I Methodology CH01_5745.indd 1 8/25/10 11:49:28 AM
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Methodology I CH01_5745.indd 1 8/25/10 11:49:28 AM CH01_5745.indd 2 8/25/10 11:49:28 AM DNA Stable Isotope Probing Yin Chen and J. Colin Murrell 1 DNA (now referred to as metagenomics) were also performed (e.g., see Handelsman et al., 1998; Handelsman, 2005; Chen and Murrell, 2009). These cultivation-independent analyses of environment samples demonstrated that the microorganisms studied previously in pure culture are only a minor fraction of the whole microbial community occurring in nature. The discovery of the enormous diversity of microorganisms in the environment raises the question, what are the functions of these microorganisms in situ? A number of novel approaches have been developed to link microbial identity to their environmental function (reviewed by Wagner, 2009). DNA stable isotope probing (DNA-SIP) is one such method. It was originally developed to determine the identities of active one-carbon utilizers (methylotrophs) in the environment (Radajewski et al., 2000). The DNA-SIP technique relies on the incorporation of stable isotopes into newly synthesized DNA of microorganisms incubated with specific isotope-labeled substrate and subsequent isopycnic centrifugation to separate stable isotope-labeled DNA (“heavy” DNA) from unlabeled background DNA (“light” DNA), followed by identification and characterization of labeled “heavy” DNA from target microbes. Since its development, DNA-SIP has INTRODUCTION: OVERVIEW INCLUDING BASIC CONCEPTS Microbiologists realized that there was an urgent need to develop novel approaches in order to understand the diversity of microorganisms when it was realized that only 0.1% to 10% of microorganisms present in the environment can be cultivated in the laboratory (Amann et al., 1995). A number of new cultivation and isolation techniques have been developed since then to cultivate microorganisms present in the environment (reviewed by Zengler, 2009). The last few decades have also witnessed rapid developments in culture-independent molecular methods (reviewed by Wagner, 2009), originally pioneered by Norman Pace and coworkers, who used the 16S rRNA gene as a marker to analyze the diversity of 16S rRNA gene sequences that can be retrieved from environmental DNA samples by PCR (Pace, 1991). Cloning and phylogenetic analyses of so-called “functional genes” (genes encoding key enzymes involved in biogeochemical cycling processes) and subsequent retrieval of larger DNA fragments and whole community sequences from environmental Yin Chen and J. Colin Murrell, Department of Biological Sciences,The University of Warwick,Warwick, CV4 7AL, United Kingdom. 3 CH01_5745.indd 3 8/25/10 11:49:28 AM 4 n chen and murrell been applied in many studies (Fig. 1) (also see <QU1> chapters **–**). The first studies focused on the application of DNA-SIP in finding novel uncultivated microorganisms involved in the metabolism of specific substrates in the environment, such as methane and methanol (Radajewski et al., 2000; 2002; Morris et al., 2002). The results were indeed interesting in that novel 16S rRNA gene sequences unrelated to previously known methylotrophs were identified. Radajewski and colleagues used 13C-labeled methane and methanol to investigate the microorganisms using these compounds in a forest soil (Radajewski et al., 2000; 2002). They found that besides “classical” (i.e., extant and well-characterized) methanotrophs and methylotrophs, 16S rRNA gene sequences related to Acidobacterium were also found in the “heavy” DNA, suggesting a potential role for these bacteria in C1 utilization. Morris and colleagues then used 13C-labeled methane to investigate methanotrophs present in a peat soil, and they found 16S rRNA gene sequences related to Betaproteobacteria in the “heavy” DNA (Morris et al., 2002).The results were surprising since no known methanotrophs are found in the Betaproteobacteria group. These early reports confirmed that DNA-SIP technique can not only link microbial identity (through the cloning and sequencing of 16S rRNA genes and functional genes involved in aerobic methane/methanol oxidation pathways) to function, but also revealed that novel uncultivated C1-utilizing microorganisms are still present in the environment, awaiting cultivation. Subsequently, DNA-SIP was applied to investigate the metabolism of a diverse range of compounds in a number of different environments (Table 1). Madsen and colleagues at Cornell University pioneered the use of DNA-SIP to identify microorganisms involved in bioremediation of toxic environmental compounds (Table 1 and chapter **). Using a low dose (50 ppm) of [13C]naphthalene in an in situ label- <QU2> ing experiment within 8 h, Padmanabhan et al. (2003) demonstrated that Pseudomonas, Acineto­ bacter, and Variovoras were the major naphthalene degraders in that soil. Joen and colleagues revisited the same site and used a higher [13C] naphthalene dose to label the soil over a longer period (54 h), and in addition to Pseudomonas and Variovoras, they found that the majority of the 16S rRNA gene sequences retrieved from the “heavy” DNA were related to Polaromonas (Jeon et al., 2003).They subsequently isolated a strain, CJ2, whose 16S rRNA gene showed high sequence identity to the sequences from the major clones from the “heavy” DNA.The naphthalene dioxygenase (nahAc) gene amplified from this strain clustered into a clade of sequences that were commonly found in naphthalenecontaminated groundwater, but not present in previously cultivated strains, thus confirming the prevalent microorganism in naphthalene 40 30 20 Year CH01_5745.indd 4 2009 2008 2007 2006 2005 2004 2003 2002 0 2001 10 2000 Number of papers in Scopus 50 Figure 1 The numbers of papers published on stable-isotope probing since the first publication of DNA-SIP in 2000. Search was carried out using the Scopus database using key words “stable isotope probing” or “stableisotope probing.” 8/25/10 11:49:29 AM 1. dna stable isotope probing n 5 bioremediation. Encouraged by these early applications of DNA-SIP in bioremediation, a number of studies have subsequently been carried out (Table 1). Most studies have concerned microorganisms involved in the metabolism of benzene-related compounds (Table 1; also see <QU3> chapter 10), although other toxic compounds, such as methyl chloride and methyl bromide, have also been used in DNA-SIP experiments (Miller et al., 2004; Borodina et al., 2005). DNA-SIP has also been used to investigate plant-microbe interactions (Lu et al., 2005; Table 1; also see chapter 9). Lu and colleagues, in an elegant study, used 13CO2 to label rice <QU3> Table 1 Key studies using DNA-SIP for identifying active microorganisms from diverse habitats Substrate Habitat Phylogenetic groups identified Marker genesa Reference Methanotrophs 13CH 4 Peat soil Methylosinus, Methylocystis, uncultivated methanotrophs from RA-14 group, Methylobacter, Methylomonas, novel Betaproteobacteria 16S rRNA, pmoA, Morris et al., mmoX, mxaF 2002 13CH 4 Soda lake sediment Gammaproteobacterial methanotrophs, Methylophilaceae 16S rRNA, pmoA, Lin et al., 2004 mmoX 13CH 4 Movile Cave water and microbial mat Alphaproteobacterial and Gammaproteobacterial methanotrophs, Hyphomicrobium, Methylophilus 16S rRNA, pmoA, Hutchens et al., mmoX, mxaF 2004 13CH 4 Forest soil Methylocystis 16S rRNA, pmoA 13CH 4 Landfill soil originally from a peatbog Methylobacter, Methylomonas, Methylocystis, Methylocella 16S rRNA, pmoA, Cébron et al., mmoX 2007a 13CH 4 Landfill soil Methylobacter, Methylomicrobium, Methylocystis 16S rRNA, pmoA 13CH 4 Landfill soil with worms Methylobacter, Methylosarcina, Methylocystis, Methylomonas, Cytophaga 16S rRNA, pmoA, Hery et al., 2008 mmoX 13CH 4 Methylomicrobium, uncultivated Sediment from beneath a Lophelia Gammaproteobacteria, Methylophaga, Hyphomicrobium pertusa reef 13 Coal mine soil 13 Methylococcaceae, Hyphomicrobiaceae, Activated sludge under denitrifying Methylophilaceae conditions 13 Peat soil CH4 CH4 CH4 Methylosinus, Methylocystis, Methylobacter, Methylosoma, Methylococcus, Methylocella, Methylopila, Hyphomicrobium Methylocystis, Methylocella Dumont et al., 2006 Cébron et al., 2007b 16S rRNA, pmoA, Jensen et al., 2008 mxaF 16S rRNA, pmoA, Han et al., 2008 mmoX, mxaF 16S rRNA, pmoA Osaka et al., 2008 16S rRNA, pmoA Chen et al., 2008 Methylotrophs (continues) CH01_5745.indd 5 8/25/10 11:49:29 AM 6 n chen and murrell Table 1 Key studies using DNA-SIP for identifying active microorganisms from diverse habitats (continued) Substrate Habitat Phylogenetic groups identified Marker genesa Reference 13CH OH 3 Forest soil Uncultivated Alphaproteobacterial methylotrophs, Acidobacterium 16S rRNA, mxaF Radajewski et al., 2000 13CH OH 3 Forest soil Methylocella, Methylocapsa, Methylocystis, Rhodoblastus, Acidobacterium 16S rRNA, mxaF Radajewski et al., 2002 13CH OH 3 Active sludge Methylophilaceae 16S rRNA Ginige et al., 2004 13CH OH 3 Rice field soil Methylobacterium, Methylophilaceae 16S rRNA Lueders et al., 2004a 13C-labeled methanol, methyl­ amine, formalde­ hyde, formate Lake sediment Methylophylaceae, Sphingomonadales