Molecular phylogeny of type II methane-oxidizing bacteria isolated from various environments

Jürgen Heyer1, Valery F. Galchenko2 and Peter F. Dunfield1

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


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Type II methane-oxidizing bacteria (MOB) were isolated from diverse environments, including rice paddies, pristine and polluted freshwaters and sediments, mangrove roots, upland soils, brackish water ecosystems, moors, oil wells, water purification systems and livestock manure. Isolates were identified based on morphological traits as either Methylocystis spp., Methylosinus sporium or Methylosinus trichosporium. Molecular phylogenies were constructed based on nearly complete 16S rRNA gene sequences, and on partial sequences of genes encoding PmoA (a subunit of particulate methane monooxygenase), MxaF (a subunit of methanol dehydrogenase) and MmoX (a subunit of soluble methane monooxygenase). The maximum pairwise 16S rDNA difference between isolates was 4·2%, and considerable variability was evident within the Methylocystis (maximum difference 3·6%). Due to this variability, some of the published ‘specific’ oligonucleotide primers for type II MOB exhibit multiple mismatches with gene sequences from some isolates. The phylogenetic tree constructed from pmoA gene sequences closely mirrored that constructed from 16S rDNA sequences, and both supported the presently accepted taxonomy of type II MOB. Contrary to previously published phylogenetic trees, morphologically distinguishable species were generally monophyletic based on pmoA or 16S rRNA gene sequences. This was not true for phylogenies constructed from mmoX and mxaF gene sequences. The phylogeny of mxaF gene sequences suggested that horizontal transfer of this gene may have occurred across type II MOB species. Soluble methane monooxygenase could not be detected in many Methylocystis strains either by an enzyme activity test (oxidation of naphthalene) or by PCR-based amplification of an mmoX gene.

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 AJ458994–AJ459052, AJ459053–AJ459100 and AJ458511–AJ458535, respectively. Where multiple strains contained identical sequences, only one has been deposited.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Methane-oxidizing bacteria (MOB) are a phylogenetically diverse group defined by the ability to use methane as a sole carbon and energy source. Eleven known genera of MOB are divided into two groups, type I ({gamma}-Proteobacteria) and type II ({alpha}-Proteobacteria). These types differ in many characteristics, including carbon assimilation pathways and the arrangement of intracellular membranes (Bowman et al., 1993 ; Bowman, 2000 ). The type I group is more phylogenetically diverse, but members of the type II group are as often isolated from natural systems, or detected using cultivation-independent molecular techniques (e.g. McDonald et al., 1996 ; Costello & Lidstrom, 1999 ; Henckel et al., 1999 ; Wise et al., 1999 ; Auman et al., 2000 ; Horz et al., 2001 ; Steinkamp et al., 2001 ). Recognized species within the type II group were until recently limited to Methylosinus sporium, Methylosinus trichosporium, Methylocystis parvus and Methylocystis echinoides. Two newly described MOB, Methylocella palustris (Dedysh et al., 2000 ) and Methylocapsa acidiphila (Dedysh et al., 2002 ), also belong to the {alpha}-Proteobacteria line of descent. Presently these are grouped together with the Methylocystis/Methylosinus group as type II MOB. However they have several unique morphological and physiological characteristics, and based on 16S rRNA phylogeny are evolutionarily more closely related to some non-methanotrophic bacteria than to the previously known type II MOB.

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Isolation.
Except where indicated, all strains were isolated by the authors and co-workers in the past 30 years. Soil and water samples were obtained from diverse environments as listed in Table 1. The enrichment procedure for MOB was as described by Heyer et al. (1984) . Briefly, a serial 10x dilution series of the samples was made in a mineral salts medium (‘medium 10’) containing (per l): 0·1 g MgSO4.7H2O; 0·5 g NH4Cl; 0·7 g Na2HPO4.2H2O; 0·3 g KH2PO4; 0·01 g CaCl2.6H2O; 0·005 g FeSO4.7H2O; and 1 ml trace elements solution (see below). The final pH was 7·0. One millilitre of trace elements solution contained: 0·44 mg ZnSO4.7H2O; 0·20 mg CuSO4.5H2O; 0·17 mg MnSO4 . 2H2O; 0·06 mg Na2MoO4.2H2O; 0·10 mg H3BO3; and 0·08 mg CoCl2.6H2O. In some cases 10 ml l-1 of a sterile-filtered (0·2 µm) vitamin solution was added to the medium after autoclaving. This 10 ml aliquot contained: 20 µg folic acid; 20 µg biotin; 100 µg pyridoxine hydrochloride (vitamin B6); 50 µg thiamin hydrochloride (vitamin B1); 50 µg DL-Ca-pantothenate; 50 µg riboflavin (vitamin B2); and 1 µg vitamin B12.


