Identification of cellulolytic bacteria in soil by stable isotope probing

Plant residues, mainly made up of cellulose, are the largest fraction of organic carbon material in terrestrial ecosystems. Soil microorganisms are mainly responsible for the transfer of this carbon to the atmosphere, but their contribution is not accurately known. The aim of the present study was to identify bacterial populations that are actively involved in cellulose degradation, using the DNA-stable isotope probing (DNA-SIP) technique. ¹³C-cellulose was produced by Acetobacter xylinus and incubated in soil for 7, 14, 30 and 90 days. Total DNA was extracted from the soil, the ¹³C-labelled (heavy) and unlabelled (light) DNA fractions were separated by ultracentrifugation, and the structure of active bacterial communities was analysed by bacterial-automated ribosomal intergenic spacer analysis (B-ARISA) and characterized with denaturing gradient gel electrophoresis (DGGE). Cellulose degradation was associated with significant changes in bacterial community structure issued from heavy DNA, leading to the appearance of new bands and increase in relative intensities of other bands until day 30. The majority of bands decreased in relative intensity at day 90. Sequencing and phylogenetic analysis of 10 of these bands in DGGE profiles indicated that most sequences were closely related to sequences from organisms known for their ability to degrade cellulose or to uncultured soil bacteria.     

Protein-based stable isotope probing

We describe a stable isotope probing (SIP) technique that was developed to link microbe-specific metabolic function to phylogenetic information. Carbon ((13)C)- or nitrogen ((15)N)-labeled substrates (typically with >98% heavy label) were used in cultivation experiments and the heavy isotope incorporation into proteins (protein-SIP) on growth was determined. The amount of incorporation provides a measure for assimilation of a substrate, and the sequence information from peptide analysis obtained by mass spectrometry delivers phylogenetic information about the microorganisms responsible for the metabolism of the particular substrate. In this article, we provide guidelines for incubating microbial cultures with labeled substrates and a protocol for protein-SIP. The protocol guides readers through the proteomics pipeline, including protein extraction, gel-free and gel-based protein separation, the subsequent mass spectrometric analysis of peptides and the calculation of the incorporation of stable isotopes into peptides. Extraction of proteins and the mass fingerprint measurements of unlabeled and labeled fractions can be performed in 2-3 d.     

Validation of heavy-water stable isotope probing for the characterization of rapidly responding soil bacteria

Rapid 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 (H2(18)O-SIP) to identify bacteria that respond to soil rewetting. First, we conducted experiments to address uncertainties regarding the H2(18)O-SIP method. Using liquid chromatography-mass spectroscopy (LC-MS), we determined that oxygen from H2(18)O was incorporated into all structural components of DNA. Although this incorporation was uneven, we could effectively separate 18O-labeled and unlabeled DNAs derived from laboratory cultures and environmental samples that were incubated with H2(18)O. We found no evidence for ex vivo exchange of oxygen atoms between DNA and extracellular H2O, suggesting that 18O incorporation into DNA is relatively stable. Furthermore, the rate of 18O incorporation into bacterial DNA was high (within 48 to 72 h), coinciding with pulses of CO2 generated from soil rewetting. Second, we examined shifts in the bacterial composition of grassland soils following rewetting, using H2(18)O-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 of Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria increased, while the relative recovery of Chloroflexi and Deltaproteobacteria decreased. Together, our results suggest that H2(18)O-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.     

Identification of nitrogen-incorporating bacteria in petroleum-contaminated arctic soils by using [15N]DNA-based stable isotope probing and pyrosequencing

Arctic 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 soil in situ to 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 and alkB gene C quantitative PCR data to measure the presence of various phylogenetic groups in fractions at different buoyant densities. Several families of Proteobacteria and Actinobacteria that are directly involved in petroleum degradation incorporated the added nitrogen in contaminated soils, but it was the DNA of Sphingomonadaceae that was most enriched in ¹⁵N. 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.     

