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

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.     

Using Stable Isotope Probing to Characterize Differences Between Free-Living and Sediment-Associated Microorganisms in the Subsurface

Aquifers are subterranean reservoirs of freshwater with heterotrophic bacterial communities attached to the sediments and free-living in the groundwater. In the present study, mesocosms were used to assess factors controlling the diversity and activity of the subsurface bacterial community. The assimilation of ¹³C, derived from ¹³C-acetate, was monitored to determine whether the sediment-associated and free-living bacterial community would respond similarly to the presence of protozoan grazers. We observed a dynamic response in the sediment-associated bacterial community and none in the free-living community. The disparity in these observations highlights the importance of the sediment-associated bacterial community in the subsurface carbon cycle.     

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.     

Stable Isotope Ratios and Forensic Analysis of Microorganisms

In the aftermath of the anthrax letters of 2001, researchers have been exploring various analytical signatures for the purpose of characterizing the production environment of microorganisms. One such signature is stable isotope ratios, which in heterotrophs, are a function of nutrient and water sources. Here we discuss the use of stable isotope ratios in microbial forensics, using as a database the carbon, nitrogen, oxygen, and hydrogen stable isotope ratios of 247 separate cultures of Bacillus subtilis 6051 spores produced on a total of 32 different culture media. In the context of using stable isotope ratios as a signature for sample matching, we present an analysis of variations between individual samples, between cultures produced in tandem, and between cultures produced in the same medium but at different times. Additionally, we correlate the stable isotope ratios of carbon, nitrogen, oxygen, and hydrogen for growth medium nutrients or water with those of spores and show examples of how these relationships can be used to exclude nutrient or water samples as possible growth substrates for specific cultures.     

Characterization of para-Nitrophenol-Degrading Bacterial Communities in River Water by Using Functional Markers and Stable Isotope Probing

Microbial degradation is a major determinant of the fate of pollutants in the environment. para-Nitrophenol (PNP) is an EPA-listed priority pollutant with a wide environmental distribution, but little is known about the microorganisms that degrade it in the environment. We studied the diversity of active PNP-degrading bacterial populations in river water using a novel functional marker approach coupled with [¹³C₆]PNP stable isotope probing (SIP). Culturing together with culture-independent terminal restriction fragment length polymorphism analysis of 16S rRNA gene amplicons identified Pseudomonas syringae to be the major driver of PNP degradation in river water microcosms. This was confirmed by SIP-pyrosequencing of amplified 16S rRNA. Similarly, functional gene analysis showed that degradation followed the Gram-negative bacterial pathway and involved pnpA from Pseudomonas spp. However, analysis of maleylacetate reductase (encoded by mar), an enzyme common to late stages of both Gram-negative and Gram-positive bacterial PNP degradation pathways, identified a diverse assemblage of bacteria associated with PNP degradation, suggesting that mar has limited use as a specific marker of PNP biodegradation. Both the pnpA and mar genes were detected in a PNP-degrading isolate, P. syringae AKHD2, which was isolated from river water. Our results suggest that PNP-degrading cultures of Pseudomonas spp. are representative of environmental PNP-degrading populations.     

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

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.     

