Stable Isotope Probing for Microbial Iron Reduction in Chocolate Pots Hot Spring, Yellowstone National Park

ABSTRACTChocolate Pots hot springs (CP) is a circumneutral-pH Fe-rich geothermal feature located in Yellowstone National Park. Previous Fe(III)-reducing enrichment culture studies with CP sediments identified close relatives of known dissimilatory Fe(III)-reducing bacterial (FeRB) taxa, includingGeobacterandMelioribacter. However, the abundances and activities of such organisms in the native microbial community are unknown. Here, we used stable isotope probing experiments combined with 16S rRNA gene amplicon and shotgun metagenomic sequencing to gain an understanding of thein situFe(III)-reducing microbial community at CP. Fe-Si oxide precipitates collected near the hot spring vent were incubated with unlabeled and13C-labeled acetate to target active FeRB. We searched reconstructed genomes for homologs of genes involved in known extracellular electron transfer (EET) systems to identify the taxa involved in Fe redox transformations. Known FeRB taxa containing putative EET systems (Geobacter,Ignavibacteria) increased in abundance under acetate-amended conditions, whereas genomes related toIgnavibacteriumandThermodesulfovibriothat contained putative EET systems were recovered from incubations without electron donor. Our results suggest that FeRB play an active role in Fe redox cycling within Fe-Si oxide-rich deposits located at the hot spring vent.IMPORTANCEThe identification of past near-surface hydrothermal environments on Mars emphasizes the importance of using modern Earth environments, such as CP, to gain insight into potential Fe-based microbial life on other rocky worlds, as well as ancient Fe-rich Earth ecosystems. By combining stable carbon isotope probing techniques and DNA sequencing technology, we gained insight into the pathways of microbial Fe redox cycling at CP. The results suggest that microbial Fe(III) oxide reduction is prominentin situ, with important implications for the generation of geochemical and stable Fe isotopic signatures of microbial Fe redox metabolism within Fe-rich circumneutral-pH thermal spring environments on Earth and Mars.

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Pyrobaculum yellowstonensis Strain WP30 Respires on Elemental Sulfur and/or Arsenate in Circumneutral Sulfidic Geothermal Sediments of Yellowstone National Park

Applied and Environmental Microbiology ◽

10.1128/aem.01095-15 ◽

2015 ◽

Vol 81 (17) ◽

pp. 5907-5916 ◽

Cited By ~ 13

Author(s):

Z. J. Jay ◽

J. P. Beam ◽

A. Dohnalkova ◽

R. Lohmayer ◽

B. Bodle ◽

...

Keyword(s):

Yellowstone National Park ◽

National Park ◽

Elemental Sulfur ◽

De Novo ◽

Hot Spring ◽

Content Type ◽

Physiological Attributes ◽

And Function ◽

Metagenome Sequence

ABSTRACTThermoproteales(phylumCrenarchaeota) populations are abundant in high-temperature (>70°C) environments of Yellowstone National Park (YNP) and are important in mediating the biogeochemical cycles of sulfur, arsenic, and carbon. The objectives of this study were to determine the specific physiological attributes of the isolatePyrobaculum yellowstonensisstrain WP30, which was obtained from an elemental sulfur sediment (Joseph's Coat Hot Spring [JCHS], 80°C, pH 6.1, 135 μM As) and relate this organism to geochemical processes occurringin situ. Strain WP30 is a chemoorganoheterotroph and requires elemental sulfur and/or arsenate as an electron acceptor. Growth in the presence of elemental sulfur and arsenate resulted in the formation of thioarsenates and polysulfides. The complete genome of this organism was sequenced (1.99 Mb, 58% G+C content), revealing numerous metabolic pathways for the degradation of carbohydrates, amino acids, and lipids. Multiple dimethyl sulfoxide-molybdopterin (DMSO-MPT) oxidoreductase genes, which are implicated in the reduction of sulfur and arsenic, were identified. Pathways for thede novosynthesis of nearly all required cofactors and metabolites were identified. The comparative genomics ofP. yellowstonensisand the assembled metagenome sequence from JCHS showed that this organism is highly related (∼95% average nucleotide sequence identity) toin situpopulations. The physiological attributes and metabolic capabilities ofP. yellowstonensisprovide an important foundation for developing an understanding of the distribution and function of these populations in YNP.

