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J. Theron a; T. E. Cloete aa Department of Microbiology and Plant Pathology, University of Pretoria, 0002 Pretoria, South Africa.

Online Publication Date: 01 January 2000

Theron, J. and Cloete, T. E.(2000)'Molecular Techniques for Determining Microbial Diversity and CommunityStructure in Natural Environments',Critical Reviews in Microbiology,26:1,37 57

10.1080/10408410091154174

http://dx.doi.org/10.1080/10408410091154174

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Critical Reviews in Microbiology, 26(1):3757 (2000)

I. INTRODUCTION

Historically, it has been very difficult todetermine the diversity of microorganismsthat constitute natural and artificial ecosys-tems. Unlike most eukaryotes, few bacteriacan be recognized on the basis of morpho-

logical features. The classic approach to enu-merate bacteria in environmental samples hasbeen culture-dependent techniques combinedwith a simultaneous or subsequent differen-tiation of the isolates based on batteries ofphysiological and biochemical tests. How-ever, culture-dependent methods do not accu-

rately reflect the actual bacterial communitystructure, but rather the selectivity of growthmedia for certain bacteria. Furthermore, (1) alltechniques rely on cultivation and are timeconsuming and expensive as are the physio-logical and biochemical differentiation tests;(2) after many generations necessary to form

plate colonies, the organism may deviate fromits physiology, and possibly even from geno-typic mix, of the population in nature; (3) onlya minor fraction (0.1 to 10%) of the bacteriacan be cultivated using standard techniques;and (4) it offers a very limited insight into thespatial distribution of the microorganisms.14

Molecular Techniques for DeterminingMicrobial Diversity and Community Structure

in Natural EnvironmentsJ. Theron and T. E. Cloete

Department of Microbiology and Plant Pathology, University of Pretoria, 0002 Pretoria,South Africa

Correspondence: Prof. T. E. Cloete, Department of Microbiology and Plant Pathology, University of

Pretoria, Pretoria 0002

Dr. J. Theron, Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria 0002

ABSTRACT: The ability to quantify the number and kinds of microorganisms within a commu-

nity is fundamental to the understanding of the structure and function of an ecosystem. The simplemorphology of most microbes provides few clues for their identification and physiological traits

are often ambiguous. In addition, many organisms resist cultivation, which is essential to their

characterization. Recombinant DNA techniques have provided a means whereby many of the obsta-

cles associated with cultivation and description can be overcome and subsequently has allowed

many new insights into the complexity of natural microbial communities. Molecular approaches

based on 16S ribosomal RNA (rRNA) sequence analysis allow direct investigation of the com-

munity structure, diversity, and phylogeny of microorganisms in almost any environment, while

quantification of the individual types of microorganisms or entire microbial communities may be

addressed by nucleic acid hybridization techniques. Furthermore, the use of fluorescently labeled

population-specific rRNA probes allows microscopic examination of individual cells in complex

microbial assemblages as well as their interactions in situ. In this review, we discuss strategies

for characterizing microbial communities without the need for cultivation.

Key Words: microbial diversity, community structure, molecular techniques, 16S rDNA se-

quencing, polymeric chain reaction, nucleic acid hybridization, polymorphism-based assays.


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Fluorescent antibody techniques offeredmajor advances in aut-ecological studies ofmicroorganisms.59 These methods alloweddirect identification and enumeration of indi-vidual bacteria in environmental samples with-out requirement of prior growth in culture

media. The technique involves the use of spe-cific antibodies raised against surface markersof defined pure cultures that are either labeleddirectly with fluorescent dye molecules or viaa fluorescent secondary antibody. Althoughthis approach has yielded important insightsinto the spatial distribution of microorganisms,it has the following limitations:10,11 (1) ex-pression of the antigen may be influenced byenvironmental factors; (2) false-positive andfalse-negative results may be obtained due to

cross-reactivity or lack of reaction; (3) non-specific binding of antibodies may result inhigh levels of background fluorescence; and(4) production of specific antibodies requiresa pure culture of the organism of interest.

