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Analysis of methanotrophic bacteria in Movile Cave by stable isotope probing
Abstract
Movile Cave is an unusual groundwater ecosystem that is supported by in situ chemoautotrophic production. The cave atmosphere contains 1-2% methane (CH4), although much higher concentrations are found in gas bubbles that keep microbial mats afloat on the water surface. As previous analyses of stable carbon isotope ratios have suggested that methane oxidation occurs in this environment, we hypothesized that aerobic methane-oxidizing bacteria (methanotrophs) are active in Movile Cave. To identify the active methanotrophs in the water and mat material from Movile Cave, a microcosm was incubated with a 10%13CH4 headspace in a DNA-based stable isotope probing (DNA-SIP) experiment. Using improved centrifugation conditions, a 13C-labelled DNA fraction was collected and used as a template for polymerase chain reaction amplification. Analysis of genes encoding the small-subunit rRNA and key enzymes in the methane oxidation pathway of methanotrophs identified that strains of Methylomonas, Methylococcus and Methylocystis/Methylosinus had assimilated the 13CH4, and that these methanotrophs contain genes encoding both known types of methane monooxygenase (MMO). Sequences of non-methanotrophic bacteria and an alga provided evidence for turnover of CH4 due to possible cross-feeding on 13C-labelled metabolites or biomass. Our results suggest that aerobic methanotrophs actively convert CH4 into complex organic compounds in Movile Cave and thus help to sustain a diverse community of microorganisms in this closed ecosystem.
... Here, an active redox interface is created on both the water's surface and cave walls, colonized by floating microbial mats and biofilms. Most of our knowledge of this ecosystem comes from studies of trophic chain components such as microbial mats associated with the surface of the hydrothermal water and its close proximity [19][20][21][22]. In addition, the rich endemic invertebrates of Movile Cave live in the proximity of the sulfidic water. ...
... Assimilatory and dissimilatory nitrate reduction to ammonium were predicted in all datasets, emphasizing the ability of the microbial communities to provide bioavailable N for the other trophic links in the Movile Cave ecosystem. Methanotrophs were also postulated as primary producers in the water and microbial mats [19][20][21]24, 60] as 1-2% methane concentration was highlighted, especially in the Air-Bells [57]. In this study we were able to assemble methanotrophic MAGs from both lower and upper galleries. ...
... Interestingly Methylomirabilales-affiliated MAG was one of the most abundant MAGs in PMV7. The presence of Methylococcales (Methylococcus, Methylomonas, Methylocaldum) and Rhizobiales (Methylocystis/Methylosinus, Methylocella) methanotrophs was previously postulated in the microbial matsbased on pMMOA-sequence clones from a CH 4 -enriched culture [19,20]. The genus Methylocella comprises facultative methanotrophs that utilize multicarbon compounds (acetate, pyruvate, succinate, malate, and ethanol) [61]. ...
Background
Movile Cave (SE Romania) is a chemoautotrophically-based ecosystem fed by hydrogen sulfide-rich groundwater serving as a primary energy source analogous to the deep-sea hydrothermal ecosystems. Our current understanding of Movile Cave microbiology has been confined to the sulfidic water and its proximity, as most studies focused on the water-floating microbial mat and planktonic accumulations likely acting as the primary production powerhouse of this unique subterranean ecosystem. By employing comprehensive genomic-resolved metagenomics, we questioned the spatial variation, chemoautotrophic abilities, ecological interactions and trophic roles of Movile Cave’s microbiome thriving beyond the sulfidic-rich water.
Results
A customized bioinformatics pipeline led to the recovery of 106 high-quality metagenome-assembled genomes from 7 cave sediment metagenomes. Assemblies’ taxonomy spanned 19 bacterial and three archaeal phyla with Acidobacteriota , Chloroflexota , Proteobacteria , Planctomycetota , Ca. Patescibacteria, Thermoproteota , Methylomirabilota, and Ca. Zixibacteria as prevalent phyla. Functional gene analyses predicted the presence of CO 2 fixation, methanotrophy, sulfur and ammonia oxidation in the explored sediments. Species Metabolic Coupling Analysis of metagenome-scale metabolic models revealed the highest competition-cooperation interactions in the sediments collected away from the water. Simulated metabolic interactions indicated autotrophs and methanotrophs as major donors of metabolites in the sediment communities. Cross-feeding dependencies were assumed only towards 'currency' molecules and inorganic compounds (O 2 , PO 4 ³⁻ , H ⁺ , Fe ²⁺ , Cu ²⁺ ) in the water proximity sediment, whereas hydrogen sulfide and methanol were assumedly traded exclusively among distant gallery communities.
Conclusions
These findings suggest that the primary production potential of Movile Cave expands way beyond its hydrothermal waters, enhancing our understanding of the functioning and ecological interactions within chemolithoautotrophically-based subterranean ecosystems.
... In the absence of photosynthesis, most cave ecosystems rely on a combination of imported surface carbon and in situ chemosynthesis. One potential source of carbon in caves is methane and methane-derived carbon has been shown to drive food-web interactions in the chemosynthetic ecosystem of Movile cave (Sarbu et al., 1996;Hutchens et al., 2004;Kumaresan et al., 2018). Karst caves have also been identified as a sink for atmospheric methane with studies showing they have a consistently lower than atmospheric levels (Mattey et al., 2013;Waring et al., 2017). ...
... A number of culture-independent studies have focused particularly on methane cycling within cave ecosystems using molecular ecology tools targeting phylogenetic and functional biomarker genes (Hutchens et al., 2004;Karwautz et al., 2017;Zhao et al., 2018;Cheng et al., 2021). atmMOB have been detected in a variety of cave environments. ...
... The Movile cave sediment metagenome was also enriched in methylotrophic methanogen Methanomassiliicoccus, which is known to utilize methanol for methanogenesis in wetland soil (Narrowe et al., 2019). MOB release methanol extracellularly during the oxidation of methane and the role of MOBderived methanol in methane production needs to be elucidated (Hutchens et al., 2004). Whilst the wet saprolite sample from Diamantina (Diamantina_P7) harbored the second highest number of methanogens reads (Figure 1), the lowest abundance of methanogen reads was retrieved from the dry saprolite metagenome of Diamantina cave (Diamantina_P3). ...
Karst ecosystems represent up to 25% of the land surface and recent studies highlight their potential role as a sink for atmospheric methane. Despite this, there is limited knowledge of the diversity and distribution of methane-oxidizing bacteria (MOB) or methanogens in karst caves and the sub-surface environment in general. Here, we performed a survey of 14 shotgun metagenomes from cave ecosystems covering a broad set of environmental conditions, to compare the relative abundance and phylogenetic diversity of MOB and methanogens, targeting biomarker genes for methane monooxygenase (pmoA and mmoX) and methyl-coenzyme M reductase (mcrA). Taxonomic analysis of metagenomes showed 0.02–1.28% of classified reads were related to known MOB, of which Gammaproteobacterial MOB were the most abundant making up on average 70% of the surveyed caves’ MOB community. Potential for biogenic methane production in caves was also observed, with 0.008–0.39% of reads classified to methanogens and was dominated by sequences related to Methanosarcina. We have also generated a cave ecosystems protein database (CEPD) based on protein level assembly of cave metagenomes that can be used to profile genes of interest.
... Here, an active redox interface is created on both the water's surface and the walls of the cave, which are colonized by floating microbial mats and biofilms. Most of our understanding of this ecosystem comes from studies on trophic chain components such as microbial mats associated with the hydrothermal water surface and its close proximity [19][20][21][22]. In addition, the rich endemic invertebrate fauna of Movile Cave gravitates in the proximity of the sulfidic water, being most abundant in the Air-Bells and Lake Room [15,19] where the redox potential is higher, particularly in the Air-Bells water surface [23]. ...
... The copyright holder for this preprint (which this version posted May 20, 2022. ; https://doi.org/10.1101/2022.05.19.492637 doi: bioRxiv preprint Methanotrophs were also postulated as primary producers in the water and microbial mats [19][20][21]24, 60] as 1-2 % methane concentration was highlighted, especially in the Air-Bells [57]. In this study we were able to assemble methanotrophic MAGs from both lower and upper galleries. ...
... In this study we were able to assemble methanotrophic MAGs from both lower and upper galleries. These were affiliated to the uncultured [19,20]. The genus Methylocella comprises facultative methanotrophs that utilize multicarbon compounds (acetate, pyruvate, succinate, malate, and ethanol) [61]. ...
Background
Movile Cave (Dobrogea, SE Romania) hosts a subterranean chemoautotrophically-based ecosystem supported by a sulfidic thermal aquifer analogous to the deep-sea hydrothermal ecosystems. Our current understanding of Movile Cave microbiology has been confined to the thermal water proximity (no more than 2 m distant), with most studies focusing on the water-floating mat, which likely acts as the primary production powerhouse in this sulfidic ecosystem. To gain more insightful information on the functioning of the sulfidic Movile Cave ecosystem, we employed a metagenomics-resolved approach to reveal the microbiome diversity, metabolic potential, and interactions and infer its roles within the food webs in the sediments beyond the sulfidic thermal waters.
Results
A customized bioinformatics pipeline led to the recovery of 106 high-quality metagenome-assembled genomes from 7 cave sediment metagenomes. Assemblies’ taxonomy spanned 19 bacterial and three archaeal phyla with Acidobacteriota, Chloroflexota, Proteobacteria, Planctomycetota, Ca . Patescibacteria, Thermoproteota, Methylomirabilota , and Ca . Zixibacteria as prevalent phyla. Functional gene analyses allowed prediction of CO 2 fixation, methanotrophy, sulfur and ammonia oxidation as possibly occurring in the explored sediments. Species Metabolic Coupling Analysis of metagenome-scale metabolic models revealed the highest competition-cooperation interactions in the sediments collected at the farthest distance from the sulfidic water. As a result of simulated metabolic interactions, autotrophs and methanotrophs were hypothesized as major donors of exchanged metabolites in the sediment communities. Cross-feeding dependencies were assumed only towards ‘currency’ molecules and inorganic compounds (O 2 , PO 4 ³⁻ , H ⁺ , Fe ²⁺ , Cu ²⁺ ) in the sediment nearby sulfidic water, whereas hydrogen sulfide and methanol are predictably traded exclusively among communities dwelling in the distant gallery.
Conclusions
These findings suggest that the primary production potential of the Movile Cave expands way beyond its hydrothermal waters, enhancing our understanding of ecological interactions inside chemolithoautotrophically based subterranean ecosystems and their functioning.
... This results in a constant flow occurring tens of centimeters to 1 m below the water surface at flow rates of about 5 L/s [5], which causes a continuous slow-motion movement of the surface waters. The ecosystem relies on biomass production by prokaryotes that derive metabolic energy from the oxidation of hydrogen sulfide (H 2 S), methane (CH 4 ) and ammonium (NH 4 + ) and assimilate carbon from the cave's atmospheric methane and carbon dioxide [1,6,7]. Common electron acceptors are dioxygen (O 2 ), nitrate (NO 3 ), sulfate (SO 4 2− ) and ferric iron (Fe 3+ ). ...
... Experiments administering isotope-doped substrates for growth at various locations in the cave followed by isotopic measurements of the resulting biomass will be more definitive on this count. This type of experiment focusing on methanotrophs and performed on added microcosms indeed revealed active assimilation into biomass [7]. ...
Movile Cave, situated in Romania close to the Black Sea, constitutes a distinct and challenging environment for life. Its partially submerged ecosystem depends on chemolithotrophic processes for its energetics, which are fed by a continuous hypogenic inflow of mesothermal waters rich in reduced chemicals such as hydrogen sulfide and methane. We sampled a variety of cave sublocations over the course of three years. Furthermore, in a microcosm experiment, minerals were incubated in the cave waters for one year. Both endemic cave samples and extracts from the minerals were subjected to 16S rRNA amplicon sequencing. The sequence data show specific community profiles in the different subenvironments, indicating that specialized prokaryotic communities inhabit the different zones in the cave. Already after one year, the different incubated minerals had been colonized by specific microbial communities, indicating that microbes in Movile Cave can adapt in a relatively short timescale to environmental opportunities in terms of energy and nutrients. Life can thrive, diversify and adapt in remote and isolated subterranean environments such as Movile Cave.
... 2%) and CH 4 (1-2%) [18]. The microbial communities at these sites were found to include sulfur oxidizing bacteria such as Beggiatoa, Sulfurospirillum, Thiobacillus, Thiomonas, Thioploca, Thiothrix, and Thiovirga [19][20][21] methylotrophs such as Methylomonas, Methylococcus and Methylocystis [22], Methylotenera, Methylophilus, and Methylovorus [19,20]; ammonia and nitrite oxidizers such as Nitrosomonas, Nitrospira and Nitrotoga [20] and the methanogenic Archaea Methanobacterium [23] and Methanosarcina [24]. Of these, activity was confirmed only for a few taxa [20,[22][23][24][25] while for others it was inferred based on phylogenetic association. ...
... The microbial communities at these sites were found to include sulfur oxidizing bacteria such as Beggiatoa, Sulfurospirillum, Thiobacillus, Thiomonas, Thioploca, Thiothrix, and Thiovirga [19][20][21] methylotrophs such as Methylomonas, Methylococcus and Methylocystis [22], Methylotenera, Methylophilus, and Methylovorus [19,20]; ammonia and nitrite oxidizers such as Nitrosomonas, Nitrospira and Nitrotoga [20] and the methanogenic Archaea Methanobacterium [23] and Methanosarcina [24]. Of these, activity was confirmed only for a few taxa [20,[22][23][24][25] while for others it was inferred based on phylogenetic association. In the lower level of Movile Cave, at and directly below the water surface (<3 mm), we observed a loose floating veil ( Fig. 2 and Supplementary video 1). ...
Thiovulum spp. (Campylobacterota) are large sulfur bacteria that form veil-like structures in aquatic environments. The sulfidic Movile Cave (Romania), sealed from the atmosphere for ~5 million years, has several aqueous chambers, some with low atmospheric O 2 (~7%). The cave’s surface-water microbial community is dominated by bacteria we identified as Thiovulum . We show that this strain, and others from subsurface environments, are phylogenetically distinct from marine Thiovulum . We assembled a closed genome of the Movile strain and confirmed its metabolism using RNAseq. We compared the genome of this strain and one we assembled from public data from the sulfidic Frasassi caves to four marine genomes, including Candidatus Thiovulum karukerense and Ca . T. imperiosus, whose genomes we sequenced. Despite great spatial and temporal separation, the genomes of the Movile and Frasassi Thiovulum were highly similar, differing greatly from the very diverse marine strains. We concluded that cave Thiovulum represent a new species, named here Candidatus Thiovulum stygium. Based on their genomes, cave Thiovulum can switch between aerobic and anaerobic sulfide oxidation using O 2 and NO 3 ⁻ as electron acceptors, the latter likely via dissimilatory nitrate reduction to ammonia. Thus, Thiovulum is likely important to both S and N cycles in sulfidic caves. Electron microscopy analysis suggests that at least some of the short peritrichous structures typical of Thiovulum are type IV pili, for which genes were found in all strains. These pili may play a role in veil formation, by connecting adjacent cells, and in the motility of these exceptionally fast swimmers.
