Duplicate Copies of Genes Encoding Methanesulfonate ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 2002, p. 289–296 0099-2240/02/$04.00⫹0 DOI: 10.1128/AEM.68.1.289–296.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Vol. 68, No. 1

Duplicate Copies of Genes Encoding Methanesulfonate Monooxygenase in Marinosulfonomonas methylotropha Strain TR3 and Detection of Methanesulfonate Utilizers in the Environment Nardia J. Baxter,1 Julie Scanlan,1 Paolo De Marco,2 Ann P. Wood,3 and J. Colin Murrell1* Department of Biological Sciences, University of Warwick, Coventry CV4 7AL,1 and Division of Life Sciences, King’s College London, London SE1 9NN,3 United Kingdom, and IBMC-Universidade do Porto R. Campo Alegre, 823 4150-180 Porto, Portugal2 Received 1 May 2001/Accepted 2 October 2001

Marinosulfonomonas methylotropha strain TR3 is a marine methylotroph that uses methanesulfonic acid (MSA) as a sole carbon and energy source. The genes from M. methylotropha strain TR3 encoding methanesulfonate monooxygenase, the enzyme responsible for the initial oxidation of MSA to formaldehyde and sulfite, were cloned and sequenced. They were located on two gene clusters on the chromosome of this bacterium. A 5.0-kbp HindIII fragment contained msmA, msmB, and msmC, encoding the large and small subunits of the hydroxylase component and the ferredoxin component, respectively, of the methanesulfonate monooxygenase, while a 6.5-kbp HindIII fragment contained duplicate copies of msmA and msmB, as well as msmD, encoding the reductase component of methanesulfonate. Both sets of msmA and msmB genes were virtually identical, and the derived msmA and msmB sequences of M. methylotropha strain TR3, compared with the corresponding hydroxylase from the terrestrial MSA utilizer Methylosulfonomonas methylovora strain M2 were found to be 82 and 69% identical. The msmA gene was investigated as a functional gene probe for detection of MSA-utilizing bacteria. PCR primers spanning a region of msmA which encoded a unique Rieske [2Fe-2S] binding region were designed. These primers were used to amplify the corresponding msmA genes from newly isolated Hyphomicrobium, Methylobacterium, and Pedomicrobium species that utilized MSA, from MSA enrichment cultures, and from DNA samples extracted directly from the environment. The high degree of identity of these msmA gene fragments, compared to msmA sequences from extant MSA utilizers, indicated the effectiveness of these PCR primers in molecular microbial ecology. cytoplasmic enzyme, methanesulfonate monooxygenase, which is responsible for the initial oxidation of methanesulfonate to formaldehyde and sulfite (19). This enzyme has been purified from Methylosulfonomonas methylovora strain M2 and consists of four components, all of which are required for monooxygenase activity (15). The hydroxylase component consists of two subunits, MsmA and MsmB, of 48 and 20 kDa, probably arranged in an ␣3␤3 conformation in native form (38). Spectral analysis showed the presence of a Rieske [2Fe-2S] center (38) and probably a mononuclear iron site. The electron transfer component is a ferredoxin (MsmC) of 13.7 kDa which contains a Rieske [2Fe-2S] center (16). The purified holoenzyme probably comprises two identical subunits (7, 16). The reductase component, which is specific for NADH, has to date only been partially purified and is unstable (38). It is a single polypeptide of ca. 38 kDa. The genes encoding methanesulfonate monooxygenase, designated msmABCD, are clustered on the Methylosulfonomonas chromosome and probably constitute an operon for the coordinate expression of methanesulfonate monooxygenase. These genes have been fully sequenced. msmA and msmB encode the ␣- and ␤-subunits of the hydroxylase which show significant identity with other mono- and dioxygenases (7). msmA, however, encodes an unusually long 26-amino-acid sequence CXHX26-CXXH between the two highly conserved cysteine-histidine residues of the Rieske motif. All other oxygenases

Dimethyl sulfide is a major source of organic sulfur, arising principally from the degradation of dimethylsulfoniopropionate, an osmolyte found in marine algae. In the atmosphere it is oxidized by OH and NO3 radicals to sulfur dioxide and methanesulfonic acid (MSA). MSA is a very stable strong acid and a key intermediate of the biogeochemical sulfur cycle that is deposited on the Earth in rain and snow and by dry deposition (20, 21, 28). Although MSA is a chemically stable compound, it can be used as a sulfur source by some bacteria (reviewed in reference 6) and also as a sole carbon and energy source by specialized aerobic methylotrophic bacteria (reviewed in reference 21). The first soil bacterium to be isolated on methanesulfonate as the sole growth substrate (1) belonged to a novel genus in the ␣-subdivision of Proteobacteria, Methylosulfonomonas methylovora strain M2 (17). Subsequently, novel marine isolates were obtained comprising another phylogenetically distinct genus within the ␣-Proteobacteria, Marinosulfonomonas methylotropha strain TR3 (44). More recently, Hyphomicrobium and Methylobacterium species which grow on methanesulfonate have also been isolated (8; this study). Methylosulfonomonas methylovora strain M2 and M. methylotropha strain TR3 contain an inducible, * Corresponding author. Mailing address: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom. Phone: 44-24-76-523553. Fax: 44-24-76-523568. E-mail: cmurrell @bio.warwick.ac.uk. 289

