scholarly journals Arctic tundra soil bacterial communities active at subzero temperatures detected by stable isotope probing

2019 ◽  
Vol 96 (2) ◽  
Author(s):  
Preshita S Gadkari ◽  
Lora R McGuinness ◽  
Minna K Männistö ◽  
Lee J Kerkhof ◽  
Max M Häggblom
Keyword(s):  
Stable Isotope ◽  
16S Rrna ◽  
Arctic Tundra ◽  
Stable Isotope Probing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Subzero Temperature ◽  
Tundra Soil ◽  
Arctic Soils ◽  
Subzero Temperatures

ABSTRACT Arctic soils store vast amounts of carbon and are subject to intense climate change. While the effects of thaw on the composition and activities of Arctic tundra microorganisms has been examined extensively, little is known about the consequences of temperature fluctuations within the subzero range in seasonally frozen or permafrost soils. This study identified tundra soil bacteria active at subzero temperatures using stable isotope probing (SIP). Soils from Kilpisjärvi, Finland, were amended with 13C-cellobiose and incubated at 0, −4 and −16°C for up to 40 weeks. 16S rRNA gene sequence analysis of 13C-labelled DNA revealed distinct subzero-active bacterial taxa. The SIP experiments demonstrated that diverse bacteria, including members of Candidatus Saccharibacteria, Melioribacteraceae, Verrucomicrobiaceae, Burkholderiaceae, Acetobacteraceae, Armatimonadaceae and Planctomycetaceae, were capable of synthesising 13C-DNA at subzero temperatures. Differences in subzero temperature optima were observed, for example, with members of Oxalobacteraceae and Rhizobiaceae found to be more active at 0°C than at −4°C or −16°C, whereas Melioribacteriaceae were active at all subzero temperatures tested. Phylogeny of 13C-labelled 16S rRNA genes from the Melioribacteriaceae, Verrucomicrobiaceae and Candidatus Saccharibacteria suggested that these taxa formed subzero-active clusters closely related to members from other cryo-environments. This study demonstrates that subzero temperatures impact active bacterial community composition and activity, which may influence biogeochemical cycles.

scholarly journals Stable-Isotope Probing Identifies Uncultured Planctomycetes as Primary Degraders of a Complex Heteropolysaccharide in Soil

2015 ◽  
Vol 81 (14) ◽  
pp. 4607-4615 ◽  
Author(s):  
Xiaoqing Wang ◽  
Christine E. Sharp ◽  
Gareth M. Jones ◽  
Stephen E. Grasby ◽  
Allyson L. Brady ◽  
...  
Keyword(s):  
Stable Isotope ◽  
16S Rrna ◽  
Soil Bacteria ◽  
Stable Isotope Probing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Content Type ◽  
Degrading Bacteria ◽  
Aerobic Soil

ABSTRACTThe exopolysaccharides (EPSs) produced by some bacteria are potential growth substrates for other bacteria in soil. We used stable-isotope probing (SIP) to identify aerobic soil bacteria that assimilated the cellulose produced byGluconacetobacter xylinusor the EPS produced byBeijerinckia indica. The latter is a heteropolysaccharide comprised primarily ofl-guluronic acid,d-glucose, andd-glycero-d-mannoheptose.13C-labeled EPS and13C-labeled cellulose were purified from bacterial cultures grown on [13C]glucose. Two soils were incubated with these substrates, and bacteria actively assimilating them were identified via pyrosequencing of 16S rRNA genes recovered from13C-labeled DNA. Cellulose C was assimilated primarily by soil bacteria closely related (93 to 100% 16S rRNA gene sequence identities) to known cellulose-degrading bacteria. However,B. indicaEPS was assimilated primarily by bacteria with low identities (80 to 95%) to known species, particularly by different members of the phylumPlanctomycetes. In one incubation, members of thePlanctomycetesmade up >60% of all reads in the labeled DNA and were only distantly related (<85% identity) to any described species. Although it is impossible with SIP to completely distinguish primary polysaccharide hydrolyzers from bacteria growing on produced oligo- or monosaccharides, the predominance ofPlanctomycetessuggested that they were primary degraders of EPS. Other bacteria assimilatingB. indicaEPS included members of theVerrucomicrobia, candidate division OD1, and theArmatimonadetes. The results indicate that some uncultured bacteria in soils may be adapted to using complex heteropolysaccharides for growth and suggest that the use of these substrates may provide a means for culturing new species.