Subsequently, various recombinant DNAtechniques for the identification of bacteriahave been developed that are independent ofcultivation methods. Pace et al.1 and Olsenet al.10 outlined an approach based on identifi-cation of microorganisms via 16S rRNA genesequences determined directly from the bio-mass without cultivation. The rRNA moleculescomprise highly conserved sequence domainsinterspersed with more variable regions.12,13

Consequently, comparative analyses of rRNAsequences can identify so-called signature se-quence motifs on various taxonomic levelsthat are targets for an evolutionary-based iden-tification.4,1416 Advantages using the rRNAsinclude: (1) their occurrence in high copy num-bers of usually more than 1000 in any livingcell; (2) their sequences can be retrieved from

the samples of interest without prior cultiva-tion; and (3) 16S rRNA sequences have beendetermined for a large fraction of bacterialspecies.4,10,18

The use of recombinant DNA techniqueshave overcome many of the stumbling blocksof cultivation. It provides ways of charac-

terizing mixed microbial communities on amolecular basis in a relatively unbiased way,thereby enhancing the science and practice ofmicrobial ecology. In this article, we describerecombinant DNA technologies applied tothe analysis of mixed microbial populations.

The various approaches and tools used inthese analysis are outlined in Figure 1.

II. MOLECULAR TECHNIQUES

A. 16S rDNA Sequencing Approach

Initially, the analysis of the diversity ofnatural microbial populations relied on directextraction, purification, and sequencing of 5S

rRNA molecules from environmental sam-ples.1921 Although these studies yielded inter-esting insights, the information content of the120 nucleotides long 5S rRNA is relativelysmall, and its paucity of independently vary-ing nucleotide positions limits its usefulnessto less complex ecosystems. An average bacte-rial 16S rRNA molecule has a length of approxi-mately 1500 nucleotides and thus containsconsiderably more information for reliableanalyses than the 5S rRNA molecule. Conse-quently, the use of the larger rRNA moleculesfor studies in microbial ecology was suggest-ed.10 In addition, the development of robustDNA cloning techniques and the polymerasechain reaction (PCR) have facilitated higher-resolution analyses of more complex commu-nities using 16S rRNA sequence analysis.

1. Construction of 16S rRNA GeneClone Libraries

The starting point for the recovery ofrRNA sequence information from nucleic acidextracted from environmental samples is theefficient extraction of nucleic acids of suffi-cient quality for use in subsequent procedures.Various methods are available for the extrac-tion and purification of nucleic acids from a


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FIGURE

1.

Com

monlyusedmolecularapproachesin

microbialecology.


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wide range of environmental samples. Theseare usually based on chemical and/or physi-cal disruption of cells combined with treat-ments to remove contaminating materials, suchas humic acids and metals that can inhibitthe efficiency of subsequent enzymatic reac-

tions. Any one of three basic approaches canthen be used to obtain rRNA gene clones fromthe total community nucleic acids.

Initial approaches relied on shotgun clon-ing of the size-fractionated community DNAinto bacteriophage lambda,1,10,22 after whichthe library is screened for the presence ofrRNA genes. This is a laborious procedure,as rRNA genes will only represent a smallfraction (0.125 to 0.3%) of the total clones.22

However, this approach provides the most

unbiased estimate of community diversity,and the library is also a source of genes otherthan those encoding rRNA. A second approachis to use the enzyme reverse transcriptase anduniversal or group-specific primers to synth-esize single-stranded DNA complementaryto rRNA in the community.23,24 A PCR stepwith rDNA-specific primers following reversetranscription may also be used to synthesizedouble-stranded rDNA for cloning.11 Start-ing the sequence retrieval from rRNA whencompared with DNA has the advantage thatdue to the smaller size of rRNA more rigor-ous nucleic acid extraction techniques can beapplied.25 Furthermore, the resulting commu-nity profile will offer some reflection of themost metabolically active organisms, becausecells that produce more RNA will be betterrepresented in the clone library than meta-bolically inactive cells.26

The simplest and currently the most widelyadopted method to obtain 16S rRNA genesfrom the environment is through the use of

