Max-Planck-Institute for Terrestrial Microbiology, Karl-von-Frisch Strasse, 35043 Marburg, Germany1
Institute of Microbiology, Russian Academy of Sciences, Moscow, 117312, Russia2
Author for correspondence: Peter Dunfield. Tel: +49 6421 178 733. Fax: +49 6421 178 809. e-mail: dunfield{at}mailer.uni-marburg.de
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ABSTRACT |
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Keywords: Methylosinus, Methylocystis, methane monooxygenase, pmoA, mmoX
Abbreviations: MOB, methane-oxidizing bacteria; sMMO, soluble methane monooxygenase
The GenBank accession numbers for the nearly complete 16S rRNA gene sequences for the isolates are AJ458466 to AJ458510. Partial sequences of the pmoA, mxaF and mmoX genes have been deposited under the accession numbers AJ458994AJ459052, AJ459053AJ459100 and AJ458511AJ458535, respectively. Where multiple strains contained identical sequences, only one has been deposited.
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INTRODUCTION |
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Phylogenetic trees constructed based on 16S rRNA sequences often fail to reveal any clear organization of the type II Methylocystis/Methylosinus group. The different species are not always monophyletic (e.g. Bowman, 2000 ; Murrell & Radajewski, 2000
). This may be due to the scarcity of public-domain sequences available from pure cultures, and possibly also to errors in the sequences which are available. The lack of a large, error-free 16S rRNA sequence database has also made difficult the task of designing oligonucleotide probes to target particular MOB types or species, probes which could be applied to the cultivation-independent study of these bacteria by such techniques as retrieval of environmental 16S rRNA gene sequences and fluorescence in situ hybridization.
The recognition that several genes encoding key enzymes involved in methane oxidation could be used as phylogenetic markers has provided a useful toolbox for the cultivation-independent identification of MOB in various habitats. Those presently in use are: 1) the pmoA gene, which encodes the active-site-containing subunit of the particulate methane monooxygenase enzyme (pMMO) and is universal to all known MOB (Holmes et al., 1995 ; McDonald & Murrell, 1997a
), with the possible exception of Methylocella palustris [pmoA was not detected in Methylocella palustris using current primer sets or by hybridization with a pmoA fragment from Methylococcus capsulatus (Dedysh et al., 2000
)]; 2) the mmoX gene, which encodes a subunit of soluble methane monooxygenase (sMMO) and is present in only some MOB strains (McDonald et al., 1995
; Auman et al., 2000
); and 3) the mxaF gene, which encodes a subunit of methanol dehydrogenase. This last enzyme is universal to methylotrophic bacteria and therefore also universal to, but not unique to, MOB (McDonald & Murrell, 1997b
). Phylogenetic trees constructed based on partial sequences of any of these three genes show similar topological patterns to the phylogenetic trees constructed from 16S rRNA sequences, indicating that there has been little horizontal transfer of these functional genes and that the methanotrophic phenotype probably evolved from a common ancestor. The phylogenies constructed based on pmoA, mmoX or mxaF gene sequences therefore reflect the evolution of species rather than simply of the genes, and sequencing of gene products amplified by PCR from environmental DNA extracts can provide some information on which MOB are present in that environment. It is, however, unclear to what scale of resolution these sequences are useful. Most can easily distinguish type I from type II MOB, but more reference sequences from pure cultures are necessary to demonstrate whether particular species can be mapped to particular monophyletic clusters of these genes.
The aim of the present work was to develop better databases of 16S rRNA, pmoA, mmoX and mxaF sequences derived from pure cultures of type II MOB. Recently a similar exercise was performed for ammonia oxidizers (Purkhold et al., 2001 ). This provided a better phylogenetic framework for this group and, for example, dispelled the impression that most sequences retrieved from environmental samples stemmed from novel uncultivated bacteria. Developing a better database for MOB should help to: 1) determine at what scale of taxonomic resolution the morphologically recognized species correlate with taxa recognized by molecular sequence data; 2) provide a better database to correlate environmental gene sequences retrieved in cultivation-independent studies with strains characterized in pure culture; and 3) facilitate the design and evaluation of oligonucleotide probes targeting specific taxonomic or physiological groups, for use in cultivation-independent molecular ecology studies. To these ends a large collection of MOB was first isolated from diverse environments, and selected cultures identified based on morphological characteristics and on molecular phylogenies. The present paper presents the results of the type II MOB. The type I MOB in the collection will be described in a later study.
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METHODS |
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Morphological identification.
