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At least 19 recordsLinked to original sources
PubMed2001

Changes in activity and community structure of methane-oxidizing bacteria over the growth period of rice.

The activity and community structure of methanotrophs in compartmented microcosms were investigated over the growth period of rice plants. In situ methane oxidation was important only during the vegetative growth phase of the plants and later became negligible. The in situ activity was not directly correlated with methanotrophic cell counts, which increased even after the decrease in in situ activity, possibly due to the presence of both vegetative cells and resting stages. By dividing the microcosms into two soil and two root compartments it was possible to locate methanotrophic growth and activity, which was greatest in the rhizoplane of the rice plants. Molecular analysis by denaturing gradient gel electrophoresis and fluorescent in situ hybridization (FISH) with family-specific probes revealed the presence of both families of methanotrophs in soil and root compartments over the whole season. Changes in community structure were detected only for members of the Methylococcaceae and could be associated only with changes in the genus Methylobacter and not with changes in the dominance of different genera in the family Methylococcaceae. For the family Methylocystaceae stable communities in all compartments for the whole season were observed. FISH analysis revealed evidence of in situ dominance of the Methylocystaceae in all compartments. The numbers of Methylococcaceae cells were relatively high only in the rhizoplane, demonstrating the importance of rice roots for growth and maintenance of methanotrophic diversity in the soil.

Colony Count, Microbial↗
PubMed2000

Evolution of Bacterial Diversity during Enrichment of PCP-Degrading Activated Soils.

The microbiota of completely mixed soil slurry was acclimated with pentachlorophenol (PCP) or with a wood preservative mixture (WPM) containing several pollutants such as PCP and petroleum hydrocarbons. The impact of these compounds on the bacterial diversity was studied by using molecular tools. PCR amplifications of the 16S ribosomal RNA gene sequences (rDNA) were carried out with total DNA extracted from soil slurry samples taken at different time points during the enrichment process of the PCP and WPM reactors. The composition of these PCR products, reflecting the bacterial diversity, was monitored by the single-strand-conformation polymorphism (SSCP) method. Our results showed that the complexity of the SSCP profiles in the PCP reactor decreased significantly during the enrichment process, whereas they remained complex in the WPM reactor. PCR-amplified 16S rDNA libraries were generated from each reactor. The SSCP method was used to rapidly screen several clones of these libraries to find specific single-strand DNA migration profiles. In the PCP-activated soil, 96% of examined clones had the same SSCP profile, and sequences of representative clones were related to the genus Sphingomonas, suggesting that the enrichment with PCP resulted in a selection of little phylogenetic diversity. Four different SSCP profiles were observed with the 68 examined clones from the WPM reactor. Representative clones of these profiles were related to Methylocystaceae or Rhizobiaceae, to sulfur-oxidizing symbionts, to the genus Acinetobacter, and to the genus Sphingomonas. We also cloned and sequenced PCR-amplified DNA related to the pcpB gene, coding for the Sphingomonas PCP-4-monooxygenase and detected in both reactors after two weeks of enrichment. Of the 16 examined clones, deduced amino acid sequences of 13 clones were highly related to the Sphingomonas sp. strain UG30 pcpB. The three remaining pcpB clones were not closely related to the three known Sphingomonas pcpB.

Journal Article↗
PubMed2001

Group-specific 16S rRNA targeted probes for the detection of type I and type II methanotrophs by fluorescence in situ hybridisation.

The study of methane-oxidising bacteria (methanotrophs) is of special interest, because of their role in the natural reduction of methane emissions from many different sources. Therefore new probes were developed to detect specifically either type I (Methylococcaceae) or type II methanotrophs (Methylocystaceae). The probes have shown high specificity in fluorescence in situ hybridisations (FISH), as demonstrated by parallel hybridisation of target and reference strains as well as sequence data analysis. With these probes, methanotrophs were detected in soil and root samples from rice microcosms, demonstrating their applicability even in a complex environmental matrix.

Gram-Negative Aerobic Rods and Cocci↗
PubMed2001

Family- and genus-level 16S rRNA-targeted oligonucleotide probes for ecological studies of methanotrophic bacteria.