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examined to date contain a Rieske motif of CXH-X16-18CXXH (26), and this region in MsmA may therefore be unique to methanesulfonate utilizers containing this methanesulfonate monooxygenase. msmC is probably most closely related to the ferredoxin gene from toluene dioxygenases (16). msmD, encoding the reductase, has significant identities with reductases that use FAD and NADH (7). We report here on the cloning and sequencing of the corresponding msm gene cluster from the marine methanesulfonate utilizer M. methylotropha strain TR3 and the development of functional gene probes centered around the unique Rieske center encoding region of msmA which can be used to detect the presence of methanesulfonate-utilizing bacteria in the environment. MATERIALS AND METHODS Enrichment and isolation methods. Enrichments for MSA-utilizing bacteria were performed in MinE medium (33) containing 15 mM methanesulfonate (sodium salt) with the addition of sodium chloride 2% (wt/vol) for marine enrichments. Sterile phosphate buffer (pH 6.8; 1.2 g of K2HPO4 and 0.624 g of KH2PO4 per liter) and methanesulfonate were added after autoclaving at 121°C for 15 min. Soil from a field at Warwick University was mixed with sterile distilled water, and a 1-ml aliquot was added to 50 ml of MinE. A 1-ml aliquot of seawater from the Bristol Channel off the south coast of Wales was added to 50 ml of MinE containing NaCl. All enrichments were incubated at 30°C for 7 days, after which a sample was streaked onto MinE plates containing 1.5% (wt/vol) agar and 15 mM MSA (plus NaCl for marine enrichments). A 1-ml aliquot of the enrichment was also transferred to fresh liquid medium and then reincubated. The remainder of the enrichment was centrifuged and stored as a frozen pellet at ⫺20°C. Individual colonies were selected and purified on MinE plates containing 15 mM MSA. When pure, the cultures were streaked onto MinE plates with or without 15 mM MSA. Isolates that were able to grow in the absence of MSA were discarded. Colonies that exhibited MSA-dependent growth were kept for further analysis. Hyphomicrobium sp. strain P2 was isolated and cultured as described by De Marco et al. (8). Strain APW2, isolated from used compost (which had grown tomato plants), was grown at 15°C in the standard basal salt medium as described by Padden et al. (34), containing 15 mM MSA. Growth of M. methylotropha strain TR3 and other isolates. M. methylotropha strain TR3 was grown on 15 mM MSA in marine mineral salt medium as described by Thompson et al. (44). Strain APW2 was cultivated in the isolation medium for this organism at room temperature (22 to 23°C). Hyphomicrobium, Pedomicrobium, and Methylobacterium strains were cultivated on MinE agar containing 15 mM MSA at 30°C. Growth of MSA utilizers on other carbon sources. Cells were taken from MinE agar plates and suspended in liquid MinE without MSA. The cell suspensions were added to 5 ml of MinE containing either 15 mM MSA, 15 mM ethanesulfonate, 0.1% (vol/vol) methanol, 0.1% (vol/vol) formate, or 0.1% (vol/ vol) methylamine as the carbon sources. The cells suspensions were incubated at 30°C then examined for growth after 1 and 2 weeks by measurements of the optical density at 540 nm. DNA extraction. Total genomic DNA was extracted from M. methylotropha strain TR3 according to the method of Oakley and Murrell (32). Small-scale plasmid preparations of recombinant clones from Escherichia coli were obtained by alkaline lysis, and large-scale preparations of plasmid DNA were purified by using CsCl gradients as described by Sambrook et al. (39). DNA was extracted from pure cultures and enrichment cultures of MSA utilizers by using the method of Marmur (25). DNA, previously isolated from a Danish forest soil, which was used for the retrieval by PCR of msmA sequences directly from the environment, was kindly donated by Ian McDonald (University of Warwick). Cloning of the msm gene cluster. Restriction endonuclease digests of M. methylotropha strain TR3 DNA were carried out by using endonucleases supplied by Gibco-BRL according to the manufacturer’s recommended procedures. DNA was visualized on 1% (wt/vol) agarose gels and Southern transferred onto Hybond-N nylon membranes (Amersham UK) as described by Sambrook et al. (39). Chromosomal DNA fragments containing the msm genes were identified by probing Southern blots of DNA from M. methylotropha strain TR3 with the 2-kbp SstI DNA fragment from Methylosulfonomonas methylovora strain M2 containing msmABC genes, previously characterized by De Marco et al. (7). This probe was

