Bailey, M

Kilham, K

Lappin-Scott, H

Murrell, C

Prosser, J

Whitby, C

Whiteley, A

University of Essex Research Repository

Stable Isotope Probing Links Taxonomywith Function in Microbial CommunitiesMicrobial ecologists have a culture-independent means for analyzingsources of metabolic activities among complex mixtures of microbesCorinne Whitby, Mark Bailey, Andrew Whiteley, Colin Murrell, Ken Kilham, Jim Prosser, andHilary Lappin-ScottStable isotope probing (SIP) is provingto be a powerful tool for molecularmicrobial ecologists, providing a cul-ture-independent means of investigat-ing populations that conduct particu-lar metabolic processes in specific environments.Moreover, this alternative means for linkingtaxonomic identity with functionmay also assistmicrobiologists in developing strategies for iso-lating elusive microorganisms that are involvedin biogeochemical processes.One approach relies on density gradient cen-trifugation to separate organisms on the basis ofhaving different guanidine-cytosine (GC) con-tents. Furthermore, with steadily increasingavailability of 13C-enriched substrates and thepotential of using other isotopes such as 15N,microbial ecologists can now address challeng-ing questions surrounding natural processes.Several New Approaches to StudyingMicrobes in Ecological SettingsMicrobial ecologists face ongoing challengeswhen trying to identify members of microbialcommunities that are directly involved in partic-ular metabolic processes in specific natural envi-ronments. Traditionally, microbiologists weretrained to isolate and culture microorganismsfrom samples. However, relying on this ap-proach can bias results in favor of rapidly grow-ing microorganisms, which may not be respon-sible for the specific metabolic processes ofinterest.Advances in molecular techniques are provid-ing alternative strategies for microbial ecologiststo characterize organisms within particular hab-itats. In some instances, ecosystem function canbe inferred by relating the microbial groups tothe physical, chemical, and biological character-istics of the particular environment in whichthey are growing. This strategy can work well insimple, structured environments where local mi-crobial communities are dominated by only afew morphologically distinct organisms. Inmore complex communities where themicrobialpopulations are heterogeneous, it remains diffi-cult to unravel specific ecological roles played byparticular organisms within a group.Several recently developed techniques relatephylogeny to ecosystem functions, providing in-sights into ecological questions. One example ismicroautoradiography, which when combinedwith fluorescent in situ hybridization (FISH) ofrRNA signatures, determines the uptake of spe-cific radiochemicals by individual cells. How-ever, FISH requires large numbers of probes toprovide taxonomic resolution equivalent to thatof rRNA-based cloning or fingerprint tech-niques.Another approach relies on using lipid bi-omarkers to analyze functionally active compo-nents within a microbial community. Thismethod uses stable carbon isotopes to label spe-cific lipid biomarkers, such as phospholipidfatty acids (PLFA). Samples are incubated with a13C-enriched substrate, and the biomarkers areextracted and analyzed by isotope ratio massspectrometry. An advantage of PLFA analysis isthat it provides information on biomass of dif-ferent groups. There is also the possibility ofobtaining composite PLFA profiles from mix-tures of uncultivated microbes. Despite an in-creasing knowledge of signature lipids that canCorinne Whitby is aPostdoctoral Re-searcher at the Uni-versity of Exeter,United Kingdom;Mark Bailey is theScience Director forbiodiversity, Centrefor Ecology and Hy-drology, Oxford,United Kingdom;Andrew Whiteley isthe Section Headfor biodiversity,Centre for Ecologyand Hydrology, Ox-ford, United King-dom; Colin Murrellis a Professor inMicrobiology at theUniversity of War-wick, United King-dom; Ken Killham isa Professor of SoilMicrobiology at theUniversity of Aber-deen, United King-dom; Jim Prosser isa Professor in Envi-ronmental Microbi-ology at the Univer-sity of Aberdeen,United Kingdom;and Hilary Lappin-Scott is a Professorof EnvironmentalMicrobiology at theUniversity of Exeter,United Kingdom.Volume 71, Number 4, 2005 / ASM News Y 169be used to identify specific microbial groups,however, this method is limited in its resolution,and hence, applicability.Michael Friedrich and colleagues at the MaxPlanck Institute for Terrestrial Ecology, Mar-burg, Germany, recently developed techniquesfor enhancing the sensitivity of DNA- and RNA-based SIP, including quantitative analysis ofisopycnic centrifugation