scholarly journals Stable Isotope Probing Analysis of Interactions between Ammonia Oxidizers

2010 ◽  
Vol 76 (8) ◽  
pp. 2468-2477 ◽  
Author(s):  
Maria Tourna ◽  
Thomas E. Freitag ◽  
James I. Prosser
Keyword(s):  
Stable Isotope ◽  
16S Rrna ◽  
Microbial Communities ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Ammonia Concentration ◽  
Stable Isotope Probing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
High Ammonia

ABSTRACT The response of natural microbial communities to environmental change can be assessed by determining DNA- or RNA-targeted changes in relative abundance of 16S rRNA gene sequences by using fingerprinting techniques such as denaturing gradient gel electrophoresis (DNA-DGGE and RNA-DGGE, respectively) or by stable isotope probing (SIP) of 16S rRNA genes following incubation with a 13C-labeled substrate (DNA-SIP-DGGE). The sensitivities of these three approaches were compared during batch growth of communities containing two or three Nitrosospira pure or enriched cultures with different tolerances to a high ammonia concentration. Cultures were supplied with low, intermediate, or high initial ammonia concentrations and with 13C-labeled carbon dioxide. DNA-SIP-DGGE provided the most direct evidence for growth and was the most sensitive, with changes in DGGE profiles evident before changes in DNA- and RNA-DGGE profiles and before detectable increases in nitrite and nitrate production. RNA-DGGE provided intermediate sensitivity. In addition, the three molecular methods were used to follow growth of individual strains within communities. In general, changes in relative activities of individual strains within communities could be predicted from monoculture growth characteristics. Ammonia-tolerant Nitrosospira cluster 3b strains dominated mixed communities at all ammonia concentrations, and ammonia-sensitive strains were outcompeted at an intermediate ammonia concentration. However, coexistence of ammonia-tolerant and ammonia-sensitive strains occurred at the lowest ammonia concentration, and, under some conditions, strains inhibited at high ammonia in monoculture were active at high ammonia in mixed cultures, where they coexisted with ammonia-tolerant strains. The results therefore demonstrate the sensitivity of SIP for detection of activity of organisms with relatively low yield and low activity and its ability to follow changes in the structure of interacting microbial communities.

scholarly journals Field-Based Stable Isotope Probing Reveals the Identities of Benzoic Acid-Metabolizing Microorganisms and Their In Situ Growth in Agricultural Soil

2008 ◽  
Vol 74 (13) ◽  
pp. 4111-4118 ◽  
Author(s):  
Graham M. Pumphrey ◽  
Eugene L. Madsen
Keyword(s):  
Stable Isotope ◽  
16S Rrna ◽  
Benzoic Acid ◽  
Stable Isotope Probing ◽  
Heavy Fraction ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
In Situ Growth ◽  
Probable Number

ABSTRACT We used a combination of stable isotope probing (SIP), gas chromatography-mass spectrometry-based respiration, isolation/cultivation, and quantitative PCR procedures to discover the identity and in situ growth of soil microorganisms that metabolize benzoic acid. We added [13C]benzoic acid or [12C]benzoic acid (100 μg) once, four times, or five times at 2-day intervals to agricultural field plots. After monitoring 13CO2 evolution from the benzoic acid-dosed soil, field soils were harvested and used for nucleic acid extraction and for cultivation of benzoate-degrading bacteria. Exposure of soil to benzoate increased the number of culturable benzoate degraders compared to unamended soil, and exposure to benzoate shifted the dominant culturable benzoate degraders from Pseudomonas species to Burkholderia species. Isopycnic separation of heavy [13C]DNA from the unlabeled fraction allowed terminal restriction fragment length polymorphism (T-RFLP) analyses to confirm that distinct 16S rRNA genes were localized in the heavy fraction. Phylogenetic analysis of sequenced 16S rRNA genes revealed a predominance (15 of 58 clones) of Burkholderia species in the heavy fraction. Burkholderia sp. strain EBA09 shared 99.5% 16S rRNA sequence similarity with a group of clones representing the dominant RFLP pattern, and the T-RFLP fragment for strain EBA09 and a clone from that cluster matched the fragment enriched in the [13C]DNA fraction. Growth of the population represented by EBA09 during the field-dosing experiment was demonstrated by using most-probable-number-PCR and primers targeting EBA09 and the closely related species Burkholderia hospita. Thus, the target population identified by SIP not only actively metabolized benzoic acid but reproduced in the field upon the addition of the substrate.

