Detection and enumeration of methanotrophs in acidic Sphagnum peat by 16S rRNA fluorescence in situ hybridization, including the use of newly developed oligonucleotide probes for Methylocella palustris.

Two 16S rRNA-targeted oligonucleotide probes, Mcell-1026 and Mcell-181, were developed for specific detection of the acidophilic methanotroph Methylocella palustris using fluorescence in situ hybridization (FISH). The fluorescence signal of probe Mcell-181 was enhanced by its combined application with the oligonucleotide helper probe H158. Mcell-1026 and Mcell-181, as well as 16S rRNA oligonucleotide probes with reported group specificity for either type I methanotrophs (probes M-84 and M-705) or the Methylosinus/Methylocystis group of type II methanotrophs (probes MA-221 and M-450), were used in FISH to determine the abundance of distinct methanotroph groups in a Sphagnum peat sample of pH 4.2. M. palustris was enumerated at greater than 10(6) cells per g of peat (wet weight), while the detectable population size of type I methanotrophs was three orders of magnitude below the population level of M. palustris. The cell counts with probe MA-221 suggested that only 10(4) type II methanotrophs per g of peat (wet weight) were present, while the use of probe M-450 revealed more than 10(6) type II methanotroph cells per g of the same samples. This discrepancy was due to the fact that probe M-450 targets almost all currently known strains of Methylosinus and Methylocystis, whereas probe MA-221, originally described as group specific, does not detect a large proportion of Methylocystis strains. The total number of methanotrophic bacteria detected by FISH was 3.0 (+/-0.2) x 10(6) cells per g (wet weight) of peat. This was about 0.8% of the total bacterial cell number. Thus, our study clearly suggests that M. palustris and a defined population of Methylocystis spp. were the predominant methanotrophs detectable by FISH in an acidic Sphagnum peat bog.

Detection of methanotroph diversity on roots of submerged rice plants by molecular retrieval of pmoA, mmoX, mxaF, and 16S rRNA and ribosomal DNA, including pmoA-based terminal restriction fragment length polymorphism profiling.

The diversity of methanotrophic bacteria associated with roots of submerged rice plants was assessed using cultivation-independent techniques. The research focused mainly on the retrieval of pmoA, which encodes the alpha subunit of the particulate methane monooxygenase. A novel methanotroph-specific community-profiling method was established using the terminal restriction fragment length polymorphism (T-RFLP) technique. The T-RFLP profiles clearly revealed a more complex root-associated methanotrophic community than did banding patterns obtained by pmoA-based denaturing gradient gel electrophoresis. The comparison of pmoA-based T-RFLP profiles obtained from rice roots and bulk soil of flooded rice microcosms suggested that there was a substantially higher abundance of type I methanotrophs on rice roots than in the bulk soil. These were affiliated to the genera Methylomonas, Methylobacter, Methylococcus, and to a novel type I methanotroph sublineage. By contrast, type II methanotrophs of the Methylocystis-Methylosinus group could be detected with high relative signal intensity in both soil and root compartments. Phylogenetic treeing analyses and a set of substrate-diagnostic amino acid residues provided evidence that a novel pmoA lineage was detected. This branched distinctly from all currently known methanotrophs. To examine whether the retrieval of pmoA provided a complete view of root-associated methanotroph diversity, we also assessed the diversity detectable by recovery of genes coding for subunits of soluble methane monooxygenase (mmoX) and methanol dehydrogenase (mxaF). In addition, both 16S rRNA and 16S ribosomal DNA (rDNA) were retrieved using a PCR primer set specific to type I methanotrophs. The overall methanotroph diversity detected by recovery of mmoX, mxaF, and 16S rRNA and 16S rDNA corresponded well to the diversity detectable by retrieval of pmoA.

Hydrogenophilus hirschii sp. nov., a novel thermophilic hydrogen-oxidizing beta-proteobacterium isolated from Yellowstone National Park.