PCR. rRNA genes can be amplified directlyfrom the total community DNA using rRNA-specific primers and then cloned using stan-dard methods.27 By taking advantage of thehighly conserved nature of rRNA, universalprimers capable of annealing to rRNA genesfrom all three domains (Archaea,Bacteria,

Eukarya) or primers designed to amplify rRNAgenes from a particular group of organismscan be used.3,17,28

Following PCR amplification, the ampli-fied products can be cloned. Commerciallyavailable kits exploit the fact that PCR-ampli-

fied products have an overhanging 3 deoxy-adenosine residue at each end when certainDNA polymerases are used. This allows clon-ing of the product into a sequencing-readyvector containing a complementary deoxy-thymidine overhang, in many cases withoutrequiring the product to be purified or furthermodified. Alternatively, the PCR products canbe cloned by filling overhanging residuesfollowed by blunt-end ligation procedures.29

2. Screening of Clone Librariesfor rRNA Genes

Contrary to shotgun gene clone libraries,the majority of clones in PCR-based gene li-braries will contain rRNA sequences. To avoidunnecessary sequence determinations, redun-dant clones in both types of libraries need tobe eliminated using the following strategies:(1) single-nucleotide sequencing;24,30 (2) dotor colony hybridization using species orgroup-specific probes;3,10,31,32 (3) restrictionfragment length polymorphisms (RFLP) ofpurified plasmid DNA;31,33 and (4) colonyPCR (using sequencing primers with prim-ing sites that flank the insert DNA).34

3. Sequencing

The rapid screening and analysis of largegene clone libraries have been facilitated by

automated DNA sequence systems.35 Com-plete sequencing of the cloned rRNA genes isfacilitated by the presence of conserved se-quence domains.36 Because of their length (15to 20 nucleotides) and their universality, oli-gonucleotide primers can be designed that per-mit sequencing of the complete rRNA gene.10,20,37


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Once a sequence database has been gen-erated from a clone library, the sequences canbe compared to each other as well as previ-ously published sequences or databases38,39 toindicate the kinds of microorganisms presentin the sample. Therefore, the technique provides

a list of 16S rRNA sequences as surrogateidentification of individuals from the sample.

B. PCR and Related Techniques

The expanding rRNA sequence databasecontains several thousand sequences. By com-parative sequence analysis of the more vari-able regions of the 16S rRNA molecule, it ispossible to design oligonucleotides of vary-

ing phylogenetic resolution. These can be usedin the detection and enumeration of specificgroups of bacteria by PCR40 or combined withthe use of specific oligonucleotide probes.41

PCR provides a way of amplifying nu-cleic acid sequences that might be in low abun-dance in a complex mixture of whole cellextracts. A thermostable DNA polymerase,often the Taq enzyme from Thermus aquati-cus, is used to amplify the specific DNA se-quence of interest. The target sequence isdefined by two sequence-specific oligonucle-otide primers that flank the target sequenceand that anneal to the complementary strandsof the target sequence. During the PCR pro-cess, repetitive cycles of DNA denaturation,annealing of the oligonucleotide primers tothe target DNA, and extension of the primersacross the target sequence results in increas-ingly greater quantities of target sequence.

1. PCR with Specific Primers

The presence of universally conserved5 and 3 sequences37 allows both the recov-ery of rRNA sequences as cDNA and ampli-fication of nearly complete 16S rRNA genesextracted from natural samples. Specific prim-ers have been used to amplify fragments of

rRNA operons in order to detect specific or-ganisms or groups of organisms in environ-mental samples.4245 Although the method isboth specific and sensitive, such standard PCRreactions are not quantitative.