Methanotrophic bacteria play a major role in the global carbon cycle, degrade xenobiotic pollutants, and have the potential for a variety of biotechnological applications. To facilitate ecological studies of these important organisms, we developed a suite of oligonucleotide probes for quantitative analysis of methanotroph-specific 16S rRNA from environmental samples. Two probes target methanotrophs in the family Methylocystaceae (type II methanotrophs) as a group. No oligonucleotide signatures that distinguish between the two genera in this family, Methylocystis and Methylosinus, were identified. Two other probes target, as a single group, a majority of the known methanotrophs belonging to the family Methylococcaceae (type I/X methanotrophs). The remaining probes target members of individual genera of the Methylococcaceae, including Methylobacter, Methylomonas, Methylomicrobium, Methylococcus, and Methylocaldum. One of the family-level probes also covers all methanotrophic endosymbionts of marine mollusks for which 16S rRNA sequences have been published. The two known species of the newly described genus Methylosarcina gen. nov. are covered by a probe that otherwise targets only members of the closely related genus Methylomicrobium. None of the probes covers strains of the newly proposed genera Methylocella and "Methylothermus," which are polyphyletic with respect to the recognized methanotrophic families. Empirically determined midpoint dissociation temperatures were 49 to 57 degrees C for all probes. In dot blot screening against RNA from positive- and negative-control strains, the probes were specific to their intended targets. The broad coverage and high degree of specificity of this new suite of probes will provide more detailed, quantitative information about the community structure of methanotrophs in environmental samples than was previously available.

Alphaproteobacteria↗
PubMed2004

Isolation and characterisation of an isoproturon-mineralising Methylopila sp. TES from French agricultural soil.

Using enrichment culture three isoproturon (IPU) mineralising bacterial isolates were isolated from a French agricultural soil mineralising up to 50% of the initially added 14C-ring labelled IPU within only eight days. These isolates showed similar metabolic (BIOLOG GN) and amplified rDNA restriction (ARDRA) profiles. Partial 16S rDNA sequencing revealed that they were identical and identified as Methylopila sp TES. This strain harbours a large plasmid (220 kb) putatively bearing essential IPU-degrading genes as demonstrated by a curing experiment. Methylopila sp. TES transformed IPU and its known metabolites to CO2 and biomass but did not degrade chlorotoluron, monolinuron, diuron and linuron.

Agriculture↗
PubMed2005

Possibilities for controlling a PHB accumulation process using various analytical methods.

Poly-beta-hydroxybutyrate (PHB) and other polyesters can be produced by various species of bacteria. Of the possible carbon sources, methane could prove to be one of the most suitable substrates for the manufacture of PHB. The methanotrophic strain Methylocystis sp. GB 25 DSM 7674 was applied in order to accumulate PHB in a rapid, non-sterile process. Cultivation was performed in two stages: a continuous growth phase (dilution rate 0.17 h(-1)) and a PHB accumulation phase under deficiency conditions of an essential nutrient (e.g. phosphorus) in batch culture. The PHB content of the biomass was as high as 51%; efficiency was the highest during the first 5 h of the product formation process. The PHB produced is of very high quality, having a high molecular mass of up to 2.5 x 10(6) Da. In order to monitor and control the process, a rapid analysis method based upon turbidimetry in the visible range (438 nm) was applied. Moreover, the PHB content of the biomass was determined using an FT-IR-spectroscopic method with ATR sampling and multivariate calibration. We achieved a value of 1.4% as the best standard error of cross validation. The nitrogen content of the PHB final product (a product quality parameter) was estimated by spectroscopic method in the visible range.

Biomass↗
PubMed2003

Cometabolic degradation of chlorinated aromatic compounds.

The degradation of chlorobenzene was investigated with the specially chosen strain Methylocystis sp. GB 14 DSM 12955, using 23 ml headspace vials and in a soil column filled with quaternary aquifer material from a depth of 20 m. A long-term experiment was carried out in this column, situated in a mobile test unit at a contaminated location in Bitterfeld (Germany). Groundwater polluted by chlorobenzene was continuously fed through the column, through which a mixture comprising 4% CH(4) and 96% air was bubbled. Chlorobenzene was oxidized by up to 80% under pure culture conditions in the model experiments and was completely degraded under the mixed culture conditions of the column experiments. Over a period of 4 months, the stability of the biological system was monitored regularly by analyzing the sMMO activity as well as by classical microbiological and molecular biological methods.

