Enhanced sensitivity of DNA- and rRNA-based stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation gradients

Stable isotope probing (SIP) of nucleic acids allows the detection and identification of active members of natural microbial populations that are involved in the assimilation of an isotopically labelled compound into nucleic acids. SIP is based on the separation of isotopically labelled DNA or rRNA by isopycnic density gradient centrifugation. We have developed a highly sensitive protocol for the detection of 'light' and 'heavy' nucleic acids in fractions of centrifugation gradients. It involves the fluorometric quantification of total DNA or rRNA, and the quantification of either 16S rRNA genes or 16S rRNA in gradient fractions by real-time PCR with domain-specific primers. Using this approach, we found that fully 13C-labelled DNA or rRNA of Methylobacterium extorquens was quantitatively resolved from unlabelled DNA or rRNA of Methanosarcina barkeri by cesium chloride or cesium trifluoroacetate density gradient centrifugation respectively. However, a constant low background of unspecific nucleic acids was detected in all DNA or rRNA gradient fractions, which is important for the interpretation of environmental SIP results. Consequently, quantitative analysis of gradient fractions provides a higher precision and finer resolution for retrieval of isotopically enriched nucleic acids than possible using ethidium bromide or gradient fractionation combined with fingerprinting analyses. This is a prerequisite for the fine-scale tracing of microbial populations metabolizing 13C-labelled compounds in natural ecosystems.     

Thermoanaerobacteriaceae oxidize acetate in methanogenic rice field soil at 50°C

Rice field soils contain a thermophilic microbial community. Incubation of Italian rice field soil at 50°C resulted in transient accumulation of acetate, but the microorganisms responsible for methane production from acetate are unknown. Without addition of exogenous acetate, the δ(13)C of CH(4) and CO(2) indicated that CH(4) was exclusively produced by hydrogenotrophic methanogenesis. When exogenous acetate was added, acetoclastic methanogenesis apparently also operated. Nevertheless, addition of [2-(13)C]acetate (99% (13)C) resulted in the production not only of (13)C-labelled CH(4) but also of CO(2), which contained up to 27% (13)C, demonstrating that the methyl group of acetate was also oxidized. Part of the (13)C-labelled acetate was also converted to propionate which contained up to 14% (13)C. The microorganisms capable of assimilating acetate at 50°C were targeted by stable isotope probing (SIP) of ribosomal RNA and rRNA genes using [U-(13)C] acetate. Using quantitative PCR, (13)C-labelled bacterial ribosomal RNA and DNA was detected after 21 and 32 days of incubation with [U-(13)C]acetate respectively. In the heavy fractions of the (13)C treatment, terminal restriction fragments (T-RFs) of 140, 120 and 171 bp length predominated. Cloning and sequencing of 16S rRNA showed that these T-RFs were affiliated with the bacterial genera Thermacetogenium and Symbiobacterium and with members of the Thermoanaerobacteriaceae. Similar experiments targeting archaeal RNA and DNA showed that Methanocellales were the dominant methanogens being consistent with the operation of syntrophic bacterial acetate oxidation coupled to hydrogenotrophic methanogenesis. After 17 days, however, Methanosarcinacea increasingly contributed to the synthesis of rRNA from [U-(13)C]acetate indicating that acetoclastic methanogens were also active in methanogenic Italian rice field soil under thermal conditions.     

Detection of monochlorobenzene metabolizing bacteria under anoxic conditions by DNA-stable isotope probing

Cultivation-independent analyses were applied to study the structural diversity of the bacterial community which developed in groundwater inoculated microcosms actively metabolizing monochlorobenzene (MCB) under anaerobic conditions. Addition of ¹³C-labelled MCB demonstrated that the community produced ¹³CO₂ as a metabolite at slightly increasing rates over a period of 1,051 days while no ¹³C-methane evolved. Genetic profiles of partial 16S rRNA genes generated with the single-strand conformation polymorphism (SSCP) technique by PCR from directly extracted total DNA revealed that, despite the long incubation period, six replicate microcosms were characterized by almost the same microbial members. Nine distinguishable contributors to the SSCP-profiles were characterized by DNA sequencing, revealing the presence of different members from the phyla Proteobacteria, Fibrobacteres and from the candidate division OD1. DNA-stable isotope probing (SIP) was applied to distinguish the actual MCB metabolizing bacteria from the other community members. This study reveals for the first time the structural diversity of an anaerobic MCB metabolizing bacterial community. However, it also demonstrates the limitations of SIP to detect bacteria slowly metabolizing carbon sources under anaerobic conditions.     