APPL. ENVIRON. MICROBIOL. labeled by using the dioxigenin-11-UTP random primer labeling kit (DIG; Boehringer Mannheim). Membranes were prehybridized and hybridized in a Hybaid oven at 50°C in standard buffer (5⫻ SSC [1⫻ SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 0.1% [wt/vol] N-lauroylsarcosine, 0.02% [wt/vol] sodium dodecyl sulfate, 1% blocking reagent) according to the manufacturer’s instructions. Final washes were routinely performed with 2⫻ SSC at 50°C unless stated otherwise. Blots were developed by using the chemiluminescent substrate CSPD or CDP-Star (Roche). The genomic probing of Southern blots of M. methylotropha strain TR3 with msmA from Methylosulfonomonas methylovora strain M2 identified two HindIII DNA fragments of 5.0 and 6.5 kbp which were target fragments for cloning. HindIII DNA fragments of 4.3 to 7 kbp were excised from agarose gels and purified by using Geneclean II (BIO 101). Plasmid vector DNA (pUC19) was cut to completion with HindIII and dephosphorylated with calf intestinal phosphatase (Boehringer Mannheim) according to the manufacturer’s recommended method. HindIII fragments of M. methylotropha strain TR3 chromosomal DNA were ligated to dephosphorylated HindIII cut pUC19 vector by using T4 ligase (Gibco-BRL) and transformed into E. coli strain MC1061 (4) by standard methods (39). Recombinants were selected on Luria-Bertani agar plates containing 50 ␮g of ampicillin/ml. A master plate of ca. 200 recombinants of the M. methylotropha strain TR3 HindIII library were picked, and mini-plasmid preparations were performed from pools of 10 colonies. The plasmid pools were restricted with HindIII, electrophoresed in agarose gels, and then blotted by Southern transfer onto nylon membrane (Hybond-N; Amersham). Recombinants containing msm genes were identified by probing them with the 2-kbp SstI fragment containing the msm genes from Methylosulfonomonas methylovora strain M2, as described earlier (7). DNA sequencing and sequence analysis. CsCl-purified plasmid DNA was used as a sequencing template. Synthetic custom-made oligonucleotides (Gibco-BRL) were used to sequence msmABCD genes on plasmids pJS1 and pJS2 by using a Taq dye deoxy terminator cycle sequencing kit and a Model 373A DNA sequencing system gel apparatus (Applied Biosystems). Sequence analysis was performed by using the Genetics Computer Group Wisconsin Package, version 8. BLAST 2.0 homology searches (12) were performed by the internet facility at National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov /BLAST). Since the 3⬘ region of msmD was missing from the 6.5-kb fragment, primers were designed in order to PCR amplify and then sequence this region from the chromosome of M. methylotropha strain TR3. PCR of the msmD gene was performed with primers P4rvM2red (5⬘-ATAAGAATGCGGCCGCTCAGCCG AACITGTCGTAGCGTAT-3⬘) and P4fwM2red (5⬘-CCGGAATTCATGACTT CTTTAGCAAGAGCTGACG-3⬘) with an annealing temperature of 55°C, and the 50-␮l reaction mixture contained 1 nM forward and reverse primers, 50 mM KCl, 20 mM Tris-HCl (pH 8.4), 5 mM MgCl2, 200 ␮M concentrations of each deoxynucleoside triphosphate, 0.05% W-1 (Gibco-BRL), 4% formamide, 250 ng of M. methylotropha strain TR3 DNA, and 1.25 U of Taq polymerase (GibcoBRL). The fragment was amplified as follows: 5 min of denaturation, followed by 35 cycles of 89°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min, with a final extension period of 7 min. Identification of MSA utilizers. After Gram staining and morphological examination of newly isolated MSA utilizers, the 16S rRNA gene from each isolate was PCR amplified with the kingdom-specific primers f27 and r1492 (23). The PCR product was cloned into the TOPO TA Cloning kit (Invitrogen), analyzed by restriction fragment length polymorphism (RFLP) with EcoRI, and then sequenced as previously described. The resulting sequence was used to identify isolates by using the internet facility at the NCBI (http://www.ncbi.nlm.nih.gov /BLAST). PCR of the msmA genes from pure cultures, enrichments, and the environment. Primers designed to amplify the region of the msmA gene that encoded the unique Rieske [2Fe-2S] binding sites were based on the msmA sequences of Methylosulfonomonas methylovora strain M2 and M. methylotropha strain TR3. The forward primer ForA (5⬘-TGAATGGGTTGATAGCCG-3⬘) and the reverse primer B1rev2 (5⬘-CCACTGGTTCGGCGGCAGATG-3⬘) were used to amplify a 783-bp fragment of msmA from pure isolates and enrichment DNA from this investigation and Rold Forest (Denmark) soil DNA (kindly donated by Ian McDonald, Warwick University). The 50-␮l PCR mixture contained the following: 1⫻ PCR buffer, 200 ␮M concentrations of each deoxynucleoside triphosphate, 2.5 mM MgCl2, 10 nM forward and reverse primers, 1.25 U of Taq polymerase, and 250 ng of DNA or 5 ␮l of boiled cell lysate. The PCR program was as follows: denaturation for 5 min at 94°C, followed by 30 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 1.5 min, with a final extension period of 15

