14 Molecular Methods for Assessing and Manipulating the Diversity of Microbial Populations and Processes Søren J. Sørensen, Anne Kirstine Mu ¨ ller, Lars H. Hansen, and Lasse Dam Rasmussen University of Copenhagen, Copenhagen, Denmark Julia R. de Lipthay Geological Survey of Denmark and Greenland, Copenhagen, Denmark Tamar Barkay Cook College, Rutgers University, New Brunswick, New Jersey I. INTRODUCTION Because most soil bacteria cannot grow on standard laboratory media, a discrepancy of several orders of magnitude between direct microscopic- and viable-cell counts results (1). The reason for this discrepancy is one of the most important questions in microbial ecology (2). This difference most likely reflects the imperfections of our culturing tech- niques, although attempts to improve these techniques produce only a minimal quantitative change (3). In addition, some bacteria, while remaining viable, may lose the ability to grow on media on which they are routinely cultured in response to certain environmental stresses. This suggests that stress may induce a viable but nonculturable (VBNC) physio- logical state (4). This inability to culture most bacteria present in natural soils has, until recently, impaired studies of the relationships between the structure and function of soil microbial communities. One such relationship is flat between soil enzyme activities and the microbial populations that express the genes encoding for these enzymes. This short- coming may now be remedied by the application of a rapidly growing number of molecu- lar-based techniques that allow detection, enumeration, and characterization of microbial populations in natural environments but that do not depend on cultivation. This evolution has been facilitated by the studies of Carl Woese and coworkers, who introduced the concept of 16S ribosomal deoxyribonucleic acid–(rDNA)–based molecular phylogeny (5) and its application to the analysis of microbial communities in their natural habitats (6). The introduction of specific detection techniques and the development of finger- printing techniques for analysis of complex communities have provided the means for Copyright © 2002 Marcel Dekker, Inc. determination of the biodiversity of microbial communities without the bias of cultur- ability. Yet, applying molecular techniques to the analysis of soil microbial communities is a big challenge requiring substantial method development before these techniques be- come generally applicable. Nevertheless, it is clear that we now have an opportunity to link the functional analysis of soils with community composition. This opens up the opportunities to address a new range of questions of the sort, Who is doing what, when, and why?, and for the first time will allow the coupling of enzyme activities to the phy- siological state of specific microbial populations within the soil environment. The first part of this chapter describes different molecular approaches for the analysis of micro- bial communities responsible for major enzyme activities in soil. In addition, these molecu- lar approaches present opportunities for the introduction of new enzyme activities into soils by the deliberate release of recombinant bacteria. This strategy has a great potential in the bioremediation of disrupted and polluted environments and is the subject of the second part of this chapter. Although this review primarily deals with descriptions of bacteria in soil environments, the ideas are relevant to other microbes and to other environ- ments. II. A HOLISTIC APPROACH TO SOIL COMMUNITY DESCRIPTION The composition of complex communities can be described by using a biomarker that is present in all bacteria but shows variation among taxa or functional groups. Several macromolecules, such as nucleic acid (ribonucleic acid [RNA] and DNA) and phospho- lipid fatty acid (PLFA), the major constituents of the membrane of all living cells except the Archaea, have been used frequently as biomarkers in environmental studies. Whole- community PLFA profiles are very useful in studies that define similarities or differences among microbial communities but give less information on the organisms accounting for these similarities or differences. In 1999 Zelles (7) reviewed the use of PLFA in the analy- sis of soil communities. Therefore, the discussion is focused on nucleic acid–based tech- niques. A vast number of methods have been developed to analyze the DNA and RNA that are recovered directly from soil samples. The