Methylophylaceae, Methylophylaceae, Holophaga/Geothrix, Xanthomonadaceae 16S rRNA, pmoA, Nercessian et al., fae 2005 13CH OH 3 Activated sludge Methylophilaceae, Hyphomicrobiaceae 16S rRNA, nirS, nirK Osaka et al., 2006 13CH OH, [13C] 3 Coastal sea water Methylophaga, novel Gammaproteobacteria 16S rRNA, mxaF Neufeld et al., 2007c 13CH OH 3 Coastal sea water Methylophaga 16S rRNA, mxaF Neufeld et al., 2008a 13C-labeled Surface sea water methanol, mono­ methylamine, dime­ thylamine, methyl bromide, and dimethyl sulfide Methylophaga, Uncultivated Gammaproteobacteria, Rhodobacteraceae, CytophagaFlexibacter-Bacteroides group 16S rRNA Neufeld et al., 2008b 13CH OH 3 Methyloversatilis, Hyphomicrobium 16S rRNA Baytshtok et al., 2009 Methylophaga, Alphaproteobacterial methanotrophs 16S rRNA, pmoA Moussard et al., 2009 methylamine Activated sludge 13C-labeled Marine estuary methanol, methyl­ sediment amine, and methane Methyl halide utilizers 13CH Cl 3 Soil Rhodobacter, Lysobacter, Nocardioides 16S rRNA, cmuA Miller et al., 2004 13 CH3Br Soil Burkholderia 16S rRNA, cmuA Miller et al., 2004 13CH Cl 3 Soil Hyphomicrobium, Aminobacter cmuA Borodina et al., 2005 Pollutant degraders [13C]phenol [13C6]naphthalene 13C-caffeine Soil Pseudomonas, Pantoea, Acinetobacter, Enterobacter, Stenotrophomonas, Alcaligenes Pseudomonas, Acinetobacter, Variovorax, Acinetobacter, Enterobacter, Stenotrophomonas, Pantoea 16S rRNA Padmanabhan et al., 2003 [13C6]naphthalene Coal tar waste contaminated aquifer Polaromonas naphthalenivorans 16S rRNA Jeon et al., 2003 CH01_5745.indd 6 8/25/10 11:49:29 AM 1. dna stable isotope probing n 7 Habitat Phylogenetic groups identified Marker genesa [13C]phenol Agriculture soil Kocuria, Staphylococcus, Pseudomonas 16S rRNA DeRito et al. 2005 [13C7]benzoate Marine sediment or contaminated sediment — nosZ Gallagher et al., 2005 13C-labeled naphthalene Soil Acidovoras, Pseudomonas, Intrasporangium 16S rRNA Yu and Chu, 2005 [12C6]salicylate, [13C]naphthalene phenanthrene Bioreactor treating PAHcontaminated soil Acidovoras, Pseudomonas, Ralstonia 16S rRNA Singleton et al., 2005 [13C]pyrene bioreactor-treated soil Sphingomonas, uncultivated Betaand Gammaproteobacteria 16S rRNA Singleton et al., 2006 13C-labeled 2,4dichlorophenoxyacetic acid Agriculture soil Betaproteobacteria related to Ramlibacter (Comamonadaceae) 16S rRNA Cupples and Sims, 2007 [13C]phenanthrene, [13C]pyrene PAHcontaminated soil Acidovorax 16S rRNA Singleton et al., 2007 13C-polychlorinated biphenyls Pine tree soil Pseudonocardia, Kirbella, Nocardiodes, Sphingomonas 16S rRNA, ARHD Leigh et al., 2007 [13C6]benzene Coal gasification soil Deltaproteobacteria, Clostridia, Actinobacteria 16S rRNA Kunapuli et al., 2007 [13C]pyrene PAHcontaminated soil Uncultivated Gammaproteobacteria 16S rRNA Jones et al., 2008 [13C]benzene benzenedegrading sulfidogenic consortium enrichment An uncultivated bacterium from the 16S rRNA family Desulfobacteraceae Oka et al., 2008 acid PAHcontaminated soil Ralstonia, Pseudomonas 16S rRNA Powell et al., 2008 acid Agriculture field soil Burkholderia 16S rRNA Pumphrey and Madsen, 2008 [13C]benzene Freshwater sediment Pelomonas 16S rRNA Liou et al., 2008 [13C6]benzene anaerobic benzenedegrading enrichment culture Cryptanaerobacter, Pelotomaculum, uncultivated Epsilonproteobacteria 16S rRNA Herrmann et al., 2009 [13C12]biphenyl PCBcontaminated soil Hydrogenophaga 16S rRNA, bphA Uhlik et al., 2009 [13C]biphenyl PCBcontaminated river sediment Achromobacter, Pseudomonas 16S rRNA, bphA Sul et al., 2009 Substrate 13C -salicylic 6 13C-benzoic Reference (continues) CH01_5745.indd 7 8/25/10 11:49:29 AM 8 n chen and murrell Table 1 Key studies using DNA-SIP for identifying active microorganisms from diverse habitats (continued) Substrate Habitat Phylogenetic groups identified Marker genesa Trichosporon 18S-28S internal transcribed spacer region Reference [13C]phenol Agriculture soil Ring-13C6-toluene Agriculture soil Candidate phylum TM7 16S rRNA Luo et al., 2009 Ring-15N3hexahydro-1,3,5trinitro-1,3,5triazine (RDX) Groundwater Actinobacteria, Alphaproteobacteria, Gammaproteobacteria 16S rRNA, xplA Roh et al., 2009 [13C]acetate Activated sludge Comamonadaceae, Rhodocyclaceae 16S rRNA Ginige et al., 2005 [13C]acetate Activated sludge Comamonadaceae, Rhodocyclaceae, Rhodobacteraceae 16S rRNA, nirS, nirK Osaka et al., 2006 [13C]acetate Soil Syntrophus, Propionibacterium, Geo­ bacter, Methanosaeta, Methanosarcina 16S rRNA Chauhan and Ogram, 2006b [13C]acetate Arsenic contaminated aquifer sediments Sulfurospirillum, Desulfotomaculum, Geobacter 16S rRNA, arrA Lear et al., 2007 [13C]acetate Groundwater Proteobacteria, Firmicutes 16S rRNA Longnecker et al., 2009 DeRito and Madsen, 2009 Acetate utilizers Polysaccharide utilizers [13C]cellulose Soil Dyella, Mesorhizobium, Sphingomonas, Myxobacteria 16S rRNA Haichar et al., 2007 13C-labeled wheat residue Soil Betaproteobacteria and Gammaproteo­ bacteria 16S rRNA Bernard et al., 2007 [13C]cellulose Municipal soil waste Firmicutes, Bacteroidetes, Gamma­ proteo­bacteria 16S rRNA Li et al., 2009 13C-labeled Copper contaminated soil Betaproteobacteria 16S rRNA Bernard et al., 2009 Freshwater marshes Pelotomaculum, Syntrophobacter, Smithella propionica, sulfatereducing prokaryotes, Pelobacter, Methanosarcina Syntrophospora, Syntrophomonas, Pelospora, sulfate-reducing prokaryotes, Methanosarcina 16S rRNA Chauhan and Ogram, 2006a Soil Arthrobacter, Pseudomonas, Acinetobacter, Massilia, Flavobacterium, Pedobacter 16S rRNA Padmanabhan et al., 2003 16S rRNA, nifH Buckley et al., 2007b wheat residue Fatty acids degraders [13C]propionate [13C]butyrate Glucose utilizers [13C]glucose Microorganisms in nitrogen metabolism 15N 2 CH01_5745.indd 8 Soil Rhizobiales, Actinobacteria, Alphaproteobacteria 8/25/10 11:49:29 AM 1. dna stable isotope probing n 9 Substrate Habitat Phylogenetic groups identified Marker genesa Reference 15N 2 Soil Rhizobiales, Methylosinus, Methylocystis, novel bacteria nifH Buckley et al., 2008 K213CO3 Lake sediment Nitrosomonas 16S rRNA Whitby et al., 2001 Na213CO3 Water sediment Nitrosomonas, Nitrospira 16S rRNA Freitag et al., 2006 13CO 2 Agricultural soil Nitrospira 16S rRNA Jia and Conrad, 2009 NaH13CO3 Movile cave water Nitrosomonas, Nitrospira, Candidatus and microbial mat “Nitrotoga” 16S rRNA, amoA Chen et al., 2009 rbcL Warwrik et al., 2009 15 Seawater N-labeled ammon­ium, nitrate, urea and glutamic acid Synechococcus, diatoms Microorganisms in sulfur metabolism 13C-labeled Enrichment of glucose, acetate and marine sediment slurry pyruvate Desulfococcus, Desulfosarcina, Desulfobacter, the candidate division JS1, Firmicutes, novel bacteria 16S rRNA Webster et al., 2006 13NaHCO 3 Thiobacillus/Halothiobacillus, Thiobacter 16S rRNA, soxB Chen et al., 2009 Geobacter Acidobacteria, Firmicutes, Deltaproteobacteria, Betaproteobacteria 16S rRNA Burkhardt et al., 2009 Movile Cave water and microbial mat Iron-reducing bacteria [13C]ethanol [13C]acetate Iron-rich, Uranium contaminated soil Plant-microbe interaction 13CO 2 Rice root Methanosarcinaceae, rice cluster-1 Archaea, Methanobacteriales 16S rRNA Lu et al., 2005 13CO 2 Soil grown with different plant species Myxococcus, Enterobacter, Rhizobiales 16S rRNA Haichar et al., 2008 13CO 2 Soil grown with Rhizobiaceae, Syncephalis depressa Arabidopsis thaliana 16S rRNA, 18S rRNA Bressan et al., 2009 13 Potato cultivars Acinetobacter and Acidovorax (active bacterial endophyte) 16S rRNA Rasche et al., 2009 Rice field soil Cercozoa 16S rRNA Lueders et al., 2004b 16S rRNA Chauhan et al., 2009 CO2 Bacterial predators 13 CH3OH 13C-labeled Freshwater estuary Bdellovibrio-like organisms Gammaproteobacteria apmoA, particulate methane monooxygenase subunit A; mmoX, soluble methane monooxygenase subunit; mxaF, methanol dehydrogenase large subunit; fae, formaldehyde activating enzyme; cmuA, chloromethane utilization gene subunit A; norZ, nitric oxide reductase subunit; ARDH, aromatic ring hydroxylating dioxygenase; bphA, benzoate-para-hydroxylase; xplA, RDX-degrading catabolic gene; nirS, nitrite reductase; nirK, nitrite reductase; arrA, As(V) respiratory reductase gene; nifH, nitrogenase reductase subunit; amoA, ammonium monooxygenase subunit A; rbcL, ribulose-bisphosphate carboxylase large subunit; soxB, thiosulfate-oxidizing Sox enzyme complex subunit.9 CH01_5745.indd 9 8/25/10 11:49:29 AM 10 n chen and murrell plants in order to identify methanogens in the vicinity of the roots of rice plants that produced methane through hydrogenotrophic methanogenesis or through acetate cleavage (Lu et al., 2005). They demonstrated the activity of the RC-I lineage of methanogens, which at that <QU4> time had no cultured representatives, the Metha­ nosarcinaceae and the Methanobacteriaceae during rice root incubations with 13CO2.They showed that RC-I methanogens were responsible for production of methane from H2 and CO2 and that Methanosarcinaceae might contribute to both hydrogen- and acetate-dependent methane production. Another interesting study used DNASIP to identify active bacterial endophytes in potatoes (Rasche et al., 2009). These authors incubated two cultivars of Solanum