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Table 1. MOB isolated in this study, with strain designation, location and date of sampling, and species characterization

 
Tubes were incubated at 30 °C in closed glass chambers containing a gas mixture of 20% (v/v) CH4, 5% CO2 and 75% air. Samples of the terminal positive dilution steps were streaked on plates of medium 10 solidified by adding 1·5% Bacto agar (Difco), and grown under the same gas mixture. Single colonies growing on plates were selected with the aid of a stereo microscope and streaked on new plates, and this repeated until pure cultures were obtained. Purity was verified by phase-contrast microscopy of liquid cell culture, by streaking onto Nutrient Agar (Difco) diluted 1:1 with H2O, and onto plates of mineral salts medium incubated in an atmosphere containing no CH4. Isolated cultures were maintained by periodic transfer (6–12 month intervals).

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 (0·6–1·5 µm diameter) or short, slightly bent rods (1·2–3·5x0·8–1·5 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 (2–5 µm), thick (0·8–1·3 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 (1·5–3 µm), thick (0·8–1·2 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 {alpha}-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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Table 2. Oligonucleotide primers used in this study for the PCR amplification of genes from DNA extracts of MOB cultures

 
It has been shown that at least some type II MOB contain a second pmoA-like gene (pmoA2) which possesses only about 30% nucleotide sequence identity to the pmoA that has been well-studied in pure cultures of type II MOB (Dunfield et al., 2002 ). However, the ‘normal’ pmoA gene is favoured by the A189/A682 oligonucleotide primer set and ambiguous nucleotide assignments were rarely evident in our sequencing analyses. In the few cases where many ambiguities in nucleotide assignment of pmoA sequences were evident, the PCR was repeated with a higher annealing temperature (67 °C) and most ambiguities disappeared.

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.


   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Phylogeny and taxonomy of type II MOB isolates
With few exceptions, the type II MOB isolates could be identified based on visible morphological and cultural traits as Methylosinus sporium, Methylosinus trichosporium or Methylocystis sp. Some isolates deviated in one or more characteristics from the standard species morphologies outlined in Methods. These exceptions are noted in Table 1. For example, Methylocystis strains KS7 and KS8a were motile, a characteristic not previously observed in any Methylocystis strain. However, other characteristics of KS7 and KS8a (e.g. shape, cyst formation) were typical for Methylocystis. Other atypical strains included two unusually long and slender Methylosinus strains (KS21, D15a), some Methylocystis strains which were large, aggregated, rod-shaped cells and formed viscous colonies on agar plates (F10V2a, KS30, KS31) and several red-pigmented or yellow-pigmented Methylosinus trichosporium strains (SM6, KS24b, I4/1, I3/4). The red pigment of strain SM6 has been characterized chemically (Strauss & Berger, 1983 ). No strains of Methylocella- or Methylocapsa-like bacteria were isolated, probably due to enrichment biases caused by the medium used (Dedysh et al., 2000 , 2002 ). Therefore, further discussion of type II MOB will primarily be limited to the Methylocystis/Methylosinus group.

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 Jukes–Cantor 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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Fig. 1. Maximum-likelihood phylogenetic tree showing the relative positions of 16S rDNA sequences of type II MOB isolates, along with the non-MOB Beijerinckia indica, Afipia genospecies 6, Rhodoblastus acidophilus, and three sequences (UP1, UP3 and LO13.7) retrieved from soil by cultivation-independent methods (Radajewski et al., 2000 ; Reay et al., 2001 ). All sequences are from this study except where a GenBank accession number is given in parentheses after the name. Some sequences with <0·1% sequence difference are grouped for simplicity. The root was composed of 24 sequences from (non-methanotrophic) {alpha}-Proteobacteria. The distance bar represents 0·1 substitution per nucleotide position.

