scholarly journals Stable-Isotope Probing of Microorganisms Thriving at Thermodynamic Limits: Syntrophic Propionate Oxidation in Flooded Soil

2004 ◽  
Vol 70 (10) ◽  
pp. 5778-5786 ◽  
Author(s):  
Tillmann Lueders ◽  
Bianca Pommerenke ◽  
Michael W. Friedrich
Keyword(s):  
Stable Isotope ◽  
Dry Weight ◽  
Stable Isotope Probing ◽  
Great Promise ◽  
Natural Environments ◽  
Anoxic Environments ◽  
Propionate Oxidation ◽  
Thermodynamic Limits ◽  
And Function ◽  
Syntrophic Associations

ABSTRACT 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 H2-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 [13C]propionate (<10 mM) and the oxidation of ∼30 μmol of 13C-labeled substrate per g dry weight of soil, we found that archaeal nucleic acids were 13C 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” 13C-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 13C label, suggesting that these methanogens were directly involved in syntrophic associations and/or thriving on the [13C]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.

scholarly journals Syntrophic Growth on Formate: a New Microbial Niche in Anoxic Environments

2008 ◽  
Vol 74 (19) ◽  
pp. 6126-6131 ◽  
Author(s):  
Jan Dolfing ◽  
Bo Jiang ◽  
Anne M. Henstra ◽  
Alfons J. M. Stams ◽  
Caroline M. Plugge
Keyword(s):  
Experimental Evidence ◽  
Pure Culture ◽  
Narrow Range ◽  
Methanogenic Archaea ◽  
Anoxic Environments ◽  
Fermentative Bacteria ◽  
Thermodynamic Limits ◽  
Substrate Concentrations ◽  
Syntrophic Associations

ABSTRACT Anaerobic syntrophic associations of fermentative bacteria and methanogenic archaea operate at the thermodynamic limits of life. The interspecies transfer of electrons from formate or hydrogen as a substrate for the methanogens is key. Contrary requirements of syntrophs and methanogens for growth-sustaining product and substrate concentrations keep the formate and hydrogen concentrations low and within a narrow range. Since formate is a direct substrate for methanogens, a niche for microorganisms that grow by the conversion of formate to hydrogen plus bicarbonate—or vice versa—may seem unlikely. Here we report experimental evidence for growth on formate by syntrophic communities of (i) Moorella sp. strain AMP in coculture with a thermophilic hydrogen-consuming Methanothermobacter species and of (ii) Desulfovibrio sp. strain G11 in coculture with a mesophilic hydrogen consumer, Methanobrevibacter arboriphilus AZ. In pure culture, neither Moorella sp. strain AMP, nor Desulfovibrio sp. strain G11, nor the methanogens grow on formate alone. These results imply the existence of a previously unrecognized microbial niche in anoxic environments.

scholarly journals Stable Isotope Probing of RNA Combining Phylogenetic Microarrays and High Resolution Secondary Ion Mass Spectrometry to Link Composition and Function in Microbial Systems

2010 ◽  
Vol 16 (S2) ◽  
pp. 426-427
Author(s):  
X Mayali ◽  
PK Weber ◽  
EL Brodie ◽  
S Mabery ◽  
PD Hoeprich ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Resolution ◽  
Stable Isotope ◽  
Secondary Ion Mass Spectrometry ◽  
Stable Isotope Probing ◽  
Ion Mass Spectrometry ◽  
And Function ◽  
Microbial Systems ◽  
Secondary Ion

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

DNA-based stable isotope probing: a link between community structure and function

2009 ◽  
Vol 407 (12) ◽  
pp. 3611-3619 ◽  
Author(s):  
Ondrej Uhlík ◽  
Katerina Jecná ◽  
Mary Beth Leigh ◽  
Martina Macková ◽  
Tomas Macek
Keyword(s):  
Community Structure ◽  
Stable Isotope ◽  
Structure And Function ◽  
Stable Isotope Probing ◽  
And Function

scholarly journals A Putatively New Family of Alphaproteobacterial Chloromethane Degraders from a Deciduous Forest Soil Revealed by Stable Isotope Probing and Metagenomics

2021 ◽  
Author(s):  
Eileen Kröber ◽  
Saranya Kanukollu ◽  
Sonja Wende ◽  
Francoise Bringel ◽  
Steffen Kolb
Keyword(s):  
Stable Isotope ◽  
Forest Soil ◽  
Deciduous Forest ◽  
Stratospheric Ozone ◽  
Mineral Soil ◽  
Dry Weight ◽  
Stable Isotope Probing ◽  
Montreal Protocol ◽  
Sequencing Analysis ◽  
Gene Markers