tuberosum (Merkur and Desiree) with 13CO2 (350 ppm) for 4 days. Community profiling revealed that although many bacteria species were detected, Acinetobacter and Acidovorax were the dominant bacteria in cultivars Merkur and Desiree, respectively. These bacteria, which exhibit plantbeneficial functions, were found previously in potatoes, and these results demonstrated that bacterial endophytes found in above-ground potato tissues directly metabolize organic matter from plants (Rasche et al., 2009). Another important application of DNA-SIP is to investigate tropic interactions in the environ<QU3> ment (Table 1; also see chapter 11). Lueders and colleagues used 13C-labeled methanol to investigate organisms involved in methanol turnover in a rice field soil (Lueders et al., 2004b). After 43 days of labeling, eukaryote 18S rRNA gene sequences were found in the “heavy” DNA, including those from fungi related to Fusarium and Aspergillus and soil flagellates Cercozoa. Cercozoa are known bacterial predators, thus suggesting a role of these protozoa in grazing methylotrophic bacteria that used [13C]methanol. Rather than using 13C-labeled compounds, Chauhan and colleagues used 13C-labeled bacteria in a microcosm experiment to identify potential predators of bacteria in a river-dominated subtropical estuary located in the Florida Panhandle (Chauhan et al., 2009). The results indicated that Bdellovibrio-like organisms were heavily CH01_5745.indd 10 labeled in the “heavy” DNA. Bdellovibrio-like organisms are obligate and relatively nonspecific predators for Gram-negative bacteria, and therefore the results indicated that predation by Bdellovibrio-like organisms may be an important factor in controlling bacterial communities in aquatic systems. Interestingly, 16S rRNA gene sequences related to Bdellovibrio were also obtained in one of the earliest DNA-SIP experiments by Morris and colleagues (Morris et al., 2002) Besides carbon, nitrogen and oxygen are also components of DNA. Soon after the development of 13C-based DNA-SIP, 15N- and 18O-based DNA-SIP studies were also reported (Cardisch et al., 2005; Cupples et al., 2007; Buckley et al., 2007a, 2007b; Schwartz, 2007). The major obstacle for 15N-based DNA-SIP was insufficient separation of 15N-labeled and unlabeled DNA due to the lower nitrogen content in DNA compared with its carbon content. Studies showed that although it was possible to separate 15N-labeled and 14N-labeled DNA from a single microorganism, there was potential overlap of 15N-labeled DNA with background community DNA (unlabeled) due to the variation of GC content in microorganisms present in the natural environment (Cardisch et al., 2005; Cupples et al., 2007). An intelligent solution was presented by Buckley and colleagues to resolve this problem (Buckley et al., 2007a).They used a second round of ultracentrifugation with “heavy” DNA from the first round but added bis-benzimide to eliminate the effect of GC content on DNA buoyant density.This method proved to be successful when used to separate 15N-labeled Escherichia coli DNA (low GC content) and 14C-labeled Pseudomonas aeruginosa DNA (high GC content), which do overlap in fractions after the first round ultracentrifugation (Buckley et al., 2007a).The applications of 15N-DNA-SIP have been summarized in Table 1 (see also chapter **). The possibility of using 18O-DNA-SIP has also been investigated using <QU3> labeled and unlabeled E. coli DNA (Schwartz, 2007). Although successful, the application of 18O-DNA-SIP is still in its early phase due to the substantial exchange of 18O atoms between 8/25/10 11:49:29 AM 1. dna stable isotope probing n 11 1 Sampling 2 Incubation with stable isotopelabeled compounds (e.g., 13CH4). Measuring substrate utilization activity and estimation of the incorporation of stable isotopes 3 DNA extraction, isopycnic centrifugation, and fractionation 4 Density measurement (e.g., using a digital refractometer), “heavy” DNA identification (e.g., DNA fingerprint analysis) 5 Characterization of “heavy” DNA (PCR, cloning, sequencing, metagenomic library construction, high throughput sequencing, etc.) Figure 2 An overview of key steps in DNA-SIP. water and cellular components such as ATP (also <QU3> see chapter **). METHODS There are five major steps in DNA-SIP: (i) choice of environment, (ii) stable isotope CH01_5745.indd 11 incubations with environmental samples, (iii) DNA extraction and isopycnic separation of stable isotope-labeled DNA from unlabeled DNA, (iv) identification of “heavy” DNA, and (v) characterization of “heavy” DNA. Major steps in DNA-SIP experiments are illustrated in Fig. 2. 8/25/10 11:49:31 AM 12 n chen and murrell Sampling Although it is quite often that environmental samples are taken to laboratories to set up incubation for the sake of convenience, some studies have been performed with the labeling being carried out in situ (e.g., Jeon et al., 2003; Pumphrey and Madsen, 2008). It is sensible to bear in mind that the activity of environmental microorganisms may change dramatically even in a short time period (Janssen, 2008).Therefore, it is better to transport intact soil cores to the laboratories at ambient temperature and set up SIP incubations as soon as possible rather than freeze or store samples. fosmid vectors (Dumont et al., 2006; Neufeld et al., 2008a; Chen et al., 2008). Extracted DNA can then be loaded onto a cesium chloride (CsCl) gradient for isopycnic centrifugation and separation of labeled “heavy” DNA from unlabeled background DNA (“light” DNA). Since those target microorganisms that feed on the labeled substrate will incorporate heavy isotope into their newly synthesized DNA, their DNA will become “heavier” and thus can be separated from the “light” DNA. Full details and tips on exactly how to set up CsCl gradients (concentrations, densities, etc) can be found in Neufeld et al. (2007b). Incubation Once the samples are transported to the laboratory, incubation with the desired isotope-labeled compounds should be immediately carried out. The concentration of the substrate and incubation time should be selected carefully not only to minimize carbon flow from primary utilizers to secondary utilizers (a phenomenon known as cross-feeding), but also to ensure sufficient incorporation of labeling into microbial DNA. It is thus necessary to monitor the utilization of the substrate during microcosm incubations by measuring the concentration of the substance as it is consumed. In our experience, incorporation of ~50 mmol of 13C per g (for soil/ sediment samples) or ~5 mmol of 13C per ml (for water samples) is sufficient to detect active microorganisms above background. This assumes that at least half of the carbon is incorporated into microbial biomass (the remainder might be released as 13CO2 during microcosm incubations). Identification and Characterization of “Heavy” DNA A number of methods are available to identify “heavy” DNA after isopycnic centrifugation. Measuring the density of each fraction using a refractometer or simply by weighing a known volume using a digital analytical balance is straightforward, and “heavy” DNA is usually located in the fraction(s) with a density of ~1.725 g ml–1. DNA fingerprinting analysis (e.g., denaturing gradient gel electrophoresis [DGGE]) is also commonly used to compare DNA fingerprints of each fraction, which helps to identify the “heavy” fractions (see review by Neufeld et al., 2007b). 13C-carrier DNA (such as 13C-DNA from yeast) can also be used, and in this case, the identification of “heavy” DNA is easier since the 13C-carrier DNA can be easily identified (Gallagher et al., 2005) visually or by PCR-based 16S rRNA gene assays on gradient fractions. Using isotope ratio mass spectrometry (IRMS) is the most direct way to confirm the present of isotope-labeled DNA; however, this method relies on the availability of an IRMS instrument and requires a relatively large volume of DNA sample. Another method to identify “heavy” DNA is to use a DNA-staining chemical such as ethidium bromide; this method is rarely used now due to the lack of sensitivity (see below), and one should also bear in mind that the preparation of the CsCl density gradient is different between methods that do and do not use ethidium bromide (detailed Isopycnic Centrifugation Once the SIP incubation is terminated, DNA can then be extracted from the incubated samples using standard methods.The subsequent analysis of the “heavy” DNA needs to be taken into account when choosing a method for DNA extraction from incubated samples. For example, it is necessary to minimize shearing if a large insert library is desirable, i.e., cloning of “heavy” DNA into bacterial artificial chromosome (BAC) or CH01_5745.indd 12 8/25/10 11:49:31 AM 1. dna stable isotope probing n 13 methodology is given by Neufeld et al., 2007b). Finally, the “heavy” DNA is often subjected to PCR amplification of 16S rRNA genes and other “functional” genes, which can be used to assess the identity of target microorganisms that utilize the stable isotope-labeled substrate (reviewed by Dumont and Murrell, 2005). In addition, the “heavy” DNA can also be used for making a metagenomic