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

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.Polychlorinated biphenyls (PCBs) are very stable chloroorganic compounds with the general formula C₁₂H_10-xCl_x. Mixtures of PCBs have been used as coolants and lubricants in transformers, capacitors, and other electrical equipment as they do not burn easily and are good insulators. It is estimated that some 1.5 million tons of PCBs were produced up to 1988 worldwide (11; http://www.atsdr.cdc.gov/cercla; http://www.epa.gov/epawaste/hazard/tsd/pcbs/pubs/about.htm). Although production of these compounds was stopped, due to their long-term persistence, many sites all over the world are still contaminated with PCBs. Moreover, not only do PCBs threaten human health in the vicinity of the contaminated area, but lower PCB congeners volatilize and migrate to places far from where they were originally released (2, 3, 16). Also, their metabolic products have environmental significance; activities of both plants and microorganisms result in formation of different intermediates and final products whose toxicity can in some cases be even higher than that of the original toxicant (24, 26; http://www.atsdr.cdc.gov/cercla).Physical-chemical methods used for the removal of PCBs often cause further natural disturbance and pollution; in contrast, biological methods of removal (i.e., bioremediation) are less expensive and more environmentally sound and thus have aroused much interest (7). These methods include the use of microorganisms and also exploitation of plants (i.e., phytoremediation) (19) and the cooperation of plants with microorganisms in the rhizosphere (i.e., rhizoremediation) (21). These bioremediation options also include the use of genetically modified bacteria (6) and/or plants (18, 23). PCBs were only recently introduced into the environment, and no completely efficient pathways for the aerobic bacterial degradation of all of these compounds have evolved (34); however, lower chlorinated PCB congeners can be degraded via the pathway that is used by aerobic bacteria to degrade biphenyl (35). Therefore, metabolism of biphenyl as a potential cometabolite of PCBs was the subject of this study.The biphenyl degradation pathway is the same in all aerobic bacteria, and enzymes of this pathway degrade biphenyl in four steps into benzoate and 2-hydroxypenta-2,4-dienoate (21). The first enzyme of the pathway, biphenyl dioxygenase, has broad substrate specificity and thus permits degradation of biphenyl-related compounds (9). Substrates for biphenyl dioxygenase comprise, in addition to biphenyl itself, other diphenyl or benzene skeletons with several substituents, including halogens and bicyclic or tricyclic fused heterocyclic aromatics (35). These substrates also include certain natural compounds, including some plant flavonoids, phenols, or terpenes (10). Bacteria capable of metabolizing biphenyl are thus pervasive members of many microbial communities in vegetated soil.As reported previously (20), there are two main problems with introduction of a new population of degrading or genetically modified microorganisms to enhance the biodegradation of PCBs in a contaminated environment: legislative barriers and the inability of strains added to the soil to survive. Therefore, the use of microorganisms for bioremediation of contaminated sites is not likely to be successful. Hence, understanding the biodegradative processes in the natural communities is necessary for planning remediation strategies. Identification of members of the community potentially responsible for the degradative process has recently been enabled by DNA-based stable isotope probing (SIP), as reviewed previously; therefore, this technique has become an efficient tool in microbial ecology (33). In this study, by tracking the transfer of ¹³C from [¹³C]biphenyl into bacterial DNA, it was possible to identify biphenyl-metabolizing bacteria in PCB-contaminated soil. To analyze how the bacterial diversity can be changed by introduction of a plant and subsequent cultivation in a greenhouse, bacteria in the rhizosphere of horseradish (Armoracia rusticana) cultivated in a contaminated soil were studied.     

Stable Isotope Probing Analysis of Interactions between Ammonia Oxidizers

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.Molecular characterization of natural microbial communities has demonstrated the existence of novel high-level taxonomic groups with no cultured representatives and with significant diversity within phylogenetic and functional groups already established through analysis of organisms in laboratory culture. Autotrophic ammonia-oxidizing bacteria (AOB) exemplify the latter situation. Their low growth rates and the limited number of readily measured phenotypic characteristics available for identification of these organisms necessitate the use of molecular techniques for characterization of their diversity in natural environments. Phylogenetic analysis of 16S rRNA gene sequences places the majority of cultivated autotrophic bacterial ammonia oxidizers in a monophyletic group within the Betaproteobacteria (8, 26). Amplification and phylogenetic analysis of 16S rRNA gene sequences from enrichment cultures of ammonia oxidizers and sequences of environmental clones (31) suggest the existence of novel groups with no cultivated representative and considerable diversity within those represented by pure cultures.Increased awareness of microbial diversity has raised questions regarding links between species diversity and functional diversity, functional redundancy, and the influence of environmental conditions on the activities of representatives of different phylotypes. For ammonia-oxidizing bacteria, relationships exist between broad phylogenetic groups and the environments from which laboratory isolates were obtained, which are linked, in some cases, to differences in physiological characteristics (11). There is also evidence of links between the relative abundance of different ammonia oxidizer groups and environmental conditions (1, 13, 14, 18, 21, 23, 34), suggesting selection for organisms with particular physiological characteristics. In one study (36), a combination of molecular and physiological studies has demonstrated links between species diversity, functional diversity, and soil nitrification kinetics. However, for ammonia oxidizers and other groups, there is little direct evidence about which strains within diverse communities are active under particular conditions or the extent of competition for substrates.Stable isotope probing (SIP) (24, 27) of nucleic acids provides direct evidence of which members of mixed communities are active. This involves addition of substrates labeled with a stable isotope (most commonly ¹³C), extraction of nucleic acids, separation of ¹²C- and ¹³C-labeled nucleic acids by density gradient centrifugation, and subsequent molecular analysis. Sequences amplified from ¹³C-labeled DNA or RNA are derived from organisms actively assimilating the substrate. This approach has been used to identify organisms that utilize methane or methanol (4, 19), organic compounds (15, 20), or CO₂ (6, 9) in microcosms and those that assimilate plant root exudates in the field (28). SIP therefore links phylogeny to ecosystem function and has identified established and novel groups by utilizing labeled compounds in complex soil communities. The technique also enables in situ physiological studies and investigation of interactions between organisms in mixed cultures belonging to the same functional group. For autotrophic betaproteobacterial ammonia oxidizers, amplification of 16S rRNA genes from ¹³C-labeled DNA during incubation with [¹³C]CO₂ has the potential for discriminating which strains are active under specific conditions. Assessment of the discriminatory ability of this approach in complex natural environments requires studies under controlled and well-characterized conditions. The first aim of this study was, therefore, to assess the ability of SIP to discriminate activities of different members of simple mixed communities in comparison with direct measurement of product concentration and DNA- and RNA-denaturing gradient gel electrophoresis (DGGE). The second was to determine whether the activities of members of mixed communities of ammonia-oxidizing bacteria, in particular, their ability to grow at high ammonia concentrations, could be predicted from their physiological characteristics in monoculture. Of particular interest was whether strains with low ammonia tolerance are competitive at low ammonia concentrations. Mixed cultures were assembled from pure culture representatives of Nitrosospira clusters 0, 3a, and 3b (26, 36), which are frequently found in soil environments, and from enrichment cultures containing representatives of these clusters with heterotrophic contaminants. Other criteria for choice of community members were similarities in specific growth rate and cultivation conditions to enable meaningful competition experiments.     

