1,721,155 research outputs found
Stable isotope probing - linking microbial identity to function
No full textStable 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 C-13, 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 C-13-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
Community-level analysis: key genes of aerobic methane oxidation
No full textAerobic methane?oxidizing bacteria (methanotrophs) are a diverse group of bacteria that are currently represented by 13 recognized genera. They play a major role in the global methane cycle and are widespread in nature with representatives found in soils, freshwater, seawater, freshwater and marine sediments, peat bogs and at extremes of temperature, salinity, and pH. There has been an interest in methanotrophs for their potential in bioremediation processes. Methanotroph diversity and ecology are often studied using the “functional” genes pmoA, mmoX, and mxaF, encoding subunits of the particulate methane monooxygenase, soluble methane monooxygenase, and the methanol dehydrogenase, respectively. This chapter describes methods used to detect and analyze these functional gene
Duplication of the mmoX gene in Methylosinus sporium: cloning, sequencing and mutational analysis
No full textThe soluble methane monooxygenase (sMMO) is a key enzyme for methane oxidation, and is found in only some methanotrophs, including Methylosinus sporium 5. sMMO expression is regulated at the level of transcription from a ? 54 promoter by a copper-switch, and is only expressed when the copper-to-biomass ratio during growth is low. Extensive phylogenetic and genetic analyses of sMMOs and other soluble di-iron monooxygenases reveal that these enzymes have only been acquired relatively recently through horizontal gene transfer. In this study, further evidence of horizontal gene transfer was obtained, through cloning and sequencing of the genes encoding the sMMO enzyme complex plus the regulatory genes mmoG and mmoR, and identification of a duplicate copy of the mmoX gene in Ms. sporium. mmoX encodes the ? subunit of the hydroxylase of the sMMO enzyme, which constitutes the active site ( Prior & Dalton, 1985 ). The mmoX genes were characterized at the molecular and biochemical levels. Although both copies were transcribed, only mmoX copy 1 was essential for sMMO activity. Construction of an sMMO? mutant by marker-exchange mutagenesis gave some possible insights into the role of the water-soluble pigment in siderophore-mediated iron acquisition. Finally, the amenability of Ms. sporium to genetic manipulation was demonstrated by complementing the sMMO? mutant by heterologous expression of sMMO genes from Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath), and it was shown that Ms. sporium could be used as an alternative model organism for molecular analysis of MMO regulatio
Methodological considerations for the use of stable isotope probing in microbial ecology
No full textStable isotope probing (SIP) is a method used for labeling uncultivated microorganisms in environmental samples or directly in field studies using substrate enriched with stable isotope (e.g., 13C). After consumption of the substrate, the cells of microorganisms that consumed the substrate become enriched in the isotope. Labeled biomarkers, such as phospholipid-derived fatty acid (PLFA), ribosomal RNA, and DNA can be analyzed with a range of molecular and analytical techniques, and used to identify and characterize the organisms that incorporated the substrate. The advantages and disadvantages of PLFA-SIP, RNA-SIP, and DNA-SIP are presented. Using examples from our laboratory and from the literature, we discuss important methodological considerations for a successful SIP experiment
Enterobacteriaceae facilitate the anaerobic degradation of glucose by a forest soil
No full textAnoxic micro zones that occur in soil aggregates of oxic soils may be temporarily extended after rainfall and thus facilitate the anaerobic degradation of organic compounds in soils. The microbial degradation of glucose by anoxic slurries of a forest soil yielded acetate, CO2, H-2, succinate, and ethanol, products indicative of mixed acid fermentation. Prokaryotes involved in this process were identified by time-resolved 16S rRNA gene-targeted stable isotope probing with [C-13-U]-glucose. All labeled phylotypes from the C-13-enriched 16S rRNA gene were most closely related to Rahnella and Ewingella, enterobacterial genera known to catalyze mixed acid fermentation. These results indicate that facultative aerobes, in particular Enterobacteriaceae, (1) can outcompete obligate anaerobes when conditions become anoxic in forest soils and (2) may be involved in the initial decomposition of monosaccharides in anoxic micro zones of aerated forest soils
Involvement of MmoR and MmoG in the transcriptional activation of soluble methane monooxygenase genes in Methylosinus trichosporium OB3b
No full textMethanotrophs oxidize methane to methanol using the enzyme methane monooxygenase. Methylosinus trichosporium OB3b has two such enzymes: a membrane-bound particulate methane monooxygenase (pMMO) and a soluble, cytoplasmic methane monooxygenase (sMMO). In methanotrophs possessing both enzymes, the expression of the genes encoding sMMO and pMMO is regulated by copper ions, with sMMO expressed solely when copper is limiting. Virtually nothing is known about the specific machinery involved in the copper-regulated transcription of mmo genes except the identification of two proteins necessary for the expression: a Sigma 54-dependent transcriptional activator, MmoR, and a putative GroEL-like chaperone, MmoG. Genes encoding mmoR and mmoG are located immediately upstream of those encoding sMMO in the genome of M. trichosporium OB3b. Here, we use a green fluorescent protein promoter probe vector to show that nearly the complete intergenic DNA sequence between mmoG and mmoX is absolutely required for transcriptional activation. Furthermore, we used gel-shift assays to demonstrate that both MmoR and MmoG were required for protein binding to this region of DNA
Analysis of methanotrophic bacteria in Movile Cave by stable isotope probing