library, for direct high-throughput sequencing and to reconstruct metabolic pathways (reviewed by Chen and Murrell, 2009; also see chapter 6). PROBLEMS AND PITFALLS OF THE TECHNIQUE; ADVANTAGES AND DISADVANTAGES DNA-SIP experiments need to be implemented carefully in order to maximize achievable information and to avoid misinterpretation of resulting data. Here we highlight key considerations that need to be taken into account when setting up a DNA-SIP experiment and interpreting the data. Availability of Stable IsotopeLabeled Compounds Before one designs a DNA-SIP experiment, it is sensible to bear in mind that a suitable stable isotope-labeled compound may not always be commercially available or that it might be extremely expensive. In particular, if one is interested in the metabolism of more complex compounds (such as phenanthrene, cellulose, or others), fully 13C-labeled complex compounds such as these can be difficult to synthesize chemically and are thus costly. The alternative is to carry out in-house chemical synthesis; obviously, this depends on the availability of fully labeled 13C precursors as well as expertise in synthetic chemistry. However, partially 13C-labeled compounds may be available and can be used, in theory, for DNA-SIP (depending on the metabolic pathway of such compounds, i.e., whether the stable isotopelabeled carbon/nitrogen is incorporated into DNA); however, their use is not recommended since there may be insufficient incorporation of stable isotope into cell biomass, which CH01_5745.indd 13 further complicates subsequent identification of “heavy” DNA. However, very recently, partially labeled compounds have been used successfully in DNA-SIP studies (Table 1; Luo et al., 2009; Roh et al., 2009). Luo and colleagues used ringlabeled 13C6-toluene (mono-methylbenzene) in a DNA-SIP experiment with an agriculture soil and, interestingly, they found that bacteria from Candidate phylum TM7 were the major toluene degraders in this soil (Luo et al., 2009). Bacteria from the TM7 group are known to inhabit a wide range of environments, including soils, activated sludge, termite guts, and the human oral cavity. Currently, there is no cultured isolate, and therefore knowledge of the metabolism of this group of bacteria is lacking (Hugenholtz et al., 2001). Using DNA-SIP, this study did however demonstrate the possibility that Can­ didate phylum TM7 is involved in toluene deg<QU3a> radation in soils. Interestingly,TM7-related 16S rRNA gene sequences have been retrieved from aquifer sediment contaminated with BTEX (acronym for benzene, toluene, ethylbenzene, and xylene) (Hugenholtz et al., 2001). Another study by Roh and colleagues used ring-labeled 15N -hexahydro-1,3,5-trinitro-1,3,5-triazine 3 (RDX) to study the key degrader of this explosive, commonly used for military purposes (Roh et al., 2009). Only the nitrogen in the ring was fully labeled with 15N, whereas nitrogen in the nitro group was not labeled, due to the fact that fully labeled 15N6-RDX is not commercially available. Cloning and sequencing of 16S rRNA genes from the “heavy” DNA fraction after the DNA-SIP experiment indicated that diverse groups of bacteria were involved in the utilization of 15N3-RDX. 16S rRNA gene sequences from known RDX degraders, such as Enterobacter cloacae, Pseudomonas fluore­ scens, and Rhodococcus spp. were identified.These two studies suggest that, in the absence of fully labeled compounds, DNA-SIP studies can be carried out using partially labeled compounds if experiments are designed carefully; however, extra care needs to be taken to examine the data critically. For example, in the study by Roh and colleagues, the clone library of 16S rRNA gene from the “heavy” DNA showed that very 8/25/10 11:49:31 AM 14 n chen and murrell diverse bacteria, including Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltapro­ teobacteria, Actinobacteria, and Clostridia, were found (Roh et al., 2009). They stated that “our results suggested that phylogenetically diverse microorganisms were capable of using RDX as a nitrogen source.” However, these bacteria identified from the “heavy” DNA may not be bona fide primary RDX utilizers. For example, the 16S rRNA gene sequences detected in the clone library included those from Methylobacter and Methylobacterium, which are well-known one-carbon utilizers.Although it is possible that these bacteria were using RDX, it is more likely that they were using one-carbon compounds as well as nitrogen compounds released by primary RDX utilizers. In fact, it has been documented that methanol, formaldehyde, and ammonium are the final products of the RDX degradation pathway (McCormick et al., 1981). Furthermore, when using partially labeled compounds, it is likely that DNA from a microorganism that only metabolizes part of the substrate that is not labeled will be absent in the “heavy” DNA. For example, it is obvious that microorganisms that only use nitro-nitrogen in the ring-labeled 15N -RDX would not be seen in this study 3 (Roh et al., 2009). Such facts need to be taken into account, since DNA-SIP using partially labeled compounds will only identify those microorganisms that incorporated labeled isotope into their DNA. Sensitivity of DNA-SIP One of the limitations of DNA-SIP is that its sensitivity is not comparable to that of other SIP techniques, such as RNA-SIP (Manefield et al., 2002), phospholipid fatty acid (PLFA)SIP (Bull et al., 2000; Boschker et al., 1998), and protein-SIP (Jehmlich et al., 2008) (Table 2). Ethidium bromide-based staining methods needed ~500 ng of DNA to visualize the “heavy” DNA band in the CsCl gradient in early DNA-SIP experiments (Neufeld et al., 2007a; Vohra and Murrell, unpublished data). The sensitivity of the staining-based method can be increased to ~100 ng in “heavy” DNA bands if SYBR Safe rather than ethidium bromide is used for staining of DNA in CsCl gradients (Martineau et al., 2008). Another way to improve the sensitivity is to use [13C]labeled carrier DNA (such as 13C-DNA from an Archeon if only the bacterial community is the focus of the study) (Gallagher et al., 2005).This also shortens incubation time considerably since the carrier DNA can be visualized as a “heavy” DNA band in CsCl gradients (carrying with it small amounts of 13C-labeled target DNA). However, the use of carrier DNA is not advisable if one wants to construct a metagenomic Table 2 Comparison of DNA, RNA, PLFA, and protein SIP SIP Advantages Disadvantages DNA-SIP • Instrument requirement is minimal • Whole genomic DNA is available for downstream analyses (e.g., functional gene analyses, genome reconstruction) • Long incubations required • Less sensitive RNA-SIP • More sensitive than DNA-SIP • Requires less labeling therefore minimizes potential cross-feeding • Only rRNA genes can be analyzed • Difficult to extract RNA from soil PLFA-SIP • Very sensitive • Can be used for quantification of relative bacterial abundance • Taxonomic assignment of PLFA is trouble­ some due to the lack of complete database • Labor intensive • Require special instrumentation ProteinSIP • Very sensitive due to the high sensitivity of mass spectrometry • Requires metagenome sequences prior to experimental set-up • Requires special instrumentation CH01_5745.indd 14 8/25/10 11:49:31 AM 1. dna stable isotope probing n 15 library using the “heavy” DNA, since the added 13C-carrier DNA will account for most of the “heavy” DNA retrieved from the CsCl gradient and further complicate the screening for genes of interest or the reconstruction of metabolic pathways of microorganisms of interest. Achievable Yield of “Heavy” DNA in DNA-SIP Experiments Since only primary utilizers of the substrate are targeted by DNA-SIP, it is advisable to use nearin situ concentrations of the target substrate and to minimize the incubation time necessary for sufficient incorporation of 13C label into DNA. As a result, only a few nanograms of “heavy” DNA can usually be retrieved from the CsCl gradient since the total amount of DNA that can be loaded into the CsCl gradient is limited (5 to 10 mg maximum; overloading of the CsCl gradient will cause precipitation of DNA from the CsCl solution).The low amount of “heavy” DNA obtained can be an issue if a metagenomic library is constructed, or if the “heavy” DNA is intended to be used for whole community shotgun sequencing.We have tested the possibility of using multiple displacement amplification (MDA) to amplify “heavy” DNA to generate large quantities of DNA for later analysis and demonstrated that there was minimum bias associated with the MDA technique if >1 ng of “heavy” DNA was used as a template in MDA (see chapter **; Neufeld et al., 2008a; Chen et al., 2008). However, as with other studies where MDA has been used, formation of chimeras in the MDA-generated DNA can occur. This can further complicate subsequent DNA sequence analyses, gene assembly, and metabolic pathway reconstruction (Lasken and Stockwell, 2007). Interpretation of DNA Sequence Data Obtained in SIP Experiments Quite often, the “heavy” DNA isolated from DNA-SIP experiments is used as a template for PCR amplification of 16S rRNA genes and “functional genes.” To assign the taxonomic identity of microorganisms into functional guilds that utilize the target stable isotope-labeled substrate is not always straightforward. Ironically, CH01_5745.indd 15 data analysis of the first DNA-SIP study is an excellent example, where Radajewski and colleagues added 13CH3OH into an acidic forest soil in a microcosm experiment (Radajewski et al., 2000). After labeling, the “heavy” DNA was subjected to PCR amplification of 16S rRNA and mxaF genes (encoding the large subunit of methanol dehydrogenase, a key enzyme in the methanol oxidation pathway in Gram-negative methylotrophs). The results were quite surprising at that time in that sequences related to extant and well-characterized methylotrophs were identified neither in the 16S rRNA gene nor in the mxaF gene clone libraries. In fact, the majority (97%) of the 16S rRNA gene sequences from the clone library were related to 16S rRNA gene sequence of Beijerinckia indica, which is not a methanol utilizer. Furthermore, all of the mxaF sequences from the clone library were related to mxaF gene sequences from uncultivated environmental microorganisms. Therefore, it was difficult to ascertain what were the major methanol utilizers in this forest soil. Subsequently, a novel methane and methanol utilizer, namely Methylocella silvestris BL2, was isolated from an acidic forest soil in Germany (Dunfield et al., 2003). Its 16S rRNA gene is highly similar (>97% identity) to the 16S rRNA genes retrieved from the DNA-SIP experiment (Radajewski et al., 2000). It is therefore tempting to speculate that the major players in the methanol DNA-SIP microcosm experiment were related to this new methane and methanol utilizer, although the mxaF gene from M. silvestris BL2 only showed ~75% identity to the mxaF sequences retrieved from the DNA-SIP experiments. However, very recently, Vorob’ev et al. (2009) isolated a new acidophilic facultative methylotroph species, Methylovirgula ligni, whose 16S rRNA gene as well as mxaF gene showed high identity (~97%) to the corresponding sequences retrieved from the methanol DNA-SIP experiments. This new information indicates that the major players in the DNA-SIP microcosm experiment were likely to have been related to Methylovirgula ligni. However, questions still remain, since in the original methanol DNA-SIP experiment, 3% of the 16S rRNA 8/25/10 11:49:31 AM 16 n chen and murrell genes retrieved from the “heavy” DNA were related to Acidobacterium spp., which are also not methanol utilizers. Are they novel methanol utilizers awaiting to be confirmed experimentally, or are they simply cross-feeding from 13C metabolites produced by primary methanol utilizers? Clearly, this warrants further investigation with the application of novel techniques to address this unsolved mystery (also see later section on future perspectives). One key issue with many DNA-SIP studies is the assignment of functions to microorganisms that are detected in low abundance in “heavy” DNA. The common concern is whether these microorganisms, present in relative low abundance in the “heavy” DNA, are actively involved in the utilization of added stable isotope-labeled substrate, or whether they are detected simply because of contamination from background community DNA with a high GC content. For example, it has been calculated theoretically and confirmed experimentally that 15N-labeled E. coli (GC content, 51%) and 14N-labeled P. aeruginosa (GC content 67%) have similar buoyant densities and could not be separated by ultracentrifugation using CsCl (Buckley et al., 2007a). Extra care needs to be taken when “rare microorganisms” are found in “heavy” DNA obtained from DNA-SIP experiments, since these microorganisms may not be true primary utilizers of the added stable isotope-labeled substrate. Unfortunately, this GC effect may be a pitfall in DNA-SIP studies where authors are quick to claim that “novel” microorganisms have been found to metabolize a particular compound with no further evidence to support their conclusions. However, this GC effect can be minimized, if not completely eliminated, from DNA-SIP experiments by using a second ultracentrifugation step. This can be carried out using the DNA from fractions that correspond to “normal”“heavy” DNA with the addition of bis-benzimide to the CsCl solution, which eliminates the effect of GC content of genomic DNA on its buoyant density (Buckley et al., 2007a).This additional centrifugation step should be carried out by researchers in future DNA-SIP studies. CH01_5745.indd 16 EXAMPLES OF THE USE OF THESE METHODS Peatlands represent one of the major sources of atmospheric methane (Gorham, 1991), but methanotrophs in peatlands are not well studied. In this study, we used DNA-SIP to analyze the active methanotroph populations in a peatland from the United Kingdom (Chen et al., 2008). Peat soils were taken from Moor House (England). Once shipped into the laboratory, 5 g was immediately incubated in a 125-ml crimp-top serum vial with 2% (vol/vol) of 13CH4. Methane consumption during this SIP incubation was followed by measuring the remaining methane present in the headspace using a gas chromatograph. The incubation was stopped when 10, 50, and 140 mmol g–1(wet weight soil) of 13CH4 were consumed, respectively. DNA was then extracted from the incubated soils and loaded into individual CsCl gradients for isopycnic centrifugation to separate labeled “heavy” DNA from “light” DNA. After centrifugation, each tube was fractionated into 12 fractions (each contained ~400 ml of CsCl solution) and the density of each fraction was measured using a digital refractometer (Reichert AR200). DNA from each fraction was then precipitated and used as template for PCR using primers targeting bacterial 16S rRNA genes (341f_GC/ 907r; Muyzer et al., 1993). Denaturing gradient gel electrophoresis (DGGE) fingerprinting analysis was then carried out to compare the fingerprints of all 12 fractions.This helps to determine the “heavy” DNA fraction(s) after fractionation. In this case, we noticed that after the consumption of 10 and 50 mmol of 13CH4 per g (wet weight) of soil during the incubation, there was no identifiable enrichment of 13C-DNA in the fractions, indicating that the incorporation of 13C label into microbial DNA was not sufficient for a successful separation of the DNA of active methanotrophs from the DNA of nonmethanotrophs (i.e., background). However, after the consumption of 140 mmol of 13CH4 per g (wet weight) of soil, we noticed there were two extra bands highlighted in CsCl gradient fraction 7 (corresponding to a density of 1.725, where the majority of “heavy” DNA is normally present) 8/25/10 11:49:31 AM 1. dna stable isotope probing n 17 compared to DGGE fingerprints of fraction 10 (corresponding to a density of 1.710, where the majority of the “light” DNA is normally present) (Fig. 3). Sequencing and analyses of these two bands demonstrated that they showed >97% identity to 16S rRNA sequences from known methanotrophs, i.e., Methylocella and Methylocystis, respectively. In conclusion, by using DNA-SIP, we demonstrated that Methylocella and Methylocystis are probably the most active methane utilizers in this peatland. FUTURE PROSPECTS There is no doubt that DNA-SIP using various labeled compounds will be applied to many different environments to reveal the functions of uncultivated microorganisms. However, DNASIP should also be combined with other molecular ecology techniques (Fig. 4) to enable environmental microbiologists to finally resolve the long-standing question,Who is doing what in the environment? Combining DNA-SIP with Metagenomics (“Focused Metagenomics”) Metagenomics is a method to study microorganisms without the prerequisite of cultivation (Handelsman et al., 1998). Genetic information from environmental samples is retrieved by large-scale sequencing of DNA extracted directly from that environment, or by PCR- and hybridization-based screens of large-insert libraries (reviewed by Handelsman, 2005). Since its development, the metagenomic approach has been widely used and has proven to be extremely powerful in elucidating microorganisms in the environment. Although it is a powerful approach, the major concern with traditional sequence-based metagenomic studies is that the functions of the associated microorganisms are still uncertain due to the lack of a direct link between the DNA sequences retrieved from the environment and the functions that may be encoded in the uncultivated microbes themselves. Recently, a combination of DNA-SIP and metagenomics (“focused metagenomics”) has been developed to act as a “filter” in isolating DNA from functionally relevant microorganisms (see chapter **; Dumont et al., 2006; Chen et al., 2008; Neufeld et al., 2008a; Kalyuzhnaya <QU3> et al., 2008). Dumont and colleagues showed for the first time that, with careful preparation, “heavy” DNA from DNA-SIP experiment could be used for making a metagenome library and that relatively large inserts (up to 30 kb) could be obtained (Dumont et al., 2006). Neufeld and colleagues then demonstrated that Figure 3 DGGE fingerprints of 16S rRNA