Methodological Considerations for the Use of Stable Isotope Probing in Microbial Ecology

Stable 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.     

Identification of Ciliate Grazers of Autotrophic Bacteria in Ammonia-Oxidizing Activated Sludge by RNA Stable Isotope Probing

It is well understood that protozoa play a major role in controlling bacterial biomass and regulating nutrient cycling in the environment. Little is known, however, about the movement of carbon from specific reduced substrates, through functional groups of bacteria, to particular clades of protozoa. In this study we first identified the active protozoan phylotypes present in activated sludge, via the construction of an rRNA-derived eukaryote clone library. Most of the sequences identified belonged to ciliates of the subclass Peritrichia and amoebae, confirming the dominance of surface-associated protozoa in the activated sludge environment. We then demonstrated that ¹³C-labeled protozoan RNA can be retrieved from activated sludge amended with ¹³C-labeled protozoa or ¹³C-labeled Escherichia coli cells by using an RNA stable isotope probing (RNA-SIP) approach. Finally, we used RNA-SIP to track carbon from bicarbonate and acetate into protozoa under ammonia-oxidizing and denitrifying conditions, respectively. RNA-SIP analysis revealed that the peritrich ciliate Epistylis galea dominated the acquisition of carbon from bacteria with access to CO₂ under ammonia-oxidizing conditions, while there was no evidence of specific grazing on acetate consumers under denitrifying conditions.Protozoa are the main consumers of bacteria in the environment, and as such they play a major role in controlling bacterial biomass (33) and regulating nutrient recycling (14). Therefore, being able to study the flow of carbon in food webs involving bacteria and protozoa can provide an insight into how protozoan grazing affects bacterial function in a wide range of systems.Protozoan grazing has the potential to alter the genotypic and phenotypic composition of bacterial communities (15, 17, 18, 35). Perhaps as a result of this, protozoan grazing has also been found to have an effect on the function of activated sludge systems, including nitrogen removal processes. Studies using eukaryotic inhibitors to remove grazers from activated sludge have reported a wide range of effects following reduced grazing pressure. These effects include an increase in turbidity or planktonic cell densities, and either no effect (21, 32), higher nitrification rates (16, 20), or lower (29, 30) rates of nitrification in the absence of grazers. These conflicting reports on the effect of predation on nitrification might be due to protozoa displaying feeding preferences or to the indirect ways in which protozoan grazing can affect bacterial processes. For example, it has been shown that the release of substances, such as vitamins and nucleotides, secreted by protozoa as metabolic by-products, can act as growth factors, which enhance bacterial activity, including nitrification (31). In addition, the presence of some types of grazers, particularly ciliates, has been shown to be closely related to a decrease in biochemical oxygen demand (27), and it has been reported that ciliates can alter water flux and help redistribute nutrients in flocs (8), which in turn might have an impact on nitrification rates.Although the data presented in the literature suggest that protozoan grazing is an important factor for activated sludge processes, there are still many questions as to whether its effects are directly linked to predation and possibly feeding preferences. In the present study we sought to identify protozoa assimilating carbon from autotrophic bacteria under ammonia-oxidizing conditions and acetate-consuming bacteria under denitrifying conditions using RNA-stable isotope probing (RNA-SIP).Since it was first developed, RNA-SIP has been successfully used to identify functional groups of bacteria responsible for different processes, including denitrification and benzene and phenol degradation (9, 10, 19, 25, 26). In these studies, the flow of carbon was tracked from soluble labeled substrates into the bacteria consuming it. However, recent studies have shown that it is possible to use this technique to track the flow of carbon across more than one trophic level, unraveling some of the interactions observed in microbial food webs (11, 13, 23, 24). These studies exemplify the broad range of questions that can be addressed with SIP and have begun to define the boundaries beyond which SIP has limited utility.     