No full textMovile Cave is an unusual groundwater ecosystem that is supported by in situ chemoautotrophic production. The cave atmosphere contains 1-2% methane (CH4), although much higher concentrations are found in gas bubbles that keep microbial mats afloat on the water surface. As previous analyses of stable carbon isotope ratios have suggested that methane oxidation occurs in this environment, we hypothesized that aerobic methane-oxidizing bacteria (methanotrophs) are active in Movile Cave. To identify the active methanotrophs in the water and mat material from Movile Cave, a microcosm was incubated with a 10%(CH4)-C-13 headspace in a DNA-based stable isotope probing (DNA-SIP) experiment. Using improved centrifugation conditions, a C-13-labelled DNA fraction was collected and used as a template for polymerase chain reaction amplification. Analysis of genes encoding the small-subunit rRNA and key enzymes in the methane oxidation pathway of methanotrophs identified that strains of Methylomonas, Methylococcus and Methylocystis/Methylosinus had assimilated the (CH4)-C-13, and that these methanotrophs contain genes encoding both known types of methane monooxygenase (MMO). Sequences of non-methanotrophic bacteria and an alga provided evidence for turnover of CH4 due to possible cross-feeding on C-13-labelled metabolites or biomass. Our results suggest that aerobic methanotrophs actively convert CH4 into complex organic compounds in Movile Cave and thus help to sustain a diverse community of microorganisms in this closed ecosystem
Links between methane oxidation rates and methanotroph community composition in a pine forest soil
No full textThe main gap in our knowledge about what determines the rate of CH4 oxidation in forest soils is the biology of the microorganisms involved, the identity of which remains unclear. In this study, we used stable-isotope probing (SIP) following 13CH4 incorporation into phospholipid fatty acids (PLFAs) and DNA/RNA, and sequencing of methane mono-oxygenase (pmoA) genes, to identify the influence of variation in community composition on CH4 oxidation rates. The rates of 13C incorporation into PLFAs differed between horizons, with low 13C incorporation in the organic soil and relatively high 13C incorporation into the two mineral horizons. The microbial community composition of the methanotrophs incorporating the 13C label also differed between horizons, and statistical analyses suggested that the methanotroph community composition was a major cause of variation in CH4 oxidation rates. Both PLFA and pmoA-based data indicated that CH4 oxidizers in this soil belong to the uncultivated ‘upland soil cluster α’. CH4 oxidation potential exhibited the opposite pattern to 13C incorporation, suggesting that CH4 oxidation potential assays may correlate poorly with in situ oxidation rates. The DNA/RNA-SIP assay was not successful, most likely due to insufficient 13C-incorporation into DNA/RNA. The limitations of the technique are briefly discussed
Identification of active methanotrophs in a landfill cover soil through detection of expression of 16S rRNA and functional genes
No full textActive methanotrophs in a landfill soil were revealed by detecting the 16S rRNA of methanotrophs and the mRNA transcripts of key genes involved in methane oxidation. New 16S rRNA primers targeting type I and type II methanotrophs were designed and optimized for analysis by denaturing gradient gel electrophoresis. Direct extraction of RNA from soil enabled the analysis of the expression of the functional genes: mmoX, pmoA and mxaF, which encode subunits of soluble methane monooxygenase, particulate methane monooxygenase and methanol dehydrogenase respectively. The 16S rRNA polymerase chain reaction (PCR) primers for type I methanotrophs detected Methylomonas, Methylosarcina and Methylobacter sequences from both soil DNA and cDNA which was generated from RNA extracted directly from the landfill cover soil. The 16S rRNA primers for type II methanotrophs detected primarily Methylocella and some Methylocystis 16S rRNA genes. Phylogenetic analysis of mRNA recovered from the soil indicated that Methylobacter, Methylosarcina, Methylomonas, Methylocystis and Methylocella were actively expressing genes involved in methane and methanol oxidation. Transcripts of pmoA but not mmoX were readily detected by reverse transcription polymerase chain reaction (RT-PCR), indicating that particulate methane monooxygenase may be largely responsible for methane oxidation in situ
The impact of burning and Calluna removal on below-ground methanotroph diversity and activity in a peatland soil
No full textMethanotroph community structure and activity was investigated in a peat soil in which the above-ground vegetation was burned repeatedly during the last 50 years, and in soil unburned since 1954. Regular burning (every 10 years) was found to have no obvious impact on the potential methane-uptake capacity; however, a lower abundance of type I methanotrophs relative to type II methanotrophs in the frequently burned soils was observed using pmoA (encoding a key polypeptide of particulate methane monooxygenase) microarray analyses. Denaturing gradient gel electrophoresis of bacterial 16S rRNA genes indicated that the total bacterial community, and not just the methanotrophs, was affected by the burning. The regular burning also resulted in a decreased abundance of Calluna vegetation relative to mixed grasses in the peatland plots. In a separate mesocosm experiment, Calluna plants and their roots were removed from the peat soils for a growing season (from February 2006 to November 2006). It was shown that removal of Calluna from the soil greatly decreased the methane-uptake capacity of the soils, although no obvious impact on the methanotroph population structure was observed. Real-time PCR quantification of pmoA genes showed that the abundance of methanotrophs in barren soil (without Calluna vegetation) was about fivefold less than in the control soil (with Calluna vegetation). These findings indicate that the methanotroph community is strongly influenced by the above-ground vegetation cover. Burning of Calluna seemed to favour the type II metbanotrophs, whereas the removal of the Calluna cover did not appear to affect the relative abundance of methanotroph genera but caused a uniform decrease in the size of methanotroph populations. (C) 2008 Elsevier B.V. All rights reserved
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