Stable isotope probing analysis of the influence of liming on root exudate utilization by soil microorganisms

Rhizosphere microorganisms play an important role in soil carbon flow, through turnover of root exudates, but there is little information on which organisms are actively involved or on the influence of environmental conditions on active communities. In this study, a 13CO2 pulse labelling field experiment was performed in an upland grassland soil, followed by RNA-stable isotope probing (SIP) analysis, to determine the effect of liming on the structure of the rhizosphere microbial community metabolizing root exudates. The lower limit of detection for SIP was determined in soil samples inoculated with a range of concentrations of 13C-labelled Pseudomonas fluorescens and was found to lie between 10(5) and 10(6) cells per gram of soil. The technique was capable of detecting microbial communities actively assimilating root exudates derived from recent photo-assimilate in the field. Denaturing gradient gel electrophoresis (DGGE) profiles of bacteria, archaea and fungi derived from fractions obtained from caesium trifluoroacetate (CsTFA) density gradient ultracentrifugation indicated that active communities in limed soils were more complex than those in unlimed soils and were more active in utilization of recently exuded 13C compounds. In limed soils, the majority of the community detected by standard RNA-DGGE analysis appeared to be utilizing root exudates. In unlimed soils, DGGE profiles from 12C and 13C RNA fractions differed, suggesting that a proportion of the active community was utilizing other sources of organic carbon. These differences may reflect differences in the amount of root exudation under the different conditions.     

Identification of acetate-utilizing Bacteria and Archaea in methanogenic profundal sediments of Lake Kinneret (Israel) by stable isotope probing of rRNA

Acetate is an important intermediate in the decomposition of organic matter in anoxic freshwater sediments. Here, we identified distinct microorganisms active in its oxidation and transformation to methane in the anoxic methanogenic layers of Lake Kinneret (Israel) profundal sediment by rRNA-based stable isotope probing (RNA-SIP). After 18 days of incubation with amended [U-(13)C]acetate we found that archaeal 16S rRNA was (13)C-labelled to a far greater extent than bacterial rRNA. We identified acetoclastic methanogens related to Methanosaeta concilii as being most active in the degradation and assimilation of acetate. Oxidation of the acetate-methyl group played only a minor role, but nevertheless 'heavy'(13)C-labelled bacterial rRNA templates were identified. 'Heavy' bacteria were mainly affiliated with the Betaproteobacteria (mostly Rhodocyclales and Nitrosomonadales), the Nitrospira phylum (related to 'Magnetobacterium bavaricum' and Thermodesulfovibrio yellowstonii), and also with the candidate phylum 'Endomicrobia'. However, the mode of energy gain that allowed for the assimilation of (13)C-acetate by these bacterial groups remains unknown. It may have involved syntrophic oxidation of acetate, reduction of chlorinated compounds, reduction of humic substances, fermentation of organic compounds, or even predation of (13)C-labelled Methanosaeta spp. In summary, this SIP experiment shows that acetate carbon was predominantly consumed by acetoclastic methanogens in profundal Lake Kinneret sediment, while it was also utilized by a small and heterogeneous community of bacteria.     