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Nanoarchaeota, Their Sulfolobales Host, and Nanoarchaeota Virus Distribution across Yellowstone National Park Hot Springs

Applied and Environmental Microbiology ◽

10.1128/aem.01539-15 ◽

2015 ◽

Vol 81 (22) ◽

pp. 7860-7868 ◽

Cited By ~ 34

Author(s):

Jacob H. Munson-McGee ◽

Erin K. Field ◽

Mary Bateson ◽

Colleen Rooney ◽

Ramunas Stepanauskas ◽

...

Keyword(s):

Single Cell ◽

Yellowstone National Park ◽

National Park ◽

Hot Springs ◽

Hot Spring ◽

Genome Comparison ◽

Fish Analysis ◽

Content Type ◽

Cell Genome ◽

Single Cell Genome

ABSTRACTNanoarchaeotaare obligate symbionts with reduced genomes first described from marine thermal vent environments. Here, both community metagenomics and single-cell analysis revealed the presence ofNanoarchaeotain high-temperature (∼90°C), acidic (pH ≈ 2.5 to 3.0) hot springs in Yellowstone National Park (YNP) (United States). Single-cell genome analysis of two cells resulted in two nearly identical genomes, with an estimated full length of 650 kbp. Genome comparison showed that these two cells are more closely related to the recently proposedNanobsidianus stetterifrom a more neutral YNP hot spring than to the marineNanoarchaeum equitans. Single-cell and catalyzed reporter deposition-fluorescencein situhybridization (CARD-FISH) analysis of environmental hot spring samples identified the host of the YNPNanoarchaeotaas aSulfolobalesspecies known to inhabit the hot springs. Furthermore, we demonstrate thatNanoarchaeotaare widespread in acidic to near neutral hot springs in YNP. An integrated viral sequence was also found within oneNanoarchaeotasingle-cell genome and further analysis of the purified viral fraction from environmental samples indicates that this is likely a virus replicating within the YNPNanoarchaeota.

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Refining the Application of Microbial Lipids as Tracers of Staphylococcus aureus Growth Rates in Cystic Fibrosis Sputum

Journal of Bacteriology ◽

10.1128/jb.00365-18 ◽

2018 ◽

Vol 200 (24) ◽

Cited By ~ 7

Author(s):

Cajetan Neubauer ◽

Ajay S. Kasi ◽

Nora Grahl ◽

Alex L. Sessions ◽

Sebastian H. Kopf ◽

...

Keyword(s):

Cystic Fibrosis ◽

Fatty Acids ◽

Microbial Community ◽

Stable Isotope ◽

Community Composition ◽

Growth Rates ◽

Microbial Community Composition ◽

Stable Isotope Probing ◽

Content Type ◽

Lung Infections

ABSTRACT Chronic lung infections in cystic fibrosis (CF) could be treated more effectively if the effects of antimicrobials on pathogens in situ were known. Here, we compared changes in the microbial community composition and pathogen growth rates in longitudinal studies of seven pediatric CF patients undergoing intravenous antibiotic administration during pulmonary exacerbations. The microbial community composition was determined by counting rRNA with NanoString DNA analysis, and growth rates were obtained by incubating CF sputum with heavy water and tracing incorporation of deuterium into two branched-chain (“anteiso”) fatty acids (a-C15:0 and a-C17:0) using gas chromatography-mass spectrometry (GC/MS). Prior to this study, both lipids were thought to be specific for Staphylococcaceae; hence, their isotopic enrichment was interpreted as a growth proxy for Staphylococcus aureus. Our experiments revealed, however, that Prevotella is also a relevant microbial producer of a-C17:0 fatty acid in some CF patients; thus, deuterium incorporation into these lipids is better interpreted as a more general pathogen growth rate proxy. Even accounting for a small nonmicrobial background source detected in some patient samples, a-C15:0 fatty acid still appears to be a relatively robust proxy for CF pathogens, revealing a median generation time of ∼1.5 days, similar to prior observations. Contrary to our expectation, pathogen growth rates remained relatively stable throughout exacerbation treatment. We suggest two straightforward “best practices” for application of stable-isotope probing to CF sputum metabolites: (i) parallel determination of microbial community composition in CF sputum using culture-independent tools and (ii) assessing background levels of the diagnostic metabolite. IMPORTANCE In chronic lung infections, populations of microbial pathogens change and mature in ways that are often unknown, which makes it challenging to identify appropriate treatment options. A promising tool to better understand the physiology of microorganisms in a patient is stable-isotope probing, which we previously developed to estimate the growth rates of S. aureus in cystic fibrosis (CF) sputum. Here, we tracked microbial communities in a cohort of CF patients and found that anteiso fatty acids can also originate from other sources in CF sputum. This awareness led us to develop a new workflow for the application of stable-isotope probing in this context, improving our ability to estimate pathogen generation times in clinical samples.