Biodegradation, Environmental↗
PubMed2003

Amplification of marine methanotrophic enrichment DNA with 16S rDNA PCR primers for type II alpha proteobacteria methanotrophs.

Type II alpha proteobacteria methanotrophs are capable of a wide range of cometabolic transformations of chlorinated solvents and polycyclic aromatic hydrocarbons (PAHs), and this activity has been exploited in many terrestrial bioremediation systems. However, at present, all known obligately marine methanotrophic isolates are Type I gamma proteobacteria which do not have this activity to the extent of Type II methanotrophs. In previous work in our laboratory, determining the presence of Type II alpha proteobacteria methanotrophs in marine enrichment cultures that co-metabolized PAHs required a more sensitive assay. 16S rDNA PCR primers were designed based on oligonucleotide probes for serine pathway methanotrophs and serine pathway methylotrophs with an approximate amplification fragment size of 870 base pairs. Comparison of the primers using double primer BLAST searches in established nucleotide databases showed potential amplification with all Methylocystis and Methylosinus spp., as well as potential amplification with Methylocella palustrus. DNA from Methylosinus trichosporium OB3b, a Type II methanotroph, amplified with the primers with a fragment size of approximately 850 base pairs, whereas DNA extracted from Methylomonas methanica, a Type I methanotroph, did not. The primers were used to amplify DNA extracted from two marine methanotrophic enrichment cultures: a low nitrogen/low copper enrichment to select for Type II methanotrophs and a high nitrogen/high copper enrichment to select for Type I methanotrophs. Although DNA from both cultures amplified with the PCR primers, amplification was stronger in cultures that were specifically enriched for Type II methanotrophs, suggesting the presence of higher numbers of Type II methanotrophs. These results provide further evidence for the existence of Type II marine methanotrophs, suggesting the possibility of exploiting cometabolic activity in marine systems.

Biodegradation, Environmental↗
PubMed2006

Methylocystis rosea sp. nov., a novel methanotrophic bacterium from Arctic wetland soil, Svalbard, Norway (78 degrees N).

A Gram-negative, rod-shaped, non-motile, non-spore-forming, pink-pigmented bacterium, SV97T, was isolated from a wetland soil near Ny-Alesund, Svalbard Islands, Norway (78 degrees N). On the basis of 16S rRNA gene sequence similarity, strain SV97T was shown to belong to the Alphaproteobacteria and was highly related to a number of non-characterized Methylocystis strains with GenBank accession nos AJ458507 and AJ458502 (100 %) and AF177299, AJ458510, AJ458467, AJ458471, AJ431384, AJ458475, AJ458484, AJ458501 and AJ458466 (99 %). The most closely related type strains were Methylocystis parvus OBBP(T) (97.2 %) and Methylocystis echinoides IMET 10491T (97%). The closest related recognized species within the genus Methylosinus was Methylosinus sporium NCIMB 11126T (96.0% similarity). Chemotaxonomic and phenotypic data (C(18:1)omega8 as the major fatty acid, non-motile, no rosette formation) supported the affiliation of strain SV97T to the genus Methylocystis. The results of DNA-DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain SV97(T) from the two recognized Methylocystis species. Strain SV97T therefore represents a novel species, for which the name Methylocystis rosea sp. nov. is proposed, with the type strain SV97T (= DSM 17261T = ATCC BAA-1196T).

Arctic Regions↗
PubMed2006

Characterizing a stable methane-utilizing mixed culture used in the synthesis of a high-quality biopolymer in an open system.