Quantitative fluorescence in situ hybridization analysis of microbial consortia from a biogenic gas field in Alaska's Cook Inlet basin

Filter-collected production water samples from a methane-rich gas field in the Cook Inlet basin of Alaska were investigated using whole-cell rRNA-targeted fluorescence in situ hybridization (FISH) and 16S rRNA tag pyrosequencing. Both techniques were consistent in determining the microbial community composition, including the archaeal or bacterial dominance of samples. The archaeal community is dominated by the obligate methylotrophic methanogen genus Methanolobus as well as the nutritional generalist methanogen genus Methanosarcina, which is capable of utilizing acetate, CO(2), and methyl-bearing compounds. The most-abundant bacterial groups are Firmicutes, notably of the Acetobacterium genus, and Cytophaga-Flexibacter-Bacteroides species (CFBs) affiliated with the order Bacteroidales. We observed spatial variation among samples in both the percentage of members of Archaea compared to that of members of Bacteria and the dominant members of the bacterial community, differences which could not be explained with the available geochemical data. Based upon the microbial community composition and the isotopic signature of methane associated with the Cook Inlet basin site, we propose a simplified reaction network beginning with the breakdown of coal macromolecules, followed by fermentation and methylotrophic and acetoclastic methane production.     

Effect of light regimes on the utilisation of an exogenous carbon source by freshwater biofilm bacterial communities

This study reports the novel use of nucleic acid stable isotope probing (NA-SIP) to identify metabolically active ([¹³C]-acetate assimilating) bacteria in freshwater biofilms. Currently, a little is known of the factors affecting the structure and activity of these complex microbial biofilm communities, although it is likely that they are influenced by riparian vegetation through attenuation of light and alteration of allochthonous inputs of carbon. NA-SIP was used to investigate the effect of varying light regimes on [¹³C]-acetate assimilating bacteria within laboratory biofilm microcosms. Differences in clone libraries of 16S rRNA and rRNA genes from ¹³C-labelled and unlabelled nucleic acids indicated differential uptake of acetate and the rapid transfer of ¹³C to organisms at a higher trophic level. Biofilm communities incubated in the dark changed least over time and retained a significant fraction of phototrophic organisms. Incubation under elevated light caused the greatest change in bacterial community structure. Contrary to expectation, a complete loss of chlorophyll containing organisms occurred within this treatment, challenging current thinking that elevated light promotes communities dominated by photoautotrophs in nutrient enriched environments.     

Stable-isotope probing of microorganisms thriving at thermodynamic limits: syntrophic propionate oxidation in flooded soil

Propionate is an important intermediate of the degradation of organic matter in many anoxic environments. In methanogenic environments, due to thermodynamic constraints, the oxidation of propionate requires syntrophic cooperation of propionate-fermenting proton-reducing bacteria and H(2)-consuming methanogens. We have identified here microorganisms that were active in syntrophic propionate oxidation in anoxic paddy soil by rRNA-based stable-isotope probing (SIP). After 7 weeks of incubation with [(13)C]propionate (<10 mM) and the oxidation of approximately 30 micromol of (13)C-labeled substrate per g dry weight of soil, we found that archaeal nucleic acids were (13)C labeled to a larger extent than those of the bacterial partners. Nevertheless, both terminal restriction fragment length polymorphism and cloning analyses revealed Syntrophobacter spp., Smithella spp., and the novel Pelotomaculum spp. to predominate in "heavy" (13)C-labeled bacterial rRNA, clearly showing that these were active in situ in syntrophic propionate oxidation. Among the Archaea, mostly Methanobacterium and Methanosarcina spp. and also members of the yet-uncultured "rice cluster I" lineage had incorporated substantial amounts of (13)C label, suggesting that these methanogens were directly involved in syntrophic associations and/or thriving on the [(13)C]acetate released by the syntrophs. With this first application of SIP in an anoxic soil environment, we were able to clearly demonstrate that even guilds of microorganisms growing under thermodynamic constraints, as well as phylogenetically diverse syntrophic associations, can be identified by using SIP. This approach holds great promise for determining the structure and function relationships of further syntrophic or other nutritional associations in natural environments and for defining metabolic functions of yet-uncultivated microorganisms.     