Use of Field-Based Stable Isotope Probing To Identify Adapted Populations and Track Carbon Flow through a Phenol-Degrading Soil Microbial Community

The goal of this field study was to provide insight into three distinct populations of microorganisms involved in in situ metabolism of phenol. Our approach measured ¹³CO₂ respired from [¹³C]phenol and stable isotope probing (SIP) of soil DNA at an agricultural field site. Traditionally, SIP-based investigations have been subject to the uncertainties posed by carbon cross-feeding. By altering our field-based, substrate-dosing methodologies, experiments were designed to look beyond primary degraders to detect trophically related populations in the food chain. Using gas chromatography-mass spectrometry (GC/MS), it was shown that ¹³C-labeled biomass, derived from primary phenol degraders in soil, was a suitable growth substrate for other members of the soil microbial community. Next, three dosing regimes were designed to examine active members of the microbial community involved in phenol metabolism in situ: (i) 1 dose of [¹³C]phenol, (ii) 11 daily doses of unlabeled phenol followed by 1 dose of [¹³C]phenol, and (iii) 12 daily doses of [¹³C]phenol. GC/MS analysis demonstrated that prior exposure to phenol boosted ¹³CO₂ evolution by a factor of 10. Furthermore, imaging of ¹³C-treated soil using secondary ion mass spectrometry (SIMS) verified that individual bacteria incorporated ¹³C into their biomass. PCR amplification and 16S rRNA gene sequencing of ¹³C-labeled soil DNA from the 3 dosing regimes revealed three distinct clone libraries: (i) unenriched, primary phenol degraders were most diverse, consisting of α-, β-, and γ-proteobacteria and high-G+C-content gram-positive bacteria, (ii) enriched primary phenol degraders were dominated by members of the genera Kocuria and Staphylococcus, and (iii) trophically related (carbon cross-feeders) were dominated by members of the genus Pseudomonas. These data show that SIP has the potential to document population shifts caused by substrate preexposure and to follow the flow of carbon through terrestrial microbial food chains.     

Stable Isotope Probing with 15N Achieved by Disentangling the Effects of Genome G+C Content and Isotope Enrichment on DNA Density

Stable isotope probing (SIP) of nucleic acids is a powerful tool that can identify the functional capabilities of noncultivated microorganisms as they occur in microbial communities. While it has been suggested previously that nucleic acid SIP can be performed with ¹⁵N, nearly all applications of this technique to date have used ¹³C. Successful application of SIP using ¹⁵N-DNA (¹⁵N-DNA-SIP) has been limited, because the maximum shift in buoyant density that can be achieved in CsCl gradients is approximately 0.016 g ml⁻¹ for ¹⁵N-labeled DNA, relative to 0.036 g ml⁻¹ for ¹³C-labeled DNA. In contrast, variation in genome G+C content between microorganisms can result in DNA samples that vary in buoyant density by as much as 0.05 g ml⁻¹. Thus, natural variation in genome G+C content in complex communities prevents the effective separation of ¹⁵N-labeled DNA from unlabeled DNA. We describe a method which disentangles the effects of isotope incorporation and genome G+C content on DNA buoyant density and makes it possible to isolate ¹⁵N-labeled DNA from heterogeneous mixtures of DNA. This method relies on recovery of “heavy” DNA from primary CsCl density gradients followed by purification of ¹⁵N-labeled DNA from unlabeled high-G+C-content DNA in secondary CsCl density gradients containing bis-benzimide. This technique, by providing a means to enhance separation of isotopically labeled DNA from unlabeled DNA, makes it possible to use ¹⁵N-labeled compounds effectively in DNA-SIP experiments and also will be effective for removing unlabeled DNA from isotopically labeled DNA in ¹³C-DNA-SIP applications.     