To obtain quantitative data from PCR-based

analyses, statistical methods based on mostprobable number (MPN) estimations havebeen used.44,46,47 In MPN-PCR, DNA extractsare diluted before PCR amplification and lim-its are set on the number of genes in the sam-ple by reference to known control dilutions.Another way to quantify PCR-amplified prod-ucts for comparison is to include an internalcontrol in the PCR reaction.4850 Here, a knownamount of target DNA is added to a PCR re-action containing DNA from the mixed mi-

crobial population. The known target DNA iscomplementary to the same primers and thuscompetes with the target sequences in the ex-tracted DNA sample. By preparing a dilutionseries of the known and unknown DNA spe-cies, it is possible to quantify the amount ofproduct produced from the complementary genein the extracted DNA. The known DNA tar-get can be generated by cloning the gene ofinterest or purifying the PCR-amplified prod-uct, after which a deletion is introduced togive a differently sized PCR product.41

There exist many variations of the PCRtechnique. The sensitivity and specificity ofthe PCR may be improved by adopting anested approach. The initial amplification iscarried out with a pair of primers that are notorganism specific, whereafter a second roundof amplification is conducted on the productusing primers specific for an organism withtarget sites internal to the first primer pair.51,52

C. Nucleic Acid HybridizationTechniques

The easiest way of detecting specific nucleicacid sequences is through direct hybridizationof a probe to microbial nucleic acid extracts.Whole cell DNA or RNA is extracted from


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the environmental sample and fixed to a nylonor nitrocellulose membrane. Bacterial colo-nies can also be replica-plated from agar platesto membranes and their nucleic acids exposedin situ following lysis for subsequent hybrid-ization. Probes may be used to detect genes in

the bacterial genome (Southern blots) or to de-tect mRNA or rRNA (Northern blots).41 Forthe in situ identification of individual wholecells, it is necessary to make the cells perme-able to oligonucleotide probes hybridizing withrRNA.17

These hybridization techniques rely on thespecific binding of nucleic acid probes to com-plementary DNA or RNA (target nucleic acid).These probes are single strands of nucleic acidwith the potential of carrying detectable marker

molecules highly specific to complementarytarget sequences, even if these sequences ac-count for only a small fraction of the targetnucleic acid. Either DNA or RNA can serveas a nucleic acid probe, but for a number ofreasons (e.g., ease of synthesis and stability),most studies have employed DNA probes.2

D. GENOMIC DNA HYBRIDIZATIONTECHNIQUES

Nucleic acid hybridization methods suchas DNA reassociation and reciprocal hybrid-ization of community DNA provides limitedinformation of specific sequence content. Fur-thermore, the extent to which these methodscan be used to estimate species diversity andindividual population identity varies with themethod and community.

1. DNA Reassociation

DNA reassociation kinetics have been usedto investigate genomic sequence complexity53

and to assess the diversity of natural micro-bial communities.54,55 In these analyses, com-munity DNA isolated from the environmentis denatured and then allowed to reanneal.

The principle of DNA renaturation kineticsis that the rate of hybridization is propor-tional to the concentration of complemen-tary DNA sequences and inversely propor-tional to the total length of different sequencesin the sample.2,56 Thus, as microbial commu-

nity diversity (heterogenecity) increases (e.g.,there are a greater number of unique ge-nomes), the rate of reassociation of DNAextracted from the community decreases.57

Experimentally, DNA reassociation is mea-sured over time and the fraction of reassoci-ated DNA (C/Co) is expressed as a functionof Cot, where Co is the initial molar concen-tration of nucleotides in single-stranded DNAand tis the time in seconds. The plot of thisrelationship is referred to as a Cot curve. The

reaction rate constant, k, can be expressed as1/Cot1/2, where t1/2 denotes the time in secondsrequired for 50% reassociation. Under definedconditions, most importantly temperature andmonovalent ion concentration, Cot1/2 is pro-portional to the complexity (e.g., number ofunique genomes) of the DNA.58 The conclu-sion from DNA reassociation studies is gen-erally that natural microbial ecosystems arevery complex.2,54

DNA reassociation can provide a usefulmeasure of community structure.57 However,one concern in the interpretation of DNA re-association estimates is a reduction in the rateof reassociation resulting from impurities inthe DNA sample. It is also important to evalu-ate changes in reassociation kinetics that mightresult from the use of different extraction andpurification techniques.