scholarly journals Biphenyl-Metabolizing Bacteria in the Rhizosphere of Horseradish and Bulk Soil Contaminated by Polychlorinated Biphenyls as Revealed by Stable Isotope Probing

2009 ◽  
Vol 75 (20) ◽  
pp. 6471-6477 ◽  
Author(s):  
Ondrej Uhlik ◽  
Katerina Jecna ◽  
Martina Mackova ◽  
Cestmir Vlcek ◽  
Miluse Hroudova ◽  
...  
Keyword(s):  
Microbial Community ◽  
Stable Isotope ◽  
Polychlorinated Biphenyls ◽  
Stable Isotope Probing ◽  
Amino Acid Sequences ◽  
16S Rrna Genes ◽  
Bulk Soil ◽  
Rrna Genes ◽  
Soil Environment ◽  
Α Subunits

ABSTRACT DNA-based stable isotope probing in combination with terminal restriction fragment length polymorphism was used in order to identify members of the microbial community that metabolize biphenyl in the rhizosphere of horseradish (Armoracia rusticana) cultivated in soil contaminated with polychlorinated biphenyls (PCBs) compared to members of the microbial community in initial, uncultivated bulk soil. On the basis of early and recurrent detection of their 16S rRNA genes in clone libraries constructed from [13C]DNA, Hydrogenophaga spp. appeared to dominate biphenyl catabolism in the horseradish rhizosphere soil, whereas Paenibacillus spp. were the predominant biphenyl-utilizing bacteria in the initial bulk soil. Other bacteria found to derive carbon from biphenyl in this nutrient-amended microcosm-based study belonged mostly to the class Betaproteobacteria and were identified as Achromobacter spp., Variovorax spp., Methylovorus spp., or Methylophilus spp. Some bacteria that were unclassified at the genus level were also detected, and these bacteria may be members of undescribed genera. The deduced amino acid sequences of the biphenyl dioxygenase α subunits (BphA) from bacteria that incorporated [13C]into DNA in 3-day incubations of the soils with [13C]biphenyl are almost identical to that of Pseudomonas alcaligenes B-357. This suggests that the spectrum of the PCB congeners that can be degraded by these enzymes may be similar to that of strain B-357. These results demonstrate that altering the soil environment can result in the participation of different bacteria in the metabolism of biphenyl.

scholarly journals Active Ammonia Oxidizers in an Acidic Soil Are Phylogenetically Closely Related to Neutrophilic Archaeon

2013 ◽  
Vol 80 (5) ◽  
pp. 1684-1691 ◽  
Author(s):  
Baozhan Wang ◽  
Yan Zheng ◽  
Rong Huang ◽  
Xue Zhou ◽  
Dongmei Wang ◽  
...  
Keyword(s):  
16S Rrna ◽  
Buoyant Density ◽  
Stable Isotope Probing ◽  
Acidic Soil ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Molecular Evidence ◽  
Ammonia Oxidizing Bacteria ◽  
Content Type ◽  
Metabolic Versatility

