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.

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 Identification of Nitrogen-Incorporating Bacteria in Petroleum-Contaminated Arctic Soils by Using [15N]DNA-Based Stable Isotope Probing and Pyrosequencing

2011 ◽  
Vol 77 (12) ◽  
pp. 4163-4171 ◽  
Author(s):  
Terrence H. Bell ◽  
Etienne Yergeau ◽  
Christine Martineau ◽  
David Juck ◽  
Lyle G. Whyte ◽  
...  
Keyword(s):  
Stable Isotope ◽  
16S Rrna ◽  
Contaminated Soils ◽  
Sequence Data ◽  
Stable Isotope Probing ◽  
Hydrocarbon Contamination ◽  
The Arctic ◽  
Phylogenetic Groups ◽  
Content Type ◽  
Arctic Soils

ABSTRACTArctic soils are increasingly susceptible to petroleum hydrocarbon contamination, as exploration and exploitation of the Arctic increase. Bioremediation in these soils is challenging due to logistical constraints and because soil temperatures only rise above 0°C for ∼2 months each year. Nitrogen is often added to contaminated soilin situto stimulate the existing microbial community, but little is known about how the added nutrients are used by these microorganisms. Microbes vary widely in their ability to metabolize petroleum hydrocarbons, so the question becomes: which hydrocarbon-degrading microorganisms most effectively use this added nitrogen for growth? Using [15N]DNA-based stable isotope probing, we determined which taxonomic groups most readily incorporated nitrogen from the monoammonium phosphate added to contaminated and uncontaminated soil in Canadian Forces Station-Alert, Nunavut, Canada. Fractions from each sample were amplified with bacterial 16S rRNA and alkane monooxygenase B (alkB) gene-specific primers and then sequenced using lage-scale parallel-pyrosequencing. Sequence data was combined with 16S rRNA andalkBgene C quantitative PCR data to measure the presence of various phylogenetic groups in fractions at different buoyant densities. Several families ofProteobacteriaandActinobacteriathat are directly involved in petroleum degradation incorporated the added nitrogen in contaminated soils, but it was the DNA ofSphingomonadaceaethat was most enriched in15N. Bacterial growth in uncontaminated soils was not stimulated by nutrient amendment. Our results suggest that nitrogen uptake efficiency differs between bacterial groups in contaminated soils. A better understanding of how groups of hydrocarbon-degraders contribute to the catabolism of petroleum will facilitate the design of more targeted bioremediation treatments.

scholarly journals Metagenomic Analysis of the Bioremediation of Diesel-Contaminated Canadian High Arctic Soils

PLoS ONE ◽  
2012 ◽  
Vol 7 (1) ◽  
pp. e30058 ◽  
Author(s):  
Etienne Yergeau ◽  
Sylvie Sanschagrin ◽  
Danielle Beaumier ◽  
Charles W. Greer
Keyword(s):  
High Arctic ◽  
Metagenomic Analysis ◽  
Canadian High Arctic ◽  
Arctic Soils

scholarly journals Family- and Genus-Level 16S rRNA-Targeted Oligonucleotide Probes for Ecological Studies of Methanotrophic Bacteria

2001 ◽  
Vol 67 (10) ◽  
pp. 4726-4733 ◽  
Author(s):  
Jay Gulledge ◽  
Azeem Ahmad ◽  
Paul A. Steudler ◽  
William J. Pomerantz ◽  
Colleen M. Cavanaugh
Keyword(s):  
16S Rrna ◽  
Environmental Samples ◽  
Quantitative Information ◽  
Negative Control ◽  
Type I ◽  
Methanotrophic Bacteria ◽  
Oligonucleotide Probes ◽  
Ecological Studies ◽  
Dot Blot ◽  
The Family