... These represent a copious food source for the numerous consumers thriving in this groundwater ecosystem [7]. Various types of Archaea [10], sulfur-oxidizing bacteria [8], methanotrophs [9,10,[22][23][24], or nitrifying and denitrifying bacteria [10], have been identified in Movile Cave. Representatives of a newly described strain of Thiovulum swim actively at the water surface and gather in loose veils [25]. ...
... New technological advances in research methods allow for better understanding of how life can prosper even in such extreme environments, like Movile Cave, in total darkness, low pH, hypoxia and anoxia, high sulfide-, CH 4 , and CO 2 concentrations. Microbiological research has evolved significantly from characterization of enzymes produced by microbes and cultivation of sulfide oxidizers [63], to the first molecular characterization of microbial communities by basic fingerprinting techniques and generation of clone libraries [10,22], to the nowadays Next-Generation Sequencing approaches that allow the examination of tens of thousands of sequences or complete genomes [25,64]. Regarding the invertebrates, new and undescribed species are no longer a great surprise, but they have to be identified and studied before their possible disappearance. ...
Movile Cave hosts one of the world’s most diverse subsurface invertebrate communities. In the absence of matter and energy input from the surface, this ecosystem relies entirely on in situ primary productivity by chemoautotrophic microorganisms. The energy source for these microorganisms is the oxidation of hydrogen sulfide provided continuously from the deep thermomineral aquifer, alongside methane, and ammonium. The microbial biofilms that cover the water surface, the cave walls, and the sediments, along with the free-swimming microorganisms, represent the food that protists, rotifers, nematodes, gastropods, and crustacean rely on. Voracious water-scorpions, leeches, and planarians form the peak of the aquatic food web in Movile Cave. The terrestrial community is even more diverse. It is composed of various species of worms, isopods, pseudoscorpions, spiders, centipedes, millipedes, springtails, diplurans, and beetles. An updated list of invertebrate species thriving in Movile Cave is provided herein. With 53 invertebrate species (21 aquatic and 32 terrestrial), of which 38 are endemic for this unusual, but fascinating environment, Movile Cave is the first known chemosynthesis-based groundwater ecosystem. Therefore, Movile Cave deserves stringent attention and protection.
... The genome was extracted using a DNA extraction kit (QIAGEN, Valencia, CA, USA). The 16S ribosomal RNA gene was amplified using 27F/1492R primers [49,50], and the methane monooxygenase gene was amplified using A189F/A682R [47], mmox1/mmox2 [51], and mmox206F/mmox886R [52] primers. PCR conditions were as previously described [47,51,52]. ...
... The 16S ribosomal RNA gene was amplified using 27F/1492R primers [49,50], and the methane monooxygenase gene was amplified using A189F/A682R [47], mmox1/mmox2 [51], and mmox206F/mmox886R [52] primers. PCR conditions were as previously described [47,51,52]. Phylogenetic trees based on 16S rRNA genes and pmoA genes aligned using Bioedit were constructed using the neighbor-joining method [53] in MEGA 7 [54] based on the Kimura 2-parameter model and Poisson model using 1000 replicates, respectively. ...
Methane-oxidizing bacteria are crucial players in controlling methane emissions. This study aimed to isolate and characterize a novel wetland methanotroph to reveal its role in the wetland environment based on genomic information. Based on phylogenomic analysis, the isolated strain, designated as B8, is a novel species in the genus Methylocystis. Strain B8 grew in a temperature range of 15 °C to 37 °C (optimum 30–35 °C) and a pH range of 6.5 to 10 (optimum 8.5–9). Methane, methanol, and acetate were used as carbon sources. Hydrogen was produced under oxygen-limited conditions. The assembled genome comprised of 3.39 Mbp and 59.9 mol% G + C content. The genome contained two types of particulate methane monooxygenases (pMMO) for low-affinity methane oxidation (pMMO1) and high-affinity methane oxidation (pMMO2). It was revealed that strain B8 might survive atmospheric methane concentration. Furthermore, the genome had various genes for hydrogenase, nitrogen fixation, polyhydroxybutyrate synthesis, and heavy metal resistance. This metabolic versatility of strain B8 might enable its survival in wetland environments.
... Bins 9 and 14 were assigned to bacterial taxa associated with predation of microbial cells (Myxococcota and Bdellovibrionaceae respectively). This labelling of predatory bacteria was previously observed in SIP experiments performed with 13 C methane or 13 C enriched prey cells and various terrestrial innocula (Barnett et al., 2016;Hutchens et al., 2004;Lueders et al., 2004). The methylotrophs enriched in these experiments were exclusively Gram-negative, effectively generating an abundant source of labelled carbon for this specific metabolic guild of microbe. ...
Metagenome assembled genomes (MAGs), generated from sequenced ¹³C‐labelled DNA from ¹³C‐methanol enriched soils, were binned using an ensemble approach. This method produced a significantly larger number of higher‐quality MAGs compared to direct binning approaches. These MAGs represent both the primary methanol utilizers and the secondary utilizers labelled via cross‐feeding and predation on the labelled methylotrophs, including numerous uncultivated taxa. Analysis of these MAGs enabled the identification of multiple metabolic pathways within these active taxa that have climatic relevance relating to nitrogen, sulfur and trace gas metabolism. This includes denitrification, dissimilatory nitrate reduction to ammonium, ammonia oxidation and metabolism of organic sulfur species. The binning of viral sequence data also yielded extensive viral MAGs, identifying active viral replication by both lytic and lysogenic phages within the methanol‐enriched soils. These MAGs represent a valuable resource for characterizing biogeochemical cycling within terrestrial environments.
... Although MOB likely played important roles on N 2 -fixation in the two studied soils under CH 4 -feeding microcosm, some non-MOB diazotrophs might also contribute to BNF by forming symbiotic relationships (cross-feeding) with MOB (Hutchens et al., 2004;Lueders et al., 2004), as revealed by the enrichment of non-MOB-related nifH genes in 13 Clabeled DNA (Fig. 3). It is likely that these diazotrophic communities grew on methanotrophy-derived organic C while fixing N 2 as N source. ...
... They are mesophiles, show optimum growth at 25-30 • C and most species are halotolerant [56]. To date, they have been found in various ecosystems including lake and river sediments [57][58][59][60][61], seawater [8], peatlands and other wetlands [62][63][64][65], wastewater, groundwater, and coal mines [56], and organic-mineral sediments from copper mines [66]. The diversity of environments from which Methylomonas spp. ...
Simple Summary
Deep subsurfaces such as caves and mines are extreme environments inhabited by specialized microorganisms that are able to cope with the adverse conditions caused by low moisture, and high pressure. Our research confirmed that one such site is the rocks surrounding the salt beds in the Wieliczka Salt Mine, where methane-oxidizing bacteria are present. We demonstrated methanotrophic activity in both natural and mineral-supplemented rock material. In the next stage of our research, we confirmed that the methanotrophic community can be cultured on a mineral substrate, using methane as the sole source of carbon and energy source. The issues addressed in this study contribute to our understanding of the biodiversity of microorganisms inhabiting the extreme subsurface biosphere but also provide insights into changes in biodiversity during the isolation of bacterial communities from this type of ecosystem. This information can be valuable for the acquisition of microorganisms with potential applications in biotechnology.
Abstract
The rocks surrounding Wieliczka salt deposits are an extreme, deep subsurface ecosystem that as we studied previously harbors many microorganisms, including methanotrophs. In the presented research bacterial community structure of the Wieliczka Salt Mine was determined as well as the methanotrophic activity of the natural microbiome. Finally, an enrichment culture of methane-consuming methanotrophs was obtained. The research material used in this study consisted of rocks surrounding salt deposits in the Wieliczka Salt Mine. DNA was extracted directly from the pristine rock material, as well as from rocks incubated in an atmosphere containing methane and mineral medium, and from a methanotrophic enrichment culture from this ecosystem. As a result, the study describes the composition of the microbiome in the rocks surrounding the salt deposits, while also explaining how biodiversity changes during the enrichment culture of the methanotrophic bacterial community. The contribution of methanotrophic bacteria ranged from 2.614% in the environmental sample to 64.696% in the bacterial culture. The methanotrophic enrichment culture was predominantly composed of methanotrophs from the genera Methylomonas (48.848%) and Methylomicrobium (15.636%) with methane oxidation rates from 3.353 ± 0.105 to 4.200 ± 0.505 µmol CH4 mL⁻¹ day⁻¹.
... Putative colonies of methanotrophs that formed on the solid plates were assessed for the presence of genes encoding methanotrophy. DNA was extracted from the individual colonies, and PCR-amplified fragments of methanotrophic genes (pmoA gene; A189F/mb661R [32], mmoX gene; mmoX206F/mmoX886R [33]) and the 16S rRNA gene (27F/1492R and 20F/958R [34]) were examined. The isolated methanotrophic colonies were used for further culturing and research. ...
Strain 16-5 T , a mesophilic methanotroph of the genus Methylococcus , was isolated from rice field soil sampled in Chungcheong Province, Republic of Korea. Strain 16-5 T had both particulate and soluble methane monooxygenases and could only grow on methane and methanol as electron donors. Strain 16-5 T cells are Gram-negative, white to light tan in color, non-motile, non-flagellated, diplococcoid to cocci, and have the typical type I intracytoplasmic membrane system. Strain 16-5 T grew at 18–38 °C (optimum, 27 °C) and at pH 5.0–8.0 (optimum, pH 6.5–7.0). C 16 : 1 ω7 c (38.8%), C 16 : 1 ω 5 c (18.8%), C 16 : 1 ω 6 c (16.8%) and C 16 : 0 (16.9%) were the major fatty acids, and phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and an unidentified phospholipid were the major polar lipids. The main respiratory quinone was methylene-ubiquinone-8. Strain 16-5 T displayed the highest 16S rRNA gene sequence similarities to other taxonomically recognized members of the genus Methylococcus , i.e. Methylococcus capsulatus Texas T (98.62%) and Methylococcus geothermalis IM1 T (98.49 %), which were its closest relatives. It did, however, differ from all other taxonomically described Methylococcus species due to some phenotypic differences, most notably its inability to grow at temperatures above 38 °C, where other Methylococcus species thrive. Its 4.34 Mbp-sized genome has a DNA G+C content of 62.47 mol%, and multiple genome-based properties such as average nucleotide identity and digital DNA-DNA hybridization value distanced it from its closest relatives. Based on the data presented above, this strain represents the first non-thermotolerant species of the genus Methylococcus . The name Methylococcus mesophilus sp. nov. is proposed, and 16-5 T (=JCM 35359 T =KCTC 82050 T ) is the type strain.
... Detection of microorganisms is usually based on the sequences of the small subunit ribosomal RNA (rRNA) genes (16S for prokaryotes) because rRNA genes are highly conserved and contain a level of variability that allows the identification of microorganisms detected by their sequences and the possibility of performing phylogenetic analyses with their closest relatives. A variety of methods have been used to analyze these sequences, including polymerase chain reaction (PCR)-based fingerprinting methods, such as DGGE (denaturing gradient gel electrophoresis) [32,33], T-RFLP (terminal restriction fragment length polymorphism) [33,34], clone library construction [35], quantitative PCR assays (including those targeting functional genes of interest), sequencing, and the use of stable isotope probing methods [36,37]. DNA sequencing approaches are very useful for phylogenetic identification [38], and more recently, next-generation sequencing (NGS) tools on a variety of platforms, such as Roche FLX 454 pyrosequencing [39], Illumina [40][41][42], and SOLiD and Ion Torrent PGM [43,44], have been applied to the study of cave microorganisms [16]. ...
Pristine environments, such as caves, are unique habitats that are isolated from human activity and are exposed to extreme environmental conditions. These environments are rich sources of microbial diversity, and the microorganisms that thrive in these conditions have developed unique survival skills. One such skill is the biosynthesis of secondary metabolites with potential bioactivities, which provide the organisms with a competitive advantage in these extreme environments. The isolation and characterization of microbial strains from the surfaces of pristine cave environments are important for exploring the biotechnological potential of these organisms. These studies can reveal new products with antibacterial, antifungal, anti-inflammatory, antioxidant, and anticancer activities, among others. In addition, the identification of specific compounds responsible for these biological activities can contribute to the development of new drugs and products for sustainable biotechnological applications. Recent developments in genomics, bioinformatics, chemoinformatics, metabolic engineering, and synthetic biology have opened new possibilities for drug discovery, making the exploration of bacterial secondary metabolites more promising. In recent years, several bacteria with bioactive potential have been described, and several compounds with bioactivity have been identified. These findings are essential for the development of new drugs and products for the benefit of society. This paper discusses the potential of microorganisms found in pristine cave surfaces as a source of new metabolites with bioactivity that could have sustainable biotechnological applications. The authors suggest that more research should be conducted in these environments to better understand the microorganisms and the biosynthesis of these metabolites and to identify new compounds and metabolic pathways that could be of interest for the development of new drugs and products. The aim is to highlight the importance of these habitats as a potential source of new bioactive compounds that could be used for sustainable biotechnological applications.
... Fluorescent oligonucleotide probes based on 16S rRNA (Bourne et al., 2000;Dedysh et al., 2003;Kalyuzhnaya et al., 2006), and PCR-based assays targeting 16S rRNA, pmoA, mmoX, and methanol dehydrogenase gene, mxaF (Chen et al., 2008;Ghashghavi et al., 2017;Hutchens et al., 2004;Kip et al., 2011;Lau et al., 2007;Redmond et al., 2010) are commonly used to identify and investigate the diversity and abundance of acidophilic methanotrophs in different environments. With the PCR-based assays, many uncultured clones T A B L E 1 Characteristics of isolated acidophilic methanotrophs. ...