msm GENES IN MARINOSULFONOMONAS METHYLOTROPHA TR3

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TABLE 1. Comparison between the MSA monooxygenase gene sequences and predicted protein sequences from Methylsulfonomonas methylovora strain M2 and M. methylotropha strain TR3. Identity or similarity

Identity (gene) Identity (protein) Similarity (protein)

FIG. 1. Restriction maps of msm gene clusters from M. methylotropha strain TR3 clones pJS1 and pJS2. No ORFs were located upstream (5⬘) of msmA on pJS2.

min. The PCR products were either cloned into the TOPO TA Cloning kit (Invitrogen) and then analyzed by RFLP with EcoRI before sequencing or were sequenced directly. All sequencing was conducted as described previously. Accession numbers of the msm gene cluster from M. methylotropha strain TR3. The two gene clusters were submitted to GenBank. The accession number for the gene cluster from pJS1 is AF354805 and that for pJS2 is AF360864.

RESULTS AND DISCUSSION The M. methylotropha strain TR3 msm gene clusters. Probing of the M. methylotropha strain TR3 chromosome with msmABC from Methylosulfonomonas methylovora strain M2 indicated that there were two copies of the methanesulfonate monooxygenase genes. Two HindIII fragments of 5.0 and 6.5 kbp which hybridized with the probe were cloned as plasmids pJS1 and pJS2, respectively, and then sequenced (both strands). Within the genome of M. methylotropha strain TR3, two copies of msmA and msmB but only one copy of msmC and msmD were found. Contained on pJS1 were five open reading frames (ORFs), identified by BLAST analysis as msmA, msmB, and msmC, and two unidentified ORFs (Fig. 1). There were only three ORFs on pJS2: msmA, msmB, and msmD (Fig. 1). Neither of the putative transport genes, msmE or msmF, which were located upstream (5⬘) from the methanesulfonate monooxygenase gene cluster in Methylosulfonomonas methylovora strain M2 (7), was found in association with msm genes in M. methylotropha strain TR3. In each of the cloned clusters, all ORFs were transcribed in the same direction, and a bias toward guanine and cytosine in the third codon position was observed.

% Identity or similarity of gene: msmA

msmB

msmC

msmD

82 82 89

69 64 82

65 59.5 77

67 60 74.5

msmA from M. methylotropha strain TR3. msmA encodes the large subunit of the hydroxylase component of MSA monooxygenase (7). The nucleotide sequence of the two 1,248-bp copies of msmA in M. methylotropha strain TR3 were identical. msmA encoded a 416-amino-acid polypeptide with a theoretical pI of 5.83 and molecular mass of 48,373 Da. The molecular mass was comparable to that of MsmA from Methylosulfonomonas methylovora strain M2, which is 48,145 Da (38). A comparison of the msmA genes from M. methylotropha strain TR3 and Methylosulfonomonas methylovora strain M2 is shown in Table 1. The percentage identity between the two DNA sequences was 82%, and at the amino acid level the sequences were 82% identical (89% similar; Table 1). As observed with Methylosulfonomonas methylovora strain M2 (7), M. methylotropha strain TR3 contained the unique Rieske [2Fe-2S] binding motif with a 26-amino-acid spacer (see below). MsmA had moderate sequence similarity to the large subunits of toluene 2,3-dioxygenase (47), the benzene 1,2-dioxygenase from Pseudomonas putida (18), and the biphenyl dioxygenase from Burkholderia cepacia (9) (Table 2). msmB from M. methylotropha strain TR3. msmB encoded the small subunit of the hydroxylase component of methanesulfonate monooxygenase (7). There was only one base difference between the two copies of the 546-bp msmB, which had no effect on the predicted protein sequence. msmB encoded a polypeptide of 182 amino acids with a theoretical pI of 5.96 and a molecular mass of 20,422 Da. The predicted molecular mass was very similar to the 20,479-Da MsmB from Methylosulfonomonas methylovora strain M2 (38). msmB from M. methylotropha strain TR3 had 69% identity to msmB from Methylosulfonomonas methylovora strain M2 (64% identity and 82% similarity at the amino acid level). Sequence analysis of MsmB revealed significant similarity with XylY of toluate 1,2dioxygenase from P. putida (13) and BenB of benzoate 1,2dioxygenase from Acinetobacter calcoaceticus (29) as shown in Table 2. msmC from M. methylotropha strain TR3. msmC encodes the ferredoxin component of the methanesulfonate monooxygenase (7). The only copy of the 372-bp msmC gene from M. methylotropha strain TR3 was located on pJS1. It encoded a 124-amino-acid polypeptide with a theoretical pI of 4.0 and a molecular mass of 14,126 Da. msmC from M. methylotropha strain TR3 had 65% identity at the nucleotide level to msmC from Methylosulfonomonas methylovora strain M2 and 60% identity (77% similarity) at the amino acid level (Table 1). TmoC of toluene-4-monooxygenase from Pseudomonas mendocina (46) and Bpa3 of biphenyl dioxygenase from Pseudomonas sp. KKS102 (10), which encode the ferredoxin compo-