ABSTRACT Methanotrophic bacteria play a major role in the global carbon cycle, degrade xenobiotic pollutants, and have the potential for a variety of biotechnological applications. To facilitate ecological studies of these important organisms, we developed a suite of oligonucleotide probes for quantitative analysis of methanotroph-specific 16S rRNA from environmental samples. Two probes target methanotrophs in the family Methylocystaceae(type II methanotrophs) as a group. No oligonucleotide signatures that distinguish between the two genera in this family,Methylocystis and Methylosinus, were identified. Two other probes target, as a single group, a majority of the known methanotrophs belonging to the familyMethylococcaceae (type I/X methanotrophs). The remaining probes target members of individual genera of theMethylococcaceae, includingMethylobacter, Methylomonas,Methylomicrobium, Methylococcus, andMethylocaldum. One of the family-level probes also covers all methanotrophic endosymbionts of marine mollusks for which 16S rRNA sequences have been published. The two known species of the newly described genus Methylosarcina gen. nov. are covered by a probe that otherwise targets only members of the closely related genus Methylomicrobium. None of the probes covers strains of the newly proposed generaMethylocella and “Methylothermus,” which are polyphyletic with respect to the recognized methanotrophic families. Empirically determined midpoint dissociation temperatures were 49 to 57°C for all probes. In dot blot screening against RNA from positive- and negative-control strains, the probes were specific to their intended targets. The broad coverage and high degree of specificity of this new suite of probes will provide more detailed, quantitative information about the community structure of methanotrophs in environmental samples than was previously available.

Methylohalobius crimeensis gen. nov., sp. nov., a moderately halophilic, methanotrophic bacterium isolated from hypersaline lakes of Crimea

2005 ◽  
Vol 55 (5) ◽  
pp. 1817-1826 ◽  
Author(s):  
Jürgen Heyer ◽  
Ursula Berger ◽  
Martin Hardt ◽  
Peter F. Dunfield
Keyword(s):  
Fatty Acids ◽  
16S Rrna ◽  
Gene Sequence ◽  
Rrna Gene ◽  
Type I ◽  
Methanotrophic Bacteria ◽  
Rrna Gene Sequence ◽  
Methanotrophic Bacterium ◽  
Hypersaline Lakes ◽  
Novel Genus And Species

A novel genus and species are proposed for two strains of methanotrophic bacteria isolated from hypersaline lakes in the Crimean Peninsula of Ukraine. Strains 10KiT and 4Kr are moderate halophiles that grow optimally at 1–1·5 M (5·8–8·7 %, w/v) NaCl and tolerate NaCl concentrations from 0·2 M up to 2·5 M (1·2–15 %). This optimum and upper limit are the highest for any methanotrophic bacterium known to date. The strains are Gram-negative, aerobic, non-pigmented, motile, coccoid to spindle-shaped bacteria that grow on methane or methanol only and utilize the ribulose monophosphate pathway for carbon assimilation. They are neutrophilic (growth occurs only in the range pH 6·5–7·5) and mesophilic (optimum growth occurs at 30 °C). On the basis of 16S rRNA gene sequence phylogeny, strains 10KiT and 4Kr represent a type I methanotroph within the ‘Gammaproteobacteria’. However, the 16S rRNA gene sequence displays <91·5 % identity to any public-domain sequence. The most closely related methanotrophic bacterium is the thermophilic strain HB. The DNA G+C content is 58·7 mol%. The major phospholipid fatty acids are 18 : 1ω7 (52–61 %), 16 : 0 (22–23 %) and 16 : 1ω7 (14–20 %). The dominance of 18 : 1 over 16 : 0 and 16 : 1 fatty acids is unique among known type I methanotrophs. The data suggest that strains 10KiT and 4Kr should be considered as belonging to a novel genus and species of type I methanotrophic bacteria, for which the name Methylohalobius crimeensis gen. nov., sp. nov. is proposed. Strain 10KiT (=DSM 16011T=ATCC BAA-967T) is the type strain.

scholarly journals Nutrient Amendments in Soil DNA Stable Isotope Probing Experiments Reduce the Observed Methanotroph Diversity

2006 ◽  
Vol 73 (3) ◽  
pp. 798-807 ◽  
Author(s):  
Aur�lie C�bron ◽  
Levente Bodrossy ◽  
Nancy Stralis-Pavese ◽  
Andrew C. Singer ◽  
Ian P. Thompson ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Stable Isotope Probing ◽  
Population Diversity ◽  
Type I ◽  
Mineral Salts ◽  
Bacterial Populations ◽  
Soil Dna ◽  
Gradient Gel Electrophoresis ◽  
Soil Water Conditions