Methanotrophs have been identified and isolated from acidic environments such as wetlands, acidic soils, peat bogs, and groundwater aquifers. Due to their methane (CH4) utilization as a carbon and energy source, acidophilic methanotrophs are important in controlling the release of atmospheric CH4, an important greenhouse gas, from acidic wetlands and other environments. Methanotrophs have also played an important role in the biodegradation and bioremediation of a variety of pollutants including chlorinated volatile organic compounds (CVOCs) using CH4 monooxygenases via a process known as cometabolism. Under neutral pH conditions, anaerobic bioremediation via carbon source addition is a commonly used and highly effective approach to treat CVOCs in groundwater. However, complete dechlorination of CVOCs is typically inhibited at low pH. Acidophilic methanotrophs have recently been observed to degrade a range of CVOCs at pH < 5.5, suggesting that cometabolic treatment may be an option for CVOCs and other contaminants in acidic aquifers. This paper provides an overview of the occurrence, diversity, and physiological activities of methanotrophs in acidic environments and highlights the potential application of these organisms for enhancing contaminant biodegradation and bioremediation.
... For instance, methanotrophs showed a significant reduction of connections between them and with other nonmethane-cycling microbes in pasture soils ( Figure 4). Although not autotrophs, methanotrophs are the base of food webs acting as an accessible carbon source for heterotrophs (Hutchens et al., 2004;Kalyuzhnaya et al., 2013;Karwautz et al., 2018). In this regard, a network meta-analysis conducted by Methanotrophs that carry the soluble form of the enzyme MMO (sMMO) were depleted by LUC from forest to pasture, whereas those harbouring the particulate form of this enzyme (pMMO) were enhanced (Figures 2 and 5). ...
Deforestation threatens the integrity of the Amazon biome and the ecosystem services it provides, including greenhouse gas mitigation. Forest-to-pasture conversion has been shown to alter the flux of methane gas (CH4 ) in Amazonian soils, driving a switch from acting as a sink to a source of atmospheric CH4 . This study aimed to better understand this phenomenon by investigating soil microbial metagenomes, focusing on the taxonomic and functional structure of methane-cycling communities. Metagenomic data from forest and pasture soils were combined with in situ CH4 fluxes and soil edaphic factors measurements and analysed using multivariate statistical approaches. We found a significantly higher abundance and diversity of methanogens in pasture soils. As inferred by co-occurrence networks, these microorganisms seem to be less interconnected within the soil microbiota in pasture soils. Metabolic traits were also different between land uses, with increased hydrogenotrophic and methylotrophic pathways of methanogenesis in pasture soils. Land-use change also induced shifts in taxonomic and functional traits of methanotrophs, with bacteria harboring genes encoding the soluble form of methane monooxygenase enzyme (sMMO) depleted in pasture soils. Redundancy analysis and multimodel inference revealed that the shift in methane-cycling communities was associated with high pH, organic matter, soil porosity, and micronutrients in pasture soils. These results comprehensively characterize the effect of forest-to-pasture conversion on the microbial communities driving the methane-cycling microorganisms in the Amazon rainforest, which will contribute to the efforts to preserve this important biome.
... Although MOB likely played important roles on N 2 -fixation in the two studied soils under CH 4 -feeding microcosm, some non-MOB diazotrophs might also contribute to BNF by forming symbiotic relationships (cross-feeding) with MOB (Hutchens et al., 2004;Lueders et al., 2004), as revealed by the enrichment of non-MOB-related nifH genes in 13 Clabeled DNA (Fig. 3). It is likely that these diazotrophic communities grew on methanotrophy-derived organic C while fixing N 2 as N source. ...
... Such high concentration of CH 4 could be of biological origin due to the following reason: air in soil and caves could be involved in methanogenesis (Mc-Donough et al., 2016), and the use of CO 2 to produce methane is the most common metabolic pathway known in methanogens (Liu and Whitman, 2008); the high concentration of CO 2 (i.e., substrate for methanogenesis) in XYC might provide basis for high methane through methanogenesis. In addition, high concentration of CH 4 was ever found to co-exist with high CO 2 in caves (Hutchens et al., 2004). However, other sources could not be excluded for the methane in the studied caves due to the lack of stable carbon isotopic characteristic of methane and microbial methane production rate test, which awaits further investigation. ...
There is limited knowledge about microbial communities and their ecological functions in karst caves with high CO2 concentrations. Here, we studied the microbial community compositions and functions in Shuiming Cave ("SMC", CO2 concentration 3303 ppm) and Xueyu Cave ("XYC", CO2 concentration 8753 ppm) using Illumina MiSeq high-throughput sequencing in combination with BIOLOG test. The results showed that Proteobacteria, Actinobacteria, and Bacteroidetes were dominant phyla in these two caves, and Thaumarchaeota was the most abundant in the rock wall samples of SMC. The microbial diversity in the water samples decreased with increasing HCO3- concentration, and it was higher in XYC than that in SMC. The microbial community structures in the sediment and rock wall samples were quite different between the two caves. High concentrations of CO2 can reduce the microbial diversity on the rock walls in karst caves, probably through changing microbial preference for different types of carbon sources and decreasing the microbial utilization rate of carbon sources. These results expanded our understanding of microbial community and its response to environments in karst caves with high CO2 .
... The phylum Acidobacteriota was equally distributed in all caves, ranging from 3.02% in "Grotta di Monte Corruccio" (sample GMC_5) and 12.39% in "Grotta del Santo" (sample GS_2A). Several studies have detected the presence of acidobacterial 16S rRNA gene sequences in caves (e.g., Holmes et al. [63]; Hutchens et al. [64]; Chelius and Moore [65]; Engel et al. [66]). However, their role is still poorly understood [67]. ...
While microbial communities in limestone caves across the world are relatively understood, knowledge of the microbial composition in lava tubes is lagging behind. These caves are found in volcanic regions worldwide and are typically lined with multicolored microbial mats on their walls and ceilings. The Mount Etna (Sicily, S-Italy) represents one of the most active volcanos in the world. Due to its outstanding biodiversity and geological features, it was declared Natural Heritage of Humanity by the UNESCO in 2013. Despite the presence of more than 200 basaltic lava tubes, the microbial diversity of these hypogean systems has never been investigated so far. Here, we investigated bacterial communities in four lava tubes of Mount Etna volcano. Field emission scanning electron microscopy (FESEM) was carried out for the morphological characterization and detection of microbial features. We documented an abundant presence of microbial cells with different morphotypes including rod-shaped, filamentous, and coccoidal cells with surface appendages, resembling actinobacteria reported in other lava tubes across the world. Based on 16S rRNA gene analysis, the colored microbial mats collected were mostly composed of bacteria belonging to the phyla Actinomycetota, Pseudomonadota, Acidobacteriota, Chloroflexota, and Cyanobacteria. At the genus level, the analysis revealed a dominance of the genus Crossiella, which is actively involved in biomineralization processes, followed by Pseudomonas, Bacillus, Chujaibacter, and Sphingomonas. The presence of these taxa is associated with the carbon, nitrogen, and ammonia cycles, and some are possibly related to the anthropic disturbance of these caves. This study provides the first insight into the microbial diversity of the Etna volcano lava tubes, and expands on previous research on microbiology of volcanic caves across the world.
... By exuding metabolites derived from methane-C, methanotrophs can use carbon in a high-methane environment and maintain a food web composed of organisms with multiple nutrient levels (Hutchens et al., 2004;Agasild et al., 2014;Karwautz et al., 2018). Methylotrophs can use C1 compounds, such as methanol (CH 3 OH), an intermediary product of methane oxidation, thus facilitating cross-feeding and co-occurrence with methanotrophs (Krause et al., 2017). ...
The extent to which propagule limitation can govern the responses of microbially-mediated processes (such as methane oxidation) to sudden environmental changes, is poorly understood. Here, we compared the ability of the methanotroph community in lakeshore soils of two lakes to respond to an experimental increase in salinity. One set of samples was taken from lakeshore soils of a freshwater lake (Yang Lake), the other from a slightly brackish lake (Qinghai Lake), both on the Tibetan Plateau. Samples were incubated in microcosms by adding ∼ 5 % ¹³CH4 or ¹²CH4 and different concentrations of NaCl solution. DNA stable-isotope probing (DNA-SIP) followed by high-throughput sequencing was used to determine how the active methanotrophic populations differed in lakeshore soils with different salinity levels. Samples from saline and freshwater lake initially showed much-reduced methane oxidation ability and methanotrophic activity at increased salinity. For the freshwater samples with the salinity of 25 to 50 g/L after NaCl addition, there was no adaptation and increase in methanotrophy after 7 days. By contrast, samples from the brackish lake showed an initial depression of methane oxidation, followed by greatly increased rates after several days. Sequencing revealed that this recovery of methanotrophy in the brackish lake samples was associated with a major switchover in composition of active methanotroph community. In particular, the relative abundance of Type Ⅰa methanotrophs became more abundant at increased salinity. It appears that in this freshwater lake environment, isolation from any nearby high-salinity-tolerant bacterial sources has prevented the possibility of full adaptation to a high salinity change in the environment, and only a moderate salinity adaptation is possible by species-sorting from within the existing community. By contrast, in the higher-salinity environment, the highly salinity-tolerant Methylomicrobium was able to break the establishment limitation in the high salinity environment and become the dominant methanotroph. Our study provides an instance of propagule limitation preventing adaptation to changed conditions.
... For instance, methanotrophs showed a signi cant reduction of connections between them and with other nonmethane-cycling microbes in pasture soils (Fig. 4). Although not autotrophs, methanotrophs are base of food webs acting as an accessible carbon source for heterotrophs [90][91][92]. In this regard, a network meta-analysis conducted by Ho et al. [43] revealed a CH 4 -derived food web in diverse ecosystems, in which methylotrophs were always present underlying the cross-feeding between methanotrophs and methylotrophs, whereas non-methanotrophic communities were site-speci c and not consistent among the diverse ecosystems. ...
Deforestation threatens the integrity of the Amazon biome and the ecosystem services it provides. Most of this deforested land is converted to pastures for cattle raising. Early studies revealed that forest-to-pasture conversion alters the flux of methane gas (CH4) in Amazonian soils, driving a switch from acting as a sink to a source of atmospheric CH4. We sought to better understand this phenomenon by investigating the soil microbial metagenomes, focusing on the taxonomic and functional structure of methane-cycling communities.
... In contrast, in continental sulphidic cave ecosystems (SCE), which share their highly unusual nature with sulphuric deep-sea hydrothermal vents, fundamental studies on invertebrate biota are still scarce 5,[10][11][12][13][14][15] . An exception is the first discovered cave ecosystem of this type, the Movile Cave in Romania, the microorganism communities of which have been exhaustively studied since its discovery [5][6][16][17] . ...
Sulphidic cave ecosystems are remarkable evolutionary hotspots that have witnessed adaptive radiation of their fauna represented by extremophile species having particular traits. Ostracods, a very old group of crustaceans, exhibit specific morphological and ecophysiological features that enable them to thrive in groundwater sulphidic environments. Herein, we report a peculiar new ostracod species Pseudocandona movilaensis sp. nov. thriving in the chemoautotrophic sulphidic groundwater ecosystem of Movile Cave (Romania). The new species displays a set of homoplastic features specific for unrelated stygobitic species, for e.g., triangular carapace in lateral view with reduced postero–dorsal part and simplification of limb chaetotaxy (i.e., loss of some claws and reduction of secondary male sex characteristics), driven by a convergent or parallel evolution during or after colonization of the groundwater realm. P. movilaensis sp. nov. thrives exclusively in sulphidic meso-thermal waters (21°C) with high concentrations of sulphides, methane, and ammonium. Based on the geometric morphometrics-based study of the carapace shape and molecular phylogenetic analyses based on the COI marker (mtDNA), we discuss the phylogenetic relationship and evolutionary implication for the new species to thrive in groundwater sulphidic groundwater environments.
... The pmoA gene was targeted with the primers A189 f (5ʹ-GGNGACTGG GACTTCTGG-3ʹ) and A682 (5ʹ-GAASGCNGAGAAGAASGC-3ʹ) [57] with annealing at 55°. For Eb1 we performed PCRs for the mmoX gene which was targeted with the primers mmoX-206 f_(5ʹ-ATCGC BAARGAATAYGCSCG-3ʹ) and mmoX-886 r (5ʹ-ACCCANGGCTCGA CYTTGAA-3ʹ) [58] with annealing at 61°C. The nifH gene of Eb1 was targeted with the primers Pol f (5ʹ-TGCGAYCCSAARGCB GACTC-3ʹ) and Pol r (5ʹ-ATSGCCATCATYTCRCCGGA-3ʹ) [59] with annealing at 55°C. ...
Three strains of methanotrophic bacteria (EbAT, EbBT and Eb1) were isolated from the River Elbe, Germany. These Gram-negative, rod-shaped or coccoid cells contain intracytoplasmic membranes perpendicular to the cell surface. Colonies and liquid cultures appeared bright-pink. The major cellular fatty acids were 12:0 and 14:0, in addition in Eb1 the FA 16:1ω5t was also dominant. Methane and methanol were utilized as sole carbon sources by EbBT and Eb1, while EbAT could not use methanol. All strains oxidize methane using the particulate methane monooxygenase. Only EbBT contains an additional soluble methane monooxygenase. The strains grew optimally at 15 – 25°C and at pH 6 and 8. Based on 16S rRNA gene analysis recovered from the full genome, the phylogenetic position of EbAT is robustly outside any species clade with its closest relatives being Methylomonas sp. MK1 (98.24 %) and Methylomonas sp. 11b (98.11 %). Its closest type strain is Methylomonas methanica NCIMB11130 (97.91%). The 16S rRNA genes of EbBT are highly similar to Methylomonas methanica strains with Methylomonas methanica R-45371 as the closest relative (99.87 % sequence identity). However, average nucleotide identity (ANI) and digital DNA-DNA-hybridization (dDDH) values reveal it as distinct species. The DNA G+C contents were 51.07 mol% and 51.5 mol% for EbAT and EbBT, and 50.7 mol% for Eb1, respectively. Strains EbAT and EbBT are representing two novel species within the genus Methylomonas. For strain EbAT we propose the name Methylomonas albis sp. nov (LMG 29958, JCM 32282) and for EbBT, we propose the name Methylomonas fluvii sp. nov (LMG 29959, JCM 32283). Eco-physiological descriptions for both strains are provided. Strain Eb1 (LMG 30323, JCM 32281) is a member of the species Methylovulum psychrotolerans. This genus is so far only represented by two isolates but Eb1 is the first isolate from a temperate environment; so, an emended description of the species is given.