Stimulated saprotrophic fungi in arable soil extend their activity to the rhizosphere and root microbiomes of crop seedlings

Saprotrophic fungi play an important role in ecosystem functioning and plant performance, but their abundance in intensively managed arable soils is low. Saprotrophic fungal biomass in arable soils can be enhanced with amendments of cellulose-rich materials. Here, we examined if sawdust-stimulated saprotrophic fungi extend their activity to the rhizosphere of crop seedlings and influence the composition and activity of other rhizosphere and root inhabitants. After growing carrot seedlings in sawdust-amended arable soil, we determined fungal and bacterial biomass and community structure in roots, rhizosphere and soil. Utilization of root exudates was assessed by stable isotope probing (SIP) following ¹³CO₂-pulse-labelling of seedlings. This was combined with analysis of lipid fatty acids (PLFA/NLFA-SIP) and nucleic acids (DNA-SIP). Sawdust-stimulated Sordariomycetes colonized the seedling's rhizosphere and roots and actively consumed root exudates. This did not reduce the abundance and activity of bacteria, yet higher proportions of α-Proteobacteria and Bacteroidia were seen. Biomass and activity of mycorrhizal fungi increased with sawdust amendments, whereas exudate consumption and root colonization by functional groups containing plant pathogens did not change. Sawdust amendment of arable soil enhanced abundance and exudate-consuming activity of saprotrophic fungi in the rhizosphere of crop seedlings and promoted potential beneficial microbial groups in root-associated microbiomes.     

A comparison of stable-isotope probing of DNA and phospholipid fatty acids to study prokaryotic functional diversity in sulfate-reducing marine sediment enrichment slurries

Marine sediment slurries enriched for anaerobic, sulfate-reducing prokaryotic communities utilizing glucose and acetate were used to provide the first comparison between stable-isotope probing (SIP) of phospholipid fatty acids (PLFA) and DNA (16S rRNA and dsrA genes) biomarkers. Different 13C-labelled substrates (glucose, acetate and pyruvate) at low concentrations (100 microM) were used over a 7-day incubation to follow and identify carbon flow into different members of the community. Limited changes in total PLFA and bacterial 16S rRNA gene DGGE profiles over 7 days suggested the presence of a stable bacterial community. A broad range of PLFA were rapidly labelled (within 12 h) in the 13C-glucose slurry but this changed with time, suggesting the presence of an active glucose-utilizing population and later development of another population able to utilize glucose metabolites. The identity of the major glucose-utilizers was unclear as 13C-enriched PLFA were common (16:0, 16:1, 18:1omega7, highest incorporation) and there was little difference between 12C- and 13C-DNA 16S rRNA gene denaturing gradient gel electrophoresis (DGGE) profiles. Seemingly glucose, a readily utilizable substrate, resulted in widespread incorporation consistent with the higher extent of 13C-incorporation (approximately 10 times) into PLFA compared with 13C-acetate or 13C-pyruvate. 13C-PLFA in the 13C-acetate and 13C-pyruvate slurries were similar to each other and to those that developed in the 13C-glucose slurry after 4 days. These were more diagnostic, with branched odd-chain fatty acids (i15:0, a15:0 and 15:1omega6) possibly indicating the presence of Desulfococcus or Desulfosarcina sulfate-reducing bacteria (SRB) and sequences related to these SRB were in the 13C-acetate-DNA dsrA gene library. The 13C-acetate-DNA 16S rRNA gene library also contained sequences closely related to SRB, but these were the acetate-utilizing Desulfobacter sp., as well as a broad range of uncultured Bacteria. In contrast, analysis of DGGE bands from 13C-DNA demonstrated that the candidate division JS1 and Firmicutes were actively assimilating 13C-acetate. Denaturing gradient gel electrophoresis also confirmed the presence of JS1 in the 13C-DNA from the 13C-glucose slurry. These results demonstrate that JS1, originally found in deep subsurface sediments, is more widely distributed in marine sediments and provides the first indication of its metabolism; incorporation of acetate and glucose (or glucose metabolites) under anaerobic, sulfate-reducing conditions. Here we demonstrate that PLFA- and DNA-SIP can be used together in a sedimentary system, with low concentrations of 13C-substrate and overlapping incubation times (up to 7 days) to provide complementary, although not identical, information on carbon flow and the identity of active members of an anaerobic prokaryotic community.     