ABSTRACTAll cultivated ammonia-oxidizing archaea (AOA) within theNitrososphaeracluster (former soil group 1.1b) are neutrophilic. Molecular surveys also indicate the existence ofNitrososphaera-like phylotypes in acidic soil, but their ecological roles are poorly understood. In this study, we present molecular evidence for the chemolithoautotrophic growth ofNitrososphaera-like AOA in an acidic soil with pH 4.92 using DNA-based stable isotope probing (SIP). Soil microcosm incubations demonstrated that nitrification was stimulated by urea fertilization and accompanied by a significant increase in the abundance of AOA rather than ammonia-oxidizing bacteria (AOB). Real-time PCR analysis ofamoAgenes as a function of the buoyant density of the DNA gradient following the ultracentrifugation of the total DNA extracted from SIP microcosms indicated a substantial growth of soil AOA during nitrification. Pyrosequencing of the total 16S rRNA genes in the “heavy” DNA fractions suggested that archaeal communities were labeled to a much greater extent than soil AOB. Acetylene inhibition further showed that13CO2assimilation by nitrifying communities depended solely on ammonia oxidation activity, suggesting a chemolithoautotrophic lifestyle. Phylogenetic analysis of both13C-labeledamoAand 16S rRNA genes revealed that most of the active AOA were phylogenetically closely related to the neutrophilic strainsNitrososphaera viennensisEN76 and JG1 within theNitrososphaeracluster. Our results provide strong evidence for the adaptive growth ofNitrososphaera-like AOA in acidic soil, suggesting a greater metabolic versatility of soil AOA than previously appreciated.

scholarly journals Origin and Diversity of Metabolically Active Gut Bacteria from Laboratory-Bred Larvae of Manduca sexta (Sphingidae, Lepidoptera, Insecta)

2008 ◽  
Vol 74 (23) ◽  
pp. 7189-7196 ◽  
Author(s):  
Nicole Brinkmann ◽  
Rainer Martens ◽  
Christoph C. Tebbe
Keyword(s):  
16S Rrna ◽  
Manduca Sexta ◽  
Metabolic Activity ◽  
Stable Isotope Probing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Bacterial Cells ◽  
Rt Pcr ◽  
Tobacco Leaves ◽  
Active Bacteria

ABSTRACT Cultivation-independent analyses based on genetic profiling of partial bacterial 16S rRNA genes by PCR-single-strand conformation polymorphism (PCR-SSCP), reverse transcriptase (RT)-PCR-SSCP of the 16S rRNA itself, and stable isotope probing (SIP), followed by RT-PCR-SSCP, were applied to characterize the diversity of metabolically active bacteria in the larval gut of Manduca sexta bred on tobacco leaves under greenhouse conditions. For SIP, hatching larvae were fed with leaves from tobacco plants grown in a 13CO2-enriched atmosphere. Dominant SSCP bands were sequenced and phylogenetically analyzed. Only one major gut colonizer, an Enterococcus relative, was detected; it occurred in the heavy RNA fraction, demonstrating its metabolic activity, and it originated from eggs, where its metabolic activity was also indicated by rRNA-based SSCP profiles. In contrast, a Citrobacter sedlakii relative was detected on eggs by DNA-SSCP, but rRNA-SSCP and SIP-rRNA-SSCP were negative, suggesting that these bacterial cells were inactive. A Burkholderia relative was dominant and metabolically active on the tobacco leaves but inactive inside the gut, where it was also quantitatively reduced, as suggested by lower band intensities in the DNA-based SSCP profiles. SIP-RNA-SSCP detected another metabolically active gut bacterium (Enterobacter sp.) and more bacteria in the light RNA fraction, indicating low or no metabolic activity of the latter inside the gut. We conclude that the larval gut supported only a low diversity of metabolically active bacteria.

scholarly journals Stable Isotope Probing Analysis of the Diversity and Activity of Methanotrophic Bacteria in Soils from the Canadian High Arctic

2010 ◽  
Vol 76 (17) ◽  
pp. 5773-5784 ◽  
Author(s):  
Christine Martineau ◽  
Lyle G. Whyte ◽  
Charles W. Greer
Keyword(s):  
Stable Isotope ◽  
16S Rrna ◽  
High Arctic ◽  
Stable Isotope Probing ◽  
Type I ◽  
Methanotrophic Bacteria ◽  
Gene Sequences ◽  
Canadian High Arctic ◽  
Oxidation Rates ◽  
Arctic Soils

