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.     

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.     

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.     

Studying plant-microbe interactions using stable isotope technologies

Interactions between plants and microorganisms in the rhizosphere are complex and varied. They include the general transfer of nutrients and specific interactions mediated by the release of signalling molecules from plant roots. Until recently, understanding the nature of these interactions was limited by a reliance on traditional, cultivation-based techniques. Stable isotope probing provides the potential for cultivation-independent characterisation of organisms actively assimilating carbon derived from plant root exudate or added to the soil. Current applications have focused on interactions with relatively low-level specificity, but there is significant potential for mechanistic studies of more specific interactions, particularly if the sensitivity of the technique can be increased.     

Monitoring of microbial proteome dynamics using Raman stable isotope probing

Abnormal protein kinetics could be a cause of several diseases associated with essential life processes. An accurate understanding of protein dynamics and turnover is essential for developing diagnostic or therapeutic tools to monitor these changes. Raman spectroscopy in combination with stable isotope probes (SIP) such as carbon-13, and deuterium has been a breakthrough in the qualitative and quantitative study of various metabolites. In this work, we are reporting the utility of Raman-SIP for monitoring dynamic changes in the proteome at the community level. We have used ¹³C-labeled glucose as the only carbon source in the medium and verified its incorporation in the microbial biomass in a time-dependent manner. A visible redshift in the Raman spectral vibrations of major biomolecules such as nucleic acids, phenylalanine, tyrosine, amide I, and amide III were observed. Temporal changes in the intensity of these bands demonstrating the feasibility of protein turnover monitoring were also verified. Kanamycin, a protein synthesis inhibitor was used to assess the feasibility of identifying effects on protein turnover in the cells. Successful application of this work can provide an alternate/adjunct tool for monitoring proteome-level changes in an objective and nondestructive manner.     

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.     

Detecting active methanogenic populations on rice roots using stable isotope probing

Methane is formed on rice roots mainly by CO2 reduction. The present study aimed to identify the active methanogenic populations responsible for this process. Soil-free rice roots were incubated anaerobically under an atmosphere of H2/(13CO2) or N2/(13CO2) with phosphate or carbonate (marble) as buffer medium. Nucleic acids were extracted and fractionated by caesium trifluoroacetate equilibrium density gradient centrifugation after 16-day incubation. Community analyses were performed for gradient fractions using terminal restriction fragment polymorphism analysis (T-RFLP) and sequencing of the 16S rRNA genes. In addition, rRNA was extracted and analysed at different time points to trace the community change during the 16-day incubation. The Methanosarcinaceae and the yet-uncultured archaeal lineage Rice Cluster-I (RC-I) were predominant in the root incubations when carbonate buffer and N2 headspace were used. The analysis of [13C]DNA showed that the relative 16S rRNA gene abundance of RC-I increased whereas that of the Methanosarcinaceae decreased with increasing DNA buoyant density, indicating that members of RC-I were more active than the Methanosarcinaceae. However, an unexpected finding was that RC-I was suppressed in the presence of high H2 concentrations (80%, v/v), which during the early incubation period caused a lower CH4 production compared with that with N2 in the headspace. Eventually, however, CH4 production increased, probably because of the activity of Methanosarcinaceae, which became prevalent. Phosphate buffer appeared to inhibit the activity of the Methanosarcinaceae, resulting in lower CH4 production as compared with carbonate buffer. Under these conditions, Methanobacteriaceae were the prevalent methanogens. Our study suggests that the active methanogenic populations on rice roots change in correspondence to the presence of H2 (80%, v/v) and the type of buffer used in the system.     

Selective stimulation of type I methanotrophs in a rice paddy soil by urea fertilization revealed by RNA-based stable isotope probing

Methane-oxidizing bacteria (MOB) in soil are not only controlled by their main substrates, methane and oxygen, but also by nitrogen availability. We compared an unfertilized control with a urea-fertilized treatment and applied RNA-stable-isotope-probing to follow activity changes upon fertilization as closely as possible. Nitrogen fertilization of an Italian rice field soil increased the CH4 oxidation rates sevenfold. In the fertilized treatment, isopycnic separation of 13C-enriched RNA became possible after 7 days when 300 micromol 13CH4 g(dry soil)(-1) had been consumed. Terminal-restriction fragment length polymorphism (T-RFLP) fingerprints and clone libraries documented that the type I methanotrophic genera Methylomicrobium and Methylocaldum assimilated 13CH4 nearly exclusively. Although previous studies had shown that the same soil contains a much larger diversity of MOB, including both type I and type II, nitrogen fertilization apparently activated only a small subset of the overall diversity of MOB, type I MOB in particular.     