most detailed genetic information is obtained by sequencing the genes of interest, and one may argue that it is the most obvious method for investigating the heterogeneity of the community. Indeed, the construction of 16s rDNA clone libraries from DNA extracts of natural samples and the subsequent sequence analysis of these clones have revealed the genetic diversity of bacterial communities from many environments, including soil (8–11). These studies showed high genetic diversity and previously undescribed 16s rDNA sequences; in several only novel sequences were found. However, since this is a time-consuming and costly process, only a limited number of clones can be sequenced. In the studies mentioned 30–124 clones were sequenced, numbers that are probably too low to give an accurate overview of the genetic diversity of the microbial community. For example, Borneman and Triplett (9) found no duplicate sequences when investigating 100 clones obtained from Amazonian soil. So, although DNA sequence data provide a suitable descriptor of the many unknown species in the environment, their use is not the method of choice when investigating the dynamics of microbial communities by trying to link enzyme activity in soil to community structure. Instead the use of genetic fingerprinting techniques combined with probe hybrid- ization and sequencing of representative samples could be a better choice. Copyright © 2002 Marcel Dekker, Inc. Figure 1 Diagram of steps employed in genetic fingerprinting of soil bacterial communities. Genetic fingerprinting techniques (Fig. 1) involve extraction and purification of nu- cleic acids directly from the soil, although some investigators prefer to separate the cells from the environment prior to cell lysis (12–14). Extraction is followed by amplification of specific target sequences by using the polymerase chain reaction (PCR). When RNA is the molecule of interest, reverse transcriptase is used to transcribe the RNA sequence to complementary DNA (cDNA), which subsequently is used in PCR amplification. Exam- ination of variations in the amplified target sequences is achieved by separation techniques such as denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electro- phoresis (TGGE), amplified ribosomal DNA restriction analysis (ARDRA), or restriction fragment length polymorphism (RFLP). The steps involved in fingerprinting analyses of soil microbial community structure and function are described in the following sections. A. Nucleic Acids Community analysis may use extracts of both community DNA and RNA from the soil. Genomic DNA is present in all bacteria, active as well as dormant, and in an extracellular form that is protected by adsorption to soil particles (15). Hence, a genetic fingerprint based on community DNA may overestimate the number of intact species present in the community at the time of sampling. On the other hand, the RNA content is generally presumed to be higher in active bacteria than in inactive bacteria. With pure cultures the number of ribosomes and the amount of rRNA are almost proportional to the growth rate of the organism (16). Therefore, RNA-based analysis may provide information on metabolically active subpopulations in the microbial community. Copyright © 2002 Marcel Dekker, Inc. Teske et al. (17) found that DGGE profiles of 16S rDNA and 16S rRNA extracted from the same water column differed markedly. Further results from hybridization with group-specific probes (different groups of sulfate-reducing bacteria) suggested that certain strains played a more significant role in the community because of their activity rather than their abundance. Ribosomal RNA extraction procedures have successfully been applied to soil ecosystems (18,19). Thus, RNA-based analysis is a more appropriate choice in studies that link community structure to enzyme activity. However, recent studies showing rRNA persistance long after cell death questioned the use of rRNA in assessing metabolic activity of cells in natural samples (20). B. Genes Used as Biomarkers The most commonly used genetic marker in community analysis is the small subunit ribosomal RNA (16S rRNA) or the gene encoding for it (16S rDNA). This is a suitable genetic marker in investigations of diversity because each 16S rRNA/DNA nucleotide contains both highly conserved regions that are shared by all organisms and variable re- gions that are unique to specific organisms (or, at least, to closely related groups of organ- isms). This means that