ABSTRACT Stable isotope probing (SIP) can be used to analyze the active bacterial populations involved in a process by incorporating 13C-labeled substrate into cellular components such as DNA. Relatively long incubation times are often used with laboratory microcosms in order to incorporate sufficient 13C into the DNA of the target organisms. Addition of nutrients can be used to accelerate the processes. However, unnatural concentrations of nutrients may artificially change bacterial diversity and activity. In this study, methanotroph activity and diversity in soil was examined during the consumption of 13CH4 with three DNA-SIP experiments, using microcosms with natural field soil water conditions, the addition of water, and the addition of mineral salts solution. Methanotroph population diversity was studied by targeting 16S rRNA and pmoA genes. Clone library analyses, denaturing gradient gel electrophoresis fingerprinting, and pmoA microarray hybridization analyses were carried out. Most methanotroph diversity (type I and type II methanotrophs) was observed in nonamended SIP microcosms. Although this treatment probably best reflected the in situ environmental conditions, one major disadvantage of this incubation was that the incorporation of 13CH4 was slow and some cross-feeding of 13C occurred, thereby leading to labeling of nonmethanotroph microorganisms. Conversely, microcosms supplemented with mineral salts medium exhibited rapid consumption of 13CH4, resulting in the labeling of a less diverse population of only type I methanotrophs. DNA-SIP incubations using water-amended microcosms yielded faster incorporation of 13C into active methanotrophs while avoiding the cross-feeding of 13C.

scholarly journals Novel Biphenyl-Oxidizing Bacteria and Dioxygenase Genes from a Korean Tidal Mudflat

2011 ◽  
Vol 77 (11) ◽  
pp. 3888-3891 ◽  
Author(s):  
Tae Kwon Lee ◽  
Jaejin Lee ◽  
Woo Jun Sul ◽  
Shoko Iwai ◽  
Benli Chai ◽  
...  
Keyword(s):  
Amino Acid ◽  
Stable Isotope ◽  
16S Rrna ◽  
Amino Acid Identity ◽  
Stable Isotope Probing ◽  
Content Type ◽  
Acid Identity ◽  
Rrna Sequences ◽  
Mudflat Sediments ◽  
16S Rrna Sequences

ABSTRACTGene-targeted FLX titanium pyrosequencing integrated with stable isotope probing (SIP) using [13C]biphenyl substrate revealed that tidal mudflat sediments harbor novel aromatic ring hydroxylating dioxygenases (ARHD). More than 80% of the detected ARHD genes comprise four clades (0.5 distance) with 49 to 70% amino acid identity to sequences in public databases. The 16S rRNA sequences enriched in the13C fraction were from theBetaproteobacteria, bacilli (primarilyPaenibacillus-like), and unclassified phyla.

scholarly journals Methanotrophic bacteria in warm geothermal spring sediments identified using stable-isotope probing

2014 ◽  
Vol 90 (1) ◽  
pp. 92-102 ◽  
Author(s):  
Christine E. Sharp ◽  
Azucena Martínez-Lorenzo ◽  
Allyson L. Brady ◽  
Stephen E. Grasby ◽  
Peter F. Dunfield
Keyword(s):  
Stable Isotope ◽  
Stable Isotope Probing ◽  
Methanotrophic Bacteria ◽  
Geothermal Spring

Analysis of methanotrophic bacteria in Movile Cave by stable isotope probing

2003 ◽  
Vol 6 (2) ◽  
pp. 111-120 ◽  
Author(s):  
Elena Hutchens ◽  
Stefan Radajewski ◽  
Marc G. Dumont ◽  
Ian R. McDonald ◽  
J. Colin Murrell
Keyword(s):  
Stable Isotope ◽  
Stable Isotope Probing ◽  
Methanotrophic Bacteria ◽  
Movile Cave

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.

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