2. Reciprocal Hybridizationof Community DNA

Reciprocal hybridization of total commu-nity DNA59,60 provides an approach to deter-mine whether two samples share the same kindsof organisms, regardless of exactly their species.This is done by cross-hybridization of the com-munity DNA extracted from the two samples,


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based on the idea that significant cross-hybrid-ization of pure culture DNA occurs only be-tween identical or very closely related species.59

The prokaryotic DNA is extracted from eachsample, radioactively labeled by standard tech-niques and then hybridized under stringent con-

ditions to DNA on filters. Radioactive probesfrom each are hybridized to filters contain-ing DNA spots from the same sample (control)as well as the test sample. The result (measuredradioactivity bound to the test spot) is expressedas a percentage of the self-hybridization resultand is referred to as the similarity between thesamples. However, interpretation is complicatedby uncertainties about relative population abun-dances and hybridization kinetics as well aspossible differences in labeling efficiencies

of DNAs comprising different DNA pools.Nevertheless, the method provides a relative-ly clear assessment of community relationshipwhen the communities are nearly identical (highreciprocal hybridization similarity) or distinct(low reciprocal hybridization). Intermediatecross-hybridization values may, however, bemore difficult to interpret.57,61

E. Quantitative DNA Hybridization

Qualitative and quantitative estimates ofcommunity structure can be made using tailor-made oligonucleotide probes that bind to tar-gets with a wide range of specificities fromdomain to strain. Possibly the most importanthybridization techniques for microbial ecol-ogy, because they are the most direct, are quan-titative dot-blot hybridizations of extractednucleic acids,25 and in situ hybridization offixed environmental samples.4,18

1. Oligonucleotide Probes

According to their length, DNA probes canbe grouped as either polynucleotide probes(more than 50 nucleotides) or as oligonucle-otide probes (less than 20 nucleotides).18 The

latter are used more frequently because theyare short enough to allow for single mismatchdiscrimination of target nucleic acids and largequantities of oligonucleotides can be rapidlyand inexpensively produced. During oligo-nucleotide synthesis a variety of marker or

linker sequences can be introduced to the 5end of the oligonucleotide. The principal con-jugate fluorochromes are derivatives of fluo-rescein or rhodamine, although labels such asdigoxigenin and biotin have also been used.4,11,62

The design of rRNA-targeted group- orspecies-specific probes should be based on agood rRNA sequence database and be per-formed in a computer-assisted way using theappropriate software. A resource for such rRNAstudies is the Ribosomal Database Project,38,39

which provides aligned rRNA sequences aswell as a variety of other services such as probeanalysis and sequence similarity analysis. Theorganisms encompassed by a probe varies ac-cording to the region of the 16S rRNA select-ed as the hybridization target. Species-specificprobes complement the most variable regions,while more general probes target more con-served regions of the molecule.3,25,63 The princi-pal steps involved in the design of probes are(1) the alignment of rRNA gene sequences;(2) the identification of sequence idiosyn-crasies; (3) the synthesis and labeling of com-plementary nucleic acid probes; and (4) theexperimental evaluation and optimization ofthe probe specificities and assay sensitivitiesusing cultured reference strains.16,17,64

2. Quantitative Dot-BlotHybridization

Although the comparative analysis of

sequences retrieved from an environmentalsample yields information on the identity orrelatedness of new sequences compared withthe available sequences, it gives a minimalestimate of the microbial diversity in theexamined sample. Clone or sequence abun-dances may also be a biased measure of organ-


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ism abundance due to variations in extractionand cloning efficiencies.4,22 Furthermore, it ispossible that a sequence may have originatedfrom the pool of free DNA present in the eco-system or from contamination and not fromcells in the habitat. Thus, the abundance or

relative abundance of a certain rRNA or rDNAin the extracted nucleic acid pool can be de-termined more reliably by dot-blot hybrid-ization or of a certain clone in the library bycolony hybridization.