PCR primers with universal sequences can be designed to amplify DNA with species-/genus-specific sequences. It is also possible to analyze the genetic diversity of monophyletic groups of bacteria by designing primers specific to the group of interest, e.g., ammonia oxidizers or actinomycetes (21–23). Since some functions have a polyphyletic distribution (i.e., they are present among distantly related taxa) genes specifying the function of interest must be used as the bio- marker rather than rDNA genes (24,25). Henckel et al. (24), who investigated the methane oxidizing microbial community in rice field soil, targeted genes encoding the methane monooxygenase and methanol dehydrogenase enzymes. Likewise, genes coding for spe- cific resistances such as resistance to mercury (26) have been used as targets in genetic fingerprinting techniques. C. Fingerprinting Techniques 1. Restriction Fragment Patterns Restriction fragment length polymorphism (RFLP), also known as amplified rDNA restric- tion analysis (ARDRA), targets sequence differences in species-/group-specific regions of 16S rDNA as reflected by variable number and locations of restriction enzyme recogni- tion sites. Thus, restriction enzyme digests result in a specific number and size of DNA fragments that are distinguished after separation by gel electrophoresis. The more diverse the community is, the more elaborate are its RFLP patterns. Usually three to four different tetrameric enzymes are used to ensure a sufficient number of fragments for diversity analy- sis. The choice of restriction enzymes may greatly influence the results: e.g., 18 of the 23 correct clones found in a study of an anaerobic cyanide degrading consortium remained uncut even after treatment with four different enzymes (27). Either a mixture of PCR- amplified community 16S rDNA or clones derived by cloning this DNA may be analyzed. In both cases, PCR amplification of environmental DNA with 16S rDNA primers precedes RFLP analysis. This method was used to show differences in genetic diversity of bacterial communi- ties from extreme environments. In hypersaline ponds, RFLP performed with the 16S Copyright © 2002 Marcel Dekker, Inc. rDNAamplificationproductsofthenativecommunityshowedadecreasedeubacterial geneticdiversitywithincreasingsalinity,whereasthereversewastrueforArchaea(28). Structuralchangesinthemicrobialcommunityinsoilduetocoppercontaminationwere detectedbySmitandassociates(29)onthebasisofARDRAprofilesofisolates,clones, andsoilcommunityDNA.However,aproblemariseswhenusingRFLPintheinvestiga- tionofmorecomplexmicrobialcommunities.Inanalyzingtheamplificationproductsof communityDNA,differentsequencesresultinadifferentnumberofDNAfragments, dependingonhowmanyrestrictionsitesarepresentinaparticularsequence.Presence ofmanybandsdoesnotnecessarilyreflecthighdiversity.Insteadtheymaybeduetothe presenceofmanyrestrictionenzymesitesintheamplifiedsequences.Thisproblemis solvedbytheuseofterminalrestrictionfragmentlengthpolymorphism(T-RFLP)(30), wherebytheterminalendoftheamplificationproductislabeledwithafluorescentmarker duringPCR.Afterdigestionwithrestrictionenzymes,onlytheterminalrestrictionfrag- mentisdetected,ensuringthateachbacteriumisrepresentedbyasinglefluorescentfrag- ment.Thismethodhasbeenusedtoanalyzetheeffectoftemperatureoncommunity structureofamethanogeniccommunitybyusingArchaea-specificPCRprimers(31)and ofcommunitiesinmercurycontaminatedanduncontaminatedsoil(26).Thismethodis stillaffectedbythechoiceoftherestrictionenzymeasDNAlackingthespecificsiteare notdigested. 2.DistinguishingDeoxyribonucleicAcidMoleculesby MeltingCharacteristics AnotherapproachforfingerprintingistheanalysisofthePCRamplificationproducts bydenaturinggradientgelelectrophoresis(DGGE).Thistechnique,originallydeveloped forthedetectionofpointmutations(32–34),wasmorerecentlyappliedtostudiesof microbialgeneticdiversity(35).Unlikecommonelectrophoresis,inwhichDNAfrag- mentsareseparatedbysizedifferences,DGGEseparatesDNAfragmentsofthesame lengthaccordingtosequenceheterogeneitythatresultsindissimilarmeltingproperties. ThecomplexmixtureofamplifiedDNAiselectrophoresed(atelevatedtemperature, usually60°C)throughanacrylamidegelthatcontainsalineargradientofdenaturant concentrations(formamideandurea).DNAmigratingthroughthegelpartiallymeltsat aspecificdenaturantconcentration,dependingonitsprimarysequence.Thispartial meltingoftheDNAresultsinbranchingofthemolecule,thusloweringitsmobilityin thegel.TopreventcompletemeltingaGC-clamp(anapproximately40-bases-long