A novel thermophilic hydrogen-oxidizing bacterium, Hydrogenophilus hirschii Yel5aT (= DSM 11420T = JCM 10831T) has been isolated from the Angel Terrace Spring, Yellowstone National Park. The isolate was rod-shaped (1.0-1.5 x 0.8 microm) with a polarly inserted flagellum. Cells grew chemolithoautotrophically under an atmosphere of H2 and CO2 (80:20) in the presence of low concentrations of O2 (optimum 2.5%). Organotrophic growth occurred on complex organic substrates such as yeast extract and peptone and on organic acids. Carbohydrates and amino acids were not utilized. The strain grew between 50 and 67 degrees C; optimal growth occurred at a temperature of 63 degrees C. The pH optimum was 6.5. NaCl inhibited growth at concentrations higher than 1.5%. The major respiratory lipoquinone was ubiquinone-8. Analysis of fatty acids of Yel5aT revealed a straight-chain saturated C16:0 as the major component followed by cyclo C17:0 and cyclo C19:0. The G+C content of total DNA was 61 mol%. Phylogenetic analysis placed the strain in the beta-proteobacteria. The 16S rDNA sequence of strain Yel5aT was related to that of Hydrogenophilus thermoluteolus. To our knowledge, Hydrogenophilus hirschii is the most thermophilic micro-organism found within the proteobacteria that grows in the temperature range 50-68 degrees C.

Novel bacterial lineages at the (sub)division level as detected by signature nucleotide-targeted recovery of 16S rRNA genes from bulk soil and rice roots of flooded rice microcosms.

Using a newly developed 16S rRNA gene (rDNA)-targeted PCR assay with proposed group specificity for planctomycetes, we examined anoxic bulk soil of flooded rice microcosms for the presence of novel planctomycete-like diversity. For comparison, oxic rice roots were included as an additional sample in this investigation. The bacterial diversity detectable by this PCR assay was assessed by using a combined approach that included terminal restriction fragment length polymorphism (T-RFLP) analysis and comparative sequence analysis of cloned 16S rDNA. T-RFLP fingerprint patterns generated from rice roots contained 12 distinct terminal restriction fragments (T-RFs). In contrast, the T-RFLP fingerprint patterns obtained from the anoxic bulk soil contained 33 distinct T-RFs, a clearly higher level of complexity. A survey of 176 bulk soil 16S rDNA clone sequences permitted correlation of 20 T-RFs with phylogenetic information. The other 13 T-RFs remained unidentified. The predominant T-RFs obtained from rice roots could be assigned to members of the genus Pirellula within the Planctomycetales, while most of the T-RFs obtained from the bulk soil corresponded to novel lines of bacterial descent. Using a level of 16S rDNA sequence dissimilarity to cultured microorganisms of approximately 20% as a threshold value, we detected 11 distinct bacterial lineages for which pure-culture representatives are not known. Four of these lineages could be assigned to the order Planctomycetales, while one lineage was affiliated with the division Verrucomicrobia and one lineage was affiliated with the spirochetes. The other five lineages either could not be assigned to any of the main lines of bacterial descent or clearly expanded the known diversity of division level lineages WS3 and OP3. Our results indicate the presence of bacterial diversity at a subdivision and/or division level that has not been detected previously by the so-called universal 16S rDNA PCR assays.

Opitutus terrae gen. nov., sp. nov., to accommodate novel strains of the division 'Verrucomicrobia' isolated from rice paddy soil.

Three strains of obligately anaerobic bacteria were isolated from rice paddy soil microcosms. Comparative analysis of the 16S rRNA genes showed that these novel isolates have identical gene sequences and are members of the division 'Verrucomicrobia'. The novel strains are phenotypically and phylogenetically distinct from species described previously. One strain, PB90-1T, was characterized in more detail. The cells are cocci and are motile by means of a flagellum. Catalase and oxidase activities are absent. Growth-supporting substrates include mono-, di- and polysaccharides, while alcohols, amino acids and organic acids do not support growth. Propionate and acetate are the major end-products of fermentation. Nitrate is reduced to nitrite, but other external electron acceptors are not utilized. The G+C content of the genomic DNA is 74 mol%. This strain represents a taxon that has not yet been formally recognized, for which the name Opitutus terrae gen. nov., sp. nov. is proposed. The type strain is PB90-1T (= DSM 11246T).

A novel pmoA lineage represented by the acidophilic methanotrophic bacterium Methylocapsa acidiphila [correction of acidophila] B2.