Morphological characterization was performed at the time of isolation, and again immediately before molecular analyses to ensure that contamination of cultures did not occur. Cell form, size, motility, exospore formation, lipid cyst formation, and cell arrangement (especially rosette formation) were determined by phase-contrast microscopy of 1-week-old (exponential phase) and 2-to 3-week-old (stationary phase) liquid cultures and agar plate cultures. Agar plate cultures were also characterized based on colony form, size, surface appearance, edge, consistency and pigmentation.
Type II MOB can be distinguished on the basis of visible morphological characteristics (Whittenbury et al., 1970 ; Bowman et al., 1993
; Bowman, 2000
). The type species are Methylosinus trichosporium strain OB3b, Methylosinus sporium strain 5, Methylocystis parvus strain OBBp, and Methylocystis echinoides strain IMET 10491. Methylocystis are coccoid (0·61·5 µm diameter) or short, slightly bent rods (1·23·5x0·81·5 µm), occasionally reniform, and often contain reserve material (polyhydroxybutyrate) at the poles. They form lipid cysts (enlarged, irregularly shaped, refractive cells) in older cultures. They are non-motile (with few exceptions noted in our collection, Table 1
) and do not form rosettes. Methylosinus are motile, rod-shaped cells which form exospores and often cluster in rosettes. Methylosinus sporium differs from Methylosinus trichosporium in the form and size of cells. M. trichosporium cells are generally long (25 µm), thick (0·81·3 µm) rods which are only slightly bent and are slightly thicker at the basal pole (holdfast site during rosette formation) than at the apical pole (site of exospore formation). Methylosinus sporium cells are shorter (1·53 µm), thick (0·81·2 µm) cells which are strongly bent, often crescent-shaped and vibrioid. They are clearly thicker at the basal pole than the apical pole.
Methylocystis parvus and Methylocystis echinoides are difficult to distinguish without electron micrographs to visualize the spinae which project from the cell envelope of M. echinoides. Therefore, the identification of new Methylocystis isolates was made to the genus level only. However, molecular analyses were also performed on the type strain Methylocystis echinoides strain IMET 10491 (Table 1) which was isolated by Meyer et al. (1986)
(originally designated strain IC 493s/5), and phenotypically characterized by Bowman et al. (1993)
. In addition, molecular analyses were performed on 11 Methylocystis strains that were isolated from various sites in Russia and divided based on phenotypic traits into four morphotypes: parvus, echinoides, minimus and methanolicus. Detailed phenotypic descriptions of these strains, including morphological observations from electron microscopy, physiological tolerances (pH, temperature, salt), phospholipid fatty acid (PLFA) profiles, G+C content and various enzymic tests, have been published elsewhere (Galchenko et al., 1980
) and are also available online at http://inmi.da.ru/.
Colorimetric detection of sMMO activity.
To test for sMMO activity, the naphthalene oxidation assay was performed on 1- to 2-week-old streaked plate cultures of selected isolates (Graham et al., 1992 ). Medium was made with and without added copper. Some isolates grew poorly on the medium of Graham et al., (1992)
; in these cases the test was made on medium 10 instead. A positive assay was scored by the development of a deep purple colour, indicating the oxidation of naphthalene to
-naphthol by sMMO.
Molecular analyses.
DNA was extracted from cultures grown on agar slants using a procedure based on mechanical agitation in a FastPrep FP120 cell disrupter (Savant Instruments) of 2 ml screw-cap reaction vessels filled with a mixture of culture, 0·1 mm diameter silica-zirconium beads and a phosphate buffer (pH 8) containing 2% SDS (Henckel et al., 1999 ). Oligonucleotide primer sets listed in Table 2
were used to amplify partial gene products of 16S rDNA, pmoA, mmoX and mxaF on a PE GeneAmp PCR System 9700 temperature cycler (Perkin-Elmer Applied Biosystems). Temperature programs were as described previously (Henckel et al., 1999
), except for mmoX, which was as described by Auman et al. (2000)
. PCR mixtures (50 µl) contained 0·5 µM each primer, 1x Premix F (Epicenter Technologies), 1 µl template DNA and 1 U Taq DNA polymerase (Q Biogene). PCR products were sequenced on an ABI 373 automated sequencer (Perkin-Elmer Applied Biosystems) as described by Henckel et al. (1999)
or on an ABI Prism 377 DNA sequencer using BigDye terminator chemistry as specified by the manufacturer (Perkin-Elmer Applied Biosystems).
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Phylogenetic analyses.
Comparative sequence analyses of the nearly complete 16S rRNA genes and of the partial sequences of the various functional genes, were performed using the ARB software (Strunk & Ludwig, 1996 ). Sequencing errors were minimized by comparing each gene sequence obtained to a cumulative consensus sequence for type II MOB and double-checking all mismatches against the original ABI sequencer output. Identical sequences from different strains were input only once into each analysis.