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Use of Stable-Isotope Probing, Full-Cycle rRNA Analysis, and Fluorescence In Situ Hybridization-Microautoradiography To Study a Methanol-Fed Denitrifying Microbial Community

Applied and Environmental Microbiology ◽

10.1128/aem.70.1.588-596.2004 ◽

2004 ◽

Vol 70 (1) ◽

pp. 588-596 ◽

Cited By ~ 169

Author(s):

Maneesha P. Ginige ◽

Philip Hugenholtz ◽

Holger Daims ◽

Michael Wagner ◽

Jürg Keller ◽

...

Keyword(s):

In Situ Hybridization ◽

Microbial Community ◽

Stable Isotope ◽

Fluorescence In Situ Hybridization ◽

Clone Library ◽

Stable Isotope Probing ◽

Denitrification Rate ◽

Biological Nutrient Removal ◽

Group A

ABSTRACT A denitrifying microbial consortium was enriched in an anoxically operated, methanol-fed sequencing batch reactor (SBR) fed with a mineral salts medium containing methanol as the sole carbon source and nitrate as the electron acceptor. The SBR was inoculated with sludge from a biological nutrient removal activated sludge plant exhibiting good denitrification. The SBR denitrification rate improved from less than 0.02 mg of NO3 −-N mg of mixed-liquor volatile suspended solids (MLVSS)−1 h−1 to a steady-state value of 0.06 mg of NO3 −-N mg of MLVSS−1 h−1 over a 7-month operational period. At this time, the enriched microbial community was subjected to stable-isotope probing (SIP) with [13C]methanol to biomark the DNA of the denitrifiers. The extracted [13C]DNA and [12C]DNA from the SIP experiment were separately subjected to full-cycle rRNA analysis. The dominant 16S rRNA gene phylotype (group A clones) in the [13C]DNA clone library was closely related to those of the obligate methylotrophs Methylobacillus and Methylophilus in the order Methylophilales of the Betaproteobacteria (96 to 97% sequence identities), while the most abundant clone groups in the [12C]DNA clone library mostly belonged to the family Saprospiraceae in the Bacteroidetes phylum. Oligonucleotide probes for use in fluorescence in situ hybridization (FISH) were designed to specifically target the group A clones and Methylophilales (probes DEN67 and MET1216, respectively) and the Saprospiraceae clones (probe SAP553). Application of these probes to the SBR biomass over the enrichment period demonstrated a strong correlation between the level of SBR denitrification and relative abundance of DEN67-targeted bacteria in the SBR community. By contrast, there was no correlation between the denitrification rate and the relative abundances of the well-known denitrifying genera Hyphomicrobium and Paracoccus or the Saprospiraceae clones visualized by FISH in the SBR biomass. FISH combined with microautoradiography independently confirmed that the DEN67-targeted cells were the dominant bacterial group capable of anoxic [14C]methanol uptake in the enriched biomass. The well-known denitrification lag period in the methanol-fed SBR was shown to coincide with a lag phase in growth of the DEN67-targeted denitrifying population. We conclude that Methylophilales bacteria are the dominant denitrifiers in our SBR system and likely are important denitrifiers in full-scale methanol-fed denitrifying sludges.