... In recent years, with the rapid development of high-throughput sequencing technology, the dominant microbial species and their functions in caves have been gradually discovered (8,9). The cave microbial communities exhibited high taxonomic diversity (10,11). It has been reported that the microbial communities in Kartchner caverns (eastern North America) and Jinjia caves (eastern Asia) were similar, despite the differential geographical locations (12,13). ...
In general, the constant physicochemical conditions and limited nutrient sources over long periods in the subsurface support a stable ecosystem in karst cave. Previous studies on cave microbial ecology were mostly focused on community composition, diversity, and the relationship with local environmental factors.
... Difficulties in isolating these methanotrophs make a study of the metabolism of methanotrophs in this environment tricky. However, targeted metagenomics, combining DNA stable isotope probing (DNA-SIP) techniques with analysis of 13 Clabelled DNA by high-throughput DNA sequencing allows us to study the activity and metabolic potential of specific groups of microbes in these unusual environments (Hutchens et al., 2003;Héry et al., 2008;Avrahami et al., 2011;Khadem et al., 2011;Grob et al., 2015;Esson et al., 2016;Jameson et al., 2017;Carri on et al., 2020). The technology recovers partial or complete genomes from the available metagenomic data. ...
The Zoige wetland of the Tibetan Plateau is one of the largest alpine wetlands in the world and a major emission source of methane. Methane oxidation by methanotrophs can counteract the global warming effect of methane released in the wetlands. Understanding methanotroph activity, diversity, and metabolism at the molecular level can guide the isolation of the uncultured microorganisms and inform strategy‐making decisions and policies to counteract global warming in this unique ecosystem. Here we applied DNA stable isotope probing using 13C‐labeled methane to label the genomes of active methanotrophs, examine the methane oxidation potential, and recover metagenome‐assembled genomes (MAGs) of active methanotrophs. We found that gmmaproteobacteria of type I methanotrophs are responsible for methane oxidation in the wetland. We recovered two phylogenetically novel methanotroph MAGs distantly related to extant Methylobacter and Methylovulum. They belong to type I methanotrophs of gammaproteobacteria, contain both mxaF and xoxF types of methanol dehydrogenase (MDH) coding genes, and participate in methane oxidation via H4MPT and RuMP pathways. Overall, the community structure of active methanotrophs and their methanotrophic pathways revealed by DNA‐SIP metagenomics and retrieved methanotroph MAGs highlight the importance of methanotrophs in suppressing methane emission in the wetland under the scenario of global warming. This article is protected by copyright. All rights reserved.
... The microbiology and composition of white filaments were previously studied, but limited to a few caves and springs. The caves with major research efforts were Frasassi (Macalady et al., 2006Engel, 2007), Movile (Hutchens et al., 2004;Chen et al., 2009;Kumaresan et al., 2014;Bizic et al., 2020), and Lower Kane (Engel et al., 2003(Engel et al., , 2004(Engel et al., , 2010. A few individual reports on other caves and springs can be found in the literature (Mattison et al., 1998;Engel et al., 2001;Elshahed et al., 2003;Barton and Luiszer, 2005;Reigstad et al., 2011;Rossmassler et al., 2012). ...
The thermal spring of Fetida Cave, a still active sulfuric acid cave opening at sea level and located in Santa Cesarea Terme, southeastern Salento (Apulia region, Southern Italy) hosts abundant floating white filaments. The white filaments were mainly composed of sulfur crystals surrounded by microbial mass of the phyla Epsilonbacteraeota, Proteobacteria, Bacteroidetes, and Patescibacteria. The most abundant genus in the white filaments collected from the waters in the innermost part of the cave dominated by sulfidic exhalations was Arcobacter. This abundance can be related to the higher concentration of sulfide dissolved in water, and low oxygen and pH values. Conversely, lower Arcobacter abundances were obtained in the filaments collected in the entrance and middle part of the cave, where sulfidic water mixes with seawater, as the cave is subjected to tides and the mixing of fresh (continental) with marine water. The geochemical analysis of water and atmospheric gases confirmed these environmental constraints. In fact, higher concentrations of H2S in the air and water were recorded closest to the spring upwelling in the innermost part of the cave, and the lower ones near the cave entrance. The metabolic versatility of Arcobacter might provide a competitive advantage in the colonization of water bodies characterized by high sulfide, low oxygen, and dynamic fluid movement.
... The presence of methanotrophic bacteria in caves has been widely studied in Movile Cave, Romania, by using isolation techniques, 13 CH 4 -labelling, and 13 C-DNA analysis, and the significant importance to the ecosystem development and primary productivity has been remarked upon [28][29][30][31]. Evidence of the occurrence of methanotrophs has also been found in other caves [10,32,33]. ...
Of the several critical challenges present in environmental microbiology today, one is the assessment of the contribution of microorganisms in the carbon cycle in the Earth-climate system. Karstic subterranean ecosystems have been overlooked until recently. Covering up to 25% of the land surface and acting as a rapid CH4 sink and alternately as a CO2 source or sink, karstic subterranean ecosystems play a decisive role in the carbon cycle in terms of their contribution to the global balance of greenhouse gases. Recent data indicate that microbiota must play a significant ecological role in the biogeochemical processes that control the composition of the subterranean atmosphere, as well as in the availability of nutrients for the ecosystem. Nevertheless, there are still essential gaps in our knowledge concerning the budgets of greenhouse gases at the ecosystem scale and the possible feedback mechanisms between environmental-microclimatic conditions and the rates and type of activity of microbial communities in subterranean ecosystems. Another challenge is searching for bioactive compounds (antibiotics) used for treating human diseases. At present, there is a global health emergency and a strong need for novel biomolecules. In recent decades, great research efforts have been made to extract antibiotics from marine organisms. More recently, caves have been receiving considerable attention in search of novel antibiotics. Cave methanotrophic and heterotrophic bacteria are producers of bioactive compounds and may be potential sources of metabolites with antibacterial, antifungal or anticancer activities of interest in pharmacological and medical research, as well as enzymes with a further biotechnological use. Here we also show that bacteria isolated from mines, a still unexplored niche for scientists in search of novel compounds, can be a source of novel secondary metabolites.
... Epigenic caves are more widespread, and atmospheric to subatmospheric CH4 concentrations of 1.8 ppmv to < 0.1 ppmv have been observed in these settings (Mattey et Lennon et al., 2017). In some hypogenic caves be contrast, elevated CH4 concentrations from 2 ppmv to 1 % have been observed in association with CH4-rich springs or seeps related to fluid migration from deep hydrocarbon-bearing sedimentary rocks, i.e. seepage processes that are widespread on Earth (Sarbu et al., 1996;Hutchens et al., 2004;Jones et al., 2012;Webster et al., 2017). The dominance of epigenic karst suggests that karst is likely functioning as a CH4 sink at the global scale, but more observations are needed. ...
The air in subterranean karst cavities is often depleted in methane (CH4) relative to the atmosphere. Karst is considered a potential sink for the atmospheric greenhouse gas CH4 because its subsurface drainage networks and solution-enlarged fractures facilitate atmospheric exchange. Karst landscapes cover about 14 % of earth’s continental surface, but observations of CH4 concentrations in cave air are limited to localized studies in Gibraltar, Spain, Indiana (USA), Vietnam, Australia, and by incomplete isotopic data. To test if karst is systematically acting as a global CH4 sink, we measured the CH4 concentrations, δ13CCH4, and δ2HCH4 values of cave air from 33 caves in the USA and three caves in New Zealand. We also measured CO2 concentrations, δ13CCO2, and radon (Rn) concentrations to support CH4 data interpretation by assessing cave air residence times and mixing processes. Among these caves, 35 exhibited subatmospheric CH4 concentrations in at least one location compared to their local atmospheric backgrounds. CH4 concentrations and δ13CCH4 and δ2HCH4 values suggest that microbial methanotrophy within caves is the primary CH4 consumption mechanism as the atmosphere exchanges with subsurface air. The pattern of δ13CCH4 and δ2HCH4 values along CH4 concentration gradients in cave air provides evidence for incomplete oxidation by methanotrophy. Only 5 locations from 3 caves showed elevated CH4 concentrations compared to the atmospheric background and could be ascribed to local CH4 sources from sewage and outgassing swamp water. Several associated δ13CCH4 and δ2HCH4 values point to carbonate reduction and acetate fermentation as biochemical pathways of limited methanogenesis in karst environments and suggest that these pathways occur in the environment over large spatial scales. Our data show that karst environments function as a global CH4 sink. Estimates of CH4 flux in karst landscapes are needed in order to include the subterranean CH4 sink in climate models.
... Epigenic caves are more widespread, and atmospheric to subatmospheric CH 4 concentrations of 1.8 ppmv to <0.1 ppmv have been observed in these settings (Mattey et al., 2013;Fernandez-Cortes et al., 2015;McDonough et al., 2016;Webster et al., 2016;Lennon et al., 2017). For comparison, in some hypogenic caves elevated CH 4 concentrations from 2 ppmv to 1% have been observed in association with CH 4 -rich springs or seeps related to fluid migration from deep hydrocarbon-bearing sedimentary rocks, i.e. seepage processes that are widespread on Earth (Sarbu et al., 1996;Hutchens et al., 2004;Jones et al., 2012;Webster et al., 2017). The dominance of epigenic karst suggests these regions are functioning as a CH 4 sink at the global scale, but more observations are needed. ...
The air in subterranean karst cavities is often depleted in methane (CH4) relative to the atmosphere. Karst is considered a potential sink for the atmospheric greenhouse gas CH4 because its subsurface drainage networks and solution-enlarged fractures facilitate atmospheric exchange. Karst landscapes cover about 14% of earth’s continental surface, but observations of CH4 concentrations in cave air are limited to localized studies in Gibraltar, Spain, Indiana (USA), Vietnam, Australia, and by incomplete isotopic data. To test if karst is systematically acting as a global CH4 sink, we measured the CH4 concentrations, δ13CCH4, and δ2HCH4 values of cave air from 33 caves in the USA and three caves in New Zealand. We also measured CO2 concentrations, δ13CCO2, and radon (Rn) concentrations to support CH4 data interpretation by assessing cave air residence times and mixing processes. Among these caves, 35 exhibited subatmospheric CH4 concentrations in at least one location compared to their local atmospheric backgrounds. CH4 concentrations and δ13CCH4 and δ2HCH4 values suggest that microbial methanotrophy within caves is the primary CH4 consumption mechanism as the atmosphere exchanges with subsurface air. The pattern of δ13CCH4 and δ2HCH4 values along CH4 concentration gradients in cave air provides evidence for incomplete oxidation by methanotrophy. Only 5 locations from 3 caves showed elevated CH4 concentrations compared to the atmospheric background and could be ascribed to local CH4 sources from sewage and outgassing swamp water. Several associated δ13CCH4 and δ2HCH4 values point to carbonate reduction and acetate fermentation as biochemical pathways of limited methanogenesis in karst environments and suggest that these pathways occur in the environment over large spatial scales. Our data show that karst environments function as a global CH4 sink. Estimates of CH4 flux in karst landscapes are needed in order to include the subterranean CH4 sink in climate models.
... High molecular weight genomic DNA of strain IM1 T was extracted from a 1 l culture at the early stationary phase using a previously described protocol [31]. PCR amplification of the 16S rRNA gene and partial fragments of the pmoA and mmoX genes were performed with the primers 27F/1492R [32], A189F/mb661R and mmoX206f/mmoX886r [33], respectively. The PCR conditions used were the same as those described in the original publications. ...
A Gram-stain-negative, aerobic, non-motile and coccoid methanotroph, strain IM1T, was isolated from hot spring soil. Cells of strain IM1T were catalase-negative, oxidase-positive and displayed a characteristic intracytoplasmic membrane arrangement of type I methanotrophs. The strain possessed genes encoding both membrane-bound and soluble methane monooxygenases and grew only on methane or methanol. The strain was capable of growth at temperatures between 15 and 48 °C (optimum, 30–45 °C) and pH values between pH 4.8 and 8.2 (optimum, pH 6.2–7.0). Based on phylogenetic analysis of 16S rRNA gene and PmoA sequences, strain IM1T was demonstrated to be affiliated to the genus Methylococcus . The 16S rRNA gene sequence of this strain was most closely related to the sequences of an uncultured bacterium clone FD09 (100 %) and a partially described cultured Methylococcus sp. GDS2.4 (99.78 %). The most closely related taxonomically described strains were Methylococcus capsulatus TexasT (97.92 %), Methylococcus capsulatus Bath (97.86 %) and Methyloterricola oryzae 73aT (94.21 %). Strain IM1T shared average nucleotide identity values of 85.93 and 85.62 % with Methylococcus capsulatus strains TexasT and Bath, respectively. The digital DNA–DNA hybridization value with the closest type strain was 29.90 %. The DNA G+C content of strain IM1T was 63.3 mol% and the major cellular fatty acids were C16 : 0 (39.0 %), C16 : 1 ω7c (24.0 %), C16 : 1 ω6c (13.6 %) and C16 : 1 ω5c (12.0 %). The major ubiquinone was methylene-ubiquinone-8. On the basis of phenotypic, genetic and phylogenetic data, strain IM1T represents a novel species of the genus Methylococcus for which the name Methylococcus geothermalis sp. nov. is proposed, with strain IM1T (=JCM 33941T=KCTC 72677T) as the type strain.
The cosmos, replete with the elemental diversity of Earth, continues to cloak the existence of extraterrestrial life in mystery. Vital to the search for life is the identification of environments that can support biological processes. This review synthesizes current knowledge on the conditions necessary for life, with a special focus on extremophiles. These organisms, thriving under extreme conditions, illustrate the robust adaptability required for life beyond Earth. We examine the evolutionary adaptations on Earth to provide a framework for potential analogues on other celestial bodies. Importantly, this paper reports the initial identification of approximately three hundred three (∼ 303) sites on Mars as potential habitats intriguingly considered as potential Mars Cave Candidates (MCC) based on meticulous visual interpretations, and require detailed investigation to confirm their nature, whether as caves, other geological features, or craters. These investigations are currently ongoing, highlighting the dynamic and exploratory nature of Martian research. The findings are preliminary and serve to inform the development of robotic exploration strategies aimed at in-depth study of these environments, thereby advancing the astrobiological search for life. This review sets strategic directions for future research, aiming to refine our approach to uncovering where and how life could exist across the cosmos. By guiding future missions, both robotic and astrobiological, this work seeks to deepen our understanding of potential extraterrestrial habitats and to foster a systematic exploration of these promising sites.