The 16S rDNA treeing analysis included all complete sequences from type II MOB isolates that are available in public-domain databases, with a few exceptions. When performing phylogenetic analyses on closely related species such as type II MOB, tree topology can be greatly affected by only a few erroneous nucleotide position assignments. Therefore the earliest published sequences (GenBank accession numbers L20848, L20803, L20844, M29026, M29024, M95665) were not used, since subsequent analysis of two strains has demonstrated that the original sequences contained many errors (Dedysh et al., 2000 ). Two sequences from the present study should also be used to replace older sequences in GenBank: that of Methylocystis echinoides strain 2 (new accession number AJ458502; old accession number L20848) and that of Methylosinus trichosporium strain SM6, identified previously by its classification number in the Ukrainian Collection of Microorganisms, IMV B-3060 (new accession number AJ458477; old accession number L20845). Based on the new data, the earlier sequences contained 6·5% and 1·5% erroneous nucleotide assignments, respectively. Finally, the 16S rDNA sequence of Methylosinus pucelana (AF107461) was nearly identical (99·5%) to Methylocystis parvus strain 81; we suspect that this strain has been falsely identified as Methylosinus.
For phylogenetic analyses of pmoA, all sequences from type II MOB isolates in the public-domain database were included, along with representative sequences that were obtained from cultivation-independent studies and fall into the type II MOB cluster of pmoA sequences.
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RESULTS AND DISCUSSION |
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A phylogenetic tree based on 16S rDNA sequences and constructed using a maximum-likelihood algorithm suggests that the three morphologically identified groups of type II MOB are also each monophyletic (Fig. 1). The only exception was strain D15a, which was identified as Methylosinus trichosporium based on morphology but which groups within the Methylosinus sporium cluster based on 16S rDNA phylogeny. Possibly this strain represents an intermediate between the two species (a hypothesis supported by pmoA phylogeny, see below). Otherwise the phylogenetic analysis identified three distinct clusters which corresponded to morphologically recognizable species of type II MOB. This monophyletic nature of different type II MOB species has not always been evident in previously constructed phylogenies (e.g. Hanson & Hanson, 1996
; Bowman, 2000
; Murrell & Radajewski, 2000
). A maximum-parsimony phylogenetic tree of 16S rDNA sequences (100 bootstraps) preserved the monophyletic nature of the three groups (Methylocystis spp., Methylosinus trichosporium and Methylosinus sporium). Bootstrap values for the species cluster nodes were not high (25%, 32% and 84%, respectively); however, bootstrap values are not especially useful when evaluating a bush-like structure with a gradient of many closely related sequences. Neighbour-joining trees varied depending on the filter and distance correction used (data not shown). The neighbour-joining tree which best preserved the topology of the maximum-likelihood and maximum-parsimony trees used a JukesCantor or Felsenstein correction and incorporated no filter, which is reasonable considering that the most variable nucleotide positions are the most useful in discerning differences among closely related strains and should therefore not be filtered out (Swofford et al., 1996
). In this tree the Methylosinus sporium remained monophyletic, but some Methylosinus trichosporium strains (KS21, I4/1, I3/4, SM6) formed a separate cluster from the other Methylosinus trichosporium, branching nearest to Methylocystis F10V2a and thereby separating the Methylocystis into two clusters.
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pmoA
For each pairwise permutation of type II strains, the percentage 16S rDNA sequence difference was compared to the percentage partial pmoA gene sequence difference. The nucleotide substitution rate of the partial pmoA gene was 3·5 times the nucleotide substitution rate of 16S rDNA (i.e. percentage difference was on average 3·5 times greater). There was a strong linear relationship between pairwise pmoA and 16S rDNA differences (r2=0·90), and therefore comparative analysis of partial pmoA gene sequences yielded a tree topology largely in agreement with that produced from 16S rDNA sequences (Fig. 2). The three main groups (Methylocystis spp., Methylosinus trichosporium and Methylosinus sporium) were again monophyletic, with the possible exception of Methylosinus trichosporium strains D15a and KS21. The monophyletic nature of the three groups in the neighbour-joining analysis (Fig. 2
) was preserved in a maximum-parsimony tree with 100 bootstraps. Maximum-likelihood analysis also preserved the three groups, except that again the morphologically unusual Methylosinus strains KS21, KS24b and D15a often were not clearly assigned to either the Methylosinus sporium or Methylosinus trichosporium clusters in separate runs (bootstraps).