Diversity of anaerobic microorganisms involved in long-chain fatty acid degradation in methanogenic sludges as revealed by RNA-based stable isotope probing

Long-chain fatty acid (LCFA) degradation is a key step in methanogenic treatment of wastes/wastewaters containing high concentrations of lipids. However, despite the importance of LCFA-degrading bacteria, their natural diversity is little explored due to the limited availability of isolate information and the lack of appropriate molecular markers. We therefore investigated these microbes by using RNA-based stable isotope probing. We incubated four methanogenic sludges (mesophilic sludges MP and MBF and thermophilic sludges TP and JET) with (13)C-labeled palmitate (1 mM) as a substrate. After 8 to 19 days of incubation, we could detect (13)C-labeled bacterial rRNA. A density-resolved terminal restriction fragment length polymorphism fingerprinting analysis showed distinct bacterial populations in (13)C-labeled and unlabeled rRNA fractions. The bacterial populations in the (13)C-labeled rRNA fractions were identified by cloning and sequencing of reverse-transcribed 16S rRNA. Diverse phylogenetic bacterial sequences were retrieved, including those of members of the family Syntrophaceae, clone cluster MST belonging to the class Deltaproteobacteria, Clostridium clusters III and IV, phylum Bacteroidetes, phylum Spirochaetes, and family Syntrophomonadaceae. Although Syntrophomonadaceae species are considered to be the major fatty acid-degrading syntrophic microorganisms under methanogenic conditions, they were detected in only two of the clone libraries. These results suggest that phylogenetically diverse bacterial groups were active in situ in the degradation of LCFA under methanogenic conditions.     

Diversity of five anaerobic toluene-degrading microbial communities investigated using stable isotope probing

Time-series DNA-stable isotope probing (SIP) was used to identify the microbes assimilating carbon from [(13)C]toluene under nitrate- or sulfate-amended conditions in a range of inoculum sources, including uncontaminated and contaminated soil and wastewater treatment samples. In all, five different phylotypes were found to be responsible for toluene degradation, and these included previously identified toluene degraders as well as novel toluene-degrading microorganisms. In microcosms constructed from granular sludge and amended with nitrate, the putative toluene degraders were classified in the genus Thauera, whereas in nitrate-amended microcosms constructed from a different source (agricultural soil), microorganisms in the family Comamonadaceae (genus unclassified) were the key putative degraders. In one set of sulfate-amended microcosms (agricultural soil), the putative toluene degraders were identified as belonging to the class Clostridia (genus Desulfosporosinus), while in other sulfate-amended microcosms, the putative degraders were in the class Deltaproteobacteria, within the family Syntrophobacteraceae (digester sludge) or Desulfobulbaceae (contaminated soil) (genus unclassified for both). Partial benzylsuccinate synthase gene (bssA, the functional gene for anaerobic toluene degradation) sequences were obtained for some samples, and quantitative PCR targeting this gene, along with SIP, was further used to confirm anaerobic toluene degradation by the identified species. The study illustrates the diversity of toluene degraders across different environments and highlights the utility of ribosomal and functional gene-based SIP for linking function with identity in microbial communities.     

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.     

Functional analysis of an anaerobic m-xylene-degrading enrichment culture using protein-based stable isotope probing

A sulfate-reducing consortium maintained for several years in the laboratory with m-xylene as sole source of carbon and energy was characterized by terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of PCR-amplified 16S rRNA genes and stable isotope probing of proteins (Protein-SIP). During growth upon m-xylene or methyl-labeled m-xylene (1,3-dimethyl-(13)C(2)-benzene), a phylotype affiliated to the family Desulfobacteriaceae became most abundant. A second dominant phylotype was affiliated to the phylum Epsilonproteobacteria. In cultures grown with methyl-labeled m-xylene, 331 proteins were identified by LC-MS/MS analysis. These proteins were either not (13)C-labeled (23%) or showed a (13)C-incorporation of 19-22 atom% (13)C (77%), the latter demonstrating that methyl groups of m-xylene were assimilated. (13)C-labeled proteins were involved in anaerobic m-xylene biodegradation, in sulfate reduction, in the Wood-Ljungdahl-pathway, and in general housekeeping functions. Thirty-eight percent of the labeled proteins were affiliated to Deltaproteobacteria. Probably due to a lack of sequence data from Epsilonproteobacteria, only 14 proteins were assigned to this phylum. Our data suggest that m-xylene is assimilated by the Desulfobacteriaceae phylotype, whereas the role of the Epsilonproteobacterium in the consortium remained unclear.     