Abstract Background: Chloromethane (CH3 Cl) is the most abundant halogenated organic compound in the atmosphere and substantially responsible for the destruction of the stratospheric ozone layer. Since anthropogenic CH 3 Cl sources have become negligible with the application of the Montreal Protocol (1987), natural sources, such as vegetation and soils, have increased proportionally in the global budget. CH3 Cl-degrading methylotrophs occurring in soils might be an important and overlooked sink.Results & Conclusions: The objective of our study was to link the biotic CH3 Cl sink with the identity of active microorganisms and their biochemical pathways for CH3 Cl degradation in a deciduous forest soil. When tested in laboratory microcosms, biological CH3 Cl consumption occurred in leaf litter, senescent leaves, and organic and mineral soil horizons. Highest consumption rates, around 2 mmol CH3 Cl g -1 dry weight h -1 , were measured in organic soil and senescent leaves, suggesting that top soil layers are active (micro-)biological CH 3 Cl degradation compartments of forest ecosystems. The DNA of these [13C]-CH3 Cl-degrading microbial communities was labelled using stable isotope probing (SIP), and the corresponding taxa and their metabolic pathways studied using high-throughput metagenomics sequencing analysis. [ 13C]-labelled Metagenome-Assembled Genome closely related to the family Beijerinckiaceae may represent a new methylotroph family of Alphaproteobacteria, which is found in metagenome databases of forest soils samples worldwide. Gene markers of the only known pathway for aerobic CH3 Cl degradation, via the methyltransferase system encoded by the CH3 Cl utilisation genes (cmu), were undetected in the DNA-SIP metagenome data, suggesting that biological CH3 Cl sink in this deciduous forest soil operates by a cmu-independent metabolism.

scholarly journals Measurement error and resolution in quantitative stable isotope probing: implications for experimental design

2020 ◽  
Author(s):  
Ella T. Sieradzki ◽  
Benjamin J. Koch ◽  
Alex Greenlon ◽  
Rohan Sachdeva ◽  
Rex R. Malmstrom ◽  
...  
Keyword(s):  
Measurement Error ◽  
Experimental Design ◽  
Stable Isotope ◽  
Statistical Power ◽  
Quantitative Estimation ◽  
Cost Benefit ◽  
Stable Isotope Probing ◽  
Community Interactions ◽  
Density Fractions ◽  
And Function

AbstractQuantitative stable isotope probing (qSIP) estimates the degree of incorporation of an isotope tracer into nucleic acids of metabolically active organisms and can be applied to microorganisms growing in complex communities, such as the microbiomes of soil or water. As such, qSIP has the potential to link microbial biodiversity and biogeochemistry. As with any technique involving quantitative estimation, qSIP involves measurement error; a more complete understanding of error, precision and statistical power will aid in the design of qSIP experiments and interpretation of qSIP data. We used several existing qSIP datasets of microbial communities found in soil and water to evaluate how variance in the estimate of isotope incorporation depends on organism abundance and on the resolution of the density fractionation scheme. We also assessed statistical power for replicated qSIP studies, and sensitivity and specificity for unreplicated designs. We found that variance declines as taxon abundance increases. Increasing the number of density fractions reduces variance, although the benefit of added fractions declines as the number of fractions increases. Specifically, nine fractions appear to be a reasonable tradeoff between cost and precision for most qSIP applications. Increasing replication improves power and reduces the minimum detectable threshold for inferring isotope uptake to 5 atom%. Finally, we provide evidence for the importance of internal standards to calibrate the %GC to mean weighted density regression per sample. These results should benefit those designing future SIP experiments, and provide a reference for metagenomic SIP applications where financial and computational limitations constrain experimental scope.ImportanceOne of the biggest challenges in microbial ecology is correlating the identity of microorganisms with the roles they fulfill in natural environmental systems. Studies of microbes in pure culture reveal much about genomic content and potential functions, but may not reflect an organism’s activity within its natural community. Culture-independent studies supply a community-wide view of composition and function in the context of community interactions, but fail to link the two. Quantitative stable isotope probing (qSIP) is a method that can link the identity and function of specific microbes within a naturally occurring community. Here we explore how the resolution of density-gradient fractionation affects the error and precision of qSIP results, how they may be improved via additional replication, and cost-benefit balanced scenarios for SIP experimental design.