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TABLE 2. Comparison of the amino acid sequences of the MSA monooxygenase components (MsmABCD) and the two unknown ORFs (ORFX and ORFY) from M. methylotropha strain TR3 with sequences in the SwissProt Database Component or ORF

Polypeptide

% Amino acid Identity

Similarity

Organism

Accession no.

MsmA Tod1 BznA BphA

Toluene 2,3-dioxygenase Benzene 1,2-dioxygenase Biphenyl dioxygenase

35 35 31

55 55 48

P. putida P. putida B. cepacia

P13450 P08084 P37333

MsmB XylY BenB

Toluate 1,2-dioxygenase Benzoate 1,2-dioxygenase

40 25

62 41

P. putida A. calcoaceticus

P23100 P07770

MsmC TmoC Bpa3

Toluene-4-monooxygenase Biphenyl dioxygenase

29 25

49 41

P. mendocina Pseudomonas sp. strain KKS102

Q00458 Q52440

MsmD DmpP NdoR XylZ

Phenol hydroxylase Naphthalene 1,2-dioxygenase Toluate 1,2-dioxygenase

26 26 26

42 42 42

Pseudomonas sp. strain CF600 P. putida P. putida

P19734 Q52126 P23101

OrfX Cyc6 Cyc6

Cytochrome c6 Cytochrome c6

33 32

44 47

E. gracilis M. aeruginosa

P00119 P00112

OrfY CynR YnfL OxyR

Transcriptional activator Hypothetical transcriptional regulator Inducible activator gene

23 25 22

39 31 38

E. coli E. coli P. chrysanthemi

P27111 P77559 Q9X725

nent of these dioxygenases, had low but significant identity to MsmC from M. methylotropha strain TR3 (Table 2). msmD from M. methylotropha strain TR3. msmD encodes the reductase component of the methanesulfonate monooxygenase (7). Situated downstream (3⬘) from msmA and msmB on pJS2 was msmD (1,095 bp), encoding a 365-amino-acid polypeptide with a theoretical pI of 5.11 and a molecular mass of 40,106 Da. Within the sequence of MsmD a putative plant type [2Fe-2S] binding motif and an oxidoreductase FAD/NAD binding domain located toward the C-terminal end of the polypeptide (Fig. 2) were found. This same structure had also been observed in MsmD of Methylosulfonomonas methylovora strain M2 (7). The sequence of the msmD gene from M. methylotropha was 67% identical to that of Methylosulfonomonas methylovora strain M2 (60% identical and 74.5% similar at the derived amino acid level) (Table 1). The predicted amino acid sequence of MsmD shared similarities of 42% with DmpP of phenol hydroxylase from Pseudomonas sp. strain CF600 (30, 36), NdoR from naphthalene 1,2-dioxygenase from P. putida (41), and XylZ of toluate 1,2-dioxygenase from P. putida (13) (Table 2). ORFX and ORFY from M. methylotropha strain TR3. Upstream (5⬘) from msmA on pJS1 was an ORF, designated ORFX, which encoded an 81-amino-acid polypeptide with a theoretical pI of 4.16 and a molecular mass of 8,285 Da. This ORF has some sequence similarity with cytochrome c6 from Euglena gracilis (35) and Microcystis aeruginosa (5) (Table 2). Downstream (3⬘) of the MSA monooxygenase gene on pJS1 cluster is an ORF, designated ORFY, which encodes a 336amino-acid polypeptide, theoretically of 37,873 Da with a pI of 6.47. Downstream (3⬘) from msmD in Methylosulfonomonas