A fragment of the functional gene pmoA, which encodes the active-site polypeptide of particulate methane monooxygenase (pMMO), was PCR-amplified from DNA of the recently described acidophilic methanotrophic bacterium Methylocapsa acidiphila [corrected] B2. This methanotroph was isolated from an acidic Sphagnum peat bog and possesses a novel type III arrangement of intracytoplasmic membranes. Comparative sequence analysis revealed that the inferred peptide sequence of pmoA of Methylocapsa acidiphila [corrected] B2 belongs to a novel PmoA lineage. This lineage was only distantly related to the PmoA sequence cluster of type II methanotrophs, with identity values between 69.5% and 72%. The identity values between the PmoA of Methylocapsa acidiphila [corrected] B2 and PmoA sequences of type I methanotrophs ranged from 55.5 to 68%. However, the PmoA of this acidophilic methanotroph was more closely affiliated with the inferred peptide sequences of pmoA clones retrieved from various acidic upland soils showing atmospheric methane consumption. The PmoA identity values with these clones were 79.5-81%. Nonetheless, the apparent affinity for methane exhibited by Methylocapsa acidiphila [corrected] B2 was 1-2 microM, which is similar to values measured in other methanotrophic bacteria. This finding suggests that the pMMO of Methylocapsa acidiphila [corrected] B2 is not a novel enzyme specialized to have a high affinity for methane and that apparent "high-affinity" methane uptake is either the result of particular culture conditions or is performed by another pMMO type.

Microbiology of flooded rice paddies.

Flooded rice paddies are one of the major biogenic sources of atmospheric methane. Apart from this contribution to the 'greenhouse' effect, rice paddy soil represents a suitable model system to study fundamental aspects of microbial ecology, such as diversity, structure, and dynamics of microbial communities as well as structure-function relationships between microbial groups. Flooded rice paddy soil can be considered as a system with three compartments (oxic surface soil, anoxic bulk soil, and rhizosphere) characterized by different physio-chemical conditions. After flooding, oxygen is rapidly depleted in the bulk soil. Anaerobic microorganisms, such as fermentative bacteria and methanogenic archaea, predominate within the microbial community, and thus methane is the final product of anaerobic degradation of organic matter. In the surface soil and the rhizosphere well-defined microscale chemical gradients can be measured. The oxygen profile seems to govern gradients of other electron acceptors (e.g., nitrate, iron(III), and sulfate) and reduced compounds (e.g., ammonium, iron(II), and sulfide). These gradients provide information about the activity and spatial distribution of functional groups of microorganisms. This review presents the current knowledge about the highly complex microbiology of flooded rice paddies. In Section 2 we describe the predominant microbial groups and their function with particular regard to bacterial populations utilizing polysaccharides and simple sugars, and to the methanogenic archaea. Section 3 describes the spatial and temporal development of microscale chemical gradients measured in experimentally defined model systems, including gradients of oxygen and dissolved and solid-phase iron(III) and iron(II). In Section 4, the results of measurements of microscale gradients of oxygen, pH, nitrate-nitrite, and methane in natural rice fields and natural rice soil cores taken to the laboratory will be presented. Finally, perspectives of future research are discussed (Section 5).

Use of the T-RFLP technique to assess spatial and temporal changes in the bacterial community structure within an agricultural soil planted with transgenic and non-transgenic potato plants.

The aim of this study was to examine whether the terminal restriction fragment length polymorphism (T-RFLP) analysis represents an appropriate technique for monitoring highly diverse soil bacterial communities, i.e. to assess spatial and/or temporal effects on bacterial community structure. The T-RFLP method, a recently described fingerprinting technique, is based on terminal restriction fragment length polymorphisms between distinct small-subunit rRNA gene sequence types. This technique permits an automated quantification of the fluorescence signal intensities of the individual terminal restriction fragments (T-RFs) in a given community fingerprint pattern. The indigenous bacterial communities of three soil plots located within an agricultural field of 110 m(2) were compared. The first site was planted with non-transgenic potato plants, while the other two were planted with transgenic GUS and Barnase/Barstar potato plants, respectively. Once prior to planting and three times after planting, seven parallel samples were taken from each of the three soil plots. The T-RFLP analysis resulted in very complex but highly reproducible community fingerprint patterns. The percentage abundance values of defined T-RFs were calculated for the seven parallel samples of the respective soil plot. A multivariate analysis of variance was used to test T-RFLP data sets for significant differences. The statistical treatments clearly revealed spatial and temporal effects, as well as spacextime interaction effects, on the structural composition of the bacterial communities. T-RFs which showed the highest correlations to the discriminant factors were not those T-RFs which showed the largest single variations between the seven-sample means of individual plots. In summary, the T-RFLP technique, although a polymerase chain reaction-based method, proved to be a suitable technique for monitoring highly diverse soil microbial communities for changes over space and/or time.