GC-richsequence)isattachedtotheendofoneofthePCRprimers.Atthedenaturant concentrationatwhichthe16SrDNAiscompletelydenatured,thecomplexofsingle- strandedDNAanddouble-strandedGC-clampisalmosttotallyimmobile,resultingina DNAbandatalocationspecifictothis16SrDNAsequence(Fig.2).ByusingaGC- clamp it is possible to detect almost 100% of all possible sequence variations (33). Another approach used to create a continuous gradient in denaturing conditions is based on temper- ature in the so-called temperature gradient gel electrophoresis (TGGE). Since DGGE and TGGE are in principle the same no distinction between the two is made in the following discussion. Specific fingerprints using DGGE/TGGE have been performed with many environ- mental samples (17,35–39), including soil (40–43). Furthermore, DGGE/TGGE is an excellent tool for monitoring changes in community diversity resulting from environmen- tal disturbances. Diversity has been shown to change in agricultural soil by fumigation and pesticide treatments (40,43,44). Copyright © 2002 Marcel Dekker, Inc. Figure 2 Theoretical and actual appearance of DGGE. Arrows indicate direction of migration in the gel. One way by which DGGE/TGGE analysis may overestimate diversity is due to the presence of more than one 16S-rDNA gene in a single species. This has so far been reported only in the case of Paenibacillus polymyxa, when DGGE analysis revealed sev- eral bands (45). How this phenomenon affects the diversity analysis of microbial commu- nities still needs to be investigated, but if it is a common phenomenon, diversity may be greatly overestimated by all fingerprinting techniques based on rRNA or rDNA. On the other hand, it is not possible to distinguish more than approximately 50–100 bands in DGGE/TGGE profiles. Therefore, this analysis does not identify all the species diversity in a complex community. The sensitivity of the method was found to be limited to populations representing at least 1% of the total bacterial community (35). Newly developed dyes like SYBR-green and silver staining are more sensitive than ethidium bromide when bound to DNA and may improve the current detection limit. The number of bands that can be distinguished in a gradient gel may limit analysis of very complex bacterial communities in which high diversity is expected, e.g., soil communities. Nevertheless, changes in population composition in communities revealing more than 100 bands on DGGE gels have been reported (43). 3. Deoxyribonucleic Acid Reassociation A very different approach to diversity analysis is the DNA reassociation technique de- scribed by Torsvik and colleagues (13,46). The heterogeneity of DNA extracted from soil samples was determined by reassociation kinetics, measured spectrophotometrically, after DNA denaturation. At a given DNA concentration, the time required for reassociation is proportional to the complexity of DNA. The method has been used to estimate the number of different species in soil communities by comparing the total genomic size of the mixed soil community DNA with that of the mean genome size of cultivated bacterial isolates from the same soil (13,46). This pioneering work estimated the presence of 4000 com- pletely different bacterial genomes in 1 g of soil, equivalent to 13,000 species (13). In 1998, the method was used to investigate the impact of environmental distur- bances on community diversity (47). The genetic diversity of the microbial community was reduced 20-fold in CH 4 -perturbed soil compared with that of communities in nondis- turbed soil (38). Copyright © 2002 Marcel Dekker, Inc. D. General Problems in Community Analysis Although molecular approaches have facilitated inclusion of nonculturable microbes in community analysis, the question whether the results are representative of the total micro- bial community still needs to be addressed. The ideal analysis should of course reflect both the quantitative (‘‘how many’’) and qualitative (‘‘who’’) composition of the commu- nity. Each step in the molecular analysis could result in bias, making it difficult to interpret the results. The following section considers nucleic acid extraction and amplification as crucial steps in the accurate analysis of community diversity. 