Identification of triclosan-degrading bacteria in a triclosan enrichment culture using stable isotope probing

Triclosan, a widely used antimicrobial agent, is an emerging contaminant in the environment. Despite its antimicrobial character, biodegradation of triclosan has been observed in pure cultures, soils and activated sludge. However, little is known about the microorganisms responsible for the degradation in mixed cultures. In this study, active triclosan degraders in a triclosan-degrading enrichment culture were identified using stable isotope probing (SIP) with universally ¹³C-labeled triclosan. Eleven clones contributed from active microorganisms capable of uptake the ¹³C in triclosan were identified. None of these clones were similar to known triclosan-degraders/utilizers. These clones distributed among α-, β-, or γ-Proteobacteria: one belonging to Defluvibacter (α-Proteobacteria), seven belonging to Alicycliphilus (β-Proteobacteria), and three belonging to Stenotrophomonas (γ-Proteobacteria). Successive additions of triclosan caused a significant shift in the microbial community structure of the enrichment culture, with dominant ribotypes belonging to the genera Alicycliphilus and Defluvibacter. Application of SIP has successfully identified diverse uncultivable triclosan-degrading microorganisms in an activated sludge enrichment culture. The results of this study not only contributed to our understanding of the microbial ecology of triclosan biodegradation in wastewater, but also suggested that triclosan degraders are more phylogenetically diverse than previously reported.     

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.     

稳定同位素探针技术在有机污染物生物降解中的应用

稳定同位素探针技术(Stable isotope probing,SIP)是稳定同位素标记技术和各种分子生物学手段相结合的一系列技术总称。将其应用于探查污染物降解的功能微生物,实现了不经过分离培养直接把微生物的代谢功能、微生物间相互作用与微生物种群结合起来,从而克服了传统分离培养的缺陷,扩大了微生物资源的利用空间,具有广阔的发展前景。本文介绍了稳定同位素探针技术的基本原理和技术路线,对常规PLFA-SIP、DNA-SIP、RNA-SIP的特点进行了阐述和对比;综述了SIP在有机污染物——苯系物、多环芳烃、多氯联苯生物降解方面的研究进展,提出SIP应用于根际研究是今后该技术在生物降解研究中的一个发展方向。     

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.     

Identification of functionally active aerobic methanotrophs in sediments from an arctic lake using stable isotope probing

Arctic lakes are a significant source of the greenhouse gas methane (CH₄), but the role that methane oxidizing bacteria (methanotrophs) play in limiting the overall CH₄ flux is poorly understood. Here, we used stable isotope probing (SIP) techniques to identify the metabolically active aerobic methanotrophs in upper sediments (0–1 cm) from an arctic lake in northern Alaska sampled during ice‐free summer conditions. The highest CH₄ oxidation potential was observed in the upper sediment (0–1 cm depth) with 1.59 µmol g wet weight^?1 day^?1 compared with the deeper sediment samples (1–3 cm, 3–5 cm and 5–10 cm), which exhibited CH₄ oxidation potentials below 0.4 µmol g wet weight^?1 day^?1. Both type I and type II methanotrophs were directly detected in the upper sediment total communities using targeted primer sets based on 16S rRNA genes. Sequencing of 16S rRNA genes and functional genes (pmoA and mxaF) in the ¹³C‐DNA from the upper sediment indicated that type I methanotrophs, mainly Methylobacter, Methylosoma, Methylomonas and Methylovulum miyakonense, dominated the assimilation of CH₄. Methylotrophs, including the genera Methylophilus and/or Methylotenera, were also abundant in the ¹³C‐DNA. Our results show that a diverse microbial consortium acquired carbon from CH₄ in the sediments of this arctic lake.     

Metagenomic stable isotope probing reveals bacteriophage participation in soil carbon cycling

Soil viruses are important components of the carbon (C) cycle, yet we still know little about viral ecology in soils. We added diverse ¹³C-labelled carbon sources to soil and we used metagenomic-SIP to detect ¹³C assimilation by viruses and their putative bacterial hosts. These data allowed us to link a ¹³C-labelled bacteriophage to its ¹³C-labelled Streptomyces putative host, and we used qPCR to track the dynamics of the putative host and phage in response to C inputs. Following C addition, putative host numbers increased rapidly for 3 days, and then more gradually, reaching maximal abundance on Day 6. Viral abundance and virus:host ratio increased dramatically over 6 days, and remained high thereafter (8.42 ± 2.94). From Days 6 to 30, virus:host ratio remained high, while putative host numbers declined more than 50%. Putative host populations were ¹³C-labelled on Days 3–30, while ¹³C-labelling of phage was detected on Days 14 and 30. This dynamic suggests rapid growth and ¹³C-labelling of the host fueled by new C inputs, followed by extensive host mortality driven by phage lysis. These findings indicate that the viral shunt promotes microbial turnover in soil following new C inputs, thereby altering microbial community dynamics, and facilitating soil organic matter production.