The carbonate critical zone (CZ) is characterized by extensive groundwater-surface water exchange that leads to highly variable redox states of groundwater. Changes in redox condition may cause either production or consumption of methane (CH4), thereby providing an atmospheric source or sink of this important greenhouse gas. To assess how groundwater-surface water exchange affects redox state and CH4 cycling in the carbonate CZ, we measured CH4 concentrations and 13C isotopes in water from streams, spring systems, and wells in north-central Florida. Sampled groundwater has subsurface residence times ranging from hours at a stream sink-rise system, to months following a flood recharge event into a spring vent, to decades at springs with limited point recharge. Concentrations of CH4 ranged from 0.002 to 89 μM, with an inverse relationship in springs between subsurface residence time and CH4 concentration. Where residence time is short, low CH4 concentrations result from methanotrophy linked to elevated dissolved oxygen (DO) concentrations. Following flooding, methanotrophy occurs soon after recharge and is followed by methanogenesis as groundwater becomes increasingly reducing. Groundwater extracted from wells had CH4 concentrations greater than spring water indicating CH4 is lost during flow to spring vents. CH4 concentrations covary with δ13C-CH4 values, which supports both methanogenesis and methanotrophy with changing residence times. Mean fluxes of CH4 ranged from -0.05 to 1.0 mg m-2 d-1 at spring vents, with negative values caused by CH4 uptake in water undersaturated with respect to atmospheric concentration. Most springs are dominated by methanotrophy, limiting atmospheric evasion of CH4 produced in the carbonate CZ. We estimate CH4 emissions to be 12.6 × 10-6 Tg a-1 across all Florida springs or about two orders of magnitude less than emissions from Floridan aquifer groundwater abstraction (3041 × 10-6 Tg a-1). Although CH4 is produced in the carbonate CZ, natural attenuation limits its effects on the global carbon cycle.
A novel methane-oxidizing bacterial strain SS37A-Re T was isolated from surface soil of a rice paddy field in Japan. Cells were Gram-stain-negative, motile rods with single polar flagellum and type II intracytoplasmic membrane arrangement. The strain grew on methane or methanol as the sole carbon and energy source. It grew at 15–37 °C (optimum 25–30 °C), pH 6.0–9.0 (optimum 7.0–8.0) and with 0–0.1 % (w/w) NaCl (no growth at 0.5 % or above). Cells formed cysts, but not exospores. The results of sequence analysis of the 16S rRNA gene indicated that SS37A-Re T represented a member of the family Methylocystaceae , with the highest similarity (98.9 %) to Methylocystis parva corrig. OBBP T . Phylogenetic analysis of pmoA and mxaF genes and core genes in the genome indicated that the strain was closely related to the members of the genus Methylocystis , while the analysis of the mmoX gene indicated the close relationships with the genus Methylosinus . The values of genome relatedness between SS37A-Re T and species of the genera Methylocystis and Methylosinus were 78.6–82.5% and 21.7–24.9 % estimated by the average nucleotide identity and digital DNA–DNA hybridisation, respectively, showing the highest values with Methylocystis echinoides LMG 27198 T . The DNA G+C content was 63.2 mol% (genome). The major quinone and fatty acids were Q-8 and, C 18 : 1 (C 18 : 1 ω8 t and C 18 : 1 ω8 c ) and C 18 : 2 , respectively. On the basis of the phenotypic and phylogenetic features, the strain represents a novel species of the genus Methylocystis , for which the name Methylocystis iwaonis sp. nov. is proposed. The type strain is SS37A-Re T (=JCM 34278 T =NBRC 114996 T =KCTC 82710 T ).
Sulfidic cave ecosystems are remarkable evolutionary hotspots that have witnessed adaptive radiation of their fauna represented by extremophile species having particular traits. Ostracods, a very old group of crustaceans, exhibit specific morphological and ecophysiological features that enable them to thrive in groundwater sulfidic environments. Herein, we report a peculiar new ostracod species Pseudocandonamovilaensis sp. nov. thriving in the chemoautotrophic sulfidic groundwater ecosystem of Movile Cave (Romania). The new species displays a set of homoplastic features specific for unrelated stygobitic species, e.g., triangular carapace in lateral view with reduced postero-dorsal part and simplification of limb chaetotaxy (i.e., loss of some claws and reduction of secondary male sex characteristics), driven by a convergent or parallel evolution during or after colonization of the groundwater realm. P.movilaensis sp. nov. thrives exclusively in sulfidic meso-thermal waters (21 °C) with high concentrations of sulphides, methane, and ammonium. Based on the geometric morphometrics-based study of the carapace shape and molecular phylogenetic analyses based on the COI marker (mtDNA), we discuss the phylogenetic relationship and evolutionary implication for the new species to thrive in groundwater sulfidic groundwater environments.
Landfill cover soil is the environmental interface between landfills and the atmosphere and plays an important role in mitigating CH4 emission from landfills. Here, stable isotope probing microcosms with CH4 or CH4 and dimethyl sulfide (DMS) were carried out to characterize activity and community structure of methanotrophs in landfill cover soils under DMS stress. The CH4 oxidation activity in the landfill cover soils was not obviously influenced at the DMS concentration of 0.05%, while it was inhibited at the DMS concentrations of 0.1% and 0.2%. DMS-S was mainly oxidized to sulfate (SO42-) in the landfill cover soils. In the landfill cover soils, DMS could inhibit the expression of bacteria and decrease the abundances of pmoA and mmoX genes, while it could prompt the expression of pmoA and mmoX genes. γ-Proteobacteria methanotrophs including Methylocaldum, Methylobacter, Crenothrix and unclassified Methylococcaceae and α-Proteobacteria methanotrophs Methylocystis dominated in assimilating CH4 in the landfill cover soils. Of them, Methylobacter and Crenothrix had strong tolerance to DMS or DMS could promote the growth and activity of Methylobacter and Crenothrix, while Methylocaldum had weak tolerance to DMS and showed an inhibitory effect. Metagenomic analyses showed that methanotrophs had the genes of methanethiol oxidation and could metabolize CH4 and methanethiol simultaneously in the landfill cover soils. These findings suggested that methanotrophs might metabolize sulfur compounds in the landfill cover soils, which may provide the potential application in engineering for co-removal of CH4 and sulfur compounds.
Among fundamental research questions in subterranean biology, the role of subterranean microbiomes playing in key elements cycling is a top-priority one. Karst caves are widely distributed subsurface ecosystems, and cave microbes get more and more attention as they could drive cave evolution and biogeochemical cycling. Research have demonstrated the existence of diverse microbes and their participance in biogeochemical cycling of elements in cave environments. However, there are still gaps in how these microbes sustain in caves with limited nutrients and interact with cave environment. Cultivation of novel cave bacteria with certain functions is still a challenging assignment. This review summarized the role of microbes in cave evolution and mineral deposition, and intended to inspire further exploration of microbial performances on C/N/S biogeocycles.
Lakes are important methane (CH4) sources to the atmosphere, especially eutrophic lakes with cyanobacterial blooms accompanied by volatile sulfur compound (VSC) emissions. CH4 oxidation is a key strategy to mitigate CH4 emission from lakes. In this study, we characterized the fate of CH4-derived carbon and active microbial communities in lake sediments with CS2 used as a typical VSC, based on the investigation of CH4 and VSC fluxes from Meiliang Bay in Lake Taihu. Stable isotope probing microcosm incubation showed that the efficiency of CH4-derived carbon incorporated into organic matter was 21.1% in the sediment with CS2 existence, which was lower than that without CS2 (27.3%). SO4²⁻-S was the main product of CS2 oxidation under aerobic condition, accounting for 59.3–62.7% of the input CS2–S. CS2 and CH4 coexistence led to a decrease of methanotroph and methylotroph abundances and stimulated the production of extracellular polymeric substances. CS2 and its metabolites including total sulfur, SO4²⁻ and acid volatile sulfur acted as the main drivers influencing the active microbial community structure in the sediments. Compared with α-proteobacteria methanotrophs, γ-proteobacteria methanotrophs Methylomicrobium, Methylomonas, Crenothrix and Methylosarcina were more dominant in the sediments. CH4-derived carbon mainly flowed into methylotrophs in the first stage. With CH4 consumption, more CH4-derived carbon flowed into non-methylotrophs. CS2 could prompt more CH4-derived carbon flowing into non-methanotrophs and non-methylotrophs, such as sulfur-metabolizing bacteria. These findings can help elucidate the influence of VSCs on microorganisms and provide insights to carbon fluxes from eutrophic lake systems.
Methane-oxidizing bacteria, or methanotrophs, can use methane as their sole carbon and energy source, and have a wide range of applications including: (1) methane removal from the atmosphere, (2) pollutant degradation, and (3) valorization of methane. Such applications are strongly dependent on copper and rare earth elements (REEs) due to their central role in regulating the metabolism of methanotrophs. To fully utilize these intriguing microbes for various applications, the genetics and biochemistry of metal uptake systems in methanotrophs must thus be identified and characterized. To address this general goal, this work first sought to characterize the evolution of methanotrophs to glean insights into potential uptake systems of copper and REEs, and to develop strategies to optimize their production for industrial and medical applications. Bioinformatic analyses suggested that methylotrophs with preexisting copper uptake system(s) may have evolved into methanotrophs with the lateral gene transfer of methane monooxygenase, the critical enzyme that oxidizes methane to methanol in methanotrophs. In addition, lateral gene transfer events of methanol dehydrogenase (MeDH) with a REE active site (Xox-MeDH) were more prevalent than those of MeDH with Ca(II). This may be attributable to the higher catalytic efficiency of the former MeDH, which consequently increased the fitness of methanotrophs with multiple copies. Second, competition between methanotrophs for copper was investigated to identify “cheating” behavior amongst methanotrophs and determine how the collective activity of methanotrophs is affected. Some methanotrophs produce methanobactin (MB), a chalkophore that can strongly bind and deliver copper. It was found that methanotrophs Methylomicrobium album BG8 and Methylocystis sp. strain Rockwell, both non-MB producers, can take up MB, while Methylococcus capsulatus Bath cannot. In addition, Mmb. album BG8 was found to produce a novel chalkophore yet to be characterized. Moreover, Mmb. album BG8 and Methylocystis sp. strain Rockwell could also take up methylmercury-MB (MeHg-MB) complex and subsequently demethylate MeHg into the less toxic inorganic mercury. The results of this study provide insight into copper competition between methanotrophs and potential applications exploiting such interaction, such as MeHg remediation. Finally, the mechanism of methanotrophic-mediated MeHg demethylation was investigated. It has been found that MB serves as a delivery mechanism for MeHg into the cell of methanotrophs, where Xox-MeDH contributes to demethylating MeHg. The genes encoding for organoarsenical lyase (ArsI), responsible for cleaving the carbon-arsenic bond, and lanmodulin (LanM), a periplasmic REE-binding protein, were each knocked out in wildtype Methylosinus trichosporium OB3b. Deletion of arsI did not affect MeHg degradation in the Msn. trichosporium OB3b delarsI mutant. However, the dellanM mutant was unable to degrade MeHg under all conditions tested, suggesting lanM to be critical for MeHg degradation. In addition, a spheroplast prepared from Msn. trichosporium OB3b delmbnT mutant exhibited decreased MeHg degradation, whereas greater MeHg degradation was observed in the extract containing the periplasm and outer membrane debris. These results suggest that MeHg degradation occurs in the periplasm, where both Xox-MeDH and LanM reside. The results of this study are anticipated to contribute to our ability to utilize methanotrophy for a wide range of applications.
In the past two decades, DNA/RNA-stable isotope probing (SIP) techniques have been widely used in microbial ecology to decipher the link between microbial metabolic function and phylogenetic information. Diverse methodological strategies were employed to guarantee the successful SIP experiments. In view of the emerging literature and our experience in the optimization of methods for DNA/RNA-SIP, it is crucial to provide a methodological comparison. This review discusses methods published on DNA/RNA SIP, considering a range of experimental parameters, including the selection of nucleic acid, labelled element, substrate amount, incubation time, the speed and time of isopycnic gradient centrifugation, and the detection approach of labelled fractions and microbes after ultracentrifugation. Moreover, some scopes of environmental microorganism studied through ¹³C-DNA/RNA-SIP technology, such as microbes assimilating the inorganic carbon and low molecular weight organic acids, degrading the organic pollutants and residues were summarized in this chapter.
Aerobic methanotrophs are crucial in ombrotrophic peatlands, driving the methane and nitrogen cycles. Peat mining adversely affects the methanotrophs, but activity and community composition/abundances may recover after restoration. Considering that the methanotrophic activity and growth are significantly stimulated in the presence of other microorganisms, the methane-driven interaction network, encompassing methanotrophs and non-methanotrophs (i.e., methanotrophic interactome), may also be relevant in conferring community resilience. Yet, little is known of the response and recovery of the methanotrophic interactome to disturbances. Here, we determined the recovery of the methanotrophic interactome as inferred by a co-occurrence network analysis, comparing a pristine and restored peatland. We coupled a DNA-based stable isotope probing (SIP) approach using 13C-CH4 to a co-occurrence network analysis derived from the 13C-enriched 16S rRNA gene sequences to relate the response in methanotrophic activity to the structuring of the interaction network. Methanotrophic activity and abundances recovered after peat restoration since 2000. ‘Methylomonaceae’ was the predominantly active methanotrophs in both peatlands, but differed in the relative abundance of Methylacidiphilaceae and Methylocystis. However, bacterial community composition was distinct in both peatlands. Likewise, the methanotrophic interactome was profoundly altered in the restored peatland. Structuring of the interaction network after peat mining resulted in the loss of complexity and modularity, indicating a less connected and efficient network, which may have consequences in the event of recurring/future disturbances. Therefore, determining the response of the methane-driven interaction network, in addition to relating methanotrophic activity to community composition/abundances, provided a more comprehensive understanding of the resilience of the methanotrophs.
Importance
The resilience and recovery of microorganisms from disturbances are often determined with regard to their activity and community composition/abundances. Rarely has the response of the network of interacting microorganisms been considered, despite accumulating evidence showing that microbial interaction modulates community functioning. Comparing the methane-driven interaction network of a pristine and restored peatland, our findings revealed that the metabolically active microorganisms were less connected and formed less modular ‘hubs’ in the restored peatland, indicative of a less complex network which may have consequences with recurring disturbances and environmental changes. This also suggests that the resilience and full recovery in the methanotrophic activity and abundances do not reflect on the interaction network. Therefore, it is relevant to consider the interaction-induced response, in addition to documenting changes in activity and community composition/abundances, to provide a comprehensive understanding of the resilience of microorganisms to disturbances.
