same ecologicalniches (18, 30). Thus, methanol affinity exhibited by MxaFI andMdh2 appears to be a result of convergent evolution.

The discovery of the novel MDH enabling methylotrophy inBurkholderiales has major implications for detection of themethanol-oxidizing capability in natural microbial populations.To date, the methylotrophic Burkholderiales have been over-looked by molecular approaches as the mxaF-directed probesand primers would not detect them. However, it appears thatin some environments, Mdh2 may be the dominant methanoloxidation system. For example, a number of mdh2-like se-quences are present in the metagenome resulting from globalocean sampling (35) while no sequences closely related tomxaF sequences are present. Conversely, analysis of mxaFgene fragments amplified from some environments may over-estimate the methylotrophic capability as some of the mxaFgenes, as we demonstrated here by the examples of strainsRZ18-153 and FAM1, do not appear to code for functionalMDH, likely due to lack of expression. Thus, detection ofmxaF in environmental samples can no longer be consideredsufficient to imply that methylotrophic growth occurs.

In conclusion, this study expands our understanding of thepathways enabling methylotrophy in proteobacteria by identi-fying a novel MDH, named Mdh2, which may be at least aswidespread in natural bacterial communities as the well-stud-ied mxaFI-encoded MDH, and suggests that the methanol-oxidizing capabilities of Burkholderiales and Methylophilalesevolved independently. Our findings should have a major im-pact on environmental detection of methylotrophs, highlight-ing the role of Burkholderiales in global cycling of single-carboncompounds.

ACKNOWLEDGMENTS

We are grateful to A. Munk (Alphalyse) for help with protein iden-tification.

This work was funded by the National Science Foundation as part ofthe Microbial Observatories Program (MCB-0131957). Partial supportfor K.R.H. was provided by a grant (5 P42 ES004699) from the Na-tional Institute of Environmental Health Sciences, NIH.

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