Methylocella palustris gen. nov., sp. nov., a new methane-oxidizing acidophilic bacterium from peat bogs, representing a novel subtype of serine-pathway methanotrophs.

A new genus, Methylocella, and a new species, Methylocella palustris, are proposed for three strains of methane-oxidizing bacteria isolated from acidic Sphagnum peat bogs. These bacteria are aerobic, Gram-negative, colourless, non-motile, straight and curved rods that utilize the serine pathway for carbon assimilation, multiply by normal cell division and contain intracellular poly-beta-hydroxybutyrate granules (one at each pole). These strains use methane and methanol as sole sources of carbon and energy and are moderately acidophilic organisms with growth between pH 4.5 and pH 7.0, the optimum being at pH 5.0-5.5. The temperature range for growth is 10-28 degrees C with the optimum at 15-20 degrees C. The intracytoplasmic membrane system is different from those of type I and II methanotrophs. Cells contain an extensive periplasmic space and a vesicular membrane system connected to the cytoplasmic membrane. The strains grew only on media with a low salt content (0.2-0.5 g l(-1)). All three strains were found to possess soluble methane monooxygenase and are able to fix atmospheric nitrogen via an oxygen-sensitive nitrogenase. No products were observed in a PCR with particulate methane monooxygenase-targeted primers; hybridization with a pmoA probe was also negative. The major phospholipid fatty acids are 18:1 acids. The G+C content of the DNA is 61.2 mol%. The three strains share identical 16S rRNA gene sequences and represent a novel lineage of methane-oxidizing bacteria within the alpha-subclass of the class Proteobacteria and are only moderately related to type II methanotrophs of the Methylocystis-Methylosinus group. The three strains are most closely related to the acidophilic heterotrophic bacterium Beijerinckia indica subsp. indica (96.5% 16S rDNA sequence similarity). Collectively, these strains comprise a new species and genus Methylocella palustris gen. nov., sp. nov.; strain KT (= ATCC 700799T) is the type strain.

Identification of major subgroups of ammonia-oxidizing bacteria in environmental samples by T-RFLP analysis of amoA PCR products.

A cloning-independent method based on T-RFLP (terminal restriction fragment length polymorphism) analysis of amoA PCR products was developed to identify major subgroups of autotrophic ammonia oxidizers of the beta-subclass of the class Proteobacteria in total community DNA. Based on a database of 28 partial gene sequences encoding the active-site polypeptide of ammonia monooxygenase (amoA), defined lengths of terminal restriction fragments (= operational taxonomic units, OTUs) of amoA were predicted to correlate in TaqI-based T-RFLP analysis with phylogenetically defined subgroups of ammonia oxidizers. Members of the genus Nitrosospira showed a specific OTU of 283 bp in length, while a fragment size of 219 bp was indicative of Nitrosomonas-like sequence types including N. europaea, N. eutropha, and N. halophila. Two amoA sequence clusters designated previously as the lineages 'PluBsee' and 'Schöhsee' [Rotthauwe, J.-H., Witzel, K.-P., Liesack, W., 1997. Appl. Environ. Microbiol. 63, 4704-4712] shared a TaqI-based OTU with a fragment size of 48 bp, but sequence types of these two lineages could be differentiated by AluI-based T-RFLP analysis. A survey of various environmental samples and enrichment cultures by T-RFLP analysis and by comparative analysis of cloned amoA sequences confirmed the predicted correlations between distinct OTUs and phylogenetic information. Our data suggest that amoA-based T-RFLP analysis is a reliable tool to rapidly assess the complexity of ammonia-oxidizing communities in environmental samples with respect to the presence of major subgroups, i.e. nitrosospiras versus nitrosomonads.