Detection of active butyrate-degrading microorganisms in methanogenic sludges by RNA-based stable isotope probing

Butyrate-degrading bacteria in four methanogenic sludges were studied by RNA-based stable isotope probing. Bacterial populations in the (13)C-labeled rRNA fractions were distinct from unlabeled fractions, and Syntrophaceae species, Tepidanaerobacter sp., and Clostridium spp. dominated. These results suggest that diverse microbes were active in butyrate degradation under methanogenic conditions.     

Assimilatory nitrate utilization by bacteria on the West Florida Shelf as determined by stable isotope probing and functional microarray analysis

Dissolved inorganic nitrogen (DIN) uptake by marine heterotrophic bacteria has important implications for the global nitrogen (N) and carbon (C) cycles. Bacterial nitrate utilization is more prevalent in the marine environment than traditionally thought, but the taxonomic identity of bacteria that utilize nitrate is difficult to determine using traditional methodologies. (15) N-based DNA stable isotope probing was applied to document direct use of nitrate by heterotrophic bacteria on the West Florida Shelf. Seawater was incubated in the presence of 2 μM (15) N ammonium or (15) N nitrate. DNA was extracted, fractionated via CsCl ultracentrifugation, and each fraction was analyzed by terminal restriction fragment length polymorphism (TRFLP) analysis. TRFs that exhibited density shifts when compared to controls that had not received (15) N amendments were identified by comparison with 16S rRNA gene sequence libraries. Relevant marine proteobacterial lineages, notably Thalassobacter and Alteromonadales, displayed evidence of (15) N incorporation. RT-PCR and functional gene microarray analysis could not demonstrate the expression of the assimilatory nitrate reductase gene, nasA, but mRNA for dissimilatory pathways, i.e. nirS, nirK, narG, nosZ, napA, and nrfA was detected. These data directly implicate several bacterial populations in nitrate uptake, but suggest a more complex pattern for N flow than traditionally implied.     

Novel aerobic benzene degrading microorganisms identified in three soils by stable isotope probing

The remediation of benzene contaminated groundwater often involves biodegradation and although the mechanisms of aerobic benzene biodegradation in laboratory cultures have been well studied, less is known about the microorganisms responsible for benzene degradation in mixed culture samples or at contaminated sites. To address this knowledge gap, DNA based stable isotope probing (SIP) was utilized to identify active benzene degraders in microcosms constructed with soil from three sources (a contaminated site and two agricultural sites). For this, replicate microcosms were amended with either labeled (¹³C) or unlabeled benzene and the extracted DNA samples were ultracentrifuged, fractioned and subject to terminal restriction fragment length polymorphism (TRFLP). The dominant benzene degraders (responsible for ¹³C uptake) were determined by comparing relative abundance of TRFLP phylotypes in heavy fractions of labeled benzene (¹³C) amended samples to the controls (from unlabeled benzene amended samples). Two phylotypes (a Polaromonas sp. and an Acidobacterium) were the major benzene degraders in the microcosms constructed from the contaminated site soil, whereas one phylotype incorporated the majority of the benzene-derived ¹³C in each of the agricultural soils (“candidate” phylum TM7 and an unclassified Sphingomonadaceae).     

Detection of monochlorobenzene metabolizing bacteria under anoxic conditions by DNA-stable isotope probing

Cultivation-independent analyses were applied to study the structural diversity of the bacterial community which developed in groundwater inoculated microcosms actively metabolizing monochlorobenzene (MCB) under anaerobic conditions. Addition of ¹³C-labelled MCB demonstrated that the community produced ¹³CO₂ as a metabolite at slightly increasing rates over a period of 1,051 days while no ¹³C-methane evolved. Genetic profiles of partial 16S rRNA genes generated with the single-strand conformation polymorphism (SSCP) technique by PCR from directly extracted total DNA revealed that, despite the long incubation period, six replicate microcosms were characterized by almost the same microbial members. Nine distinguishable contributors to the SSCP-profiles were characterized by DNA sequencing, revealing the presence of different members from the phyla Proteobacteria, Fibrobacteres and from the candidate division OD1. DNA-stable isotope probing (SIP) was applied to distinguish the actual MCB metabolizing bacteria from the other community members. This study reveals for the first time the structural diversity of an anaerobic MCB metabolizing bacterial community. However, it also demonstrates the limitations of SIP to detect bacteria slowly metabolizing carbon sources under anaerobic conditions.     