methylovora strain M2 is an incomplete ORF encoding 200 amino acids which has 77% identity (85% similarity) to ORFY. The predicted ORFY polypeptide had significant sequence similarity with the transcriptional regulator proteins CynR (43) and YnfL (2) from E. coli and the hydrogen peroxide inducible activator gene OxyR from Pectobacterium chrysanthemi (E. Miguel, C. Poza-Carrion, E. Lopez-Solanilla, F. GarciaOlmedo, and P. Rodriguez-Palenzuela [SwissProt Database accession number Q9X725]) (Table 2), although its role in M. methylotropha strain TR3 and Methylosulfonomonas methylovora strain M2 is not known and would be difficult to determine at present, since there are no genetic systems available for these MSA utilizers to, for example, delete these genes by marker exchange mutagenesis. Also, attempts to express methanesulfonate monooxygenase genes in E. coli and other heterologous hosts have thus far proved unsuccessful (J. C. Murrell and W. Reichenbecher, unpublished results). Methylosulfonomonas methylovora strain M2 contains only one copy of msmABCD; therefore, the duplicate copies of msmA and msmB in M. methylotropha strain TR3 were unexpected. Although the presence of duplicate copies of enzymes is uncommon in prokaryotes (3), it is not unknown. For example, there are known to be multiple copies of methane monooxygenase genes in methanotrophs (11, 40, 42), ammonia monooxygenase genes in nitrifying bacteria (27, 31), and ribulose 1,5-bisphosphate carboxylase and oxygenase genes in Hydrogenovibrio spp. (14) and Rhodopseudomonas palustris (DOE Joint Genome Institute [http://www.jgi.doe.gov/tempweb/ JGI_microbial/html/rhodopseudomonas/rhodops_homepage .html]). The purpose of the multiple copies of these genes has not yet been fully explained. Studies of Methylococcus capsulatus Bath

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FIG. 2. Sequence alignment of MsmD from M. methylotropha strain TR3 (MsmD), Methylosulfonomonas methylovora strain M2 (MsmDm2), XylZ of the toluate 1,2-dioxygenase from P. putida (13), DmpP of the phenol hydroxylase from Pseudomonas sp. strain CF600 (30, 36), and NdoR of the naphthalene 1,2-dioxygenase from P. putida (41) which contain the plant type [2Fe-2S] binding motif (❋) and the oxidoreductase FAD/NAD binding domain (䡺).

found that the two essentially identical copies of the particulate methane monooxygenase genes, pmoCAB, were not equal. Methane oxidation was affected only when copy 2 was impaired (42). The unusual aspect of msmAB duplication is further complicated by the lack of duplication of msmC and msmD. Only Methylococcus capsulatus Bath shares a similar trait with the presence of three copies of pmoC and only two copies of pmoAB (42). The identity of the two copies of msmA and msmB would suggest that they arose from gene duplication; however, the significance of the two copies will be determined only when systems for marker exchange mutagensis in MSA utilizers have been established, and this may be difficult since many of them, particularly M. methylotropha strain TR3, grow very poorly on agar plates. Isolation and identification of MSA utilizers. Enrichments from terrestrial and marine environments yielded nine bacterial isolates which used MSA as a sole carbon and energy source. NB34 and NB35 were isolated from soil after a second enrichment with MSA following an initial enrichment on both MSA and methanol. The colonies formed by NB34 and NB35 when grown on MSA were 0.5 mm in diameter, had pink pigmentation, and were flat, shiny, and opaque. The cells were singular gram-negative rods. The BLAST results obtained with the sequences from the 16S rRNA from NB34 and NB35 indicated that these two bacteria belonged to the genus Methylobacterium. The 16S rRNA genes of both NB34 and NB35 were 99% identical to that of Methylobacterium species strain PC30.38 (P. Hugenholtz, H. G. Gwilliam, and J. A. Fuerst,

unpublished data [GenBank accession number X89904]). A Methylobacterium-like isolate strain P1 from Portugal was also found to use MSA as a sole carbon and energy source (8). NB34 and NB35 both grew on MSA as a sole carbon and energy source in liquid media. NB34 also grew, albeit poorly, on ethanesulfonate. Both isolates grew on 0.1% (vol/vol) methanol, but neither was capable of growth on methylamine or formate. Isolates NB36, NB38, and NB84 were identified by 16S rRNA sequencing as being most closely related to Hyphomicrobium methylovorum (37; T. Hamada, GenBank accession number AB016812) strains with identities of 98, 99, and 97%, respectively, to the published H. methylovorum 16S rRNA sequence. De Marco et al. (8) also isolated a Hyphomicrobiumlike bacterium, strain P2, from Portuguese soil which used MSA as a sole carbon and energy source. Phylogenetic analysis of the 16S rRNA grouped strain P2 with Hyphomicrobium facile, Hyphomicrobium sp. strain M3, and Hyphomicrobium denitrificans (8, 24, 37). Hybridization studies with msm genes from Methylosulfonomonas methylovora strain M2 indicated the presence of methanesulfonate monooxygenase genes in strain P2 (8). NB36 and NB38 were isolated from a secondary soil enrichment with MSA as a sole carbon source after an initial enrichment with MSA and formate as the carbon sources. NB84 came from a soil enrichment with MSA as the sole carbon and energy source. Colonies of NB36, NB38, and NB84 were 1 mm in diameter, flat, and opaque. Cells occurred