1. Extraction of Nucleic Acids A number of methods to extract DNA from soil samples have been developed, including cycles of freezing and thawing, sonication, sodium dodecylsulphate (SDS) treatment, boil- ing, liquid nitrogen, and bead beating (14,48–51). Whereas some investigators have evalu- ated the quantity and quality of the extracted DNA by different methods (14,52), few have compared the relative compositions of the extracted gene pool. Our own experiments have shown that DGGE profiles from soil community DNA varied markedly, depending on the extraction method (Fig. 3). Two different extraction methods were used: a sonication method (representing a gentle treatment of the cells) and a harsher bead beating method. It is very likely that the sonication method mainly extracted fragile, thin-walled cells, Figure 3 DGGE profiles of amplified 16S rDNA fragments extracted from a sandy loam soil by a bead beating method or by a sonication method. Copyright © 2002 Marcel Dekker, Inc. leavingmoresturdycellsintact.Beadbeatingmethods,ontheotherhand,yieldedDNA frommoresturdycellsandspores(52),butlossofspecificsequencescouldresultfrom shearingoftheDNAbytheratherharshprocedure(53,54).TheDGGEprofilesdiffered bothinthepositionandinthenumberofbands,clearlyshowingthatourconclusion regarding‘‘whoandhowmuchisthere’’dependsonourDNAextractionprocedure.Even whentwoDNAextractionprotocols,bothcontainingabead-millhomogenizationstep, werecompared,differentDGGEprofileswereobserved(55).Thisstudyalsoconcluded thattherelativecompositionoftheextractedgenepoolsvariedwithmethods.Therefore, thesuggestionthatrDNAampliconsvisualizedbyDGGErepresentthedominantspecies inthecommunity(35,42)needstotakeintoaccountbiasintroducedbythechosenDNA extractionmethods. 2.TheAmplificationofSpecificSequences ThenextcrucialstepinmostanalysesistheamplificationofthetargetgenesbyPCR. WhenitisappliedtocommunityDNAseveralproblemsmayresultinabiasedsynthesis ofamplificationproducts(fordetailedreviewseevonWintzingerodeetal.[56]).The amplificationefficiencyhastobethesameforallthesequencesintheDNAmixtureif thePCRproductsaretoreflectthecommunitycomposition.Itisknownthatenvironmental samplesmaycontaininhibitorsofDNAamplification,e.g.,phenoliccompounds,humic acids,andheavymetals(foranextensivelistseeTable3inWilson[57]).Amplification alsodependsonprimerspecificityandhybridizationefficiency,templateandprimercon- centration,andnumberofPCRcycles(58).EvenwhenquantitativePCRisachieved,the numberoftargetsequencesbeforeamplificationcoulddependnotonlyonthenumberof bacteriacontainingthespecificsequence,butalsoonthenumberofgenecopiesineach cell(59),whichforunculturablebacteriaisstillunknown. TheformationofchimericDNAmoleculesandotherPCRartifacts(60),aswellas crosscontamination(61,62),areadditionalproblems.Eventhoughinsituhybridization hasshownthatphylogeneticgroupsfoundbysequencingofclonesina16Slibrarywere presentintheenvironment(61),thisisnotalwaysthecase.Tanneretal.(62)showed that16SrDNAsequencesobtainedincontrolswithoutDNAtemplatecorrespondedto sequencesfoundinenvironmentalsamples.Thisfindingindicatesthatsomesequences presentincommunityDNApoolswerecontaminantsofunknownorigin.Thiscrosscon- tamination,ifprevalent,mayblurdifferencesingeneticdiversityamonghabitats. E.ComparativeStudies Fewinvestigatorshavecomparedcommunitydiversityanalysesobtainedbymolecular approachestothoseobtainedbymoretraditionalapproaches,e.g.,substrateutilization (40,44,63–66),ortotypingaccordingtocolonymorphologicalcharacteristics(65,66). Weevaluatedtheeffectofmercuryonsoilmicrobialcommunitiesbythreedifferentap- proaches:DGGE,colonymorphologicalfeatures,andsolecarbonutilizationpatterns (BIOLOG).Allmethodswereabletodetectstructuralchangesofthecommunityinthe presenceofmercury.Theeffectofmercuryonthediversityofthecommunity(hereexpressed asnumberoftypes[Table1])wasrevealedbybothDGGEandcolonymorphologicalanaly- sis. DGGE was the most sensitive of the methods, showing a reduced number of bands not only in the most contaminated soil but also in the intermediate contaminated soil. Torsvik et al. (13) reported agreement between the genetic diversity as described by DNA reassociation kinetics and by phenotypic diversity of isolated strains, and Engelen Copyright © 2002 Marcel Dekker, Inc. Table 1 A Comparison Among Different Methods That Assess Community Diversity a Number of Substrates Soil Morphotypes DGGE bands utilized A 20.7 Ϯ 1.9 57.3 Ϯ 0.7 a 15.0 Ϯ 1.2 B 23.3 Ϯ 2.2 53.3 Ϯ 1.2 a 13.3 Ϯ 0.3 C 14.7 Ϯ 0.9 b 47.0 Ϯ 1.2 a 15.7 Ϯ 1.2 a Numbers (mean Ϯ standard error [SE]) of morphotypes represented by the morphological features of 50 randomly selected colonies, bands in the DGGE profiles, and substrates utilized in the BIOLOG Ecoplates for three soils with different levels of mercury contamina- tion: soil A (7 µgHgg