Bacteria grow and transform elements at different rates, yet quantifying this variation in the environment is difficult. Determining isotope enrichment with fine taxonomic resolution after exposure to isotope tracers could help, but there are few suitable techniques. We propose a modification to Stable Isotope Probing (SIP) that enables determining the isotopic composition of DNA from individual bacterial taxa after exposure to isotope tracers. In our modification, after isopycnic centrifugation, DNA is collected in multiple density fractions, and each fraction is sequenced separately. Taxon specific density curves are produced for labeled and non-labeled treatments, from which the shift in density for each individual taxon in response to isotope labeling is calculated. Expressing each taxon’s density shift relative to that taxon’s density measured without isotope enrichment accounts for the influence of nucleic acid composition on density and isolates the influence of isotope tracer assimilation. The shift in density translates quantitatively to isotopic enrichment. Because this revision to SIP allows quantitative measurements of isotope enrichment, we propose to call it quantitative Stable Isotope Probing (qSIP). We demonstrate qSIP using soil incubations, in which soil bacteria exhibited strong taxonomic variation in ¹⁸ O and ¹³ C composition after exposure to ¹⁸ O-H 2 O or ¹³ C-glucose. Addition of glucose increased assimilation of ¹⁸ O into DNA from ¹⁸ O-H 2 O. However, the increase in ¹⁸ O assimilation was greater than expected based on utilization of glucose-derived carbon alone, because glucose addition indirectly stimulated bacteria to utilize other substrates for growth. This example illustrates the benefit of a quantitative approach to stable isotope probing.
Microbial N 2 fixation (diazotrophy) represents an important nitrogen source to oligotrophic peatland ecosystems, which are important sinks for atmospheric CO 2 and susceptible to changing climate. The objectives of this study were: (i) to determine the active microbial group and type of nitrogenase mediating diazotrophy in a ombrotrophic Sphagnum -dominated peat bog (the S1 peat bog, Marcell Experimental Forest, Minnesota, USA); and (ii) to determine the effect of environmental parameters (light, O 2 , CO 2 , CH 4 ) on potential rates of diazotrophy measured by acetylene (C 2 H 2 ) reduction and ¹⁵ N 2 incorporation. Molecular analysis of metabolically active microbial communities suggested that diazotrophy in surface peat was primarily mediated by Alphaproteobacteria ( Bradyrhizobiaceae and Beijerinckiaceae ). Despite higher dissolved vanadium (V; 11 nM) than molybdenum (Mo; 3 nM) in surface peat, a combination of metagenomic, amplicon sequencing and activity measurements indicated that Mo-containing nitrogenases dominate over the V-containing form. Acetylene reduction was only detected in surface peat exposed to light, with the highest rates observed in peat collected from hollows with the highest water content. Incorporation of ¹⁵ N 2 was suppressed 90% by O 2 and 55% by C 2 H 2 , and was unaffected by CH 4 and CO 2 amendments. These results suggest that peatland diazotrophy is mediated by a combination of C 2 H 2 - sensitive and C 2 H 2 - insensitive microbes that are more active at low O 2 and show similar activity at high and low CH 4 .
Importance
Previous studies indicate that diazotrophy provides an important nitrogen source and is linked to methanotrophy in Sphagnum -dominated peatlands. However, the environmental controls and enzymatic pathways of peatland diazotrophy, as well as the metabolically active microbial populations that catalyze this process remain in question. Our findings indicate that oxygen levels and photosynthetic activity override low nutrient availability in limiting diazotrophy, and that members of the Alphaproteobacteria ( Rhizobiales ) catalyze this process at the bog surface using the molybdenum - based form of the nitrogenase enzyme.
Bacteria grow and transform elements at different rates, yet quantifying this variation in the environment is difficult. Determining isotope enrichment with fine taxonomic resolution after exposure to isotope tracers could help, but there are few suitable techniques. We propose a modification to Stable Isotope Probing (SIP) that enables determining the isotopic composition of DNA from individual bacterial taxa after exposure to isotope tracers. In our modification, after isopycnic centrifugation, DNA is collected in multiple density fractions, and each fraction is sequenced separately. Taxon specific density curves are produced for labeled and non-labeled treatments, from which the shift in density for each individual taxon in response to isotope labeling is calculated. Expressing each taxon’s density shift relative to that taxon’s density measured without isotope enrichment accounts for the influence of nucleic acid composition on density and isolates the influence of isotope tracer assimilation. The shift in density translates quantitatively to isotopic enrichment. Because this revision to SIP allows quantitative measurements of isotope enrichment, we propose to call it quantitative Stable Isotope Probing (qSIP). We demonstrate qSIP using soil incubations, in which soil bacteria exhibited strong taxonomic variation in ¹⁸ O and ¹³ C composition after exposure to ¹⁸ O-H 2 O or ¹³ C-glucose. Addition of glucose increased assimilation of ¹⁸ O into DNA from ¹⁸ O-H 2 O. However, the increase in ¹⁸ O assimilation was greater than expected based on utilization of glucose-derived carbon alone, because glucose addition indirectly stimulated bacteria to utilize other substrates for growth. This example illustrates the benefit of a quantitative approach to stable isotope probing.
Movile Cave, recently discovered in southern Romania, contains sulfide‐rich thermal waters in submerged passages, as well as isolated air pockets. The water surfaces within the air pockets are covered by substantial microbial biofilms, while the air bells contain an abundant and diverse community of terrestrial and aquatic animal species. Based on the results of dehydrogenase activity, fecal streptococci counts, and stable carbon isotope ratios, we propose that the cave community is biologically isolated and receives little, if any, organic carbon inputs from the surface environment. Several sulfide‐oxidizing chemoautotrophic bacteria were isolated from the cave waters. One putative Thiosphaera sp. strain, LV‐43, was further characterized. The presence and high level activity of RuBisCO was clearly demonstrated in this strain.
The numbers of methane-oxidizing bacteria (methanotrophs) in the sediments of Lake Washington were estimated using three culture-independent methods. Quantitative slot-blot hybridizations were performed with type I and type II methanotroph-specific probes. These data were compared to data from quantitative hybridizations using a pmoA-specific probe and a eubacterial probe. From the combined hybridization data, the methanotroph population in Lake Washington was estimated to be 3.6 x 10(8)-7.4 x 10(8) cells/g dry weight. Methanotroph community structure and number were also investigated using polar lipid fatty acid (PLFA) analysis. Analysis of biomarker PLFAs characteristic of both type I (16:1 omega 8) and type II (18:1 omega 8) methanotrophs was used to estimate the abundance of these bacteria in Lake Washington sediments. From the PLFA data, the methanotroph population in Lake Washington was estimated to be 7.1 x 10(8)-9.4 x 10(9) cells/g dry weight. As a third method of quantitation, we calculated the methanotroph population using the total methane oxidation rate for whole cells in Lake Washington sediment to be 1.3 x 10(8)-1.2 x 10(9) cells/g dry weight. The three independent estimates of the number of methanotrophs in Lake Washington sediment agree within a two- to fourfold range. These data suggest that the three techniques used in this study detect the functionally significant population of methanotrophs in Lake Washington. Furthermore, these techniques will be useful for obtaining estimates of methanotroph abundance in additional environments.
A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
A major limitation of rRNA-targeted group-specific probes is that they may cross-react with organisms of other physiological, or even phylogenetic groups when applied to environmental samples containing unknown sequences. We have exploited the restricted physiology of methane-oxidizing bacteria to assess the specificity and efficiency of probes for this physiological type which target the 16S rRNA or genes involved in methanotroph physiology. Seawater samples were enriched for methanotrophs by addition of methane and essential nutrients. The changes in composition of the bacterial population were monitored by analysis of 16S rRNA gene libraries. Methanotroph group-specific probes failed to give a signal with samples from these enrichments even though a methanol dehydrogenase structural gene was detected. A 16S rDNA sequence that was abundant only after methane addition was recovered and found to show a close phylogenetic relationship to Methylomonas. Organisms containing this sequence were observed in enrichments by in situ hybridization. The combination of enrichment on methane and screening with the broad specificity methanol dehydrogenase probe allowed detection of novel methanotrophs that were not detected with the original suite of methanotroph group-specific probes.
The methanol dehydrogenase gene mxaF, encoding the large subunit of the enzyme, was amplified from the DNA of a number of representative methanotrophs, methyletrophs, and environmental samples by PCR using primers designed from regions of conserved amino acid sequence identified by comparison of three known sequences of the large subunit of methanol dehydrogenase. The resulting 550-bp PCR products were cloned and sequenced. Analysis of the predicted amino acid sequences corresponding to these mxaF genes revealed strong sequence conservation. Of the 172 amino acid residues, 47% were conserved among all 22 sequences obtained in this study. Phylogenetic analysis of these MxaF sequences showed that those from type I and type II methanotrophs form two distinct clusters and are separate from MxaF sequences of other gram-negative methylotrophs. MxaF sequences retrieved by PCR from DNA isolated from a blanket bog peat core sample formed a distinct phylogenetic cluster within the MxaF sequences of type II methanotrophs and may originate from a novel group of acidophilic methanotrophs which have yet to be cultured from this environment.
Bacterial strain LV43 was previously isolated from a floating microbial mat located in Movile Cave, the access point to a chemoautotrophically based groundwater ecosystem in southern Romania. This gram-negative, rod-shaped organism grows autotrophically through the oxidation of thiosulfate and sulfide, but it does not grow heterotrophically. Strain LV43 grows over a pH range of 5.0 to 9.0, with an optimum near 7.5 at 28 degrees C. The pH of the medium decreased from 7.5 to 6.5 during growth on thiosulfate. Carbon isotope fractionation values for strain LV43 were within the previously reported range of fractionation values for the overall floating microbial mat in Movile Cave and were similar to values reported for chemoautotrophic sulfur-oxidizing strains of Thiobacillus neapolitanus and Thiomicrospira sp. The 16S rRNA gene sequence of strain LV43 was determined, and phylogenetic analysis indicated that strain LV43 was most closely related to Thiobacillus thioparus and the uncultured bacterial strain Strip2, which is represented by a 16S rRNA clone obtained by direct PCR from the Stripa research mine in Sweden. This identification of strain LV43 is supported by its G+C content of 62%, which is within the range reported for strains of T. thioparus. Fluorescently labeled polyclonal antibodies specific for strain LV43 were used to locate and enumerate this strain at different locations in Movile Cave and in nearby surface-water and groundwater sources. Strain LV43 was found only at aerobic, neutral-pH sites within the cave. Strain LV43 was also found outside Movile Cave in surface waters and in groundwater believed to intercept the same sulfurous aquifer as Movile Cave.
Rice field soil with a nonsaturated water content induced CH4 consumption activity when it was supplemented with 5% CH4. After a lag phase of 3 days, CH4 was consumed rapidly until the concentration was less than 1.8 parts per million by volume (ppmv). However, the soil was not able to maintain the oxidation activity at near-atmospheric CH4 mixing ratios (i.e., 5 ppmv). The soil microbial community was monitored by performing denaturing gradient gel electrophoresis (DGGE) during the oxidation process with different PCR primer sets based on the 16S rRNA gene and on functional genes. A universal small-subunit (SSU) ribosomal DNA (rDNA) primer set and 16S rDNA primer sets specifically targeting type I methylotrophs (members of the gamma subdivision of the class Proteobacteria [gamma-Proteobacteria]) and type II methylotrophs (members of the alpha-Proteobacteria) were used. Functional PCR primers targeted the genes for particulate methane monooxygenase (pmoA) and methanol dehydrogenase (mxaF), which code for key enzymes in the catabolism of all methanotrophs. The yield of PCR products amplified from DNA in soil that oxidized CH4 was the same as the yield of PCR products amplified from control soil when the universal SSU rDNA primer set was used but was significantly greater when primer sets specific for methanotrophs were used. The DGGE patterns and the sequences of major DGGE bands obtained with the universal SSU rDNA primer set showed that the community structure was dominated by nonmethanotrophic populations related to the genera Flavobacterium and Bacillus and was not influenced by CH4. The structure of the methylotroph community as determined with the specific primer sets was less complex; this community consisted of both type I and type II methanotrophs related to the genera Methylobacter, Methylococcus, and Methylocystis. DGGE profiles of PCR products amplified with functional gene primer sets that targeted the mxaF and pmoA genes revealed that there were pronounced community shifts when CH4 oxidation began. High CH4 concentrations stimulated both type I and II methanotrophs in rice field soil with a nonsaturated water content, as determined with both ribosomal and functional gene markers.
The influence of grazing by the bacterivorous nanoflagellate Ochromonas sp. strain DS on the taxonomic and morphological structures of a complex bacterial community was studied in one-stage chemostat experiments. A bacterial community, consisting of at least 30 different strains, was fed with a complex carbon source under conditions of low growth rate (0.5 day(-1) when nongrazed) and low substrate concentration (9 mg liter(-1)). Before and after the introduction of the predator, the bacterial community composition was studied by in situ techniques (immunofluorescence microscopy and fluorescent in situ hybridization), as well as by cultivation on agar media. The cell sizes of nonspecifically stained and immunofluorescently labeled bacteria were measured by image analysis. Grazing by the flagellate caused a bidirectional change in the morphological structure of the community. Medium-size bacterial cells, which dominated the nongrazed community, were largely replaced by smaller cells, as well as by cells contained in large multicellular flocs. Cell morphological changes were combined with community taxonomic changes. After introduction of the flagellate, the dominating strains with medium-size cells were largely replaced by single-celled strains with smaller cells on the one hand and, on the other hand, by Pseudomonas sp. strain MWH1, which formed the large, floc-like forms. We assume that size-selective grazing was the major force controlling both the morphological and the taxonomic structures of the model community.
The soluble MMO (sMMO) gene clusters from group I methanotrophs were characterized. An 8.1-kb KpnI fragment from Methylomonas sp. strain KSWIII and a 7.5-kb SalI fragment from Methylomonas sp. strain KSPIII which contained the sMMO gene clusters were cloned and sequenced. The sequences of these two fragments were almost identical. The sMMO gene clusters in the fragment consisted of six open reading frames which were 52 to 79% similar to the corresponding genes of previously described sMMO gene clusters of the group II and group X methanotrophs. The phylogenetic analysis of the predicted amino acid sequences of sMMO demonstrated that the sMMOs from these strains were closer to that from M. capsulatus Bath in the group X methanotrophs than to those from Methylosinus trichosporium OB3b and Methylocystis sp. strain M in the group II methanotrophs. Based on the sequence data of sMMO genes of our strains and other methanotrophs, we designed a new PCR primer to amplify sMMO gene fragments of all the known methanotrophs harboring the mmoX gene. The primer set was successfully used for detecting methanotrophs in the groundwater of trichloroethylene-contaminated sites during in situ-biostimulation treatments.