AIMS: To characterize a methane-utilizing poly-beta-hydroxybutyrate (PHB)-producing microbial community. METHODS AND RESULTS: Three different approaches based on microbiology, analytical chemistry and molecular biology were used to determine the composition of the mixed culture. The dominant species, Methylocystis sp. GB25, represents more than 86% of the total biomass. Seven accompanying bacterial species are present in the mixed culture of which two are methylotrophic bacteria and five are utilizers of complex carbon sources. Both these groups were found to be present at the same ratio with respect to each other. Results of fatty acid analysis and PCR-DGGE fingerprints reflect the stability of the mixed-culture composition in the open system during multiple continuous growth and polymer formation processes throughout a period of 29 months. The consistently high quality of the accumulated polymer further corroborates this finding. CONCLUSION: The methane-utilizing mixed culture has the potential of self-regulation resulting in a stable composition even under non-aseptic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Avoiding the necessity of sterile conditions, as demonstrated in this paper, is an important step towards the development of a viable large-scale process for the production of PHB using cheap substrates like methane from natural or renewable sources. This is the first report characterizing a bacterial mixed culture being used for the biotechnological production of a high-value product in an open system.

Bacteria↗
PubMed2004

Molecular diversity of methanotrophs in Transbaikal soda lake sediments and identification of potentially active populations by stable isotope probing.

Soda lakes are an environment with an unusually high pH and often high salinity. To identify the active methanotrophs in the Soda lake sediments, sediment slurries were incubated with a 10% (v/v) (13)CH(4) headspace and the (13)C-labelled DNA was subsequently extracted from these sediments following CsCl density gradient centrifugation. This DNA was then used as a template for PCR amplification of 16S rRNA genes and genes encoding PmoA and MmoX of methane monooxygenase, key enzymes in the methane oxidation pathway. Phylogenetic analysis of 16S rRNA genes, PmoA and MmoX identified that strains of Methylomicrobium, Methylobacter, Methylomonas and 'Methylothermus' had assimilated the (13)CH(4). Phylogenetic analysis of PmoA sequences amplified from DNA extracted from Soda lake sediments before Stable Isotope Probing (SIP) treatment showed that a much wider diversity of both type I and type II methanotroph sequences are present in this alkaline environment. The majority of methanotroph sequences detected in the (13)C-DNA studies were from type I methanotrophs, with 50% of 16S rRNA clones and 100% of pmoA clones from both Lake Suduntuiskii Torom and Lake Gorbunka suggesting that the type I methanotrophs are probably responsible for the majority of methane oxidation in this environment.

Base Sequence↗
PubMed2006

Diversity of soluble methane monooxygenase-containing methanotrophs isolated from polluted environments.

Methanotrophs were enriched and isolated from polluted environments in Canada and Germany. Enrichments in low copper media were designed to specifically encourage growth of soluble methane monooxygenase (sMMO) containing organisms. The 10 isolates were characterized physiologically and genetically with one type I and nine type II methanotrophs being identified. Three key genes: 16S rRNA; pmoA and mmoX, encoding for the particulate and soluble methane monooxygenases respectively, were cloned from the isolates and sequenced. Phylogenetic analysis of these sequences identified strains, which were closely related to Methylococcus capsulatus, Methylocystis sp., Methylosinus sporium and Methylosinus trichosporium. Diversity of sMMO-containing methanotrophs detected in this and previous studies was rather narrow, both genetically and physiologically, suggesting possible constraints on genetic diversity of sMMO due to essential conservation of enzyme function.

Copper↗
PubMed2006

Nutrient amendments in soil DNA stable isotope probing experiments reduce the observed methanotroph diversity.