Ubiquitous Dissolved Inorganic Carbon Assimilation by Marine Bacteria in the Pacific Northwest Coastal Ocean as Determined by Stable Isotope Probing

In order to identify bacteria that assimilate dissolved inorganic carbon (DIC) in the northeast Pacific Ocean, stable isotope probing (SIP) experiments were conducted on water collected from 3 different sites off the Oregon and Washington coasts in May 2010, and one site off the Oregon Coast in September 2008 and March 2009. Samples were incubated in the dark with 2 mM ¹³C-NaHCO₃, doubling the average concentration of DIC typically found in the ocean. Our results revealed a surprising diversity of marine bacteria actively assimilating DIC in the dark within the Pacific Northwest coastal waters, indicating that DIC fixation is relevant for the metabolism of different marine bacterial lineages, including putatively heterotrophic taxa. Furthermore, dark DIC-assimilating assemblages were widespread among diverse bacterial classes. Alphaproteobacteria, Gammaproteobacteria, and Bacteroidetes dominated the active DIC-assimilating communities across the samples. Actinobacteria, Betaproteobacteria, Deltaproteobacteria, Planctomycetes, and Verrucomicrobia were also implicated in DIC assimilation. Alteromonadales and Oceanospirillales contributed significantly to the DIC-assimilating Gammaproteobacteria within May 2010 clone libraries. 16S rRNA gene sequences related to the sulfur-oxidizing symbionts Arctic96BD-19 were observed in all active DIC assimilating clone libraries. Among the Alphaproteobacteria, clones related to the ubiquitous SAR11 clade were found actively assimilating DIC in all samples. Although not a dominant contributor to our active clone libraries, Betaproteobacteria, when identified, were predominantly comprised of Burkholderia. DIC-assimilating bacteria among Deltaproteobacteria included members of the SAR324 cluster. Our research suggests that DIC assimilation is ubiquitous among many bacterial groups in the coastal waters of the Pacific Northwest marine environment and may represent a significant metabolic process.     

Sorption of Cadmium by Microorganisms in Competition with Other Soil Constituents

The fate of cadmium in soil is influenced to a great extent by microbial activity. Microorganisms were compared with abiotic soil components for their ability to sorb Cd from a liquid medium. When the same amount (on a dry weight basis) of bacterial cells (Serratia marcescens and Paracoccus sp.), clay (montmorillonite), or sand was separately incubated in 0.05 M phosphate buffer, pH 7.2, containing 10 ppm of Cd (10 μg/ml), bacterial cells removed the largest quantity of Cd. Dead cells sorbed much more Cd from the medium than live cells. A comparative study of Cd removal from the medium by seven soil bacteria and four fungi did not indicate appreciable differences. With increasing microbial biomass, the relative efficiency of 0.1 M NaOH as an extractant of sorbed Cd increased, whereas the extraction efficiency of 0.005 M DTPA (diethylenetriaminepentaacetic acid) decreased. It appeared that NaOH and DTPA extracted different chemical forms of Cd. This assumption was supported by vastly different correlation coefficients in the relative amount of Cd extracted by the two solvents.     

Stable-Isotope Probing of Microorganisms Thriving at Thermodynamic Limits: Syntrophic Propionate Oxidation in Flooded Soil

Propionate is an important intermediate of the degradation of organic matter in many anoxic environments. In methanogenic environments, due to thermodynamic constraints, the oxidation of propionate requires syntrophic cooperation of propionate-fermenting proton-reducing bacteria and H₂-consuming methanogens. We have identified here microorganisms that were active in syntrophic propionate oxidation in anoxic paddy soil by rRNA-based stable-isotope probing (SIP). After 7 weeks of incubation with [¹³C]propionate (<10 mM) and the oxidation of ~30 μmol of ¹³C-labeled substrate per g dry weight of soil, we found that archaeal nucleic acids were ¹³C labeled to a larger extent than those of the bacterial partners. Nevertheless, both terminal restriction fragment length polymorphism and cloning analyses revealed Syntrophobacter spp., Smithella spp., and the novel Pelotomaculum spp. to predominate in “heavy” ¹³C-labeled bacterial rRNA, clearly showing that these were active in situ in syntrophic propionate oxidation. Among the Archaea, mostly Methanobacterium and Methanosarcina spp. and also members of the yet-uncultured “rice cluster I” lineage had incorporated substantial amounts of ¹³C label, suggesting that these methanogens were directly involved in syntrophic associations and/or thriving on the [¹³C]acetate released by the syntrophs. With this first application of SIP in an anoxic soil environment, we were able to clearly demonstrate that even guilds of microorganisms growing under thermodynamic constraints, as well as phylogenetically diverse syntrophic associations, can be identified by using SIP. This approach holds great promise for determining the structure and function relationships of further syntrophic or other nutritional associations in natural environments and for defining metabolic functions of yet-uncultivated microorganisms.     