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.
Profiles of dissolved O2 and methane with increasing depth were generated for Lake Washington sediment, which suggested the zone of methane oxidation
is limited to the top 0.8 cm of the sediment. Methane oxidation potentials were measured for 0.5-cm layers down to 1.5 cm
and found to be relatively constant at 270 to 350 μmol/liter of sediment/h. Approximately 65% of the methane was oxidized
to cell material or metabolites, a signature suggestive of type I methanotrophs. Eleven methanotroph strains were isolated
from the lake sediment and analyzed. Five of these strains classed as type I, while six were classed as type II strains by
16S rRNA gene sequence analysis. Southern hybridization analysis with oligonucleotide probes detected, on average, one to
two copies of pmoA and one to three copies of 16S rRNA genes. Only one restriction length polymorphism pattern was shown for pmoA genes in each isolate, and in cases where, sequencing was done, the pmoA copies were found to be almost identical. PCR primers were developed formmoX which amplified 1.2-kb regions from all six strains that tested positive for cytoplasmic soluble methane mono-oxygenase (sMMO)
activity. Phylogenetic analysis of the translated PCR products with published mmoX sequences showed that MmoX falls into two distinct clusters, one containing the orthologs from type I strains and another
containing the orthologs from type II strains. The presence of sMMO-containing Methylomonas strains in a pristine freshwater lake environment suggests that these methanotrophs are more widespread than has been previously
thought.
Three particulate methane monooxygenase PCR primer sets (A189-A682, A189-A650, and A189-mb661) were investigated for their
ability to assess methanotroph diversity in soils from three sites, i.e., heath, oak, and sitka, each of which was capable
of oxidizing atmospheric concentrations of methane. Each PCR primer set was used to construct a library containing 50 clones
from each soil type. The clones from each library were grouped by restriction fragment length polymorphism, and representatives
from each group were sequenced and analyzed. Libraries constructed with the A189-A682 PCR primer set were dominated byamoA-related sequences or nonspecific PCR products with nonsense open reading frames. The primer set could not be used to assess
methanotroph diversity in these soils. A newpmoA-specific primer, A650, was designed in this study. The A189-A650 primer set demonstrated distinct biases both in clone library
analysis and when incorporated into denaturing gradient gel electrophoresis analysis. The A189-mb661 PCR primer set demonstrated
the largest retrieval of methanotroph diversity of all of the primer sets. However, this primer set did not retrieve sequences
linked with novel high-affinity methane oxidizers from the soil libraries, which were detected using the A189-A650 primer
set. A combination of all three primer sets appears to be required to examine both methanotroph diversity and the presence
of novel methane monooxygenase sequences.
The active population of low-affinity methanotrophs in a peat soil microcosm was characterized by stable-isotope probing.
“Heavy” 13C-labeled DNA, produced after microbial growth on 13CH4, was separated from naturally abundant 12C-DNA by cesium chloride density gradient centrifugation and used as a template for the PCR. Amplification products of 16S
rRNA genes and pmoA, mxaF, and mmoX, which encode key enzymes in the CH4 oxidation pathway, were analyzed. Sequences related to extant type I and type II methanotrophs were identified, indicating
that these methanotrophs were active in peat exposed to 8% (vol/vol) CH4. The 13C-DNA libraries also contained clones that were related to β-subclass Proteobacteria, suggesting that novel groups of bacteria may also be involved in CH4 cycling in this soil.
Stable-isotope probing (SIP) is a culture-independent technique that enables the isolation of DNA from micro-organisms that are actively involved in a specific metabolic process. In this study, SIP was used to characterize the active methylotroph populations in forest soil (pH 3.5) microcosms that were exposed to (13)CH(3)OH or (13)CH(4). Distinct (13)C-labelled DNA ((13)C-DNA) fractions were resolved from total community DNA by CsCl density-gradient centrifugation. Analysis of 16S rDNA sequences amplified from the (13)C-DNA revealed that bacteria related to the genera Methylocella, Methylocapsa, Methylocystis and Rhodoblastus had assimilated the (13)C-labelled substrates, which suggested that moderately acidophilic methylotroph populations were active in the microcosms. Enrichments targeted towards the active proteobacterial CH(3)OH utilizers were successful, although none of these bacteria were isolated into pure culture. A parallel analysis of genes encoding the key enzymes methanol dehydrogenase and particulate methane monooxygenase reflected the 16S rDNA analysis, but unexpectedly revealed sequences related to the ammonia monooxygenase of ammonia-oxidizing bacteria (AOB) from the beta-subclass of the PROTEOBACTERIA: Analysis of AOB-selective 16S rDNA amplification products identified Nitrosomonas and Nitrosospira sequences in the (13)C-DNA fractions, suggesting certain AOB assimilated a significant proportion of (13)CO(2), possibly through a close physical and/or nutritional association with the active methylotrophs. Other sequences retrieved from the (13)C-DNA were related to the 16S rDNA sequences of members of the Acidobacterium division, the beta-Proteobacteria and the order Cytophagales, which implicated these bacteria in the assimilation of reduced one-carbon compounds or in the assimilation of the by-products of methylotrophic carbon metabolism. Results from the (13)CH(3)OH and (13)CH(4) SIP experiments thus provide a rational basis for further investigations into the ecology of methylotroph populations in situ.
The thermomineral sulfurous waters at Mangalia in southeastern Dobrogea, Romania, have been known and used as spa facilities for well over 2,000 years (Feru and Capotà 1991). Hydrogeologieal studies performed during the last 60 years (Macovei 1912; Ciocîrdel and Protopopescu-Pache 1955; Moissiu 1968; Feru and Capotà 1991) identified a deep captive sulfurous aquifer located in Barremian-Jurassic limestones, extending 15 km to the North and 50 km to the South of Mangalia. In the Mangalia region, a system of geological faults allows the deep water to ascend toward the surface and mix with the Sarmatian oxygenated waters (Lascu et al. 1993). The biological investigation of the subsurface ecosystems associated with the sulfurous waters at Mangalia commenced in the late eighties, after the discovery of Movile Cave and its unique subterranean chemoautotrophically based ecosystem (Sarbu, 1990).
In order to investigate the food chain and energy balance of the chemolithoautotrophically-based ecosystem of the sulphur spring in Movile Cave as a model system for extraterrestrial life, a first sampling campaign was done. Microbial diversity and activity were analysed by MPN-enumeration methods and microcalorimetry, respectively. In addition, a speciation of the inorganic sulphur compounds by HPLC and IC techniques was performed. Metabolic activities were predominantly connected with thick microbial mats floating on the water surface of the cave. These mats showed an aerobic heat evolution of about 1200 μW/g and contained about 500 μmol/g elemental sulphur. In contrast, other samples collected from cave water, sediment and rock exhibited only activities of maximal 40 to 60 μW/g and contained only up to 2.5 μmol/g elemental sulphur. As the main primary producers aerobic and facultatively anaerobic sulphur oxidisers were identified at high numbers, occasionally exceeding 107 CFU/g. Methylotrophic bacteria were present in all samples at up to 106 CFU/g, indicating the important role of C1 metabolism for the cave ecosystem. Although reduced sulphur species were biologically oxidised to sulphuric acid, the pH values of the samples ranged from 6.5 to 8.2 due to the high buffering capacity of the cave walls, which consisted mainly of limestone. Surprisingly, not only neutrophilic but also extremely acidophilic bacteria were detected. Sulphate reducers were present in both aerobic and anaerobic zones. The data presented suggest a close interdependence of sulphur oxidation and reduction as well as carbon dioxide and other C1 compound metabolism in the most biologically active zone of the cave ecosystem, i.e. the floating microbial mats.
The particulate methane monooxygenase gene pmoA, encoding the 27 kDa polypeptide of the membrane-bound particulate methane monooxygenase, was amplified by PCR from DNA isolated from a blanket peat bog and from enrichment cultures established, from the same environment, using methane as sole carbon and energy source. The resulting 525 bp PCR products were cloned and a representative number of clones were sequenced. Phylogenetic analysis of the derived amino acid sequences of the pmoA clones retrieved directly from environmental DNA samples revealed that they form a distinct cluster within representative PmoA sequences from type II methanotrophs and may originate from a novel group of acidophilic methanotrophs. The study also demonstrated the utility of the pmoA gene as a phylogenetic marker for identifying methanotroph-specific DNA sequences in the environment.
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
Abstract Based on an extensive 16S rRNA sequence database for type II methanotrophic bacteria, a set of 16S rRNA-targeted oligonucleotide probes was developed for differential detection of specific phylogenetic groups of these bacteria by fluorescence in situ hybridisation (FISH). This set of oligonucleotides included a genus-specific probe for Methylocystis (Mcyst-1432) and three species-specific probes for Methylosinus sporium (Msins-647), Methylosinus trichosporium (Msint-1268) and the recently described acidophilic methanotroph Methylocapsa acidiphila (Mcaps-1032). These novel probes were applied to further characterise the type II methanotroph community that was detected in an acidic Sphagnum peat from West Siberia in a previous study (Dedysh et al. (2001) Appl. Environ. Microbiol. 67, 4850-4857). The largest detectable population of indigenous methanotrophs simultaneously hybridised with a group-specific probe targeting all currently known Methylosinus/Methylocystis spp. (M-450), with a genus-specific probe for Methylocystis spp. (Mcyst-1432), and with an additional probe (Mcyst-1261) that had been designed to target a defined phylogenetic subgroup of Methylocystis spp. The same subgroup of Methylocystis was also detected in acidic peat sampled from Sphagnum-dominated wetland in northern Germany. The population size of this peat-inhabiting Methylocystis subgroup was 2.0+/-0.1x10(6) cells g(-1) (wet weight) of peat from Siberia and 5.5+/-0.5x10(6) cells g(-1) of peat from northern Germany. This represented 60 and 95%, respectively, of the total number of methanotroph cells detected by FISH in these two wetland sites. Other major methanotroph populations were M. acidiphila and Methylocella palustris. Type I methanotrophs accounted for not more than 1% of total methanotroph cells. Neither M. trichosporium nor M. sporium were detected in acidic Sphagnum peat.
Abstract A type II methanotrophic bacterium (Methylocystis strain SC2) was isolated from a polluted aquifer and identified based on morphology and on 16S rRNA gene phylogeny. Primers targeting the particulate methane monooxygenase subunit A gene (pmoA) were used to obtain a PCR product from DNA extract of strain SC2. Denaturing gradient gel electrophoresis of this PCR product demonstrated that strain SC2 contained two very different pmoA-like genes. One gene (pmoA1) had very high sequence homology to pmoA genes of other type II methanotrophic bacteria (identical amino acid sequence to pmoA of some other Methylocystis strains). The second gene (pmoA2) possessed only 73% identity with the first gene at the nucleotide level and 68.5% identity (83% similarity) at the amino acid level. The presence of both pmoA-like genes was verified by developing specific oligonucleotide probes for each and using these in Southern hybridisation of genomic DNA. Purity of the culture was exhaustively verified with a variety of methods to ensure that both genes were present in a single genospecies. These included microscopic examination, plating on various media, denaturing gradient gel electrophoresis of PCR products of the 16S rRNA gene (universal to bacteria) and of the methanol dehydrogenase alpha-subunit gene mxaF (universal to methylotrophic bacteria), and whole-cell hybridisation with fluorescently labelled 16S rRNA-targeted oligonucleotide probes specific for the genera Methylosinus and Methylocystis, or specific for strain SC2. Reverse transcription PCR of extracted RNA suggested that the novel pmoA2 gene was not expressed during growth under standard conditions used for the cultivation of these bacteria. The presence of multiple, diverse pmoA-like genes in a single genospecies of methanotrophic bacteria implies that pmoA must be cautiously applied as a phylogenetic marker in cultivation-independent molecular ecology studies.
Archaea (archaebacteria) are a phenotypically diverse group of microorganisms that share a common evolutionary history. There are four general phenotypic groups of archaea: the methanogens, the extreme halophiles, the sulfate-reducing archaea, and the extreme thermophiles. In the marine environment, archaeal habitats are generally limited to shallow or deep-sea anaerobic sediments (free-living and endosymbiotic methanogens), hot springs or deep-sea hydrothermal vents (methanogens, sulfate reducers, and extreme thermophiles), and highly saline land-locked seas (halophiles). This report provides evidence for the widespread occurrence of unusual archaea in oxygenated coastal surface waters of North America. Quantitative estimates indicated that up to 2% of the total ribosomal RNA extracted from coastal bacterioplankton assemblages was archaeal. Archaeal small-subunit ribosomal RNA-encoding DNAs (rDNAs) were cloned from mixed bacterioplankton populations collected at geographically distant sampling sites. Phylogenetic and nucleotide signature analyses of these cloned rDNAs revealed the presence of two lineages of archaea, each sharing the diagnostic signatures and structural features previously established for the domain Archaea. Both of these lineages were found in bacterioplankton populations collected off the east and west coasts of North America. The abundance and distribution of these archaea in oxic coastal surface waters suggests that these microorganisms represent undescribed physiological types of archaea, which reside and compete with aerobic, mesophilic eubacteria in marine coastal environments.
Methanotrophs convert methane to methanol by the methane monooxygenase (MMO). It is well known that two forms of the MMO can be expressed: one form is found in the cytoplasm, or in the soluble fraction (sMMO); the other is associated with the membranes, or particulate fraction (pMMO). The sMMO has been extensively examined, and much is known about its structure and mechanism of dioxygen activation. The pMMO, however, is less well understood: the enzyme has proven difficult to purify as it loses activity once the membranes become solubilized. Furthermore, although copper is known to be important for the stability and activity of the pMMO, its role is still unclear. In a recent study, we reported the use of electron paramagnetic resonance (EPR) spectroscopy to probe the nature of the copper ions in these membranes. Two EPR signals were uncovered for the highly oxidized membranes: one set of signals arises from the type 2 Cu(II) centers, and the other has been assigned to trinuclear copper (Cu(II) clusters (H.-H. Nguyen, et al., J. Biol. Chem., 269, 14995 (1994). Here, we attempt to correlate the EPR spectra of the membrane fraction of Methylococcus capsulatus Bath with the amount of copper present in the membranes and to the activity of the pMMO as measured by the production of propylene oxide from propene by the pMMO. From these studies, we conclude that the primary role of copper is in the active site of the pMMO rather than simply a structural one.