For quantitative dot-blot hybridization25,65

the samples of interest are treated to maxi-mize cell lysis and release of nucleic acids.Quantification of a certain 16S rRNA com-pared with total 16S rRNA can then be ob-tained by dot-blot hybridization of a directly

isolated nucleic acid mixture with universaland specific oligonucleotide probes. The rela-tive abundance is calculated by dividing theamount of specific probe bound to a givensample by the amount of hybridized univer-sal probe. The relative abundance of a givenrRNA might reflect changes in the abundanceof the cell population of interest or in the cel-lular rRNA content. For several organisms,a direct linear relationship has been shownbetween growth rate and cellular ribosomecontent.4,66 The relative abundance of a rRNAthus can be interpreted as the relative impor-tance of a defined species in terms of actualmetabolic activity or potential metabolic ac-tivity, but cannot be directly translated intocell numbers.18

3. Whole CellIn Situ Hybridization

Analysis on a single cell level can pro-vide a more detailed picture than dot-blot

hybridization. Not only can the cell morphol-ogy of an uncultured microbe be determined,but also their spatial distribution in situ. Earlyapplications ofin situ nucleic acid hybridiza-tion relied on the use of isotopically labeledoligonucleotides that bound to the rRNAs ofintact, fixed cells, and following autoradiog-

raphy organisms could be phylogeneticallyidentified.15 However, microautoradiographyof such in situ hybridization probes requirelong exposure times to photographic emulsions.Moreover, the useful resolution is no less thanabout 1 m, because of the scatter of radioac-

tive disintegrations. The use of fluorescent dye-labeled rRNA-targeted oligonucleotide probesallows the detection of individual cells,67 andthis approach has been used to analyze manydifferent ecosystems.4,18 rRNA sequences andthereby phylogenetic affiliations were assignedto individual cells of hitherto uncultured endo-symbionts of protozoa68,69 and of magneto-tactic bacteria.70,71 Other applications encom-passed enumeration and spatial distribution ofbacterial populations in activated sludge7278

and in biofilms.11,7983

During whole cell in situ hybridization themorphology of the cells in the sample has tobe stabilized in order to maintain morphologi-cal integrity of the cells under harsh hybridiza-tion conditions. The cell walls and membraneshave to be permeabilized to allow free pen-etration of fluorescent oligonucleotides to theintracellular rRNA. This can be achieved withfixatives such as aldehydes and/or alcohols.4,67,70

The cells are either attached to gelatin-coatedmicroscope slides or hybridized in suspen-sion and immersed in hybridization solutioncontaining a fluorescently labeled oligonucle-otide. Following incubation at the hybridiza-tion temperature for one to several hours, toallow the probe to bind to complementaryrRNA sequences, washing steps are used toremove unbound or part of the nonspecifi-cally bound fluorescent probe, and the sampleis then viewed by epifluorescence microscopy.Probes will only bind correctly under definedhybridization conditions, and the optimiza-

tion of hybridization and washing conditionsis as important as the probe design.16,63 Sev-eral probes labeled with spectrally differentfluorochromes can be simultaneously used onone sample,18,84 while counterstaining of thefixed cells with DAPI (4,6-diamidino-2-phe-nylindole) allows total counts to be made.85


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A direct correlation between the growthrates of bacterial cells, the average ribosome con-tents and the probe-conferred fluorescence hasbeen reported.67,86 This is used to estimate thegrowth rates of individual cells in situ.87,88 Often,it is also important to obtain information about

how the functional components of an ecologi-cal system relates to the organization of thesystem. In communities that have an inherentarchitecture, such as biofilms and flocs, the ques-tion of where various organisms are locatedis of interest. These determinations are diffi-cult to make with conventional epifluorescencemicroscopy. By coupling in situ hybridizationwith fluorescently labeled rRNA-targeted oli-gonucleotide probes with confocal scanninglaser microscopy,89 it is possible to place the

labeled microbes in a three-dimensional re-construction of the intact microbial commu-nity.78,83,9093 Fluorescently labeled probes havealso been used in combination with flow cy-tometry for rapid, automated identificationand counting or collection of microorganismsin hybridized cell suspensions.9496