Spatial changes in the bacterial community structure along a vertical oxygen gradient in flooded paddy soil cores.

Molecular ecology techniques were applied to assess changes in the bacterial community structure along a vertical oxygen gradient in flooded paddy soil cores. Microsensor measurements showed that oxygen was depleted from 140 microM at the floodwater/soil interface to nondetectable amounts at a depth of approximately 2.0 mm and below. Bacterial 16S rRNA gene (rDNA)-based community fingerprint patterns were obtained from 200-microm-thick soil slices of both the oxic and anoxic zones by using the T-RFLP (terminal restriction fragment length polymorphism) technique. The fingerprints revealed a tremendous shift in the community patterns in correlation to the oxygen depletion measured with depth. 16S rDNA clone sequences recovered from the oxic or anoxic zone directly corresponded to those terminal restriction fragments which were highly characteristic of the respective zone. Comparative sequence analysis of these clones identified members of the alpha and beta subclasses of Proteobacteria as the abundant populations in the oxic zone. In contrast, members of clostridial cluster I were determined to be the predominant bacterial group in the oxygen-depleted soil. The extraction of total RNA followed by reverse transcription-PCR of the bacterial 16S rRNA and T-RFLP analysis resulted for both oxic and anoxic zones of flooded soil cores in community fingerprint patterns similar to those obtained by the rDNA-based analysis. This finding suggests that the microbial groups detected on the rDNA level are the metabolically active populations within the oxic and anoxic soil slices examined.

Characterization and identification of numerically abundant culturable bacteria from the anoxic bulk soil of rice paddy microcosms.

Most-probable-number (liquid serial dilution culture) counts were obtained for polysaccharolytic and saccharolytic fermenting bacteria in the anoxic bulk soil of flooded microcosms containing rice plants. The highest viable counts (up to 2.5 x 10(8) cells per g [dry weight] of soil) were obtained by using xylan, pectin, or a mixture of seven mono- and disaccharides as the growth substrate. The total cell count for the soil, as determined by using 4', 6-diamidino-2-phenylindole staining, was 4.8 x 10(8) cells per g (dry weight) of soil. The nine strains isolated from the terminal positive tubes in counting experiments which yielded culturable populations that were equivalent to about 5% or more of the total microscopic count population belonged to the division Verrucomicrobia, the Cytophaga-Flavobacterium-Bacteroides division, clostridial cluster XIVa, clostridial cluster IX, Bacillus spp., and the class Actinobacteria. Isolates originating from the terminal positive tubes of liquid dilution series can be expected to be representatives of species whose populations in the soil are large. None of the isolates had 16S rRNA gene sequences identical to 16S rRNA gene sequences of previously described species for which data are available. Eight of the nine strains isolated fermented sugars to acetate and propionate (and some also fermented sugars to succinate). The closest relatives of these strains (except for the two strains of actinobacteria) were as-yet-uncultivated bacteria detected in the same soil sample by cloning PCR-amplified 16S rRNA genes (U. Hengstmann, K.-J. Chin, P. H. Janssen, and W. Liesack, Appl. Environ. Microbiol. 65:5050-5058, 1999). Twelve other isolates, which originated from most-probable-number counting series indicating that the culturable populations were smaller, were less closely related to cloned 16S rRNA genes.

Comparative phylogenetic assignment of environmental sequences of genes encoding 16S rRNA and numerically abundant culturable bacteria from an anoxic rice paddy soil.