genes of key fractions from DNA-SIP. Bands that are highlighted were re­amplified and sequenced. CH01_5745.indd 17 8/25/10 11:49:31 AM 18 n chen and murrell Environmental sample Incubation with stable isotopelabeled compounds (13C, 15N) DNA-SIP (Isolation of “heavy” DNA) Identification of stable isotope enrichment at single cell level (e.g., by NanoSIMS, Raman microspectroscopy) Isolation and separation of target single cells (e.g., optical tweezers, flow cytometry) Cultivation and characterization of new species 16S rRNA gene sequences analysis from “heavy” DNA Design specific probes targeting 16S rRNA genes Single cell genomics Combining SIP with singlecell analysis techniques Conventional SIP “Focused metagenomics” (wholecommunity shotgun sequencing; reconstruction of metabolic pathways; screening for novel enzymes and secondary metabolites) Combining SIP with metagenomics Figure 4 An overview of conventional DNA-SIP, combining DNA-SIP with metagenomics (“focused metagenomics”) and combining DNA-SIP with single cell analysis techniques. near-in situ concentrations of substrate could be used for DNA-SIP experiments.The subsequent problems with low yield of “heavy” DNA could be overcome by amplifying the “heavy” DNA by multiple displacement amplification, therefore yielding a sufficient amount for making a fosmid library (Neufeld et al., 2008a). In an elegant study, Kalyuzhnaya and colleagues CH01_5745.indd 18 showed that the near-complete genome of a major one-carbon utilizer, Methylotenera mobilis, could be retrieved from the environment by performing shotgun sequencing of the “heavy” DNA from DNA-SIP experiments (Kalyuzhnaya et al., 2008). These studies demonstrated that DNA-SIP can be used in combination with metagenomics in a focused way to investigate 8/25/10 11:49:32 AM 1. dna stable isotope probing n 19 the function of a subpopulation of environmental microorganisms. We predict this approach will be adopted by more researchers in the near future. Combining DNA-SIP with SingleCell Analysis Techniques DNA-SIP is a method that relies on the incorporation of 13C label into microbial DNA, therefore focusing on a group of microorganisms that can perform the same function (i.e., uptake of the added 13C-labeled substrate). One of the increasing interests for environmental microbiologists is to understand the function of environmental microorganisms at a single-cell level.We predict that the combination of DNASIP with contemporary single-cell analysis techniques, such as Raman microspectroscopy <QU3> (see chapter **) and nano-SIMS (see chapter **), will help to determine the functions of environmental microorganisms at both the population level and the single cell level (Huang et al., 2009). In a study using SIP, Huang and colleagues found that Pseudomonas spp. and Acidovo­ rax spp. were the major naphthalene utilizers at a contaminated groundwater site (Huang et al., 2009).The 16S rRNA gene sequences retrieved from the SIP experiments were used to design specific probes targeting 16S rRNA genes of Acidovorax spp. and Pseudomonas spp. Cells that hybridized with Acidovorax-specific 16S rRNA gene probes were further analyzed by Raman microspectroscopy.The results indicated that at a low naphthalene concentration (3.8 mM), only Acidovorax spp. incorporated 13C label, whereas Pseudomonas spp. incorporated 13C label at a much higher naphthalene concentration (30 to 300 mM). Since the naphthalene concentration in the groundwater was in the few-micromolar range, this study suggested that Acidovorax spp. were the major naphthalene degraders in situ. Development of High-Throughput Technological Platforms for DNA-SIP It has been shown that DNA-SIP significantly improves gene detection frequency with environmental samples and therefore can reduce the cost of finding a novel enzyme (Schwarz et CH01_5745.indd 19 al., 2006; Dumont et al., 2006). DNA-SIP may offer considerable biotechnological potential in the future for gene mining, especially considering the urgent need for novel enzymes in industry (also see chapter**). However, one key issue needs to be solved. This is the develop- <QU3> ment of a high-throughput production line for analyzing multiple DNA-SIP incubations and subsequent 13C-DNA isolations. DNA-SIP was originally designed to analyze just a few samples simultaneously, and it can be time-consuming. Bioindustry uses high-throughput methods for screening of multiple samples. In order for DNA-SIP to be used for large-scale enzyme discovery, the development of a similar highthroughput technological platform is necessary. This is probably achievable in the future but will need close collaboration between environmental microbiologists and bioindustries. Acknowledgments Y. Chen and J. C. Murrell acknowledge NERC for financial support. References Amann, R. I., W. Ludwig, and K. H. Schleifer. 1995. Identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59:143–169. Baytshtok,V., H. Lu, H. Park, S. Kim, R.Yu, and K. Chandran. 2009. Impact of varying electron donors on the molecular microbial ecology and biokinetics of methylotrophic denitrifying bacteria. Biotechnol. Bioeng. 102:1527–1536. Bernard, L., Maron, V., C. Mougel, V. Nowak, J. Leveque, C. Marol, J. Balesdent, F. Gibiat, and L. Ranjard. 2009. Contamination of soil by copper affects the dynamics, diversity and activity of soil bacterial communities involved in wheat decomposition and carbon storage. Appl. Environ. Microbiol. 75: 7565–7569. Bernard, L., C. Mougel, P.-L. Maron, V. Nowak, J. Leveque, C. Henault, F. el Zahar Haichar, O. Berge, C. Marol, J. Balesdent, F. Gibiat, P. Lemanceau, and L. Ranjard. 2007. Dynamics and identification of soil microbial populations actively assimilating carbon from 13C-labelled wheat residue as estimated by DNA- and RNA-SIP techniques. Environ. Microbiol. 9:752–764. Borodina, E., M. J. Cox, I. R. McDonald, and J. C. Murrell. 2005. Use of DNA-stable isotope probing and functional gene probes to investigate 8/25/10 11:49:32 AM 20 n chen and murrell the diversity of methyl chloride-utilizing bacteria in soil. Environ. Microbiol. 7:1318–1328. Boschker, H. T. S., S. C. Nold, P. Wellsbury, D. Bos, W. de Graaf, R. Pel, R. J. Parkes, and T. E. Cappenberg. 1998. Direct linking of microbial populations to specific biogeochemical processes by 13C-labelling of biomarkers. Nature 392: 801–804. Bressan, M., M. A. Roncato, F. Bellvert, G. Comte, F. Z. Haichar, W. Achouak, and O. Berge. 2009. Exogenous glucosinolate produced by Arabidopsis thaliana has an impact on microbes in the rhizosphere and plant roots. ISME J. 3:1243–1257. Buckley, D. H., V. Huangyutitham, S. F. Hsu, and T. A. Nelson. 2007a. Stable isotope probing with 15N achieved by disentangling the effects of genome G+C content and isotope enrichment on DNA density. Appl. Environ. Microbiol. 73:3189–3195. Buckley, D. H., V. Huangyutitham, S. F. Hsu, and T. A. Nelson. 2007b. Stable isotope probing with 15N reveals novel noncultivated diazotrophs in soil. 2 Appl. Environ. Microbiol. 73:3196–3204. Buckley, D. H., V. Huangyutitham, S. F. Hsu, and T. A. Nelson. 2008. 15N2-DNA-stable isotope probing of diazotrophic methanotrophs in soil. Soil Bio. Biochem. 40:1272–1283. Bull, I. D., N. R. Parekh, G. H. Hall, P. Ineson, and R. P. Evershed. 2000. Detection and classification of atmospheric methane oxidizing bacteria in soil. Nature 405:175–178. Burkhardt, E., D. M. Akob, S. Bischoff, J. Sitte, J. E. Kostka, D. Banerjee, A. C. Scheinost, and K. Kusel. 2009. Impact of biostimulated redox processes on metal dynamics in an iron-rich creek soil of a former uranium mining area. Environ. Sci. Technol. 44:177–183. Cadisch, G., M. Espana, R. Causey, M. Richter, E. Shaw, J. A. W. Morgan, C. Rahn, and G. D. Bending. 2005.Technical considerations for the use of N-15-DNA stable-isotope probing for functional microbial activity in soils. Rapid Commun. Mass Spec­ trom. 19:1424–1428. Cébron, A., L. Bodrossy, Y. Chen, A. C. Singer, I. P. Thompson, J. I. Prosser, and J. C. Murrell. 2007a. Identity of active methanotrophs in landfill cover soil as revealed by DNA-stable isotope probing. FEMS Microbiol. Ecol. 62:12–23. Cébron, A., L. Bodrossy, N. Stralis-Pavese, A. C. Singer, I. P. Thompson, J. I. Prosser, and J. C. Murrell. 2007b. Nutrient amendments in soil DNA stable isotope probing experiments reduce the observed methanotroph diversity. Appl. Environ. Micro­ biol. 73:798–807. Chauhan, A., J. Cherrier, and H. N.Williams. 2009. Impact of sideways and bottom-up control factors on bacterial community succession over a tidal cycle. Proc. Natl. Acad. Sci. USA. 106:4301–4306. CH01_5745.indd 20 Chauhan, A., and A. Ogram. 2006a. Fatty acidoxidizing consortia along a nutrient gradient in the Florida Everglades. Appl. Environ. Microbiol. 72:2400–2406. Chauhan, A., and A. Ogram. 2006b. Phylogeny of acetate-utilizing microorganisms in soils along a nutrient gradient in the Florida Everglades. Appl. Environ. Microbiol. 72:6837–6840. Chen,Y., M. G. Dumont, J. D. Neufeld, L. Bodrossy, N. Stralis-Pavese, N. P. McNamara, N. Ostle, M. J. Briones, and J. C. Murrell. 