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The pmoA gene has been extensively used in cultivation-independent studies of MOB (e.g. McDonald & Murrell 1997a ; Costello et al., 1999
; Henckel et al., 1999
, 2000a
, b
; Holmes et al., 1999
; Bourne et al., 2001
; Fjellbirkeland et al., 2001
; Horz et al., 2001
; Reay et al., 2001
; Morris et al., 2002
). Sequences of pmoA which were directly retrieved from environmental samples and which group within the type II MOB cluster were included in phylogenetic analyses to demonstrate the usefulness of the database for species identification. Some of these environmental sequences (Rold1Rold4 and MR1) do not fall within the Methylosinus/Methylocystis cluster and may represent novel type II MOB species most closely related to Methylocapsa (Fig. 2
). Others (LP21, M84-P3) fall within a cluster that has been shown to represent a second pmoA-like gene present in some type II methanotrophs (Dunfield et al., 2002
). Finally, some fall clearly within one of the Methylosinus trichosporium, Methylosinus sporium or Methylocystis clusters, and can be assumed to indicate the presence of the respective species in that particular environment. For example, two pmoA sequences retrieved from Sphagnum peat, PD2 and PD3 (McDonald & Murrell, 1997a
), are most closely related to Methylocystis strain F10V2a, which was also isolated from a Sphagnum bog environment. Interestingly, despite the diversity of type II sequences from the present culture collection, most pmoA sequences recovered in cultivation-independent studies fall into two very closely related groups within the Methylocystis cluster. This suggests either that these strains are the numerically dominant MOB in several environments, or that the oligonucleotide primer systems that are widely used are biased towards the recovery of certain sequences.
mxaF
A comparative phylogenetic analysis of mxaF sequences from selected isolates is shown in Fig. 3. The phylogenetic tree does not quite show the same division into three main clusters as did the 16S rDNA and pmoA phylogenies. The unique feature of the mxaF phylogeny is that some Methylosinus trichosporium mxaF sequences group closely with some Methylocystis mxaF sequences, while others are more closely related to Methylosinus sporium mxaF sequences. The two clusters of Methylosinus trichosporium separate some species with nearly identical pmoA and 16S rDNA sequences (i.e. the bifurcation is not caused only by unusual species such as KS21 and D15a) and it therefore appears likely that horizontal transfer of this gene has occurred across type II MOB. The mxaF gene may still be useful to identify different type II MOB, but it is probably less useful than pmoA as a phylogenetic marker of evolutionary relationships.
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The results of the PCR assay of Auman et al. (2000) on all isolates are indicated in Table 1
. An mmoX gene was detected in all Methylosinus strains, but only in about half of the Methylocystis strains. Generally, the results of the naphthalene plate assay for sMMO enzyme activity agreed with the PCR-based detection of the mmoX gene, but there were exceptions (Table 3
). Several strains which possessed mmoX displayed no naphthalene oxidation activity. Two strains (Methylocystis Pi6/2 and KS30) displayed naphthalene oxidation activity but an mmoX gene could not be amplified with any of three primer sets (Table 3
). Interestingly, nearly identical strains often gave different results. For example the Methylocystis strain KS30 displayed sMMO activity, but the strain KS31 (identical 16S rDNA sequence) did not. The Methylocystis strains 62/38a, 50/42a, SK28 and 51 were all positive for mmoX and for naphthalene oxidation activity, but the very similar strains SC2 and 21/1 ( 99·9% 16S rDNA sequence identity to the above strains) were negative on both tests. The sMMO results are therefore something of a mystery. Possibly, nearly identical strains indeed differ in either possessing or lacking this enzyme, but it is also likely that neither the enzyme test nor the PCR assay are universal for this enzyme.
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Certain Methylosinus strains (KS21, D15a and possibly I4/1, I3/4, SM6 and KS24b) probably represent evolutionary intermediates to the type species, and were characterized both by unusual morphologies and by phylogenetic relationships which varied depending on the gene sequence (16S rRNA, pmoA, mxaF or mmoX) and on the treeing algorithm used. Gene sequences from these strains frequently branched on the edges of the major species clusters in phylogenetic trees.
Finally, since this culture collection incorporates functionally similar bacterial strains from diverse geographical areas, it might potentially provide some information on the question of bacterial endemism, the Is everything everywhere? question (e.g. Cho & Tiedje, 2000 ). Strains from different environments were often very similar or identical phylogenetically, indicating that these bacteria are not endemic at a level detectable by the gene sequences we determined. However, multiple isolates also coexisted in some environments (e.g. Lake Kinneret contained many diverse strains of both genera). This is a critical point because it is necessary to define a functionally unified group to study endemism. Although at first glance the obligate MOB is a good candidate group, the coexistence of multiple strains indicates that there is niche separation among them, and that differences cannot therefore be assigned to geographical factors but rather to ecological ones.
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ACKNOWLEDGEMENTS |
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Received 20 February 2002;
revised 18 April 2002;
accepted 14 May 2002.