We used both cultivation and direct recovery of bacterial 16S rRNA gene (rDNA) sequences to investigate the structure of the bacterial community in anoxic rice paddy soil. Isolation and phenotypic characterization of 19 saccharolytic and cellulolytic strains are described in the accompanying paper (K.-J. Chin, D. Hahn, U. Hengstmann, W. Liesack, and P. H. Janssen, Appl. Environ. Microbiol. 65:5042-5049, 1999). Here we describe the phylogenetic positions of these strains in relation to 57 environmental 16S rDNA clone sequences. Close matches between the two data sets were obtained for isolates from the culturable populations determined by the most-probable-number counting method to be large (3 x 10(7) to 2.5 x 10(8) cells per g [dry weight] of soil). This included matches with 16S rDNA similarity values greater than 98% within distinct lineages of the division Verrucomicrobia (strain PB90-1) and the Cytophaga-Flavobacterium-Bacteroides group (strains XB45 and PB90-2), as well as matches with similarity values greater than 95% within distinct lines of descent of clostridial cluster XIVa (strain XB90) and the family Bacillaceae (strain SB45). In addition, close matches with similarity values greater than 95% were obtained for cloned 16S rDNA sequences and bacteria (strains DR1/8 and RPec1) isolated from the same type of rice paddy soil during previous investigations. The correspondence between culture methods and direct recovery of environmental 16S rDNA suggests that the isolates obtained are representative geno- and phenotypes of predominant bacterial groups which account for 5 to 52% of the total cells in the anoxic rice paddy soil. Furthermore, our findings clearly indicate that a dual approach results in a more objective view of the structural and functional composition of a soil bacterial community than either cultivation or direct recovery of 16S rDNA sequences alone.

Vertical distribution of methanogens in the anoxic sediment of Rotsee (Switzerland).

Anoxic sediments from Rotsee (Switzerland) were analyzed for the presence and diversity of methanogens by using molecular tools and for methanogenic activity by using radiotracer techniques, in addition to the measurement of chemical profiles. After PCR-assisted sequence retrieval of the 16S rRNA genes (16S rDNA) from the anoxic sediment of Rotsee, cloning, and sequencing, a phylogenetic analysis identified two clusters of sequences and four separated clones. The sequences in cluster 1 grouped with those of Methanosaeta spp., whereas the sequences in cluster 2 comprised the methanogenic endosymbiont of Plagiopyla nasuta. Discriminative oligonucleotide probes were constructed against both clusters and two of the separated clones. These probes were used subsequently for the analysis of indigenous methanogens in a core of the sediment, in addition to domain-specific probes against members of the domains Bacteria and Archaea and the fluorescent stain 4', 6-diamidino-2-phenylindole (DAPI), by fluorescent in situ hybridization. After DAPI staining, the highest microbial density was obtained in the upper sediment layer; this density decreased with depth from (1.01 +/- 0.25) x 10(10) to (2.62 +/- 0.58) x 10(10) cells per g of sediment (dry weight). This zone corresponded to that of highest metabolic activity, as indicated by the ammonia, alkalinity, and pH profiles, whereas the methane profile was constant. Probes Eub338 and Arch915 detected on average 16 and 6% of the DAPI-stained cells as members of the domains Bacteria and Archaea, respectively. Probe Rotcl1 identified on average 4% of the DAPI-stained cells as Methanosaeta spp., which were present throughout the whole core. In contrast, probe Rotcl2 identified only 0.7% of the DAPI-stained cells as relatives of the methanogenic endosymbiont of P. nasuta, which was present exclusively in the upper 2 cm of the sediment. Probes Rotp13 and Rotp17 did not detect any cells. The spatial distribution of the two methanogenic populations corresponded well to the methane production rates determined by incubation with either [14C]acetate or [14C]bicarbonate. Methanogenesis from acetate accounted for almost all of the total methane production, which concurs with the predominance of acetoclastic Methanosaeta spp. that represented on average 91% of the archaeal population. Significant hydrogenotrophic methanogenesis was found only in the organically enriched upper 2 cm of the sediment, where the probably hydrogenotrophic relatives of the methanogenic endosymbiont of P. nasuta, accounting on average for 7% of the archaeal population, were also detected.

Desulfovibrio zosterae sp. nov., a new sulfate reducer isolated from surface-sterilized roots of the seagrass Zostera marina.

