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.     

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 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.     

Stable Isotope Ecology of Extant Tapirs from the Americas

Understanding the ecology of modern and ancient forests can help clarify the evolution of forest dwelling mammals. It is first necessary, however, to elucidate the source and extent of stable isotope variation in forest taxa. Tapirs are of particular interest because they are model organisms for identifying forest environments due to their highly conservative diet and habitat preferences. Here, stable carbon and oxygen isotopic compositions of extant tapirs are quantified to test hypotheses regarding ontogenetic diet shifts, stable isotope variation at the population level, and relationships between stable isotopes and climatic variables. A population of extant tapirs (Tapirus bairdii) demonstrates low δ¹³C variation (～2–3‰) and increased δ¹³C values in late erupting teeth, indicating that juveniles consume ¹³C deplete milk and/or browse in denser forests. Disparate δ¹⁸O values of late erupting teeth are instead reflecting seasonal variation. Extant tapir (T. bairdii, Tapirus pinchaque, Tapirus terrestris) δ¹⁸O values are constrained by climatic and geographic variables. Most notably, δ¹⁸O values of T. bairdii decrease with decreasing precipitation frequency. Tapirus terrestris is typically present in areas with greater precipitation than T. bairdii and δ¹⁸O values instead positively correlate with δ¹³C values. These data indicate that tapirs in wetter areas are getting a larger proportion of their water from leaves experiencing less evaporation in denser canopies, while T. bairdii is interpreted to increase its consumption of water via drinking when present in drier areas. An understanding of extant tapir stable isotope ecology improves ecological interpretations of these elusive mammals both today and in the past. Abstract in Spanish is available at http://www.blackwell‐synergy.com/loi/btp .     

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.     

稳定同位素标签技术在定量蛋白质组研究的应用

高通量的从蛋白质组水平进行整体的蛋白质鉴定和精确定量比较分析,在阐述生物功能以及疾病发生发展机制等方面非常重要。稳定同位素标签技术在过去的几年中获得了很大的发展,并形成了代谢引入类、化学合成类以及酶解引入类等三大类型。该文对稳定同位素标签技术的技术特点以及应用进行了简述。     

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.     

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.     

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 H₂¹⁸O is described. To test if sufficient amounts of ¹⁸O 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 H₂¹⁸O and labeled DNA was separated from [¹⁶O]DNA on a cesium chloride gradient. Soil samples were incubated with H₂¹⁸O 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 ¹⁸O can be used in the stable isotope probing of soil samples. DNA extracted from soil incubated for 21 days with H₂¹⁸O 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 H₂¹⁸O 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.     

Resolving Genetic Functions within Microbial Populations: In Situ Analyses Using rRNA and mRNA Stable Isotope Probing Coupled with Single-Cell Raman-Fluorescence In Situ Hybridization

Prokaryotes represent one-half of the living biomass on Earth, with the vast majority remaining elusive to culture and study within the laboratory. As a result, we lack a basic understanding of the functions that many species perform in the natural world. To address this issue, we developed complementary population and single-cell stable isotope (¹³C)-linked analyses to determine microbial identity and function in situ. We demonstrated that the use of rRNA/mRNA stable isotope probing (SIP) recovered the key phylogenetic and functional RNAs. This was followed by single-cell physiological analyses of these populations to determine and quantify in situ functions within an aerobic naphthalene-degrading groundwater microbial community. Using these culture-independent approaches, we identified three prokaryote species capable of naphthalene biodegradation within the groundwater system: two taxa were isolated in the laboratory (Pseudomonas fluorescens and Pseudomonas putida), whereas the third eluded culture (an Acidovorax sp.). Using parallel population and single-cell stable isotope technologies, we were able to identify an unculturable Acidovorax sp. which played the key role in naphthalene biodegradation in situ, rather than the culturable naphthalene-biodegrading Pseudomonas sp. isolated from the same groundwater. The Pseudomonas isolates actively degraded naphthalene only at naphthalene concentrations higher than 30 μM. This study demonstrated that unculturable microorganisms could play important roles in biodegradation in the ecosystem. It also showed that the combined RNA SIP-Raman-fluorescence in situ hybridization approach may be a significant tool in resolving ecology, functionality, and niche specialization within the unculturable fraction of organisms residing in the natural environment.     

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.     

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.     

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.     

Stable Hydrogen and Carbon Isotope Fractionation during Microbial Toluene Degradation: Mechanistic and Environmental Aspects

Primary features of hydrogen and carbon isotope fractionation during toluene degradation were studied to evaluate if analysis of isotope signatures can be used as a tool to monitor biodegradation in contaminated aquifers. D/H hydrogen isotope fractionation during microbial degradation of toluene was measured by gas chromatography. Per-deuterated toluene-d₈ and nonlabeled toluene were supplied in equal amounts as growth substrates, and kinetic isotope fractionation was calculated from the shift of the molar ratios of toluene-d₈ and nondeuterated toluene. The D/H isotope fractionation varied slightly for sulfate-reducing strain TRM1 (slope of curve [b] = −1.219), Desulfobacterium cetonicum (b = −1.196), Thauera aromatica (b = −0.816), and Geobacter metallireducens (b = −1.004) and was greater for the aerobic bacterium Pseudomonas putida mt-2 (b = −2.667). The D/H isotope fractionation was 3 orders of magnitude greater than the ¹³C/¹²C carbon isotope fractionation reported previously. Hydrogen isotope fractionation with nonlabeled toluene was 1.7 and 6 times less than isotope fractionation with per-deuterated toluene-d₈ and nonlabeled toluene for sulfate-reducing strain TRM1 (b = −0.728) and D. cetonicum (b = −0.198), respectively. Carbon and hydrogen isotope fractionation during toluene degradation by D. cetonicum remained constant over a growth temperature range of 15 to 37°C but varied slightly during degradation by P. putida mt-2, which showed maximum hydrogen isotope fractionation at 20°C (b = −4.086) and minimum fractionation at 35°C (b = −2.138). D/H isotope fractionation was observed only if the deuterium label was located at the methyl group of the toluene molecule which is the site of the initial enzymatic attack on the substrate by the bacterial strains investigated in this study. Use of ring-labeled toluene-d₅ in combination with nondeuterated toluene did not lead to significant D/H isotope fractionation. The activity of the first enzyme in the anaerobic toluene degradation pathway, benzylsuccinate synthase, was measured in cell extracts of D. cetonicum with an initial activity of 3.63 mU (mg of protein)⁻¹. The D/H isotope fractionation (b = −1.580) was 30% greater than that in growth experiments with D. cetonicum. Mass spectroscopic analysis of the product benzylsuccinate showed that H atoms abstracted from the toluene molecules by the enzyme were retained in the same molecules after the product was released. Our findings revealed that the use of deuterium-labeled toluene was appropriate for studying basic features of D/H isotope fractionation. Similar D/H fractionation factors for toluene degradation by anaerobic bacteria, the lack of significant temperature dependence, and the strong fractionation suggest that analysis of D/H fractionation can be used as a sensitive tool to assess degradation activities. Identification of the first enzyme reaction in the pathway as the major fractionating step provides a basis for linking observed isotope fractionation to biochemical reactions.     

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.