Characterization of growing microorganisms in soil by stable isotope probing with H218O

A new approach to characterize growing microorganisms in environmental samples based on labeling microbial DNA with H(2)(18)O is described. To test if sufficient amounts of (18)O could be incorporated into DNA to use water as a labeling substrate for stable isotope probing, Escherichia coli DNA was labeled by cultivating bacteria in Luria broth with H(2)(18)O and labeled DNA was separated from [(16)O]DNA on a cesium chloride gradient. Soil samples were incubated with H(2)(18)O for 6, 14, or 21 days, and isopycnic centrifugation of the soil DNA showed the formation of two bands after 6 days and three bands after 14 or 21 days, indicating that (18)O can be used in the stable isotope probing of soil samples. DNA extracted from soil incubated for 21 days with H(2)(18)O was fractionated after isopycnic centrifugation and DNA from 17 subsamples was used in terminal restriction fragment length polymorphism (TRFLP) analysis of bacterial 16S rRNA genes. The TRFLP patterns clustered into three groups that corresponded to the three DNA bands. The fraction of total fluorescence contributed by individual terminal restriction fragments (TRF) to a TRFLP pattern varied across the 17 subsamples so that a TRF was more prominent in only one of the three bands. Labeling soil DNA with H(2)(18)O allows the identification of newly grown cells. In addition, cells that survive but do not divide during an incubation period can also be characterized with this new technique because their DNA remains without the label.     

Linking phylogenetic identities of bacteria to starch fermentation in an in vitro model of the large intestine by RNA-based stable isotope probing

Carbohydrates, including starches, are an important energy source for humans, and are known for their interactions with the microbiota in the digestive tract. Largely, those interactions are thought to promote human health. Using 16S ribosomal RNA (rRNA)-based stable isotope probing (SIP), we identified starch-fermenting bacteria under human colon-like conditions. To the microbiota of the TIM-2 in vitro model of the human colon 7.4 g l⁻¹ of [U-¹³C]-starch was added. RNA extracted from lumen samples after 0 (control), 2, 4 and 8 h was subjected to density-gradient ultracentrifugation. Terminal-restriction fragment length polymorphism (T-RFLP) fingerprinting and phylogenetic analyses of the labelled and unlabelled 16S rRNA suggested populations related to Ruminococcus bromii, Prevotella spp. and Eubacterium rectale to be involved in starch metabolism. Additionally, 16S rRNA related to that of Bifidobacterium adolescentis was abundant in all analysed fractions. While this might be due to the enrichment of high-GC RNA in high-density fractions, it could also indicate an active role in starch fermentation. Comparison of the T-RFLP fingerprints of experiments performed with labelled and unlabelled starch revealed Ruminococcus bromii as the primary degrader in starch fermentation in the studied model, as it was found to solely predominate in the labelled fractions. LC-MS analyses of the lumen and dialysate samples showed that, for both experiments, starch fermentation primarily yielded acetate, butyrate and propionate. Integration of molecular and metabolite data suggests metabolic cross-feeding in the system, where populations related to Ruminococcus bromii are the primary starch degrader, while those related to Prevotella spp., Bifidobacterium adolescentis and Eubacterium rectale might be further involved in the trophic chain.     

Tracking activity and function of microorganisms by stable isotope probing of membrane lipids

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Identification of novel perchloroethene-respiring microorganisms in anoxic river sediment by RNA-based stable isotope probing