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singly, and some cells possessed prosthecae, which is a characteristic of Hyphomicrobium species. MSA-dependent growth of isolates NB36, NB38, and NB84 was observed on plates but not in liquid. Growth was also observed in liquid medium with methanol, methylamine, or formate as a carbon source. APW2 was isolated from used tomato plant compost after enrichment on MSA at 15°C. APW2 was identified by 16S rRNA sequencing and BLAST analysis as having 98% identity with the 16S rRNA sequence of H. methylovorum reported by Hamada (8). Compared to the other H. methylovorum isolates, APW2 had 99% identity to NB36 and NB38 and 98% identity to NB84. The rod-shaped cells of APW2 formed the characteristic prosthecae associated with Hyphomicrobium species. The colonies were 0.5 mm in diameter, round, flat, and slightly gray. APW2 grew on MSA, ethanesulfonate, methanol, methylamine, and formate as the sole carbon and energy sources. Four isolates were obtained from marine sediment. NB126 and NB127 were isolated after three subcultures into MinE medium containing MSA and 2% (wt/vol) NaCl. NB162 was isolated from a second marine sediment enrichment with MSA as the sole carbon and energy source after an initial enrichment with MSA and formate. NB126, NB127, NB162, and NB167 were small singular rod-shaped cells which formed flat white colonies 0.5 mm in diameter. Sequencing and BLAST analysis indicated that these isolates were 97% identical to Pedomicrobium fusiforme (37). NB126, NB127, and NB162 grew in liquid media with MSA as the sole carbon and energy source, and weak growth on ethanesulfonate was observed for NB126 and NB127. NB127 and NB162, but not NB126, grew on 0.1% (vol/vol) methanol. All Pedomicrobium strains grew on methylamine and formate. NB167 did not grow on MSA in liquid media but only on solid media. Weak growth of NB167 was observed on methanol, methylamine, and formate. All bacteria isolated which grow on MSA as a sole carbon and energy source are methylotrophic ␣-Proteobacteria. The presence of msmA was detected by PCR in all isolates. This is discussed further below. The results presented in this investigation suggest that the methanesulfonate monooxygenase genes are conserved across several genera. The MSA-utilizing bacteria isolated in this study came from a number of different environments, both marine and terrestrial, thus suggesting that MSA-utilizing bacteria are widespread in the environment (1, 8, 44) and therefore play a significant role in the cycling of organic sulfur in the environment. MSA can also be utilized as a sulfur source by a variety of microorganisms (22, 45). At least two different sets of genes are known to be involved in the utilization of MSA as a sulfur source: ssuD in Bacillus subtilis (45) and msuD in Pseudomonas aeruginosa (22). The amino acid sequences derived from these two genes had no significant similarity to any of the methanesulfonate monooxygenase components described in this study. Rieske region. A 783-bp fragment of msmA, the central domain of which encoded the Rieske [2Fe-2S] binding region, was PCR amplified from DNA from all isolates. When the DNA sequences of the 783-nucleotide msmA PCR product sequences from the MSA isolates were compared to msmA from Methylosulfonomonas methylovora strain M2, the Methylobacterium-like isolates were 81 to 82% identical, the H. methylovorum-like isolates were 81% identical, and the Pedomicrobium-like isolates were 79% identical. The derived