A sulfate-reducing bacterium, designated strain lacT, was isolated from surface-sterilized roots of the benthic macrophyte Zostera marina. Cells were motile by means of a single polar flagellum. Strain lacT utilized lactate, pyruvate, malate, ethanol, L-alanine, fumarate, choline and fructose with sulfate as electron acceptor. In addition, fumarate, pyruvate and fructose were also degraded without an external electron acceptor. Sulfate could be substituted with thiosulfate, sulfite and elemental sulfur. Optimal growth was observed between 32.5 and 34.5 degrees C, at an NaCl concentration of 0.2 M and in a pH range between 6.8 and 7.3. The G + C content of the DNA was 42.7 +/- 0.2 mol%. Desulfoviridin and catalase were present. Strain lacT contained c-type cytochromes. Comparative 16S rRNA gene sequence analysis and the fatty acid pattern grouped this isolate into the genus Desulfovibrio. However, strain lacT differs from all other described Desulfovibrio species on the bases of its 16S rRNA gene sequence, the G + C content, its cellular lipid pattern and the utilization pattern of substrates. These characteristics establish strain lacT (= DSM 11974T) as a novel species of the genus Desulfovibrio, for which the name Desulfovibrio zosterae sp. nov. is proposed.

High-affinity methane oxidation by a soil enrichment culture containing a type II methanotroph.

Methanotrophic bacteria in an organic soil were enriched on gaseous mixing ratios of <275 parts per million of volume (ppmv) of methane (CH4). After 4 years of growth and periodic dilution (>10(20) times the initial soil inoculum), a mixed culture was obtained which displayed an apparent half-saturation constant [Km(app)] for CH4 of 56 to 186 nM (40 to 132 ppmv). This value was the same as that measured in the soil itself and about 1 order of magnitude lower than reported values for pure cultures of methane oxidizers. However, the Km(app) increased when the culture was transferred to higher mixing ratios of CH4 (1,000 ppmv, or 1%). Denaturing gradient gel electrophoresis of the enrichment grown on <275 ppmv of CH4 revealed a single gene product of pmoA, which codes for a subunit of particulate methane monooxygenase. This suggested that only one methanotroph species was present. This organism was isolated from a sample of the enrichment culture grown on 1% CH4 and phylogenetically positioned based on its 16S rRNA, pmoA, and mxaF gene sequences as a type II strain of the Methylocystis/Methylosinus group. A coculture of this strain with a Variovorax sp., when grown on <275 ppmv of CH4, had a Km(app) (129 to 188 nM) similar to that of the initial enrichment culture. The data suggest that the affinity of methanotrophic bacteria for CH4 varies with growth conditions and that the oxidation of atmospheric CH4 observed in this soil is carried out by type II methanotrophic bacteria which are similar to characterized species.

Methanogenic archaea and CO2-dependent methanogenesis on washed rice roots.

Washed excised roots of rice (Oryza sativa) immediately started to produce CH4 when they were incubated in phosphate buffer under anoxic conditions (N2 atmosphere), with initial rates varying between 2 and 70nmolh(-1)g(-1) dry weight of root material (mean +/- SE: 20.3 +/- 5.9 nmol h(-1) g(-1) dry weight; n = 18). Production of CH4 continued for at least 500 h, with rates usually decreasing slowly. CH4 production was not significantly affected by methyl fluoride, an inhibitor of acetoclastic methanogenesis. Less than 0.5% of added [2-14C]-acetate was converted to 14CH4, and conversion of 14CO2 to 14CH4 indicated that CH4 was almost exclusively produced from CO2. Occasionally, however, especially when the roots were incubated without additional buffer, CH4 production started to accelerate after about 200h reaching rates of > 100 nmol h(-1) g(-1) dry weight. Methyl fluoride inhibited methanogenesis by more than 20% only in these cases, and the conversion of 14CO2 to 14CH4 decreased. These results indicate that CO2-dependent rather than acetoclastic methanogenesis was primarily responsible for CH4 production in anoxically incubated rice roots. Determination of most probable numbers of methanogens on washed roots showed highest numbers (10(6)g(-1) dry roots) on H2 and ethanol, i.e. substrates that support CH4 production from CO2. Numbers on acetate (10(5) g(-1) dry roots) and methanol (10(4)g(-1) dry roots) were lower. Methanogenic consortia enriched on H2 and ethanol were characterized phylogenetically by comparative sequence analysis of archaeal small-subunit (SSU) ribosomal RNA-encoding genes (rDNA). These sequences showed a high similarity to SSU rDNA clones that had been obtained previously by direct extraction of total DNA from washed rice roots. The SSU rDNA sequences recovered from the H2/CO2-using consortium either belonged to a novel lineage of methanogens that grouped within the phylogenetic radiation of the Methanosarcinales and Methanomicrobiales or were affiliated with Methanobacterium bryantii. SSU rDNA sequences retrieved from the ethanol-using consortium either grouped within the genus Methanosarcina or belonged to another novel lineage within the phylogenetic radiation of the Methanosarcinales and Methanomicrobiales. Cultured organisms belonging to either of the two novel lineages have not been reported yet.