 
Some of the Methylocystis strains studied have been classified based on intense morphological characterization as either Methylocystis parvus or Methylocystis echinoides, or as the morphotypes ‘Methylocystis minimus’ and ‘Methylocystis methanolicus (Table 1) (Galchenko et al., 1980 ). All Methylocystis parvus 16S rDNA sequences were very similar (Fig. 1). However, Methylocystis echinoides strains were more phylogenetically diverse, and strains classified as ‘minimus’, which do not show the spinae formation typical of Methylocystis echinoides, had nearly identical 16S rDNA sequences to some strains of Methylocystis echinoides. Molecular phylogeny of 16S rDNA therefore does not clearly resolve the species Methylocystis echinoides from other morphotypes of Methylocystis.

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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Fig. 2. Neighbour-joining phylogenetic tree (with Jukes–Cantor distance correction) constructed from partial pmoA sequences of type II MOB. Some sequences are grouped for simplicity. The root was composed of 10 pmoA sequences from type I MOB. The distance bar represents 0·1 substitution per nucleotide position. Bootstrap values (100 data resamplings) for the initial six nodes are given. All sequences were obtained during this study except where a GenBank accession number is given in parentheses after the name. The sequences in bold were recovered by cultivation-independent methods in various studies. There are: pAMC510 (lake water; Costello & Lidstrom, 1999 ); PD2, PD3 (peat bog; McDonald & Murrell, 1997a ); M84-P1, M84-P2, M84-P3 (ricefield soil; Horz et al., 2001 ); GP3, GP13 (various upland soils; Reay et al., 2001 ); LP17, LP21 (trichloroethylene-contaminated aquifer; Baker et al., 2001 ); Rold1, Rold3, Rold4, Rold5 (various forest soils; Holmes et al., 1999 ); MR2 (forest cambisol; Henckel et al., 2000a ); IV, V, VI (ricefield soil; Henckel et al., 1999 ), F1, F2, F3, F4 (various upland soils; Bourne et al., 2001 ); LOPA13.2, LOPA13.3, LOPA13.5, LOPB13.1, LOPB13.3 (peat soil; Morris et al., 2002 ); and MO3 (ricefield soil; Henckel et al., 2000b ).

 
Phylogenetic analysis of genes other than 16S rDNA are commonly based on derived amino acid sequences rather than on nucleotide sequences. However, because much of the information content is lost by translating nucleotide sequences, pairwise differences in 16S rDNA sequences were actually better correlated to pairwise pmoA nucleotide sequence differences (r2=0·90) than to pairwise PmoA amino acid sequence differences (r2=0·83). When dealing with closely related species, pmoA-based phylogeny is therefore more reliable when constructed based on nucleotide than on derived amino acid sequences. However, the topology of the tree remains nearly the same (data not shown).

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 (Rold1–Rold4 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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Fig. 3. Neighbour-joining tree (with Jukes–Cantor distance correction) showing the relationship of partial mxaF gene sequences of selected type II MOB isolates to those of other methylotrophic bacteria. All sequences are from this study except those where a GenBank accession number is given in parentheses after the species name. Some sequences are grouped for simplicity. Bootstrap values >70% (100 data resamplings) are shown. The scale bar represents 0·1 substitution per nucleotide position.

 
sMMO
sMMO has previously been detected in only some type II MOB. Methylocystis parvus OBBp, for example, appears not to contain this enzyme (Hanson & Hanson, 1996 ). We used both an enzymic test for sMMO activity (Graham et al., 1992 ), and PCR-based detection of the mmoX gene to indicate whether sMMO was present in selected isolates. Several available mmoX primer sets (McDonald et al., 1995 ; Miguez et al., 1997 ; Shigematsu et al., 1999 ; Auman et al., 2000 ; Horz et al., 2001 ), were first tested on a subset of 15 isolates. The primer set published by Auman et al. (2000) was the most universal of these. Other primer sets sometimes failed to amplify a product from isolates for which a detectable product was obtained using this primer set, but the opposite was never the case.