Identification of a Novel Toluene-Degrading Bacterium from the Candidate Phylum TM7, as Determined by DNA Stable Isotope Probing

The dominant bacterium responsible for carbon uptake from toluene in an agricultural soil was identified by stable isotope probing. Samples were amended with unlabeled toluene or labeled [ring-¹³C₆]toluene, and DNA was extracted over time. Sequencing indicated that the organism involved belongs to the candidate phylum TM7. Microorganisms in this candidate phylum are of particular interest because although they have been found in a variety of habitats, no stable culture of any species exists, so their general metabolic capabilities are largely unknown.The application of PCR technology has uncovered the impressive diversity of the microbial world. It has been estimated that less than half of the recognized bacterial phyla include cultured representatives (14). Here, stable isotope probing (SIP) (a method that links function to identity in mixed microbial samples) was used to identify dominant toluene degraders in an environment previously unexposed to the contaminant but likely containing a diverse microbial community of previously undiscovered toluene degraders.Soil samples were collected from a field in Michigan previously under corn production. This field received biosolids from a wastewater treatment plant 2 to 3 years before sample collection. Following collection, soils were homogenized, sieved (4-mm screen), and stored at 4°C until use (<1 year). The microcosms consisted of phosphate-buffered mineral medium (20 ml) (10) and soil (6 g [wet weight]) in serum bottles (150 ml). The bottles were sealed with rubber stoppers and an aluminum seal. The treatment groups included no-toluene controls, autoclaved controls, and samples amended with unlabeled (1 μl, 99%; Chem Service) or labeled (1 μl, ring-¹³C₆, 99%; Cambridge Isotope Laboratories) toluene. Eight samples for each treatment (two for each time point) were incubated at room temperature (∼20°C) with reciprocal shaking. The concentrations of toluene in headspace samples (200 μl) after toluene addition (∼2 h) and at each time point were determined with a gas chromatograph (Perkin Elmer) equipped with a flame ionization detector and a capillary column (DB-624 [diameter, 0.53 mm]; J&W Scientific). The injector and detector temperatures were set at 200°C, and the column temperature was 80°C.At four time points (3, 5, 6, and 8 days after toluene addition), two samples from the labeled and unlabeled treatment groups were sacrificed for soil DNA extraction using a Powersoil DNA extraction kit (MO BIO Laboratories, Inc., Carlsbad, CA). At each time point, DNAs from two microcosms were pooled, and SIP involved terminal restriction fragment length polymorphism (TRFLP) analysis of all fractions (labeled and unlabeled). Approximately 10 μg DNA (quantified with an ND-1000 spectrometer; Nanodrop) was added to Quick-Seal polyallomer tubes (13 by 51 mm, 5.1 ml; Beckman Coulter), along with a Tris-EDTA (pH 8.0)-CsCl solution. Before the tubes were sealed (Quick-Seal tube topper; Beckman Coulter), buoyant density (BD) was determined (∼1.77 g ml⁻¹) with a model AR200 digital refractometer (Leica Microsystems, Inc.) and adjusted by adding CsCl solution or Tris-EDTA buffer. The tubes were centrifuged at 178,000 × g (20°C) for 48 h in a Stepsaver 70 V6 vertical titanium rotor (eight tubes, 5.1-ml capacity each) within a Sorvall WX 80 Ultra Series centrifuge (Thermo Scientific). Following centrifugation, a fraction recovery system (Beckman Coulter) was used for fraction (150 μl) collection. The BD of each fraction was measured, and CsCl was removed by glycogen-assisted ethanol precipitation.The fractions were PCR amplified using 27F-FAM (5′-AGAGTTTGATCMTGGCTCAG [5′ end labeled with carboxyfluorescein]) and 1492R (5′-GGTTACCTTGTTACGACTT) (Operon Biotechnologies) as previously described (4). Briefly, this involved the following conditions: 94°C (5 min); 30 cycles at 94°C (30 s), 55°C (30 