gradients. Moreover,they are combining RNA- and DNA-SIP tomonitor the dynamics of methylotroph commu-nities and their trophic interactions with fungiand protozoa in rice field soils. Their experi-ments also illustrate how SIP-based techniquescan be used to analyze microbial food webs andinteractions.Stable Isotope Probing Encompassesa Broad Range of TaxaLabeled nucleic acids are biomarkers with thepotential to identify a very wide range of micro-bial taxa. For example, Colin Murrell and hiscollaborators at the University of Warwick,United Kingdom, began using SIP as a methodfor linking metabolic processes in situ directlywith specific members of particular microbialcommunities. Their analytical approach in-volves adding to environmental samples a sub-strate labeled with a stable isotope. To date,their efforts have involved 13C-labeled sub-strates, but other stable isotopes could also beused.During incubations, such labels are incorpo-rated into the biomass of organisms activelyassimilating that labeled substrate. Once nucleicacids become labeled, they can be used as bio-markers. SIP therefore provides a targeted ap-proach to the analysis of in situ ecosystem func-tions. A typical analysis entails separating thelabeled and unlabeled nucleic acids by means ofisopycnic density gradient centrifugation (Fig.1a and b).Early applications of SIP involved character-izing organisms that metabolize one-carbon(C-1) compounds in soils. In those experiments,13C-labeled methanol was added to soil micro-cosms, which were incubated aerobically. Thenthe 13C-enriched DNA was stained withethidium bromide and separated from the 12C-DNA in a cesium chloride gradient. Both labeledand unlabeled DNA fractions can be retrievedusing a needle and syringe under UV illumina-tion.As little as 20% incorporation of a 13C-labeled substrate into DNA is sufficient for re-solving 13C-DNA from 12C-DNA using this ap-proach. In this case, approximately 15 g ofethidium-bromide-stained DNA are required tovisualize distinct bands under UV illumination,and this quantity of DNA sometimes provesdifficult to recover from natural environmentssuch as oligotrophic freshwater lakes where bio-mass is low. However, Murrell and his col-leagues have shown that small amounts of 13C-DNA can be removed from gradients on a“blind” basis, when relying on PCR amplifica-tion to recover those labeled but otherwise in-visible DNA sequences.The sensitivity of the SIP method was greatlyenhanced by an rRNA-based SIP approach de-veloped at the Centre of Ecology andHydrology(CEH), Oxford, by Mike Manefield and co-workers. They can analyze as little as about 500ng of rRNA on cesium trifluoroacetate gradients(CsTFA), using equilibrium density centrifuga-tion to separate labeled and unlabeled RNAmolecules. In such CsTFA gradients, labeled andunlabeled RNA molecules are distributed overseveral fractions, and typically are detected us-ing RT-PCR and DGGE analysis.There is an advantage to targeting 16S rRNAmolecules rather than 16S rRNA genes. Themolecules in the ribosomes are present in highercopy numbers, show higher turnover rates, andare produced independently of cellular replica-F I G U R E 1(a) Density gradient of isotopically labelled DNAfrom a sediment enrichment culture incubated with[13C]-CO2 (Whitby et al., 2001.) Copyright permis-sion pending from Blackwell Publishing Ltd. (b)Density gradient of DNA (pure fractions and amixture of DNA) from Methylobacterium ex-torquens grown on either -[12C]- or -[13C]-methanol(Radajewski et al., 2000. Copyright permission ob-tained from Nature Publishing Group http://www.nature.com/)170 Y ASM News / Volume 71, Number 4, 2005tion. Since RNA molecules offer the same se-quence-based resolution for identifying an or-ganism as DNA analysis, this approach providesa useful biomarker for linkingmetabolic activitywith the organisms responsible for producing it.SIP Is Being Applied in aVariety of Ecological SettingsSIP unambiguously links taxonomic identity tometabolic function across a wide range of eco-systems. It is applicable to microorganisms in-volved in driving natural biogeochemical pro-cesses such as the cycling of carbon and nitrogenand also to those organisms involved in degrad-ing xenobiotics.Moreover, it provides a culture-independent means of investigating how com-plex microbial communities respond to changesin temperature, pH, and substrate concentrations.Specifically, Stefan Radajewski and colleaguesat Warwick have shown that SIP is useful forinvestigating methanol-consuming microorgan-isms in soil. They used 13C-labeled DNA to ana-lyzemicroorganisms in an oak forest soil that usedmethanol as a metabolic substrate