Ϫ1 dw soil), soil B (28 µgHgg Ϫ1 dw soil), and soil C (511 µgHgg Ϫ1 dw soil). b Significantly different from the others (t-test; p Ͻ .05). The t-test was only performed if analysis of variance (ANOVA) showed significant differences (p Ͻ .05). and associates (40) showed correlation between pesticide-induced changes in the genetic and functional diversity in soils by DGGE and substrate utilization patterns. On the other hand Duineveld and colleagues (63) found similar DGGE profiles of communities from rhizosphere and bulk soil, where large differences in metabolic properties existed. F. Ribonucleic Acid Hybridization Together with the increase in known 16S and 23S rRNA sequences, new hybridization techniques for studying bacterial community structure have emerged. Two of the most promising of these techniques are fluorescent in situ hybridization (FISH) and rRNA slot- or dot-blot hybridization. FISH can be used for detecting the abundance and distribution of specific bacteria at different phylogenetic levels. Using confocal scanning laser microscopy (CSLM) it is possible to visualisze the spatial distribution of the target organisms in complex environ- ments. Enumeration of fluorescently marked species is achieved by submitting the hybrid- ized cells to analysis in a fluorescence activated cell sorter (FACS) (67,68). Numerous studies have examined biofilms, using the FISH technique to identify key species and their positions in environmental matrices (69–71). These include activated sludge (72–76), sediments (77,78), soils (79), and plant roots (80). The technique requires cell fixation by paraformaldehyde, dehydration with ethanol, and incubation with one or even several fluorescent oligonucleotide probes. The probes are specific to the target or- ganisms’ rRNA. Since rRNAs are present in copious amounts in the cell, hybridization results in fluorescent signals that are easily detected under the microscope or by FACS. Oligonucleotide probes can be designed to target different levels of phylogenetic specific- ity (i.e., kingdom-, genus-, and species-specific probes). For example, Logeman and asso- ciates (82) studied microbial diversity in a nitrifying reactor system using probes designed to hybridize with either all eubacteria, all Cythophaga–Flexibacter–Bacterioides spp. groups, or only nitrifiers in the Nitrosomonas sp. cluster. Copyright © 2002 Marcel Dekker, Inc. rRNA slot- or dot-blot hybridization is a technique that, like FISH, relies on known sequences to examine the abundance of specific populations. The procedure is inexpensive compared to FISH as it does not require fluorescence microscopes; however, information about the spatial distribution of the microorganisms is lost. Total pools of rRNA isolated from natural samples or cultures are blotted onto a nylon membrane, and the rRNA is hybridized with both specific and more general oligonucleotide probes. By comparing sig- nals that have been hybridized with a more specific probe to the signals from a more general probe, a relative abundance of the target bacterial species or genus can be calculated. This approach has been used to examine microbial communities in aquatic (74,81) and soil (79,83) samples. Hybridization can also be applied to PCR-amplified rDNA. In one study (81), 353 clones from a 16S rDNA clone library representing the community of perma- nently cold marine sediments were hybridized with group- and species-specific oligonucle- otide probes. The study showed high bacterial diversity in the sediment and dominance of sulfate reducing bacteria, among them Desulfotalea spp. and other closely related species. A limitation to rRNA hybridization is the fact that oligonucleotide probes can target only species for which sequences are known. However, the number of known sequences is increasing rapidly, and now comprises more than 7336 Bacteria and 324 Archaea for 16S rRNA (84). By combining, for example, DGGE, in which 16S rRNA from complex environments can be separated in a gel on the basis of sequence, with subcloning and sequencing, new sequences can be obtained and used to design oligonucleotide probes that are relevant to the environment in question. G. How Do the Diversity and Structure of the Bacterial Community Influence the Function of the Soil System? The use of molecular approaches to describe the composition of soil bacterial communities offers an opportunity to study the relationships among community diversity and the struc- ture and function of soils. The