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MsmA sequence of PCR products from all MSA isolates was 81 to 84% identical and 91 to 92% similar to MsmA from Methylosulfonomonas methylovora strain M2. When DNA isolated from soil or marine enrichment cultures or DNA directly extracted from Rold Forest soil was used as a template in PCR with these msmA-specific PCR primers, a band of the correct size (783 bp) was obtained. These PCR products were cloned into the TOPO TA cloning vector. Recombinant clones were then analyzed by RFLP to separate them into different ecotypes. DNA sequence analysis of these msmA sequences retrieved from enrichments or directly from the environment revealed that these msmA PCR products were 77 to 79% identical to the msmA from Methylosulfonomonas methylovora strain M2, and the derived amino acid sequences were 81 to 84% identical (90 to 92% similar) to MsmA from Methylosulfonomonas methylovora strain M2. Therefore, the PCR primer set designed from the msmA sequences of Methylosulfonomonas methylovora and M. methylotropha is suitable for direct retrieval of methanesulfonate monooxygenase sequences directly from enrichment cultures and even from complex environmental samples such as soil. As with Methylosulfonomonas methylovora strain M2 and M. methylotropha strain TR3, all of the isolates obtained in this study, which included Methylobacterium, Hyphomicrobium, and Pedomicrobium strains, contained the characteristic 26-aminoacid spacer within their MsmA components. A short region of the MsmA containing the Rieske iron center (centered around amino acyl residues 86 to 119) is shown in Fig. 3. The amino acid sequence of the Rieske spacer region was also highly conserved between MsmA from all new MSA-utilizing isolates obtained during this study. The highly conserved nature of the MsmA Rieske iron center region was also noted in the derived sequences from soil and marine enrichment clones and three soil DNA clones. The corresponding region of MsmA derived from soil enrichment clones 2, 9, and 96, marine enrichment clones 110 and 157, and forest soil DNA clones 212, 232, and 240 is also shown in Fig. 3. The corresponding region of TodC1 from P. putida is shown for comparison (Fig. 3). Variation was found in only six amino acid positions within the MsmA from methanesulfonate utilizers. At position 92 there was either leucine, isoleucine, or methionine; at position 93 there was a threonine or isoleucine; at position 101 there was a leucine or phenylalanine; at position 102 there was either lysine or leucine; at position 103 there was aspartic acid or glutamic acid; and at position 108 there was glycine or an aspartic acid (Fig. 3). The significance of the extended spacer sequence, so far found only within the Rieske binding region of MSA monooxygenase and in no other oxygenase sequences, remains to be elucidated. An explanation for the longer amino acid sequence may relate to the chemical structure of the MSA molecule itself. The answer to this question may be found when the X-ray crystal structure of MSA monooxygenase is determined, work which is ongoing in our laboratory. However, this sequence feature uniquely found in the hydroxylase subunit of this enzyme makes it a diagnostic protein for MSA oxidizers. In this study we report the duplication of msm genes from the marine MSA oxidizer M. methylotropha strain TR3 and of the amplification and sequencing of a portion of msmA from seven new MSA-oxidizing bacterial strains. All of these bacteria were shown to contain homologous msm genes. These


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FIG. 3. Comparison of the region of MsmA, centered around amino acyl residues 86 to 119, containing the unique 26-amino-acid Rieske binding domain in MsmA retrieved by PCR from pure cultures, from soil and marine enrichments, and from soil DNA. Included in the comparison was the corresponding region of TodC1, which encodes the large subunit of toluene dioxygenase, from P. putida (47), which demonstrates the more common CXH-X17-CXXH Rieske motif. Variations in the amino acid sequence are indicated by boxes. Amino acids are numbered according to the MsmA sequence from Methylosulfonomonas methylovora strain M2. (For brevity, only the Rieske binding domain of MsmA is shown.)

findings give further strength to the hypothesis that all bacteria that can grow utilizing MSA as the sole source of carbon and energy do so by the action of a highly conserved MSA monooxygenase enzyme system and that they are ubiquitous in the natural environment.

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ACKNOWLEDGMENTS We acknowledge the Natural Environment Research Council for their financial support through research grant GR3-08242 and the Science and Technology Foundation (FCT) (Portugal) for a postdoctoral grant to P.D.M. We thank D. P. Kelly for very helpful discussions and comments on the manuscript. REFERENCES 1. Baker, S. C., D. P. Kelly, and J. C. Murrell. 1991. Microbial degradation of methanesulfonic acid—a missing link in the biogeochemical sulfur cycle. Nature 350:627–628. 2. Blattner, F. R., G. Plunckett III, C. A. Bloch, N. T. Perna, V. Burland, M. Riley, J. Collando-Vides, F. D. Glasner, C. K. Rode, G. F. Maythew, J. Gregor, N. W. Davis, H. A. Kirkpatrick, M. A. Goeden, D. J., Rose, B. Mau, and Y. Shao. 1997. The complete genome sequence of Escherichia coli K-12. Science 277:1453–1474. 3. Bowien, B., R. Bedbarski, B. Kusian, U. Windvo¨vel, J. Scha ¨ferjohann, and J.-G. Yoo. 1993. Genetic regulation of CO2 assimilation in chemoautotrophs, p. 418–492. In J. C. Murrell and D. P. Kelly (ed.), Microbial growth on C1 compounds. Intercept, Ltd., Andover, United Kingdom. 4. Casadaban, M. J., and S. N. Cohen. 1980. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J. Mol. Biol. 138:179–207. 5. Cohn, C. L., M. A. Hermodson, and D. W. Krogmann. 1989. The amino acid sequence of cytochrome c553 from Microcystis aeruginosa. Arch. Biochem. Biophys. 270:219–226. 6. Cook, A. M., H. Laue, and F. Junker. 1998. Microbial desulfonation. FEMS Microbiol. Rev. 22:399–419. 7. De Marco, P., P. Moradas-Fereira, T. P. Higgins, I. R. McDonald, E. M.

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