for growth.Phylogenetic analysis of the 16S rRNA bacterialgene sequences amplified from the 13C-containingDNA indicated that these soil-dwelling organismsbelonged to two phylogenetically distinct groupsof eubacteria, the -Proteobacteria andAcidobac-terium lineages.In addition to that soil ecosystem, ElenaHutchens and colleagues at the University ofWarwick used DNA-SIP to characterize activeaerobic methanotrophic bacteria in a closed en-vironment. Organisms in a natural groundwatersystem in Movile Cave, whose atmosphere con-tains 1–2% ambient methane plus higher con-centrations in gas bubbles that support thegrowth of microbial mats, were studied. Toidentify the active methanotrophs present in thewater and mat material, microcosms were incu-bated with 10% 13C-labeled methane in theheadspace. Subsequently, the 13C-DNA was an-alyzed by looking at both 16S rRNA genes andfunctional genes by using primer sets that targetmethane monooxygenase genes. The analysisindicates that strains of Methylomonas, Methy-lococcus, and Methylocystis/Methylosinus as-similate the labeled methane. These researcherssurmised that aerobic methanotrophs were ac-tively converting methane into complex organiccompounds in the cave that are used by a diversemicrobial community in this closed ecosystem.DNA-SIP has also been successfully appliedby Ju-Ling Lin and coworkers at Warwick tostudy methanotrophs in soda lake sediments.Methanotrophs in these highly alkaline environ-ments appear to be dominated by Type I meth-anotrophs of the generaMethylomicrobium andMethylobacter.SIP also Provide Insights intoBioremediationStill other groups of soil-dwelling methyl-otrophs, specifically those that degrade methylbromide and methyl chloride, can be identifiedby using SIP, according to Larry Miller and RonOremland at the U.S. Geological Survey inMenlo Park, Calif., working in collaborationwith Colin Murrell and colleagues at Warwick.Their approach entailed adding 13C-labeledmethyl bromide and chloride tomicrocosms andthen amplifying with PCR primers for 16SrRNA genes and also for cmuA, which encodesa specific methyltransferase involved in methylhalide metabolism. Their results suggest that thediversity of methyl halide degraders in soils isconsiderably greater than one would supposebased on those few strains that have been iso-lated. Hyphomicrobium spp. seem to be thedominant MeCl-utilizers in woodland soils.Corinne Whitby and colleagues at LiverpoolUniversity applied SIP to analyze one compo-nent of the nitrogen cycle, namely nitrification infreshwater sediments. In this study, 13C carbondioxide was added to sediment enrichment cul-tures to identify active members within the au-totrophic-ammonia oxidizing communities in ahypereutrophic freshwater lake. Sequence anal-ysis of the labeled fraction indicates 97% ho-mology withNitrosomonas europaea-eutropha.Maneesha Ginige, at the University of Queens-land, Australia, looked at denitrification at awastewater treatment plant. SIP combined withFISH/microautoradiography identify a micro-bial community at a wastewater treatment plantthat can use methanol as a carbon substrateduring denitrification.Researchers seeking to understand particularbioremediation processes often extensivelycharacterize genes and proteins involved in therelevant degradative biochemical pathways.Typically, this information is obtained by study-Volume 71, Number 4, 2005 / ASM News Y 171ing culturable isolates that degrade the com-pounds of interest.However, more recently the RNA-SIP ap-proach is being applied to identify key organ-isms involved in bioremediation processes. Forexample, Mike Manefield and colleagues at theCentre for Ecology and Hydrology, Oxford, areusing RNA-SIP to analyze how microbial com-munities deal with phenol, a recalcitrant com-pound that is widely distributed, highly toxic,and difficult to biodegrade. In their bioreactorstudy (Fig. 2), RNA-SIP analysis indicates thatthe percentage of 13C in RNA increases morethan 10-fold as the cells use labeled phenol,while that of DNA increases 2-fold. Subse-quently, they learned that a member of theThauera genus is critical for degrading phenoland controlling this process. This type of infor-mation could not have been obtained withoutthe use of the SIP-based method.Recognizing SeveralLimitations of SIPAlthough SIP represents an important advancein microbial ecology, using this method hassome disadvantages. For instance, re-searchers assume that microbial popu-lations do not discriminate between la-beled and unlabeled substrates but,instead, will use either one equally.However, small differences in the natu-ral abundance of 13C and 12C isotopesmay lead to preferential