ecological importance of these relationships cannot be over- stated. A great many of the examples discussed reveal the enormous microbial diversity in soils, but only a few address diversity as it relates to the functioning of the ecosystem. If they do, they have mainly done so on the basis of functional diversity (85) as defined by carbon utilization patterns in BIOLOG microtiter plates. BIOLOG measures the potential of a fraction of the community, which does not necessarily represent the numerically dominant species (86), to grow on a particular substrate rather than the actual activity of the commu- nity (87). Furthermore, most of the test substrates have no special ecological relevance. Therefore, it is difficult to relate a change in a utilization profile to the presence or absence of specific enzyme activities in the soil. Theories have been proposed to explain how species diversity is related to ecosystem function (88). For example, it has been suggested that enhanced species diversity is beneficial for ecosystem functioning (89,90). Others have proposed that the properties of the system are more dependent on the functional abilities of some species than on the total number of different species (91–93). Studies have focused mainly on plants, and only recently has attention been given to soil microbial communities (94–96). The diversity of the soil microbial community is enormous even within a small area (such as a soil aggregate or a root surface), yet not all the bacteria contribute to the observed activity. Rather, a large proportion of the cells are inactive and become active only when conditions are favorable. Therefore, there is probably a large difference between the diversity of the potentially active bacteria and that of those that are actually active, and this is reflected in differences between the potential and actual activity in soils. Copyright © 2002 Marcel Dekker, Inc. [...]... added strains carry the genetic information needed for the expression of the complete catabolic pathway This approach has been applied for the degradation of 1,2,4-trichlorobenzene (129), 4-ethylbenzoate (141 ), atrazine (130 ,142 ) phenoxyacetic acid (143 ), 3-chlorobenzoate (144 ), 3-phenoxybenzoic acid (145 ), and 2,4-dichlorophenoxyacetic acid (145 ) Although all these studies report a decline in the concentration... addition the concentration of the contaminant seems to be crucial, too Short et al (143 ) demonstrated that the presence of substrate was essential for the survival of ¨ a phenoxyacetic acid degrading inoculant, and Daane and Haggblom (131) showed that the encapsulation of the degrading strains in structures that protect them from the toxicity of the contaminant enhanced their performance Thus, although the. .. activities Solid lines in A and B, response of the microbial community to the presence of the pollutant in question; dashed lines, second application of the pollutant Copyright © 2002 Marcel Dekker, Inc affect the dynamics of the acclimation response, including chemical structure and concentration of the pollutant, presence of organic and inorganic nutrients, type and physiological state of the community,... populations that carry the required degradative capabilities, (2) induction of enzymes involved in the uptake and turnover of the pollutant, and (3) genetic adaptation The first two are manifested by previously existing subpopulations of degrading strains, whereas the process of genetic adaptation creates changes in the existing genetic pool of the microbial community and thus implies the evolution of new... were enriched by the availability of the substrate) Furthermore, these studies did not compare the survival of the donor strains with the survival of the 2,4-D degrading genes Such an analysis would allow evaluation of the premise that gene transfer to indigenous bacteria facilitates the maintenance of the catabolic capability in the community 3 Expansion of the Substrate Range of Indigenous Bacteria... describe the dominating species in the soil microbial community and to identify the active members of the community under various conditions Furthermore, they give the opportunity to introduce new genetic traits and thereby change the functional potential of the microbial community All these features have been applied successfully to soil communities There is an overwhelming amount of literature on the. .. humans and the risk they pose to the integrity of natural ecosystems, the removal of these compounds is of great