Stable isotope probing (SIP) can be used to analyze the active bacterial populations involved in a process by incorporating 13C-labeled substrate into cellular components such as DNA. Relatively long incubation times are often used with laboratory microcosms in order to incorporate sufficient 13C into the DNA of the target organisms. Addition of nutrients can be used to accelerate the processes. However, unnatural concentrations of nutrients may artificially change bacterial diversity and activity. In this study, methanotroph activity and diversity in soil was examined during the consumption of 13CH4 with three DNA-SIP experiments, using microcosms with natural field soil water conditions, the addition of water, and the addition of mineral salts solution. Methanotroph population diversity was studied by targeting 16S rRNA and pmoA genes. Clone library analyses, denaturing gradient gel electrophoresis fingerprinting, and pmoA microarray hybridization analyses were carried out. Most methanotroph diversity (type I and type II methanotrophs) was observed in non-amended SIP microcosms. Although this treatment probably best reflected the in situ environmental conditions, one major disadvantage of this incubation was that the incorporation of 13CH4 was slow and some cross-feeding of 13C occurred, thereby leading to labeling of nonmethanotroph microorganisms. Conversely, microcosms supplemented with mineral salts medium exhibited rapid consumption of 13CH4, resulting in the labeling of a less diverse population of only type I methanotrophs. DNA-SIP incubations using water-amended microcosms yielded faster incorporation of 13C into active methanotrophs while avoiding the cross-feeding of 13C.

Carbon Isotopes↗
PubMed2003

Wide distribution of a novel pmoA-like gene copy among type II methanotrophs, and its expression in Methylocystis strain SC2.

Experiments were conducted to determine if a novel pmoA-like gene (pmoA2) recently discovered in the methane-oxidizing bacterium Methylocystis strain SC2 (P. F. Dunfield, M. Tchawa Yimga, S. D. Dedysh, U. Berger, W. Liesack, and J. Heyer, FEMS Microbiol. Ecol. 41:17-26, 2002) is present in other methane-oxidizing bacteria (MOB), and if it is expressed. A newly developed primer combination (pmoA206f-pmoA703b) allowed a differential detection of pmoA1 and pmoA2. By using this primer combination, we identified pmoA2 in a wide range of type II MOB of the Methylosinus-Methylocystis group. However, screening by PCR and by Southern hybridization using a newly developed pmoA2-specific oligonucleotide probe also showed that closely related type II MOB, exhibiting 16S rRNA gene sequence identities of higher than 97%, may or may not harbor pmoA2. No pmoA2 was detected in five type I MOB tested: Methylococcus capsulatus strain Bath, Methylocaldum strain E10A, Methylobacter luteus, Methylomicrobium album, and Methylomonas strain D1a. In comparative sequence analyses, all pmoA2-like sequences formed a coherent cluster clearly distinct from pmoA1 sequences of type I and type II MOB, and from amoA sequences of the Nitrosomonas-Nitrosospira group. Phylogenetic analysis using the paml model suggested that pmoA2 is subject to strong purifying selection and therefore has an important cellular function. We probed total RNA extracts of Methylocystis strain SC2 for gene expression of pmoA. A strong signal was observed for pmoA1 in Northern hybridization, while the results obtained for pmoA2 were ambiguous. However, reverse transcription-PCR confirmed that pmoA2 was expressed, albeit at lower level than pmoA1. This provided experimental evidence that the gene product of pmoA2 may be a functionally active enzyme.

Base Sequence↗
PubMed2004

Comparative analysis of the conventional and novel pmo (particulate methane monooxygenase) operons from methylocystis strain SC2.

In addition to the conventional pmoA gene (pmoA1) encoding the active site polypeptide of particulate methane monooxygenase, a novel pmoA gene copy (pmoA2) is widely distributed among type II methanotrophs (methane-oxidizing bacteria [MOB]) (M. Tchawa Yimga, P. F. Dunfield, P. Ricke, J. Heyer, and W. Liesack, Appl. Environ. Microbiol. 69:5593-5602, 2003). Here we report that the pmoA1 and pmoA2 gene copies in the type II MOB Methylocystis strain SC2 are each part of a complete pmoCAB gene cluster (pmoCAB1, pmoCAB2). A bacterial artificial chromosome (BAC) library of strain SC2 genomic DNA was constructed, and BAC clones carrying either pmoCAB1 or pmoCAB2 were identified. Comparative sequence analysis showed that these two gene clusters exhibit low levels of identity at both the DNA level (67.4 to 70.9%) and the derived protein level (59.3 to 65.6%). In contrast, the secondary structures predicted for PmoCAB1 and PmoCAB2, as well as the derived transmembrane-spanning regions, are nearly identical. This suggests that PmoCAB2 is, like PmoCAB1, a highly hydrophobic, membrane-associated protein. A total of 190 of the 203 amino acid residues representing a highly conserved consensus sequence of the currently known PmoCAB1 and AmoCAB sequence types could be identified in PmoCAB2. The amoCAB gene cluster encodes ammonia monooxygenase and is evolutionarily related to pmoCAB. Analysis of a set of amino acid residues that allowed differentiation between conventional PmoA and AmoA provided further support for the hypothesis that pmoCAB2 encodes a functional equivalent of PmoCAB1. In experiments in which we used 5' rapid amplification of cDNA ends we identified transcriptional start sites 320 and 177 bp upstream of pmoC1 and pmoC2, respectively. Immediately upstream of the transcriptional start sites of both pmoCAB1 and pmoCAB2, sequence motifs similar to Escherichia coli sigma(70) promoters were identified.