assimilation ofcompounds containing the latter iso-tope in many biochemical processes.This, variation in 13C/12C ratios occursdue to differences in substrate, meta-bolic pathways, or environmental fac-tors.One problem in trying to labelDNA with a stable isotope is that theorganism being observed has to beactively replicating when the isotope isadded, which may not always be thecase. However, this problem can becircumvented by doing RNA-SIP in-stead.Another consideration is that indige-nous unlabeled substrates may dilutethe 13C-labeled substrate being addedto a particular environment, therebyreducing the proportion of label in-corporated into the biomass. To pro-duce detectable levels of material, it may benecessary to add excess labeled substrate forextended incubations. As with culture-basedmethods, these excesses could well bias the re-sults—for example, by promoting growth ofparticular organisms and promoting substratecross-feeding. Long incubation times can alsolead to 13C-labeled metabolites being assimi-lated by secondary users within a microbialcommunity.Production of corresponding 12C-labeledcompounds and other trophic interactionswould dilute labeled substrates in complexsystems such as soils. This problem also comesup when studying pathways in which a micro-bial consortium generates many intermediateswhile degrading compounds, making it impos-sible to unravel the role each organism plays inthe overall process. However, this may beovercome by labeling the intermediate sub-strates and tracking the flux of 13C uptakethrough different members of the consortium.Another approach would entail introducingcompounds containing different isotopes,such as 15N, 2H, and 3H.F I G U R E 2Degradation of phenol-13C6 concentration (Œ) and subsequent increase in13C atompercent of RNA (●) and DNA () in the microbial community from an industrial bioreactor.172 Y ASM News / Volume 71, Number 4, 2005SUGGESTED READINGGininge, 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 fluorescent in situ hybridization-microautoradiography to study methanol-fed denitrifyingmicrobial community. Appl. Environ. Microbiol. 70:588–605.Hutchens, E., S. Radajewski, M. G. Dumont, I. McDonald, and J. C. Murrell. 2004. Analysis of methanotrophic bacteria inMovile Cave by stable isotope probing. Environ. Microbiol. 6:111–120.Lueders, T., B. Wagner, P. Claus, and M. W. Friedrich. 2004. Stable isotope probing of rRNA and DNA reveals a dynamicmethylotroph community and trophic interactions with fungi and protozoa in oxic rice field soil. Environ. Microbiol.6:60–72.Lueders, T., M. Manefield, andM.W. Friedrich. 2004. Enhanced sensitivity of DNA- and RNA-based stable isotope probingby fractionation and quantitative analysis of isopycnic centrifugation gradients. Environ. Microbiol. 6:73–78.Lin, J.-L., S. Radajewski, B. T. Eshinimaev, Y. A. Trotsenko, I. R. McDonald, and J. C. Murrell. 2004. Molecular diversity ofmethanotrophs in Transbaikal soda lake sediments and identification of potential active populations by stable isotope probing.Environ. Microbiol. 6:1049–1060.Manefield, M., A. S. Whiteley, R. I. Griffiths, and M. J. Bailey. 2002. RNA stable isotope probing, a novel means of linkingmicrobial community function to phylogeny. Appl. Environ. Microbiol. 68:5367–5373.Manefield, M., A. S. Whiteley, N. Ostle, P. Ineson, and M. J. Bailey. 2002. Technical considerations for RNA-based stableisotope probing: an approach to associating microbial diversity with microbial community function. Rapid Commun. MassSpectrom. 16:2179–2183.Miller, L. G., K. 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 stableisotope probing to identify reactions and the responsible microorganisms. Geochim. Cosmochim. Acta 68:3271–3283.Radajewski, S., P. Ineson, N. R. Parekh, and J. C.Murrell. 2000. Stable-isotope probing as a tool in microbial ecology. Nature403:646–649.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 stable isotope probing. Microbiology 148:2331–2342.Rangel-Castro, J. I., K. Killham, N. Ostle, G. W. Nicol, I. C. Anderson, C. M. Scrimgeour, P. Ineson, A. A. Meharg, and J. I.Prosser 2005. Stable isotope probing analysis of the influence of liming on root exudate utilisation by soil microorganisms.Environ. Microbiol., in press.Whitby, C. B., G. Hall, R. Pickup, J. R. Saunders, P. Ineson, N. R. Parekh, and A. J. McCarthy. 2001. 13C incorporation intoDNA as a means of identifying the active components of ammonia oxidiser populations. Lett. Appl. Microbiol. 32:398–401.Volume 71, Number 4, 2005 / ASM News Y 173

Stable isotope probing links taxonomy with function in microbial communities

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