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Silicified Microbial Community at Steep Cone Hot Spring, Yellowstone National Park.

Microbes and Environments ◽

10.1264/jsme2.2001.125 ◽

2001 ◽

Vol 16 (2) ◽

pp. 125-130 ◽

Cited By ~ 22

Author(s):

Fumio Inagaki ◽

Yoshinobu Motomura ◽

Katsumi Doi ◽

Sachihiro Taguchi ◽

Eiji Izawa ◽

...

Keyword(s):

Microbial Community ◽

Yellowstone National Park ◽

National Park ◽

Hot Spring

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Use of Field-Based Stable Isotope Probing To Identify Adapted Populations and Track Carbon Flow through a Phenol-Degrading Soil Microbial Community

Applied and Environmental Microbiology ◽

10.1128/aem.71.12.7858-7865.2005 ◽

2005 ◽

Vol 71 (12) ◽

pp. 7858-7865 ◽

Cited By ~ 95

Author(s):

Christopher M. DeRito ◽

Graham M. Pumphrey ◽

Eugene L. Madsen

Keyword(s):

Mass Spectrometry ◽

Microbial Community ◽

Stable Isotope ◽

Soil Microbial Community ◽

Stable Isotope Probing ◽

Gas Chromatography Mass Spectrometry ◽

Rrna Gene ◽

Soil Dna ◽

Soil Microbial

ABSTRACT The goal of this field study was to provide insight into three distinct populations of microorganisms involved in in situ metabolism of phenol. Our approach measured 13CO2 respired from [13C]phenol and stable isotope probing (SIP) of soil DNA at an agricultural field site. Traditionally, SIP-based investigations have been subject to the uncertainties posed by carbon cross-feeding. By altering our field-based, substrate-dosing methodologies, experiments were designed to look beyond primary degraders to detect trophically related populations in the food chain. Using gas chromatography-mass spectrometry (GC/MS), it was shown that 13C-labeled biomass, derived from primary phenol degraders in soil, was a suitable growth substrate for other members of the soil microbial community. Next, three dosing regimes were designed to examine active members of the microbial community involved in phenol metabolism in situ: (i) 1 dose of [13C]phenol, (ii) 11 daily doses of unlabeled phenol followed by 1 dose of [13C]phenol, and (iii) 12 daily doses of [13C]phenol. GC/MS analysis demonstrated that prior exposure to phenol boosted 13CO2 evolution by a factor of 10. Furthermore, imaging of 13C-treated soil using secondary ion mass spectrometry (SIMS) verified that individual bacteria incorporated 13C into their biomass. PCR amplification and 16S rRNA gene sequencing of 13C-labeled soil DNA from the 3 dosing regimes revealed three distinct clone libraries: (i) unenriched, primary phenol degraders were most diverse, consisting of α-, β-, and γ-proteobacteria and high-G+C-content gram-positive bacteria, (ii) enriched primary phenol degraders were dominated by members of the genera Kocuria and Staphylococcus, and (iii) trophically related (carbon cross-feeders) were dominated by members of the genus Pseudomonas. These data show that SIP has the potential to document population shifts caused by substrate preexposure and to follow the flow of carbon through terrestrial microbial food chains.

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Active Soil Nitrifying Communities Revealed by In Situ Transcriptomics and Microcosm-Based Stable-Isotope Probing

Applied and Environmental Microbiology ◽

10.1128/aem.01807-20 ◽

2020 ◽

Vol 86 (23) ◽

Author(s):

Wei-Wei Xia ◽

Jun Zhao ◽

Yan Zheng ◽

Hui-Min Zhang ◽

Jia-Bao Zhang ◽

...