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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Table 3. Detection of sMMO in selected isolates by a colorimetric plate assay, and PCR-based detection of the mmoX gene encoding a subunit of the sMMO enzyme

 
The phylogenetic tree of mmoX sequences is shown in Fig. 4. Unlike 16S rDNA and pmoA phylogenies, this does not show a clear division into three main clusters corresponding to Methylosinus trichosporium, Methylosinus sporium and Methylocystis spp. Nevertheless, a plot of pairwise mmoX sequence difference versus pairwise 16S rDNA sequence difference is linear (Fig. 5), providing no evidence for horizontal gene transfer. The lack of the expected arrangement in this phylogenetic tree is probably due to the scarcity of sequences available. Many branches are represented by single species or by a few nearly identical species, and the relationships among these branches therefore cannot be clearly elucidated.



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Fig. 4. Neighbour-joining tree (with Jukes–Cantor distance correction) showing the relationship of partial mmoX gene sequences of selected type II MOB isolates to those of other methanotrophic bacteria. All sequences are from this study except those where a GenBank accession number is given in parentheses after the species name. Some sequences are grouped for simplicity. Bootstrap values >70% (100 data resamplings) are shown. The scale bar represents 0·1 substitution per nucleotide position.

 


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Fig. 5. Percentage difference in mmoX sequences versus percentage difference in 16S rDNA sequences for pairwise permutations of type II MOB isolates plus the mmoX sequence of Methylocella palustris (Dedysh et al., 2000 ).

 
Oligonucleotide probes
Another use of these databases is the design of specific oligonucleotide probes. The maximum pairwise 16S rDNA sequence difference within all type II MOB sequenced in the present study was 4·2%, within the genus Methylocystis 3·6% and within the genus Methylosinus 2·8% (Methylosinus trichosporium 2·5%, Methylosinus sporium 1·7%; when considering strain D15a as a Methylosinus sporium). Several oligonucleotides have already been developed with intended target specificity to 16S rRNA (or its encoding gene), and to functional genes of MOB. Some of these, when tested in silico against the databases created in this study, lacked the intended target specificity, but others were universal to the isolates (Table 4). The region of the 16S rRNA gene from Escherichia coli positions 433–497 seems to be especially useful for the specific and universal targeting of type II MOB. This region has been identified in recent studies as the target of: 1) the hybridization probe ‘type2 457’ (Auman et al., 2000 ), 2) the fluorescently labelled probes ‘M{alpha} 450’ and ‘M{alpha} 464’ used for in situ whole-cell hybridization with type II MOB (Eller et al., 2001 ) and 3) the PCR primer ‘Am445’ (Gulledge et al., 2001 ).


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Table 4. Oligonucleotide probes tested in silico on the databases created in this study

 
Summary
Molecular phylogenies constructed from 16S rDNA (and pmoA) sequences identified monophyletic clusters that could be correlated to morphologically recognizable groups of type II MOB: Methylosinus trichosporium, Methylosinus sporium and Methylocystis spp. However, Methylocystis echinoides could not be clearly distinguished from Methylocystis parvus and other morphotypes of Methylocystis based on molecular phylogeny. Sequence phylogeny based on pmoA sequences correlated well to sequence phylogeny based on 16S rDNA sequences, and pmoA sequences can therefore be used to identify type II MOB with some certainty to the species level. The mmoX and mxaF genes were less suitable than pmoA as phylogenetic markers at this scale; mmoX because it was detectable in few Methylocystis strains, and mxaF because it showed evidence of horizontal gene transfer.

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.


   ACKNOWLEDGEMENTS
 
Part of the funding for this project was provided by the Bundesministerium für Bildung und Forschung under the program ‘Biodiversity and Global Change’ (Biolog). We thank Nina Ringleff for excellent technical assistance, Svetlana Dedysh for communications services and Peter Frenzel for samples.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Received 20 February 2002; revised 18 April 2002; accepted 14 May 2002.