benefit to society A few processes contribute to this goal, among them (1) transport, (2) evaporation, (3) sorption, and (4) biodegradation The first three, however, only alter the physical state or the location of the contaminants In contrast, biodegradation is the primary process involved in the. .. transfer in the degradation of contaminants in bioreactors (146 ) and activated sludge (147 ) has been proposed Top et al (132) showed that transfer of two 2,4-dichlorophenoxy acetic acid (2,4D) degradation plasmids (that were unrelated to the ‘‘prototype’’ 2,4-D plasmid, pJP4, or to each other) enhanced the degradation of 2,4-D in soil In the presence of 2,4-D the plasmids were transferred to indigenous... Analysis of ammonia-oxidizing bacteria of the b subdivision of the class Proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplified 16S ribosomal DNA fragments Appl Environ Microbiol 63 :148 9 149 7, 1997 24 T Henckel, M Friedrich, R Conrad Molecular analyses of the methane-oxidizing community in rice field soil by targeting the genes of the 16 S rRNA, particulate... transformation and mineralization of xenobiotic compounds and, in the latter process, results in the elimination of the pollutant and its metabolites Abiotic degradation may occur, but it is less common and often results in incomplete decontamination and sometimes the formation of more toxic chemical intermediates (101) Enhanced biodegradation of toxic xenobiotics could be achieved by in situ manipulation . atrazine (130 ,142 ) phenoxyacetic acid (143 ), 3-chlorobenzoate (144 ), 3-phenoxybe- nzoic acid (145 ), and 2,4-dichlorophenoxyacetic acid (145 ). Although all these studies report a decline in the. Inc. rDNAamplificationproductsofthenativecommunityshowedadecreasedeubacterial geneticdiversitywithincreasingsalinity,whereasthereversewastrueforArchaea(28). Structuralchangesinthemicrobialcommunityinsoilduetocoppercontaminationwere detectedbySmitandassociates(29)onthebasisofARDRAprofilesofisolates,clones, andsoilcommunityDNA.However,aproblemariseswhenusingRFLPintheinvestiga- tionofmorecomplexmicrobialcommunities.Inanalyzingtheamplificationproductsof communityDNA,differentsequencesresultinadifferentnumberofDNAfragments, dependingonhowmanyrestrictionsitesarepresentinaparticularsequence.Presence ofmanybandsdoesnotnecessarilyreflecthighdiversity.Insteadtheymaybeduetothe presenceofmanyrestrictionenzymesitesintheamplifiedsequences.Thisproblemis solvedbytheuseofterminalrestrictionfragmentlengthpolymorphism(T-RFLP)(30), wherebytheterminalendoftheamplificationproductislabeledwithafluorescentmarker duringPCR.Afterdigestionwithrestrictionenzymes,onlytheterminalrestrictionfrag- mentisdetected,ensuringthateachbacteriumisrepresentedbyasinglefluorescentfrag- ment.Thismethodhasbeenusedtoanalyzetheeffectoftemperatureoncommunity structureofamethanogeniccommunitybyusingArchaea-specificPCRprimers(31 )and ofcommunitiesinmercurycontaminatedanduncontaminatedsoil(26).Thismethodis stillaffectedbythechoiceoftherestrictionenzymeasDNAlackingthespecificsiteare notdigested. 2.DistinguishingDeoxyribonucleicAcidMoleculesby MeltingCharacteristics AnotherapproachforfingerprintingistheanalysisofthePCRamplificationproducts bydenaturinggradientgelelectrophoresis(DGGE).Thistechnique,originallydeveloped forthedetectionofpointmutations(32–34),wasmorerecentlyappliedtostudiesof microbialgeneticdiversity(35).Unlikecommonelectrophoresis,inwhichDNAfrag- mentsareseparatedbysizedifferences,DGGEseparatesDNAfragmentsofthesame lengthaccordingtosequenceheterogeneitythatresultsindissimilarmeltingproperties. ThecomplexmixtureofamplifiedDNAiselectrophoresed(atelevatedtemperature, usually60°C)throughanacrylamidegelthatcontainsalineargradientofdenaturant concentrations(formamideandurea).DNAmigratingthroughthegelpartiallymeltsat aspecificdenaturantconcentration,dependingonitsprimarysequence.Thispartial meltingoftheDNAresultsinbranchingofthemolecule,thusloweringitsmobilityin thegel.TopreventcompletemeltingaGC-clamp(anapproximately40-bases-long GC-richsequence)isattachedtotheendofoneofthePCRprimers.Atthedenaturant concentrationatwhichthe16SrDNAiscompletelydenatured,thecomplexofsingle- strandedDNAanddouble-strandedGC-clampisalmosttotallyimmobile,resultingina DNAbandatalocationspecifictothis16SrDNAsequence(Fig.2).ByusingaGC- clamp. alter the physical state or the location of the contaminants. In contrast, biodegradation is the primary process involved in the transformation and mineralization of xenobiotic compounds and, in the latter