The halogenated compound tetrachloroethene (perchloroethene, PCE) is a persistent contaminant of aquifers, soils and sediments. Although a number of microorganisms are known to reductively dechlorinate PCE by dehalorespiration, their diversity and community structure especially in pristine environments remain elusive. In this study, we report on the detection of a novel group of dehalorespiring bacteria that reductively dechlorinate PCE to cis -dichloroethene by RNA-based stable isotope probing. Pristine river sediment was incubated at 15°C with PCE at low aqueous concentration. Upon formation of dechlorination products, the microbial community was probed with ¹³C-labelled acetate as electron donor and carbon source. Terminal restriction fragment length polymorphism (T-RFLP) analysis of density-separated 16S rRNA revealed a predominantly ¹³C-labelled bacterial population only in the microcosm with PCE in high-density gradient fractions, whereas in the control without PCE Bacteria-specific rRNA was restricted to light gradient fractions. By cloning and sequence analysis of 16S rRNA, the predominant population was identified as a novel group of bacteria within the phylum Chloroflexi . These microorganisms, designated Lahn Cluster (LC), were only distantly related to cultivated dehalorespiring Dehalococcoides spp. (92–94% sequence identity). Minor clone groups detected ¹³C-labelled and thus, potentially involved in PCE dehalorespiration, were related to β-proteobacterial Dechloromonas spp., and δ- Proteobacteria ( Geobacteraceae , Desulfobacteraceae , Desulfobulbaceae ). In contrast, clones from an ethene-producing microcosm incubated at 20°C grouped with known Dehalococcoides spp. Our results show that stable isotope probing allows targeting dehalorespiring bacteria as functional guild, and to identify novel PCE-respiring populations previously not recognized.     

Identification of critical members in a sulfidogenic benzene-degrading consortium by DNA stable isotope probing

Stable isotope probing (SIP) was used to identify the active members in a benzene-degrading sulfidogenic consortium. SIP-terminal restriction fragment length polymorphism analysis indicated that a 270-bp peak incorporated the majority of the (13)C label and is a sequence closely related to that of clone SB-21 (GenBank accession no. AF029045). This target may be an important biomarker for anaerobic benzene degradation in the field.     

Identification of anthracene degraders in leachate-contaminated aquifer using stable isotope probing

Polycyclic aromatic hydrocarbons (PAHs) are common contaminants in landfill leachate-contaminated aquifer. It is necessary to identify the microorganisms truly responsible for PAH degradation if bioremediation can be applied as an effective technology. DNA-based stable isotope probing (SIP) in combination with terminal restriction fragment length polymorphism (TRFLP) was used to identify the active anthracene degraders in the contaminated aquifer sediment. One kind of degrader was classified as Variovorax species within class ??-proteobacteria, but another belonged to unclassified bacteria. These findings also suggest novel microorganisms involved in PAH-degrading processes.     

Activity and composition of methanotrophic bacterial communities in planted rice soil studied by flux measurements, analyses of pmoA gene and stable isotope probing of phospholipid fatty acids

Methanotrophs in the rhizosphere of rice field ecosystems attenuate the emissions of CH₄ into the atmosphere and thus play an important role for the global cycle of this greenhouse gas. Therefore, we measured the activity and composition of the methanotrophic community in the rhizosphere of rice microcosms. Methane oxidation was determined by measuring the CH₄ flux in the presence and absence of difluoromethane as a specific inhibitor for methane oxidation. Methane oxidation started on day 24 and reached the maximum on day 32 after transplantation. The total methanotrophic community was analysed by terminal restriction fragment length polymorphism (T-RFLP) and cloning/sequencing of the pmoA gene, which encodes a subunit of particulate methane monooxygenase. The metabolically active methanotrophic community was analysed by stable isotope probing of microbial phospholipid fatty acids (PLFA-SIP) using ¹³C-labelled CH₄ directly added to the rhizospheric region. Rhizospheric soil and root samples were collected after exposure to ¹³CH₄ for 8 and 18 days. Both T-RFLP/cloning and PLFA-SIP approaches showed that type I and type II methanotrophic populations changed over time with respect to activity and population size in the rhizospheric soil and on the rice roots. However, type I methanotrophs were more active than type II methanotrophs at both time points indicating they were of particular importance in the rhizosphere. PLFA-SIP showed that the active methanotrophic populations exhibit a pronounced spatial and temporal variation in rice microcosms.     