scholarly journals RNA–Stable-Isotope Probing Shows Utilization of Carbon from Inulin by Specific Bacterial Populations in the Rat Large Bowel

2014 ◽  
Vol 80 (7) ◽  
pp. 2240-2247 ◽  
Author(s):  
Gerald W. Tannock ◽  
Blair Lawley ◽  
Karen Munro ◽  
Ian M. Sims ◽  
Julian Lee ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Bacterial Species ◽  
Buoyant Density ◽  
Stable Isotope Probing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Bacterial Cells ◽  
Bacterial Populations ◽  
Content Type ◽  
And Function

ABSTRACTKnowledge of the trophisms that underpin bowel microbiota composition is required in order to understand its complex phylogeny and function. Stable-isotope (13C)-labeled inulin was added to the diet of rats on a single occasion in order to detect utilization of inulin-derived substrates by particular members of the cecal microbiota. Cecal digesta from Fibruline-inulin-fed rats was collected prior to (0 h) and at 6, 12, 18 and 24 h following provision of the [13C]inulin diet. RNA was extracted from these cecal specimens and fractionated in isopycnic buoyant density gradients in order to detect13C-labeled nucleic acid originating in bacterial cells that had metabolized the labeled dietary constituent. RNA extracted from specimens collected after provision of the labeled diet was more dense than 0-h RNA. Sequencing of 16S rRNA genes amplified from cDNA obtained from these fractions showed thatBacteroides uniformis,Blautia glucerasea,Clostridium indolis, andBifidobacterium animaliswere the main users of the13C-labeled substrate. Culture-based studies of strains of these bacterial species enabled trophisms associated with inulin and its hydrolysis products to be identified.B. uniformisutilized Fibruline-inulin for growth, whereas the other species used fructo-oligosaccharide and monosaccharides. Thus, RNA–stable-isotope probing (RNA-SIP) provided new information about the use of carbon from inulin in microbiota metabolism.

Stable Isotope Probing: A Critique of Its Role in Linking Phylogeny and Function

2006 ◽  
pp. 205-216 ◽  
Author(s):  
Mike Manefield ◽  
Robert I. Griffiths ◽  
Mark J. Bailey ◽  
Andrew S. Whiteley
Keyword(s):  
Stable Isotope ◽  
Stable Isotope Probing ◽  
And Function

scholarly journals Validation of Heavy-Water Stable Isotope Probing for the Characterization of Rapidly Responding Soil Bacteria

2011 ◽  
Vol 77 (13) ◽  
pp. 4589-4596 ◽  
Author(s):  
Zachary T. Aanderud ◽  
Jay T. Lennon
Keyword(s):  
Stable Isotope ◽  
Microbial Communities ◽  
Heavy Water ◽  
Soil Bacteria ◽  
Structure And Function ◽  
Stable Isotope Probing ◽  
Rrna Genes ◽  
Relative Recovery ◽  
Water Stable Isotope ◽  
And Function

ABSTRACTRapid responses of bacteria to sudden changes in their environment can have important implications for the structure and function of microbial communities. In this study, we used heavy-water stable isotope probing (H218O-SIP) to identify bacteria that respond to soil rewetting. First, we conducted experiments to address uncertainties regarding the H218O-SIP method. Using liquid chromatography-mass spectroscopy (LC-MS), we determined that oxygen from H218O was incorporated into all structural components of DNA. Although this incorporation was uneven, we could effectively separate18O-labeled and unlabeled DNAs derived from laboratory cultures and environmental samples that were incubated with H218O. We found no evidence forex vivoexchange of oxygen atoms between DNA and extracellular H2O, suggesting that18O incorporation into DNA is relatively stable. Furthermore, the rate of18O incorporation into bacterial DNA was high (within 48 to 72 h), coinciding with pulses of CO2generated from soil rewetting. Second, we examined shifts in the bacterial composition of grassland soils following rewetting, using H218O-SIP and bar-coded pyrosequencing of 16S rRNA genes. For some groups of soil bacteria, we observed coherent responses at a relatively course taxonomic resolution. Following rewetting, the relative recovery ofAlphaproteobacteria,Betaproteobacteria, andGammaproteobacteriaincreased, while the relative recovery ofChloroflexiandDeltaproteobacteriadecreased. Together, our results suggest that H218O-SIP is effective at identifying metabolically active bacteria that influence soil carbon dynamics. Our results contribute to the ecological classification of soil bacteria while providing insight into some of the functional traits that influence the structure and function of microbial communities under dynamic soil moisture regimes.

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