ABSTRACT The melting of permafrost and its potential impact on CH4 emissions are major concerns in the context of global warming. Methanotrophic bacteria have the capacity to mitigate CH4 emissions from melting permafrost. Here, we used quantitative PCR (qPCR), stable isotope probing (SIP) of DNA, denaturing gradient gel electrophoresis (DGGE) fingerprinting, and sequencing of the 16S rRNA and pmoA genes to study the activity and diversity of methanotrophic bacteria in active-layer soils from Ellesmere Island in the Canadian high Arctic. Results showed that most of the soils had the capacity to oxidize CH4 at 4°C and at room temperature (RT), but the oxidation rates were greater at RT than at 4°C and were significantly enhanced by nutrient amendment. The DGGE banding patterns associated with active methanotrophic bacterial populations were also different depending on the temperature of incubation and the addition of nutrients. Sequencing of the 16S rRNA and pmoA genes indicated a low diversity of the active methanotrophic bacteria, with all methanotroph 16S rRNA and pmoA gene sequences being related to type I methanotrophs from Methylobacter and Methylosarcina. The dominance of type I methanotrophs over type II methanotrophs in the native soil samples was confirmed by qPCR of the 16S rRNA gene with primers specific for these two groups of bacteria. The 16S rRNA and pmoA gene sequences related to those of Methylobacter tundripaludum were found in all soils, regardless of the incubation conditions, and they might therefore play a role in CH4 degradation in situ. This work is providing new information supporting the potential importance of Methylobacter spp. in Arctic soils found in previous studies and contributes to the limited body of knowledge on methanotrophic activity and diversity in this extreme environment.

Recovering glycoside hydrolase genes from active tundra cellulolytic bacteria

2014 ◽  
Vol 60 (7) ◽  
pp. 469-476 ◽  
Author(s):  
Lee J. Pinnell ◽  
Eric Dunford ◽  
Patrick Ronan ◽  
Martina Hausner ◽  
Josh D. Neufeld
Keyword(s):  
Glycoside Hydrolase ◽  
Multiple Displacement Amplification ◽  
Arctic Tundra ◽  
Stable Isotope Probing ◽  
Glycoside Hydrolases ◽  
16S Rrna Genes ◽  
Bulk Soil ◽  
Metagenomic Analysis ◽  
Rrna Genes ◽  
Cellulolytic Bacteria

Bacteria responsible for cellulose hydrolysis in situ are poorly understood, largely because of the relatively recent development of cultivation-independent methods for their detection and characterization. This study combined DNA stable-isotope probing (DNA-SIP) and metagenomics for identifying active bacterial communities that assimilated carbon from glucose and cellulose in Arctic tundra microcosms. Following DNA-SIP, bacterial fingerprint analysis of gradient fractions confirmed isotopic enrichment. Sequenced fingerprint bands and clone library analysis of 16S rRNA genes identified active bacterial taxa associated with cellulose-associated labelled DNA, including Bacteroidetes (Sphingobacteriales), Betaproteobacteria (Burkholderiales), Alphaproteobacteria (Caulobacteraceae), and Chloroflexi (Anaerolineaceae). We also compared glycoside hydrolase metagenomic profiles from bulk soil and heavy DNA recovered from DNA-SIP incubations. Active populations consuming [13C]glucose and [13C]cellulose were distinct, based on ordinations of light and heavy DNA. Metagenomic analysis demonstrated a ∼3-fold increase in the relative abundance of glycoside hydrolases in DNA-SIP libraries over bulk-soil libraries. The data also indicate that multiple displacement amplification introduced bias into the resulting metagenomic analysis. This research identified DNA-SIP incubation conditions for glucose and cellulose that were suitable for Arctic tundra soil and confirmed that DNA-SIP enrichment can increase target gene frequencies in metagenomic libraries.

scholarly journals Impact of plants on the diversity and activity of methylotrophs in soil