Amino Acid Sequence↗
PubMed2004

pmoA-based analysis of methanotrophs in a littoral lake sediment reveals a diverse and stable community in a dynamic environment.

Diversity and community structure of aerobic methane-oxidizing bacteria in the littoral sediment of Lake Constance was investigated by cloning analysis and terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of the pmoA gene. Phylogenetic analysis revealed a high diversity of type I and type II methanotrophs in the oxygenated uppermost centimeter of the sediment. T-RFLP profiles indicated a high similarity between the active methanotrophic community in the oxic layer and the inactive community in an anoxic sediment layer at a 10-cm depth. There were also no major changes in community structure between littoral sediment cores sampled in summer and winter. By contrast, the fingerprint patterns showed substantial differences between the methanotrophic communities of littoral and profundal sediments.

Cloning, Molecular↗
PubMed2005

Analysis of methane monooxygenase genes in mono lake suggests that increased methane oxidation activity may correlate with a change in methanotroph community structure.

Mono Lake is an alkaline hypersaline lake that supports high methane oxidation rates. Retrieved pmoA sequences showed a broad diversity of aerobic methane oxidizers including the type I methanotrophs Methylobacter (the dominant genus), Methylomicrobium, and Methylothermus, and the type II methanotroph Methylocystis. Stratification of Mono Lake resulted in variation of aerobic methane oxidation rates with depth. Methanotroph diversity as determined by analysis of pmoA using new denaturing gradient gel electrophoresis primers suggested that variations in methane oxidation activity may correlate with changes in methanotroph community composition.

Aerobiosis↗
PubMed2005

Diversity of oxygenase genes from methane- and ammonia-oxidizing bacteria in the Eastern Snake River Plain aquifer.

PCR amplification, restriction fragment length polymorphism, and phylogenetic analysis of oxygenase genes were used for the characterization of in situ methane- and ammonia-oxidizing bacteria from free-living and attached communities in the Eastern Snake River Plain aquifer. The following three methane monooxygenase (MMO) PCR primer sets were used: A189-A682, which amplifies an internal region of both the pmoA gene of the MMO particulate form and the amoA gene of ammonia monooxygenase; A189-mb661, which specifically targets the pmoA gene; and mmoXA-mmoXB, which amplifies the mmoX gene of the MMO soluble form (sMMO). Whole-genome amplification (WGA) was used to amplify metagenomic DNA from each community to assess its applicability for generating unbiased metagenomic template DNA. The majority of sequences in each archive were related to oxygenases of type II-like methanotrophs of the genus Methylocystis. A small subset of type I sequences found only in free-living communities possessed oxygenase genes that grouped nearest to Methylobacter and Methylomonas spp. Sequences similar to that of the amoA gene associated with ammonia-oxidizing bacteria (AOB) most closely matched a sequence from the uncultured bacterium BS870 but showed no substantial alignment to known cultured AOB. Based on these functional gene analyses, bacteria related to the type II methanotroph Methylocystis sp. were found to dominate both free-living and attached communities. Metagenomic DNA amplified by WGA showed characteristics similar to those of unamplified samples. Overall, numerous sMMO-like gene sequences that have been previously associated with high rates of trichloroethylene cometabolism were observed in both free-living and attached communities in this basaltic aquifer.

Ammonia↗