DNA buoyant density shifts during 15N-DNA stable isotope probing

DNA-based stable isotope probing (SIP) is a novel technique for the identification of organisms actively assimilating isotopically labeled compounds. Herein, we define the limitations to using ¹⁵N-labeled substrates for SIP and propose modifications to compensate for these shortcomings. Changes in DNA buoyant density (BD) resulting from ¹⁵N incorporation were determined using cultures of disparate GC content (Escherichia coli and Micrococcus luteus). Incorporation of ¹⁵N into DNA increased BD by 0.015±0.002 g mL⁻¹ for E. coli and 0.013±0.002 g mL⁻¹ for M. luteus. The DNA BD shift was greatly increased (0.045 g mL⁻¹) when dual isotope (¹³C plus ¹⁵N) labeling was employed. Despite the limited DNA BD shift following ¹⁵N enrichment, we found the use of gradient fractionation, followed by a comparison of T-RFLP profiles from fractions of labeled and control treatments, facilitated detection of enrichment in DNA samples from either cultures or soil.     

Bacteriophages of methanotrophic bacteria

Bacteriophages of methanotrophic bacteria have been found in 16 out of 88 studied samples (underground waters, pond water, soil, gas and oil installation waters, fermentor cultural fluids, bacterial paste, and rumen of cattle) taken in different geographic zones of the Soviet Union. Altogether, 23 phage strains were isolated: 10 strains that specifically lysed only Methylosinus sporium strains, 2 strains that each lysed 1 of 5 Methylosinus trichosporium strains studied, and 11 strains that lysed Flavobacterium gasotypicum and, at the same time, 1 M. sporium strain. By fine structure, the phages were divided into two types (with very short or long noncontractile tails); by host range and serological properties, they fell into three types. One-step growth characteristics of the phages differed only slightly; the latent period varied from 6 to 8 h, the rise period varied from 4 to 6 h, and the average burst size was 100. All phages had guanine- and cytosine-rich double-stranded deoxyribonucleic acid consisting of common nitrogen bases. The molecular mass of the deoxyribonucleic acid as determined by restriction endonuclease analysis was 29.4 X 10(6) for M. sporium phages and 44 X 10(6) for F. gasotypicum phages. By all of the above-mentioned properties, all phages within each of the groups were completely identical to one another, but differed from phages of other groups. Bacteriophages lysing M. sporium and M. trichosporium GB2 were identical to phages M1 and M4, respectively, which were isolated earlier in the German Democratic Republic on the same methanotrophic species.     

Anaerobic methyl tert-butyl ether-degrading microorganisms identified in wastewater treatment plant samples by stable isotope probing

Anaerobic methyl tert-butyl ether (MTBE) degradation potential was investigated in samples from a range of sources. From these 22 experimental variations, only one source (from wastewater treatment plant samples) exhibited MTBE degradation. These microcosms were methanogenic and were subjected to DNA-based stable isotope probing (SIP) targeted to both bacteria and archaea to identify the putative MTBE degraders. For this purpose, DNA was extracted at two time points, subjected to ultracentrifugation, fractioning, and terminal restriction fragment length polymorphism (TRFLP). In addition, bacterial and archaeal 16S rRNA gene clone libraries were constructed. The SIP experiments indicated bacteria in the phyla Firmicutes (family Ruminococcaceae) and Alphaproteobacteria (genus Sphingopyxis) were the dominant MTBE degraders. Previous studies have suggested a role for Firmicutes in anaerobic MTBE degradation; however, the putative MTBE-degrading microorganism in the current study is a novel MTBE-degrading phylotype within this phylum. Two archaeal phylotypes (genera Methanosarcina and Methanocorpusculum) were also enriched in the heavy fractions, and these organisms may be responsible for minor amounts of MTBE degradation or for the uptake of metabolites released from the primary MTBE degraders. Currently, limited information exists on the microorganisms able to degrade MTBE under anaerobic conditions. This work represents the first application of DNA-based SIP to identify anaerobic MTBE-degrading microorganisms in laboratory microcosms and therefore provides a valuable set of data to definitively link identity with anaerobic MTBE degradation.