Microbiome ◽  
2020 ◽  
Vol 8 (1) ◽  
Author(s):  
Michael C. Macey ◽  
Jennifer Pratscher ◽  
Andrew T. Crombie ◽  
J. Colin Murrell
Keyword(s):  
Stable Isotope ◽  
Plant Growth ◽  
Molecular Probes ◽  
Stable Isotope Probing ◽  
16S Rrna Genes ◽  
Bulk Soil ◽  
Rrna Genes ◽  
Methylotrophic Bacteria ◽  
Volatile Organic ◽  
Plant Exudates

Abstract Background Methanol is the second most abundant volatile organic compound in the atmosphere, with the majority produced as a metabolic by-product during plant growth. There is a large disparity between the estimated amount of methanol produced by plants and the amount which escapes to the atmosphere. This may be due to utilisation of methanol by plant-associated methanol-consuming bacteria (methylotrophs). The use of molecular probes has previously been effective in characterising the diversity of methylotrophs within the environment. Here, we developed and applied molecular probes in combination with stable isotope probing to identify the diversity, abundance and activity of methylotrophs in bulk and in plant-associated soils. Results Application of probes for methanol dehydrogenase genes (mxaF, xoxF, mdh2) in bulk and plant-associated soils revealed high levels of diversity of methylotrophic bacteria within the bulk soil, including Hyphomicrobium, Methylobacterium and members of the Comamonadaceae. The community of methylotrophic bacteria captured by this sequencing approach changed following plant growth. This shift in methylotrophic diversity was corroborated by identification of the active methylotrophs present in the soils by DNA stable isotope probing using 13C-labelled methanol. Sequencing of the 16S rRNA genes and construction of metagenomes from the 13C-labelled DNA revealed members of the Methylophilaceae as highly abundant and active in all soils examined. There was greater diversity of active members of the Methylophilaceae and Comamonadaceae and of the genus Methylobacterium in plant-associated soils compared to the bulk soil. Incubating growing pea plants in a 13CO2 atmosphere revealed that several genera of methylotrophs, as well as heterotrophic genera within the Actinomycetales, assimilated plant exudates in the pea rhizosphere. Conclusion In this study, we show that plant growth has a major impact on both the diversity and the activity of methanol-utilising methylotrophs in the soil environment, and thus, the study contributes significantly to efforts to balance the terrestrial methanol and carbon cycle.

scholarly journals Stable Isotope Probing with 15N2 Reveals Novel Noncultivated Diazotrophs in Soil

2007 ◽  
Vol 73 (10) ◽  
pp. 3196-3204 ◽  
Author(s):  
Daniel H. Buckley ◽  
Varisa Huangyutitham ◽  
Shi-Fang Hsu ◽  
Tyrrell A. Nelson
Keyword(s):  
Nitrogen Fixation ◽  
Stable Isotope ◽  
Gene Diversity ◽  
Stable Isotope Probing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Free Living ◽  
Natural Systems ◽  
Wide Range ◽  
Microbial Groups

ABSTRACT Biological nitrogen fixation is a fundamental component of the nitrogen cycle and is the dominant natural process through which fixed nitrogen is made available to the biosphere. While the process of nitrogen fixation has been studied extensively with a limited set of cultivated isolates, examinations of nifH gene diversity in natural systems reveal the existence of a wide range of noncultivated diazotrophs. These noncultivated diazotrophs remain uncharacterized, as do their contributions to nitrogen fixation in natural systems. We have employed a novel 15N2-DNA stable isotope probing (5N2-DNA-SIP) method to identify free-living diazotrophs in soil that are responsible for nitrogen fixation in situ. Analyses of 16S rRNA genes from 15N-labeled DNA provide evidence for nitrogen fixation by three microbial groups, one of which belongs to the Rhizobiales while the other two represent deeply divergent lineages of noncultivated bacteria within the Betaproteobacteria and Actinobacteria, respectively. Analysis of nifH genes from 15N-labeled DNA also revealed three microbial groups, one of which was associated with Alphaproteobacteria while the others were associated with two noncultivated groups that are deeply divergent within nifH cluster I. These results reveal that noncultivated free-living diazotrophs can mediate nitrogen fixation in soils and that 15N2-DNA-SIP can be used to gain access to DNA from these organisms. In addition, this research provides the first evidence for nitrogen fixation by Actinobacteria outside of the order Actinomycetales.

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