s), and 72°C (1.5 min); and 72°C (5 min). The presence of PCR products was confirmed by gel electrophoresis. PCR products were purified with a QIAquick PCR purification kit (Qiagen, Inc.) (∼150 ng) and digested with HaeIII (New England Biolabs). In addition, three other enzymes (MspI, MseI, and HincII; New England Biolabs) were used for digestion of a number of heavy fractions. DNA fragments were separated by capillary electrophoresis (ABI Prism 3100 genetic analyzer; Applied Biosystems) at the Research Technology Support Facility at Michigan State University. Data were analyzed with GeneScan software (Applied Biosystems), and the percent abundance of each fragment was determined (18). Heavy-fraction ¹³C-labeled DNAs (day 8 fractions with BD values of 1.744 g ml⁻¹) were amplified, as described above, with unlabeled primers and cloned into Escherichia coli TOP10 by using a TOPO TA cloning kit (Invitrogen Corporation). E. coli clones were grown on Luria-Bertani medium solidified with 15 g agar liter⁻¹ in the presence of 50 μg ampicillin liter⁻¹ for 16 h at 37°C. Colonies with inserts were verified by PCR with primers M13 forward (5′-TGTAAAACGACGGCCAGT-3′) and M13 reverse (5′-AACAGCTATGACCATG-3′), plasmids were extracted from the positive clones with a QIAprep miniprep system (Qiagen, Inc.), and the insertions were sequenced (using primers M13 forward and M13 reverse) at the Research Technology Support Facility at Michigan State University. The partial 16S rRNA gene sequences obtained were aligned and edited with Chromas Pro (Technelysium, Pty. Ltd.). The Ribosomal Database Project (Center for Microbial Ecology, Michigan State University) analysis tool “classifier” (16) was utilized to assign taxonomic identity. In addition, the Ribosomal Database Project classifier checked the deposited sequence for chimeras.Toluene removal occurred rapidly, starting after 3 days and reaching completion after 8 days (Table ​(Table1).1). The low percentage of recoveries was likely caused by toluene sorption to the soil; however, the difference between the controls and samples clearly illustrates a biological removal mechanism. DNAs extracted over time (on days 3, 5, 6, and 8) from both labeled and unlabeled samples were subjected to ultracentrifugation, fractionation, and TRFLP on every fraction. TRFLP analyses indicated that one fragment (394 bp) was highly enriched in the heavy fractions (>1.732 g ml⁻¹) obtained from [¹³C]toluene microcosms but not in fractions with similar BD values obtained from the unlabeled controls, and further, the level of enrichment increased with time (Fig. ​(Fig.1).1). At day 3, the fragment was present in only one fraction TRFLP profile, indicating a low natural relative abundance of this organism. The peak TRFLP relative abundance values for the 394-bp fragment were 11.3%, 41.5%, and 62.1% on days 5, 6, and 8, respectively (Fig. ​(Fig.2).2). Consistent with this trend, the BD of this fragment also increased with time, with the peak relative abundances being found at BD values of 1.732 g ml⁻¹, 1.740 g ml⁻¹, and 1.744 g ml⁻¹ on days 5, 6, and 8, respectively (Fig. ​(Fig.2).2). These trends indicate that the organism represented by this fragment is directly involved in toluene transformation. A number of other TRFLP peaks were found in the heavy ¹³C fractions; however, because they were also present in the heavy ¹²C fractions, they were excluded from further analysis.Open in a separate windowFIG. 1.Dominance of the 394-bp fragment over time in TRFLP profiles from heavy fractions of soil samples amended with unlabeled (¹²C) or labeled (¹³C) toluene. The pattern is representative of other heavy-fraction samples at these time points. The BD values are given in the lower-right corners of the profiles.Open in a separate windowFIG. 2.Relative abundances of the dominant 394-bp fragment over a range of BD values from DNA extracted after 5 days (a), 6 days (b), or 8 days (c) from soil amended with either labeled (¹³C) or unlabeled (¹²C) toluene.