Identification of metolachlor mineralizing bacteria in aerobic and anaerobic soils using DNA-stable isotope probing

The influence of soil environmental factors such as aeration on the ecology of microorganisms involved in the mineralization and degradation of the popular soil-applied pre-emergent herbicide, metolachlor is unknown. To address this knowledge gap, we utilized DNA-based stable isotope probing (SIP) where soil microcosms were incubated aerobically or anaerobically and received herbicide treatments with unlabeled metolachlor or ¹³C-metolachlor. Mineralization of metolachlor was confirmed as noted from the evolution of ¹⁴CO₂ from ¹⁴C-metolachlor-treated microcosms and clearly demonstrated the efficient utilization of the herbicide as a carbon source. Terminal restriction fragment length polymorphisms (T-RFLP) bacterial community profiling performed on soil DNA extracts indicated that fragment 307 bp from aerobic soil and 212 bp from anaerobic soil were detected only in the herbicide-treated (both unlabeled metolachlor and ¹³C-metolachlor) soils when compared to the untreated control microcosms. T-RFLP profiles from the ultracentrifugation fractions illustrated that these individual fragments experienced an increase in relative abundance at a higher buoyant density (BD) in the labeled fractions when compared to the unlabeled herbicide amendment fractions. The shift in BD of individual T-RFLP fragments in the density-resolved fractions suggested the incorporation of ¹³C from labeled herbicide into the bacterial DNA and enabled the identification of organisms responsible for metolachlor uptake from the soil. Subsequent cloning and 16S rRNA gene sequencing of the ¹³C-enriched fractions implicated the role of organisms closely related to Bacillus spp. in aerobic mineralization and members of Acidobacteria phylum in anaerobic mineralization of metolachlor in soil.     

Characterization of a deep-sea microbial mat from an active cold seep at the Milano mud volcano in the Eastern Mediterranean Sea

A white, filamentous microbial mat at the Milano mud volcano in the Eastern Mediterranean Sea was sampled during the Medinaut cruise of the R/V Nadir in 1998. The composition of the mat community was characterized using a combination of phylogenetic and lipid biomarker methods. The mat sample was filtered through 0.2 and 5-microm filters to coarsely separate unicellular and filamentous bacteria. Analyses of 16S rRNA gene sequences amplified from the total community DNA from these fractions showed that similar archaeal populations were present in both fractions. However, the bacterial populations in the fractions differed from one another, and were more diverse than the archaeal ones. Lipid analysis showed that bacteria were the dominant members of the mat microbial community and the relatively low delta(13)C carbon isotope values of bulk bacterial lipids suggested the occurrence of methane- and sulfide-based chemo(auto)trophy. Consistent with this, the bacterial populations in the fractions were related to Alpha-, Gamma- and Epsilonproteobacteria, most of which were chemoautotrophic bacteria that utilize hydrogen sulfide (or reduced sulfur compounds) and/or methane. The most common archaeal 16S rRNA gene sequences were related to those of previously identified Archaea capable of anaerobic methane oxidation. Although the filamentous organisms observed in the mat were not conclusively identified, our results indicated that the Eastern Mediterranean deep-sea microbial mat community might be sustained on a combination of methane- and sulfide-driven chemotrophy.     

Stable isotope probing - linking microbial identity to function

Stable isotope probing (SIP) is a technique that is used to identify the microorganisms in environmental samples that use a particular growth substrate. The method relies on the incorporation of a substrate that is highly enriched in a stable isotope, such as (13)C, and the identification of active microorganisms by the selective recovery and analysis of isotope-enriched cellular components. DNA and rRNA are the most informative taxonomic biomarkers and (13)C-labelled molecules can be purified from unlabelled nucleic acid by density-gradient centrifugation. The future holds great promise for SIP, particularly when combined with other emerging technologies such as microarrays and metagenomics.     