2008. Revealing the uncultivated majority: combining DNA stable-isotope probing, multiple displacement amplification and metagenomic analyses of uncultivated Methylocystis in acidic peatlands. Environ. Microbiol. 10:2609–2622. Chen, Y., and J. C. Murrell. 2010. When metagenomics meets stable-isotope probing: progress and perspectives. Trends Microbiol. 18:157–163. Chen, Y., L. Wu, R. Boden, A. Hillebrand, D. Kumaresan, H. Moussard, M. Baciu, Y. Lu, and J. C. Murrell. 2009. Life without light: microbial diversity and evidence of sulfur- and ammoniumbased chemolithotrophy in Movile Cave. ISME J. 3:1093–1104. Cupples, A. M., E. A. Shaffer, J. C. Chee-Sanford, and G. K. Sims. 2007. DNA buoyant density shifts during 15N-DNA stable isotope probing. Microbiol. Res. 162:328–334. Cupples, A. M., and G. K. Sims. 2007. Identification of in situ 2,4-dichlorophenoxyacetic acid-degrading soil microorganisms using DNA-stable isotope probing. Soil Biol. Biochem. 39:232–238. Derito, C. M., and E. L. Madsen. 2009. Stable isotope probing reveals Trichosporon yeast to be active in situ in soil phenol metabolism. ISME J. 3:477–485. Derito, C. M., G. M. Pumphrey, and E. L. Madsen. 2005. Use of field-based stable isotope probing to identify adapted populations and track carbon flow through a phenol-degrading soil microbial community. Appl. Environ. Microbiol. 71:7858–7865. Dumont, M. G., and J. C. Murrell. 2005. Stable isotope probing—linking microbial identity to function. Nat. Rev. Microbiol. 3:499–504. Dumont, M. G., S. M. Radajewski, C. B. Miguez, I. R. McDonald, and J. C. Murrell. 2006. Identification of a complete methane monooxygenase operon from soil by combining stable isotope probing and metagenomics analysis. Environ. Microbiol. 8:1240–1250. Dunfield, P. F., V. N. Khmelenina, N. E. Suzina,Y. A. Trotsenko, and S. N. Dedysh. 2003. Methylo­ cella silvestris sp. nov., a novel methanotrophic bacterium isolated from an acidic forest cambisol. Int. J. Syst. Evol. Microbiol. 53:1231–1239. Freitag, T. E., L. Chang, and J. I. Prosser. 2006. Changes in the community structure and activity 8/25/10 11:49:32 AM 1. dna stable isotope probing n 21 of betaproteobacterial ammonia-oxidizing sediment bacteria along a freshwater-marine gradient. Environ. Microbiol. 8:684–696. Gallagher, E., L. McGuinness, C. Phelps, L. Y. Young, and L. J. Kerkhof. 2005. 13C-carrier DNA shortens the incubation time needed to detect benzoate-utilizing denitrifying bacteria by stable-isotope probing. Appl. Environ. Microbiol. 71:5192–5196. Ginige, M. P., P. Hugenholtz, H. Daims, M. Wagner, J. Keller, and L. L. Blackall. 2004. Use of stable-isotope probing, full-cycle rRNA analysis, and fluorescence in situ hybridization-microautoradiography to study a methanol-fed denitrifying microbial community. Appl. Environ. Microbiol. 70:588–596. Ginige, M. P., J. Keller, and L. L. Blackall. 2005. Investigation of an acetate-fed denitrifying microbial community by stable isotope probing, full-cycle rRNA analysis, and fluorescent in situ hybridizationmicroautoradiography. Appl. Environ. Microbiol. 71: 8683–8691. Gorham, E. 1991. Northern peatlands: role in the carbon cycle and probable responses to climatic warming. Ecol. Appl. 1:182–195. Handelsman, J. 2005. Metagenomics: application of genomics to uncultured microorganisms. Microbiol. Mol. Bio. Rev. 68:669–685. Handelsman, J., M. R. Rondon, S. F. Brady, J. Clardy, and R. M. Goodman. 1998. Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products. Chem. Biol. 5:245–249. Han, B., Y. Chen, G. Abell, H. Jiang, L. Bodrossy, J. Zhao, J. C. Murrell, and X. H. Xing. 2008. Diversity and activity of methanotrophs in alkaline soil from a Chinese coal mine. FEMS Microbiol. Ecol. 70:40–51. Haichar, F. E., W. Achouak,R. Christen, T. Heulin, C. Marol, M. F. Marais, C. Mougel, L. Ranjard, J. Balesdent, and O. Berge. 2007. Identification of cellulolytic bacteria in soil by stable isotope probing. Environ. Microbiol. 9:625–634. Haichar, F. E., C. Marol, O. Berge, J. I. RangelCastro, J. I. Prosser, J. Balesdent, T. Heulin, and W. Achouak. 2008. Plant host habitat and root exudates shape soil bacterial community structure. ISME J. 2:1221–1230. Héry, M., A. C. Singer, D. Kumaresan, L. Bodrossy, N. Stralis-Pavese, J. I. Prosser, I. P. Tompson, and J. C. Murrell. 2008. Effect of earthworms on the community structure of active methanotrophic bacteria in a landfill cover soil. ISME J. 2:92–104. Herrmann, S., S. Kleinsteuber, A. Chatzinotas, S. Kuppardt, T. Leuders, H. H. Richnow, and C. Vogt. 2009. Functional characterization of an anaerobic benzene-degrading enrichment culture by DNA stable-isotope probing. Environ. Microbiol. 12:401–411. CH01_5745.indd 21 Huang, W. E., A. Ferguson, A. C. Singer, K. Lawson, I. P. Thompson, R. M. Kalin, M. J. Larkin, M. M. Bailey, and A. S.Whiteley. 2009. 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. Appl. Environ. Microbiol. 75:234–241. Hugenholtz, P., G. W. Tyson, R. I. Webb, A. M. Wagner, and L. L. Blackall. 2001. Investigation of candidate division TM7, a recently recognized major lineage of the domain bacteria with no known pure-culture representatives. Appl. Environ. Microbiol. 67:411–419. Hutchens, E., S. Radajewski, M. G. Dumont, I. R. McDonald, and J. C. Murrell. 2004. Analysis of methanotrophic bacteria in Movile Cave by stableisotope probing. Environ. Microbiol. 6:111–120. Janssen, P. H. 2008. New cultivation strategies for terrestrial microorganisms. In K. Zengler (ed.), Ac­ cessing Uncultivated Microorganisms: from the Environ­ ment to Organisms and Genomes and Back. ASM Press, Washington, DC. Jehmlich, N., F. Schmidt, M. von Bergen, H.H. Richnow, and C. Vogt. 2008. Protein-based stable isotope probing (protein-SIP) reveals active species within anoxic mixed cultures. ISME J. 2:1122–1133. Jensen, S., J. D. Neufeld, N. K. Birkeland, M. Hovland, and J. C. Murrell. 2008. Methane assimilation and trophic interaction with marine Methylomicro­ bium in deep-water coral reef sediment off the coast of Norway. FEMS Microbiol. Ecol. 66:320–330. Jeon, C. O., W. Park, P. Padmanabhan, C. DeRito, J. R. Snape, and E. L. Madsen. 2003. Discovery of a bacterium, with distinctive dioxygenase, that is responsible for in situ biodegradation in contaminated sediment. Proc. Natl. Acad. Sci. USA 100:13591–13596. Jia, Z., and R. Conrad. 2009. Bacteria rather than Archaea dominate microbial ammonia oxidation in an agricultural soil. Environ. Microbiol. 11:1658–1671. Jones, M. D., D. R. Singleton, D. P. Cartensen, S. N. Powell, J. S. Swanson, F. K. Pfaender, and M. D. Aitken. 2008. Effect of incubation conditions on the enrichment of pyrene-degrading bacteria identified by stable-isotope probing in an aged, PAHcontaminated soil. Microb. Ecol. 56:341–349. Kalyuzhnaya, M. G., A. Lapidus, N. Ivanova, A. C. Copeland, A. C. McHardy, E. Szeto, A. Salamov, I. V. Grigoriev, D. Suciu, S. R. Levine, V. M. Markowitz, I. Rigoutsos, S. G. Tringe, D. C. Bruce, P. M. Richardson, M. E. Lidstrom, and L. Chistoserdova. 2008. High-resolution metagenomics targets specific functional types in complex microbial communities. Nat. Biotechnol. 26:1029–1034. 8/25/10 11:49:32 AM 22 n chen and murrell Kunapuli, U.,T. Lueders, and R. U. Meckenstock. 2007.The use of stable isotope probing to identify key iron-reducing microorganisms involved in ­anaerobic benzene degradation. ISME J. 1:643–653. Lasken, R. S., and T. B. Stockwell. 2007. Mechanism of chimera formation during the multiple displacement amplification reaction. BMC Biotechnol. 7:19. Lear, G. B. Song, A. G. Gault, D. A. Polya, and J. R. Lloyd. 2007. Molecular analysis of arsenatereducing bacteria within Cambodian sediments following amendment with acetate. Appl. Environ. Microbiol. 73:1041–1048. Leigh, M. B., V. Pellizari, O. Uhlí-k, R. Sutka, J. Rodrigues, N. E. Ostrom, J. Zhou, and J. M. Tiedje. 2007. Biphenyl-utilizing bacteria and their functional genes in a pine root zone contaminated with polychlorinated biphenyls (PCBs). ISME J. 1:134–148. Li, T., L. Mazeas, A. Sghir, G. Leblon, and T. Bouchez. 2009. Insights into networks of functional microbes catalyzing methanization of cellulose under mesophilic conditions. Environ. Microbiol. 11:889–904. Lin, J. L., S. Radajewski, B. T. Eshinimaev, Y. A. Trotsenko, I. R. McDonald, and J. C. Murrell. 2004. Molecular diversity of methanotrophs in Transbaikal soda lake sediments and identification of potential active populations by stable isotope probing. Environ. Microbiol. 6:1049–1060. Liou, J. S.-C., C. M. DeRito, and E. L. Madsen. 2008, Field-based and laboratory stable isotope probing surveys of the identities of both aerobic and anaerobic benzene-metabolizing microorganisms in freshwater sediment. Environ. Microbiol. 74:4111–4118. Longnecker, K., A. Da Costa, M. Bhatia, and E. B. Kujawinski. 2009. Effect of carbon addition and predation on acetate-assimilating bacterial cells in groundwater. FEMS Microbiol. Ecol. 70:124–138. Lu,Y.,T. Lueders,W. Michael, M.W. Friedrich, and R. Conrad. 2005. Detecting active methanogenic populations on rice roots using stable isotope probing. Environ. Microbiol. 7:326–336. Lueders, T., M. Manefield, and M. W. Friedrich. 2004a. Enhanced sensitivity of DNA- and rRNAbased stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation gradients. Environ. Microbiol. 