Novel euryarchaeotal lineages detected on rice roots and in the anoxic bulk soil of flooded rice microcosms

Because excised, washed roots of rice (Oryza sativa) immediately produce CH4 when they are incubated under anoxic conditions (P. Frenzel and U. Bosse, FEMS Microbiol. Ecol. 21:25-36, 1996), we employed a culture-independent molecular approach to identify the methanogenic microbial community present on roots of rice plants. Archaeal small-subunit rRNA-encoding genes were amplified directly from total root DNA by PCR and then cloned. Thirty-two archaeal rice root (ARR) gene clones were randomly selected, and the amplified primary structures of ca. 750 nucleotide sequence positions were compared. Only 10 of the environmental sequences were affiliated with known methanogens; 5 were affiliated with Methanosarcina spp., and 5 were affiliated with Methanobacterium spp. The remaining 22 ARR gene clones formed four distinct lineages (rice clusters I through IV) which were not closely related to any known cultured member of the Archaea. Rice clusters I and II formed distinct clades within the phylogenetic radiation of the orders "Methanosarcinales" and Methanomicrobiales. Rice cluster I was novel, and rice cluster II was closely affiliated with environmental sequences obtained from bog peat in northern England. Rice cluster III occurred on the same branch as Thermoplasma acidophilum and marine group II but was only distantly related to these taxa. Rice cluster IV was a deep-branching crenarchaeotal assemblage that was closely related to clone pGrfC26, an environmental sequence recovered from a temperate marsh environment. The use of a domain-specific oligonucleotide probe in a fluorescent in situ hybridization analysis revealed that viable members of the Archaea were present on the surfaces of rice roots. In addition, we describe a novel euryarchaeotal main line of descent, designated rice cluster V, which was detected in anoxic rice paddy soil. These results indicate that there is an astonishing richness of archaeal diversity present on rice roots and in the surrounding paddy soil.

Isolation of acidophilic methane-oxidizing bacteria from northern peat wetlands.

Acidic northern wetlands are an important source of methane, one of the gases that contributes to global warming. Methane oxidation in the surface of these acidic wetlands can reduce the methane flux to the atmosphere up to 90 percent. Here the isolation of three methanotrophic microorganisms from three boreal forest sites is reported. They are moderately acidophilic organisms and have a soluble methane monooxygenase. In contrast to the known groups of methanotrophs, 16S ribosomal DNA sequence analysis shows that they are affiliated with the acidophilic heterotrophic bacterium Beijerinckia indica subsp. indica.

Tetramethylammonium:coenzyme M methyltransferase system from methanococcoides sp

A methanogen (strain NaT1) that belongs to the family of Methanosarcinaceae and that can grow on tetramethylammonium as the sole energy source has recently been isolated. We report here that cell extracts of the archaeon catalyze the formation of methyl-coenzyme M from coenzyme M and tetramethylammonium. The activity was dependent on the presence of Ti(III) citrate and ATP, and was rapidly lost under oxic conditions. Anoxic chromatography on DEAE-Sepharose revealed that two fractions, fractions 3 and 4, were required for activity. A 50-kDa protein that together with fraction 3 catalyzed methyl-coenzyme M formation from tetramethylammonium and coenzyme M was purified from fraction 4. From fraction 3, a 22-kDa corrinoid protein and a 40-kDa protein exhibiting methylcobalamin:coenzyme M methyltransferase (MT2) activity were purified. The N-terminal amino acid sequences of these purified proteins were determined. The 40-kDa protein showed sequence similarity to MT2 isoenzymes from Methanosarcina barkeri. Cell extract of strain NaT1 grown on trimethylamine rather than on tetramethylammonium did not exhibit tetramethylammonium:coenzyme M methyltransferase activity. The strain was identified as belonging to the genus of Methanococcoides, its closest relative being Methanococcoides methylutens.