Genes encoding particulate methane monooxygenase and ammonia monooxygenase share high sequence identity. Degenerate oligonucleotide primers were designed, based on regions of shared amino acid sequence between the 27-kDa polypeptides, which are believed to contain the active sites, of particulate methane monooxygenase and ammonia monooxygenase. A 525-bp internal DNA fragment of the genes encoding these polypeptides (pmoA and amoA) from a variety of methanotrophic and nitrifying bacteria was amplified by PCR, cloned and sequenced. Representatives of each of the phylogenetic groups of both methanotrophs (alpha- and gamma-Proteobacteria) and ammonia-oxidizing nitrifying bacteria (beta- and gamma-Proteobacteria) were included. Analysis of the predicted amino acid sequences of these genes revealed strong conservation of both primary and secondary structure. Nitrosococcus oceanus AmoA showed higher identity to PmoA sequences from other members of the gamma-Proteobacteria than to AmoA sequences. These results suggest that the particulate methane monooxygenase and ammonia monooxygenase are evolutionarily related enzymes despite their different physiological roles in these bacteria.
Microbial mats discovered in a ground-water ecosystem in southern Romania contain chemoautotrophic bacteria that fix inorganic carbon, using hydrogen sulfide as an energy source. Analysis of stable carbon and nitrogen isotopes showed that this chemoautotrophic production is the food base for 48 species of cave-adapted terrestrial and aquatic invertebrates, 33 of which are endemic to this ecosystem. This is the only cave ecosystem known to be supported by in situ autotrophic production, and it contains the only terrestrial community known to be chemoautotrophically based.
The eukaryotic alga Ochromonas danica, a nutritionally versatile, mixotrophic chrysophyte, grew on phenol as the sole carbon source in axenic culture and removed the phenol carbon from the growth medium. Respirometric studies confirmed that the enzymes involved in phenol catabolism were inducible and that the alga oxidized phenol; the amount of oxygen consumed per mole of oxidized substrate was approximately 65% of the theoretical value. [U-14C]phenol was completely mineralized, with 65% of the 14C label appearing as 14CO2, approximately 15% remaining in the aqueous medium, and the rest accounted for in the biomass. Analysis of the biomass showed that 14C label had been incorporated into the protein, nucleic acid, and lipid fractions; phenol carbon is thus unequivocally assimilated by the alga. Phenol-grown cultures of O. danica converted phenols to the corresponding catechols, which were further metabolized by the meta-cleavage pathway. This surprising result was rigorously confirmed by taking the working stock culture through a variety of procedures to check that it was axenic and repeating the experiments with algal extracts. This is, as far as is known, the first definitive identification of the meta-cleavage pathway for aromatic ring degradation in a eukaryotic alga, though its incidence in other eukaryotes has been (infrequently) suggested.
After nearly 10 years of PCR-based analysis of prokaryotic small-subunit ribosomal RNAs for ecological studies it seems necessary to summarize reported pitfalls of this approach which will most likely lead to an erroneous description on the microbial diversity of a given habitat. The following article will cover specific aspects of sample collection, cell lysis, nucleic acid extraction, PCR amplification, separation of amplified DNA, application of nucleic probes and data analysis.
Comparative sequence analysis of small subunit rRNA is currently one of the most important methods for the elucidation of bacterial phylogeny as well as bacterial identification. Phylogenetic investigations targeting alternative phylogenetic markers such as large subunit rRNA, elongation factors, and ATPases have shown that 16S rRNA-based trees reflect the history of the corresponding organisms globally. However, in comparison with three to four billion years of evolution the phylogenetic information content of these markers is limited. Consequently, the limited resolution power of the marker molecules allows only a spot check of the evolutionary history of microorganisms. This is often indicated by locally different topologies of trees based on different markers, data sets or the application of different treeing approaches. Sequence peculiarities as well as methods and parameters for data analysis were studied with respect to their effects on the results of phylogenetic investigations. It is shown that only careful data analysis starting with a proper alignment, followed by the analysis of positional variability, rates and character of change, testing various data selections, applying alternative treeing methods and, finally, performing confidence tests, allows reasonable utilization of the limited phylogenetic information.
The 16S rRNA and pmoA genes from natural populations of methane-oxidizing bacteria (methanotrophs) were PCR amplified from total community DNA extracted from Lake Washington sediments obtained from the area where peak methane oxidation occurred. Clone libraries were constructed for each of the genes, and approximately 200 clones from each library were analyzed by using restriction fragment length polymorphism (RFLP) and the tetrameric restriction enzymes MspI, HaeIII, and HhaI. The PCR products were grouped based on their RFLP patterns, and representatives of each group were sequenced and analyzed. Studies of the 16S rRNA data obtained indicated that the existing primers did not reveal the total methanotrophic diversity present when these data were compared with pure-culture data obtained from the same environment. New primers specific for methanotrophs belonging to the genera Methylomonas, Methylosinus, and Methylocystis were developed and used to construct more complete clone libraries. Furthermore, a new primer was designed for one of the genes of the particulate methane monooxygenase in methanotrophs, pmoA. Phylogenetic analyses of both the 16S rRNA and pmoA gene sequences indicated that the new primers should detect these genes over the known diversity in methanotrophs. In addition to these findings, 16S rRNA data obtained in this study were combined with previously described phylogenetic data in order to identify operational taxonomic units that can be used to identify methanotrophs at the genus level.
A new method has been developed for rapidly closing a large number of gaps in a whole-genome shotgun sequencing project. The method employs multiplex PCR and a novel pooling strategy to minimize the number of laboratory procedures required to sequence the unknown DNA that falls in between contiguous sequences. Multiplex sequencing, a novel procedure in which multiple PCR primers are used in a single sequencing reaction, is used to interpret the multiplex PCR results. Two protocols are presented, one that minimizes pipetting and another that minimizes the number of reactions. The pipette optimized multiplex PCR method has been employed in the final phases of closing the Streptococcus pneumoniae genome sequence, with excellent results.
Microorganisms are responsible for driving the biogeochemical cycling of elements on Earth. Despite their importance and vast diversity, the taxonomic identity of the microorganisms involved in any specific process has usually been confined to that small fraction of the microbiota that has been isolated and cultivated. The recent coupling of molecular biological methods with stable-isotope abundance in biomarkers has provided a cultivation-independent means of linking the identity of bacteria with their function in the environment. Here we show that 13C-DNA, produced during the growth of metabolically distinct microbial groups on a 13C-enriched carbon source, can be resolved from 12C-DNA by density-gradient centrifugation. DNA isolated from the target group of microorganisms can be characterized taxonomically and functionally by gene probing and sequence analysis. Application of this technique to investigate methanol-utilizing microorganisms in soil demonstrated the involvement of members of two phylogenetically distinct groups of eubacteria; the alpha-proteobacterial and Acidobacterium lineages. Stable-isotope probing thus offers a powerful new technique for identifying microorganisms that are actively involved in specific metabolic processes under conditions which approach those occurring in situ.
Many methanotrophs contain both a soluble and a particulate methane monooxygenase. A unique metabolic switch, mediated by copper ions, regulates the expression of these enzymes. When the copper-to-biomass ratio of the cell is low, the soluble enzyme is expressed, and when the copper-to-biomass ratio is high, the particulate enzyme is expressed. A model for the mechanism of this switch is proposed.
Methane-oxidizing bacteria (methanotrophs) have attracted considerable attention over the past 30 years. They have the unique ability to use methane as sole carbon and energy source, they are found in a wide variety of environments and play a crucial role in the global methane cycle. Methanotrophs also show considerable potential for bioremediation processes such as degradation of ground water pollutants, and for production of bulk chemicals from cheap substrates. We review here the cultivation-independent molecular biological methods that are available for the detection and characterization of methanotrophs in the natural environment.
Serine biosynthesis in plants proceeds by two pathways; the glycolate pathway which is associated with photorespiration and the pathway from 3-phosphoglycerate which is presumed to take place in the plastids. The 3-phosphoglycerate pathway (phosphorylated pathway) involves three enzymes catalyzing three sequential reactions: 3-phosphoglycerate dehydrogenase (PGDH), 3-phosphoserine aminotransferase (PSAT) and 3-phosphoserine phosphatase (PSP). cDNA and genomic clones encoding these three enzymes from spinach and Arabidopsis thaliana were isolated by means of heterologous probe screening, homologous EST clones and genetic complementation in an Escherichia coli mutant. The identity of the isolated cDNAs was confirmed by functional complementation of serine auxotrophy in E. coli mutants and/or the detection of catalytic activity in the recombinant enzymes produced in E. coli. Northern blot analyses indicated the most preferential expression of these three genes in light-grown roots. In contrast, the mRNAs of two proteins involved in the glycolate pathway (H-protein of glycine decarboxylase multienzyme complex and serine hydroxymethyltransferase) accumulated to high levels in light-grown shoots. Environmental stresses, such as high salinity, flooding and low temperature, induced changes in mRNA levels of enzymes in the plastidic phosphorylated serine biosynthetic pathway but not in that of the glycolate pathway. These results indicate that the plastidic 3-phosphoglycerate pathway plays an important role in supplying serine in non-photosynthetic tissues in plants and under environmental stresses.
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.
Methane-oxidizing bacteria (methanotrophs) containing soluble methane monooxygenase (sMMO) are of interest in natural environments due to the high co-metabolic activity of this enzyme with contaminants such as trichloroethylene. We have analysed sMMO-containing methanotrophs in sediment from a freshwater lake. Environmental clone banks for a gene encoding a diagnostic sMMO subunit (mmoX) were generated using DNA extracted from Lake Washington sediment and subjected to RFLP analysis. Representatives from the six RFLP groups were cloned and sequenced, and all were found to group with Type I Methylomonas mmoX, although a majority were divergent from known Methylomonas mmoX sequences. Direct hybridization of Lake Washington sediment DNA was carried out using a series of sMMO- and Methylomonas-specific probes to assess the significance of these sMMO-containing Methylomonas-like strains in the sediment. The total sMMO-containing population and the sMMO-containing Methylomonas-like population were estimated to be similar to previous estimates for total methanotrophs and Type I methanotrophs. These results suggest that the major methanotrophic population in Lake Washington sediment consists of sMMO-containing Methylomonas-like (Type I) methanotrophs. The whole-cell TCE degradation kinetics of such a strain, LW15, isolated from this environment, were determined and found to be similar to values reported for other sMMO-containing methanotrophs. The numerical significance of sMMO-containing Methylomonas-like methanotrophs in a mesotrophic lake environment suggests that these methanotrophs may play an important role in methanotroph-mediated transformations, including co-metabolism of halogenated solvents, in natural environments.
'Pseudomonas butanovora' is capable of growth with butane via the oxidation of butane to 1-butanol, which is catalysed by a soluble butane monooxygenase (sBMO). In vitro oxidation of ethylene (an alternative substrate for sBMO) was reconstituted in the soluble portion of cell extracts and was NADH-dependent. Butane monooxygenase was separated into three components which were obligately required for substrate oxidation. The N-terminal sequences of the peptides associated with butane monooxygenase led to the cloning and sequencing of the 5797 nucleotide bmo gene cluster. Comparisons of the deduced amino acid sequences with other multicomponent monooxygenases suggest that sBMO is a multimeric hydroxylase with 61, 45 and 19 kDa subunits encoded by bmoXYZ, a 40 kDa oxidoreductase encoded by bmoC, and a 15 kDa regulatory protein encoded by bmoB. A sixth structural gene (bmoD) encodes a 9.6 kDa protein with similarity exclusively to mmoD (orfY), a putative metal centre assembly protein of the soluble methane monooxygenases. Insertional inactivation of bmoX resulted in a mutant 'P. butanovora' strain incapable of growth with butane. A putative promoter element characteristic of promoters associated with sigma(54)-dependent transcription initiation was located upstream of the bmo genes. Expression of all six genes was detected in butane-induced cells. Butane monooxygenase from 'P. butanovora' aligns most closely with non-haem carboxylate-bridged diiron monooxygenases and, moreover, contains the characteristic iron-binding motif. The structural and mechanistic implications of the high sequence identity (up to 64%) between the peptides of butane monooxygenase and methane monooxygenases are discussed.
The potential of DNA microarray technology in high-throughput detection of bacteria and quantitative assessment of their community structures is widely acknowledged but has not been fully realised yet. A generally applicable set of techniques, based on readily available technologies and materials, was developed for the design, production and application of diagnostic microbial microarrays. A microarray targeting the particulate methane monooxygenase (pmoA) gene was developed for the detection and quantification of methanotrophs and functionally related bacteria. A microarray consisting of a set of 59 probes that covers the whole known diversity of these bacteria was validated with a representative set of extant strains and environmental clones. The potential of the pmoA microarray was tested with environmental samples. The results were in good agreement with those of clone library sequence analyses. The approach can currently detect less dominant bacteria down to 5% of the total community targeted. Initial tests assessing the quantification potential of this system with artificial PCR mixtures showed very good correlation with the expected results with standard deviations in the range of 0.4-17.2%. Quantification of environmental samples with this method requires the design of a reference mixture consisting of very close relatives of the strains within the sample and is currently limited by biases inherent in environmental DNA extraction and universal PCR amplification.
Phylogeny based on ribosomal RNA sequences alone is rarely a reliable indicator of microbial function. To circumvent this problem, nucleic acid based techniques have been developed that exploit the physical properties of stable isotopes to study microbially mediated processes within complex environmental samples. Investigations using labelled substrates, or which detect variations in the natural abundance of isotopes, have thus revealed the metabolic function of microorganisms without the need to isolate them in culture.
Cultivation‐independent techniques for studying methanotroph ecology
- Murrell J.C.
Murrell, J.C., and Radajewski, S. (2000) Cultivation-independent techniques for studying methanotroph ecology. Res
Microbiol 151: 1-8.
Regulation of expression of methane monooxygenases by copper ions
- Murrell
Murrell, J.C., McDonald, I.R., and Gilbert, B. (2000) Regulation of expression of methane monooxygenases by copper
ions. Trends Microbiol 8: 221–225.
Stable-isotope probing of nucleic acids: a window to the function of uncultured microorganisms
- Radajewski
Stable-isotope probing as a tool in microbial ecology
- Radajewski