Impact of varying electron donors on the molecular microbial ecology and biokinetics of methylotrophic denitrifying bacteria

The goal of this study was to identify bacterial populations that assimilated methanol in a denitrifying sequencing batch reactor (SBR), using stable isotope probing (SIP) of ¹³C labeled DNA and quantitatively track changes in these populations upon changing the electron donor from methanol to ethanol in the SBR feed. Based on SIP derived ¹³C 16S rRNA gene clone libraries, dominant SBR methylotrophic bacteria were related to Methyloversatilis spp. and Hyphomicrobium spp. These methylotrophic populations were quantified via newly developed real‐time PCR assays. Upon switching the electron donor from methanol to ethanol, Hyphomicrobium spp. concentrations decreased significantly in accordance with their obligately methylotrophic nutritional mode. In contrast, Methyloversatilis spp. concentrations were relatively unchanged, in accordance with their ability to assimilate both methanol and ethanol. Direct assimilation of ethanol by Methyloversatilis spp. but not Hyphomicrobium spp. was also confirmed via SIP. The reduction in methylotrophic bacterial concentration upon switching to ethanol was paralleled by a significant decrease in the methanol supported denitrification biokinetics of the SBR on nitrate. In sum, the results of this study demonstrate that the metabolic capabilities (methanol assimilation and metabolism) and substrate specificity (obligately or facultatively methylotrophic) of two distinct methylotrophic bacterial populations contributed to their survival or washout in denitrifying bioreactors. Biotechnol. Bioeng. 2009;102: 1527–1536. © 2008 Wiley Periodicals, Inc.     

Identification of a complete methane monooxygenase operon from soil by combining stable isotope probing and metagenomic analysis

Stable isotope probing (SIP) allows the isolation of nucleic acids from targeted metabolically active organisms in environmental samples. In previous studies, DNA-SIP has been performed with the one-carbon growth substrates methane and methanol to study methylotrophic organisms. The methylotrophs that incorporated the labelled substrate were identified with polymerase chain reaction and sequencing of 16S rRNA and 'functional genes' for methanotrophs (mxaF, pmoA, mmoX). In this study, a SIP experiment was performed using a forest soil sample incubated with (13)CH(4), and the (13)C-DNA was purified and cloned into a bacterial artificial chromosome (BAC) plasmid. A library of 2300 clones was generated and most of the clones contained inserts between 10 and 30 kb. The library was probed for key methylotrophy genes and a 15.2 kb clone containing a pmoCAB operon, encoding particulate methane monooxygenase, was identified and sequenced. Analysis of the pmoA sequence suggested that the clone was most similar to that of a Methylocystis sp. previously detected in this forest soil. Twelve other open reading frames were identified on the clone, including the gene encoding beta-ribofuranosylaminobenzene 5'-phosphate synthase, which is involved in the biosynthesis of the 'archaeal' C(1)-carrier, tetrahydromethanopterin, which is also found in methylotrophs. This study demonstrates that relatively large DNA fragments from uncultivated organisms can be readily isolated using DNA-SIP, and cloned into a vector for metagenomic analysis.     

Molecular characterization of microbial communities in deep coal seam groundwater of northern Japan

We investigated microbial methanogenesis and community structure based on 16S rRNA gene sequences from a coal seam aquifer located 843–907 m below ground level in northern Japan; additionally, we studied the δ¹³C and δ²H (δD) of coal‐bed gases and other physicochemical parameters. Although isotopic analysis suggested a thermocatalytic origin for the gases, the microbial activity and community structure strongly implied the existence of methanogenic microbial communities in situ. Methane was generated in the enrichment cultures of the hydrogenotrophic and methylotrophic microorganisms obtained from coal seam groundwater. Methanogen clones dominated the archaeal 16S rRNA gene libraries and were mostly related to the hydrogenotrophic genus Methanoculleus and the methylotrophic genus Methanolobus. Bacterial 16S rRNA gene libraries were dominated by the clones related to the genera Acetobacterium and Syntrophus which have a symbiotic association with methanogens. LIBSHUFF analysis revealed that N₂ gas injected into the coal seam (for enhanced methane production) does not affect the coverage of archaeal and bacterial populations. However, amova analysis does provide evidence for a change in the genetic diversity of archaeal populations that are dominated by methanogens. Therefore, N₂ injection into the coal seam might affect the cycling of matter by methanogens in situ.