6:73–78. Lueders, T., B. Wagner, P. Claus, and M. W. Friedrich. 2004b. Stable isotope probing of rRNA and DNA reveals a dynamic methylotroph community and trophic interactions with fungi and protozoa in oxic rice field soil. Environ. Microbiol. 6:60–72. Luo, C., S. Xie, W. Sun, X. Li, and A. M. Cupples. 2009. Identification of a novel toluene-degrading bacterium from the candidate phylum TM7, as deter- CH01_5745.indd 22 mined by DNA stable isotope probing. Appl. Environ. Microbiol. 75:4644–4647. Manefield, M., A. S.Whiteley, R. I. Griffiths, and M. Bailey. 2002. RNA stable isotope probing, a novel means of linking microbial community function to phylogeny. Appl. Environ. Microbiol. 68:5367–5373. Martineau, C., L. G.Whyte, and C.W. Greer. 2008. Development of SYBR safeTM technique for the sensitive detection of DNA in cesium chloride density gradient for stable isotope probing assays. J. Microbiol. Methods. 73:199–202. McCormick, N. G., J. H. Cornell, and A. M. Kaplan. 1981. Biodegradation of hexahydro-1,3,5-trinitro1,3,5-triazine. Appl. Environ. Microbiol. 42:817–823. Miller, L. G., K. L. Warner, S. M. Baesman, R. S. Oremland, I. R. McDonald, S. Radajewski, and J. C. Murrell. 2004. Degradation of methyl bromide and methyl chloride in soil microcosms: use of stable C isotope fractionation and stable isotope probing to identify reactions and the responsible microorganisms. Geochim. Cosmochim. Acta 68:3271–3283. Morris, S. A., S. Radajewski, T. W. Willison, and J. C. Murrell. 2002. Identification of the functionally active methanotroph population in a peat soil microcosm by stable-isotope probing. Appl. Environ. Microbiol. 68:1446–1453. Moussard, H., N. Stralis-Pavese, L. Bodrossy, J. D. Neufeld, and J. C. Murrell. 2009. Identification of active methylotrophic bacteria inhabiting surface sediment of a marine estuary. Environ. Microbio. Rep. 1:424–433. Muyzer, G., E. C. de Waal, and A. G. Uitterlinden. 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 59:695–700. Nercessian, O., E. Noyes, M. G. Kalyuzhnaya, M. E. Lidstrom, and L. Chistoserdova. 2005. Bacterial populations active in metabolism of C1 compounds in the sediment of Lake Washington, a freshwater lake. Appl. Environ. Microbiol. 71:6885–6899. Neufeld, J.D., R. Boden, H, Moussard, H, Schäfer, and J. C. Murrell. 2008b. Substrate-specific clades of active marine methylotrophs associated with a phytoplankton bloom in a temperate coastal environment. Appl. Environ. Microbiol. 74:7321–7328. Neufeld, J. D., Y. Chen, M. G. Dumont, J. C. Murrell. 2008a. Marine methylotrophs revealed by stable-isotope probing, multiple displacement amplification and metagenomics. Environ. Microbiol. 10:1526–1535. Neufeld, J. D., M.G. Dumont, J. Vohra, and J. C. Murrell. 2007a. Methodological considerations for the use of stable isotope probing in microbial ecology. Microb. Ecol. 53:435–442. 8/25/10 11:49:32 AM 1. dna stable isotope probing n 23 Neufeld, J. D., H. Schafer, M. J. Cox, R. Boden, I. R. McDonald, and J. C. Murrell. 2007c. Stableisotope probing implicates Methylophaga spp. and novel Gammaproteobacteria in marine methanol and methylamine metabolism. ISME J. 1:480–491. Neufeld, J. D., J.Vohra, M. G. Dumont, T. Lueers, M. Manefield, M. W. Friedrich, J. C. Murrell. 2007b. DNA stable-isotope probing. Nat. Protoc. 2:860–866. Oka, A. R., C. D. Phelps, L. M. McGuinness, A. Mumford, L. Y. Young, and L. J. Kerkhof. 2008. Identification of critical members in a sulfidogenic benzene-degrading consortium by DNA stable isotope probing. Appl. Environ. Microbiol. 74:6476–6480. Osaka, T., Y. Ebie, S. Tsuneda, and Y. Ianamori. 2008. Identification of the bacterial community involved in methane-dependent denitrification in activated sludge using DNA stable-isotope probing. FEMS Microbiol. Ecol. 64:494–506. Osaka, T., S. Yoshie, S. Tsuneda, A. Hirata, N. Iwami, and Y. Inamori. 2006. Identification of acetate- or methanol-assimilating bacteria under nitrate-reducing conditions by stable-isotope probing. Microb. Ecol. 52:253–266. Pace, N. R. 1991. Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing. J. Bacteriol. 173:4371–4378. Padmanabhan, P., S. Padmanabhan, C. DeRito, A. Gray, D. Gannon, J. R. Snape, C. S.Tsai,W. Park, C. Jeon, and E. L. Madsen. 2003. Respiration of 13C-labeled substrates added to soil in the field and subsequent 16S rRNA gene analysis of 13C-labeled soil DNA. Appl. Environ. Microbiol. 69:1614–1622. Powell, S. N., D. R. Singleton, and M. D. Aitken. 2008. Effects of enrichment with salicylate on bacterial selection and PAH mineralization in a microbial community from a bioreactor treating contaminated soil. Environ. Sci.Technol. 42:4099–4105. Pumphrey, G. M., and E. L. Madsen. 2008. Fieldbased stable isotope probing reveals the identities of benzoic acid-metabolizing microorganisms and their in situ growth in agricultural soil. Appl. Environ. Microbiol. 74:4111–4118. Rasche, F., T. Leuders, M. Schloter, S. Schaefer, F. Buegger, A. Gattinger, R. C. Hood-Nowotny, and A. Sessitsch. 2009. DNA-stable isotope probing enables the identification of active bacterial endophytes in potatoes. New Phytologist. 181:802–807. Radajewski, S., P. Ineson, N. R. Parekh, and J. C. Murrell. 2000. Stable-isotope probing as a tool in microbial ecology. Nature 403:646–649. Radajewski, S., G. Webster, D. S. Reay, S. A. Morris, P. Ineson, D. B. Nedwell, J. I. Prosser, and J. C. Murrell. 2002. Identification of active methylotroph populations in an acidic forest soil by stableisotope probing. Microbiology 148:2331–2342. CH01_5745.indd 23 Roh, H., C. Yu, M. E. Fuller, and K. Chu. 2009. Identification of hexahydro-1,3,5-trinitro-1,3,5triazine-degrading microorganisms via 15N-stable ­isotope probing. Environ. Sci. Technol. 43:2505– 2511. Schwartz, E. 2007. Characterization of growing ­microorganisms in soil by stable isotope probing with H218O. Appl. Environ. Microbiol. 73:2541–2546. Schwarz, S., T. Waschkowitz, and R. Daniel. 2006. Enhancement of gene detection frequencies by combining DNA-based stable-isotope probing with the construction of metagenomic DNA libraries. World J. Microb. Biotech. 22:363–367. Singleton, D. R., M. Hunt, S. N. Powell, R. Frontera-Suau, and M. D. Aitken. 2007. Stable-isotope probing with multiple growth substrates to determine substrate specificity of uncultivated bacteria. J. Microbiol. Methods 69:180–187. Singleton, D. R., S. N. Powell, R. Sangaiah, A. Gold, L. M. Ball, and M. D. Aitken. 2005. Stableisotope probing of bacteria capable of degrading salicylate, naphthalene, or phenanthrene in a bioreactor treating contaminated soil. Appl. Environ. Microbiol. 71:1202–1209. Singleton, D. R., R. Sangaiah, A. Gold, L. M. Ball, and M. D. Aitken. 2006. Identification and quantification of uncultivated proteobacteria associated with pyrene degradation in a bioreactor treating PAH-contaminated soil. Environ. Microbiol. 10:1736–1745. Sul,W. J., J. Park, J. F. Quensen, III, J. L. Eodrigues, L. Seliger, T. V. Tsoi, G. J. Zylstra, and J. M. Tiedje. 2009. DNA-stable isotope probing integrated with metagenomics for retrieval of biphenyl dioxygenase genes from polychlorinated biphenylcontaminated river sediment. Appl. Environ. Microbiol. 75:5501–5506. Uhlik, O., K. Jecna, M. Mackova, C. Vlcek, M. Hrou­dova, K. Demnerova, V. Paces, and T. Macek. 2009. Biphenyl-metabolizing bacteria in the rhizosphere of horseradish and bulk soil contaminated by polychlorinated biphenyls as revealed by stable isotope probing. Appl. Environ. Microbiol. 75:6471–6477. Vorob’ev, A. V., W. de Boer, L. B. Folman, P. L. Bodelier, N. V. Doronina, N. E. Zuzina, Y. A. Trosenko, and S. N. Dedysh. 2009. Methylovirgula ligni gen. nov., sp. nov., an obligately acidophilic, facultatively methylotrophic bacterium with a highly divergent mxaF gene. Int. J. Syst. Evol. Microbiol. 59: 2538–2545. Wagner, M. 2009. Single-cell ecophysiology of microbes as revealed by Raman microspectroscopy or secondary ion mass spectrometry imaging. Annu. Rev. Microbiol. 63:411–429. 8/25/10 11:49:32 AM 24 n chen and murrell Warwrik, B., A. V. Callaghan, and D. A. Bronk. 2009. Use of inorganic and organic nitrogen by Syn­ echococcus spp. and diatoms on the West Florida shelf as measured using stable isotope probing. Appl. Environ. Microbiol. 75:6662–6670. Webster, G., L. C. Watt, J. Rinna, J. C. Fry, R. P. Evershed, R. J. Parkes, and A. J. Weightman. 2006. A comparison of stable-isotope probing of DNA and phospholipid fatty acids to study prokaryotic functional diversity in sulfate-reducing marine sediment enrichment slurries. Environ. Microbiol. 8:1575–1589. CH01_5745.indd 24 Whitby, C. B., G. Hall, R. Pickup, J. R. Saunders, P. Ineson, N. R. Parekh, and A. McCarthy. 2001. 13 C incorporation into DNA as a means of identifying the active components of ammonia-oxidizer populations. Lett. Appl. Microbiol. 32:398–401. Yu, C. P., and K. H. Chu. 2005. A quantitative assay for linking microbial community function and structure of a naphthalene-degrading microbial consortium. Environ. Sci.Technol. 39:9611–9619. Zengler, K. 2009. Central role of the cell in microbial ecology. Microbiol. Mol. Bio. Rev. 73:712–729. 8/25/10 11:49:32 AM