TABLE 1.

Ranges for percentages of toluene remaining in soil-liquid slurries over time

Linking Microbial Community Function to Phylogeny of Sulfate-Reducing Deltaproteobacteria in Marine Sediments by Combining Stable Isotope Probing with Magnetic-Bead Capture Hybridization of 16S rRNA

We further developed the stable isotope probing, magnetic-bead capture method to make it applicable for linking microbial community function to phylogeny at the class and family levels. The main improvements were a substantial decrease in the protocol blank and an approximately 10-fold increase in the detection limit by using a micro-elemental analyzer coupled to isotope ratio mass spectrometry to determine ¹³C labeling of isolated 16S rRNA. We demonstrated the method by studying substrate utilization by Desulfobacteraceae, a dominant group of complete oxidizing sulfate-reducing Deltaproteobacteria in marine sediments. Stable-isotope-labeled [¹³C]glucose, [¹³C]propionate, or [¹³C]acetate was fed into an anoxic intertidal sediment. We applied a nested set of three biotin-labeled oligonucleotide probes to capture Bacteria, Deltaproteobacteria, and finally Desulfobacteraceae rRNA by using hydrophobic streptavidin-coated paramagnetic beads. The target specificities of the probes were examined with pure cultures of target and nontarget species and by determining the phylogenetic composition of the captured sediment rRNA. The specificity of the final protocol was generally very good, as more than 90% of the captured 16S rRNA belonged to the target range of the probes. Our results indicated that Desulfobacteraceae were important consumers of propionate but not of glucose. However, the results for acetate utilization were less conclusive due to lower and more variable labeling levels in captured rRNA. The main advantage of the method in this study over other nucleic acid-based stable isotope probing methods is that ¹³C labeling can be much lower, to the extent that δ¹³C ratios can be studied even at their natural abundances.Linking microbial phylogeny to community function provides us with insights into the roles that microorganisms play in global elemental cycling. In recent years, stable isotope-tracing approaches combined with biomarkers have been widely applied to environmental studies (8, 27, 40). Tracking stable- or radioisotope-labeled atoms from particular substrates into components of microbial cells (biomarkers) can reveal which organisms are involved in the consumption of the substrate and also yield information on rates of specific biogeochemical transformation (8).Dissimilatory sulfate reduction is a major pathway for organic carbon mineralization in coastal marine sediments, accounting for, on average, 50% of the total carbon mineralization (18, 36). Sulfate-reducing prokaryotes are a diverse and ubiquitous component of the bacterial community. The diversity of sulfate-reducing bacteria (SRB) in marine sediments has been investigated by using clone libraries of 16S rRNA (38) and dissimilatory sulfite reductase genes (11) and by fluorescence in situ hybridization-related techniques (33, 41). Desulfobacteraceae, a group of complete-oxidizing SRB belonging to the Deltaproteobacteria, have generally been found to be a major group of SRB in marine sediments.Phospholipid-derived fatty acids (PLFA) were the first type of biomarkers to be used in combination with stable isotope probing (SIP) (8). PLFA-SIP provides high sensitivity in terms of the amount of ¹³C label needed, but the phylogenetic resolution offered is low and requires reference signatures of closely related culturable relatives (8). The main advantage of DNA- and RNA-SIP is that they offer improved phylogenetic resolution (27, 40). These two methods are based on the separation of the “heavier” ¹³C-labeled nucleic acid from unlabeled nucleic acid by density centrifugation. Subsequently, organisms incorporating the greatest proportion of label into their DNA or RNA are identified by various molecular-fingerprinting techniques or by constructing clone libraries. RNA has a higher turnover rate than DNA, resulting in faster labeling, and incubation times can therefore be substantially shortened (27, 42). RNA is also more likely to reflect the phylogenetic composition of the metabolically active community, since it is highly susceptible to chemical and enzymatic degradation, and its cellular levels are often tightly regulated (19, 32), although some prokaryotes maintain high numbers of ribosomes during starvation (13).MacGregor et al. (25, 26) developed a related approach, SIP combined with magnetic-bead capture hybridization (here called Mag-SIP), which is based on the isolation of small subunit rRNA from particular phylogenetic groups and the detection of ¹³C-labeling levels by isotope ratio mass spectrometry (IRMS). rRNA is captured by hybridization with specific biotin-labeled oligonucleotide probes, followed by retrieval of hybridized target rRNA using streptavidin-coated magnetic beads. The main advantage of Mag-SIP over other nucleic acid-based SIP methods is that in principle much lower labeling levels can be applied (about 0.001% versus >10% ¹³C, respectively), as label detection is based on IRMS methods. For instance, it has been shown that the method can be used to study the effects of oil pollution on natural δ¹³C ratios of bacterial communities in sediments (37). Moreover, Mag-SIP is not based on PCR, as the isotope ratio of the target rRNA is directly measured without amplification of nucleic acid, avoiding possible PCR artifacts. However, the large amounts (1 to 10 μg) of RNA needed for an accurate isotope ratio analysis by traditional elemental-analyzer (EA)-IRMS has limited the use of Mag-SIP to general domain-specific probes (25, 26). Recently, several methods, such as liquid chromatography (LC) combined with IRMS and spooling-wire microcombustion combined with IRMS, have been introduced that allow isotopic analysis of much smaller samples than with the traditional EA-IRMS systems (20, 43).In this study, we used the wet-oxidation interface of LC-IRMS as a micro-EA (μEA)-IRMS (20). The use of μEA-IRMS substantially lowers the detection limit of isotope ratio measurements in terms of the amount of rRNA needed for an analysis but also calls for modifications of the Mag-SIP protocol in order to decrease protocol blanks (carbon from materials and reagents used in the protocol). We tested a nested set of three biotin-labeled oligonucleotide probes to capture 16S rRNA derived from Bacteria, Deltaproteobacteria, and finally Desulfobacteraceae. The target specificities and stringencies of these probes were tested against pure cultures of both target and nontarget organisms. Moreover, phylogenetic analysis of captured 16S rRNA from environmental samples was done to check specificity and, where necessary, to adjust probe stringency. Finally, we demonstrated Mag-SIP with a study of in situ substrate use by sulfate-reducing Deltaproteobacteria in intertidal anoxic marine sediment. A generalized scheme for Mag-SIP is shown in Fig. ​Fig.11.Open in a separate windowFIG. 1.A generalized scheme for Mag-SIP. The control was sediment incubated without substrate.