Keyword(s):

Stable Isotope ◽

Stable Isotope Probing ◽

Field Conditions ◽

Nitrogen Fertilizers ◽

16S Rrna Genes ◽

Rrna Genes ◽

Content Type ◽

Nitrite Oxidizing Bacteria

ABSTRACT Long-term nitrogen field fertilization often results in significant changes in nitrifying communities that catalyze a key step in the global N cycle. However, whether microcosm studies are able to inform the dynamic changes in communities of ammonia-oxidizing bacteria (AOB) and archaea (AOA) under field conditions remains poorly understood. This study aimed to evaluate the transcriptional activities of nitrifying communities under in situ conditions, and we found that they were largely similar to those of 13C-labeled nitrifying communities in the urea-amended microcosms of soils that had received different N fertilization regimens for 22 years. High-throughput sequencing of 16S rRNA genes and transcripts suggested that Nitrosospira cluster 3-like AOB and Nitrososphaera viennensis-like AOA were significantly stimulated in N-fertilized fresh soils. Real-time quantitative PCR demonstrated that the significant increase of AOA and AOB in fresh soils upon nitrogen fertilization could be preserved in the air-dried soils. DNA-based stable-isotope probing (SIP) further revealed the greatest labeling of Nitrosospira cluster 3-like AOB and Nitrosospira viennensis-like AOA, despite the strong advantage of AOB over AOA in the N-fertilized soils. Nitrobacter-like nitrite-oxidizing bacteria (NOB) played more important roles than Nitrospira-like NOB in urea-amended SIP microcosms, while the situation was the opposite under field conditions. Our results suggest that long-term fertilization selected for physiologically versatile AOB and AOA that could have been adapted to a wide range of substrate ammonium concentrations. It also provides compelling evidence that the dominant communities of transcriptionally active nitrifiers under field conditions were largely similar to those revealed in 13C-labeled microcosms. IMPORTANCE The role of manipulated microcosms in microbial ecology has been much debated, because they cannot entirely represent the in situ situation. We collected soil samples from 20 field plots, including 5 different treatments with and without nitrogen fertilizers for 22 years, in order to assess active nitrifying communities by in situ transcriptomics and microcosm-based stable-isotope probing. The results showed that chronic N enrichment led to competitive advantages of Nitrosospira cluster 3-like AOB over N. viennensis-like AOA in soils under field conditions. Microcosm labeling revealed similar results for active AOA and AOB, although an apparent discrepancy was observed for nitrite-oxidizing bacteria. This study suggests that the soil microbiome represents a relatively stable community resulting from complex evolutionary processes over a large time scale, and microcosms can serve as powerful tools to test the theory of environmental filtering on the key functional microbial guilds.

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Cultivation-Independent Detection of Autotrophic Hydrogen-Oxidizing Bacteria by DNA Stable-Isotope Probing

Applied and Environmental Microbiology ◽

10.1128/aem.00285-11 ◽

2011 ◽

Vol 77 (14) ◽

pp. 4931-4938 ◽

Cited By ~ 8

Author(s):

Graham M. Pumphrey ◽

Anthony Ranchou-Peyruse ◽

Jim C. Spain

Keyword(s):

Stable Isotope ◽

Yellowstone National Park ◽

National Park ◽

Rhizosphere Soil ◽

Stable Isotope Probing ◽

Microbial Mat ◽

High Affinity ◽

Knallgas Bacteria ◽

Cultivation Techniques ◽

Hydrogen Oxidizing Bacteria

ABSTRACTKnallgas bacteria are a physiologically defined group that is primarily studied using cultivation-dependent techniques. Given that current cultivation techniques fail to grow most bacteria, cultivation-independent techniques that selectively detect and identify knallgas bacteria will improve our ability to study their diversity and distribution. We used stable-isotope probing (SIP) to identify knallgas bacteria in rhizosphere soil of legumes and in a microbial mat from Obsidian Pool in Yellowstone National Park. When samples were incubated in the dark, incorporation of13CO2was H2dependent. SIP enabled the detection of knallgas bacteria that were not detected by cultivation, and the majority of bacteria identified in the rhizosphere soils were betaproteobacteria predominantly related to genera previously known to oxidize hydrogen. Bacteria in soil grew on hydrogen at concentrations as low as 100 ppm. AhydBhomolog encoding a putative high-affinity NiFe hydrogenase was amplified from13C-labeled DNA from both vetch and clover rhizosphere soil. The results indicate that knallgas bacteria can be detected by SIP and populations that respond to different H2concentrations can be distinguished. The methods described here should be applicable to a variety of ecosystems and will enable the discovery of additional knallgas bacteria that are resistant to cultivation.

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