F. Polymorphism-Based Procedures

The different polymorphism-based proce-dures that have been applied to microbial eco-logical systems are generally coupled to a PCRreaction. The techniques of amplified riboso-mal DNA restriction analysis (ARDRA) anddenaturing gradient gel electrophoresis(DGGE) is specifically addressed. Two otherprotocols use a single primer to amplify frag-ments with PCR before examination on agar-ose gels or restriction. PCR amplification ofrepetitive extragenic palindromic sequences(REP-PCR) takes advantage of repetitive se-

quences found in the microbial genome.97,98

Randomly amplified polymorphic DNA(RAPD) or arbitrarily primed PCR99,100 useprimers that are not specific for a particulargene to amplify fragments. These methodsgenerate a fingerprint that is representedby a banding pattern of nucleic acid frag-

ments following separation by gel electro-phoresis. Although the detection of smalldifferences in specific DNA sequences cangive important information about communitystructure and the diversity of microbes, theyprovide little or no direct information of spe-

cific microbial population identity.

1. Amplified Ribosomal DNARestriction Analysis (ARDRA)

The amplified portions of 16S rRNA genesfrom a mixed microbial population might beof similar sizes with a particular set of oligo-nucleotide primers, but have small differencesat the nucleotide level. One way of detecting

these differences is to restrict the PCR-ampli-fied product with restriction endonucleasesand examine the pattern of restriction frag-ments.31,101104 The analytical power of thisprocedure derives from the fractionation step.The separation of DNA fragments derivedfrom different populations requires that theydiffer in sequence at the restriction endonu-clease sites, or differ in length of DNA flankedby common restriction sites. The separatedDNA fragments may also be transferred to fil-ters for hybridization with probes specific foran organism of interest, that is, are subjectedto Southern transfer.105 By design, the probefor each organism will hybridize to a fragmentof a different size and thus can be used to re-solve different environmental populations.106

2. Denaturing Gradient GelElectrophoresis (DGGE)

Denaturing gradient gel electrophoresis(DGGE)107 is a method by which fragments

of DNA of identical or near identical lengthbut different in sequence composition can beresolved electrophoretically. DGGE has beenextended to the analysis of PCR-amplified16S rRNA genes from environmental sam-ples.108111 In DGGE analysis, separation isbased on changes in electrophoretic mobility


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of DNA fragments migrating in a verticalpolyacrylamide gel containing a linearly in-creasing concentration of DNA denaturants(formamide and/or urea). As the DNA frag-ments are subjected to electrophoresis, partialmelting of the double-stranded DNA occurs

in discrete regions, the so-called melting do-mains, at a denaturant concentration specificfor the nucleotide sequence of the DNA. Themigration of the fragment therefore is severelyretarded. Sequence variation within such do-mains alters their melting behavior, and se-quence variants of the different amplificationproducts stop migrating at different positionsin the denaturing gradient.112,113

Although DGGE analysis of PCR-am-plified 16S rDNA fragments provide a rapid

method to characterize community popula-tion structure, more specific information ofpopulation composition can be obtained bysecondary analysis of the DGGE banding pat-tern. Individual bands (fragments) may be ex-cised from the gel, subjected to a second roundof PCR amplification and sequenced.111,114

Alternatively, the DNA can be transferred tonylon membranes and then challenged withgroup- and species-specific oligonucleotideprobes to identify specific populations withinthe microbial community.108,115

As DGGE is relatively rapid to performand many samples can be electrophoresed si-multaneously, the method is particularly use-ful when examining time series and populationdynamics. Once the identity of an organismassociated with any particular band has beendetermined, fluctuations in individual compo-nents of a microbial population, due to environ-mental perturbations, can be rapidly assessed.34

III. LIMITATIONS OF MOLECULARMETHODS

A. Extraction of Nucleic Acids

A major limitation of the described nucleic-based methodologies, with the exception of

whole cell in situ hybridization, is the quan-titative recovery of nucleic acids from theenvironment. Two aspects of nucleic acid re-covery deserve special mention.57 The firstaspect relates to the recovery efficiency ofthe extraction method. For example, if the total

amount of nucleic acids present in a sampleis unknown, then it is difficult to assess therecovery efficiency by any extraction method.This is compounded by the fact that sporesare more resistant to cell lysis than vegetativecells, and Gram-negative cells are more sus-ceptible to cell lysis than Gram-positive cells.It is also possible that the same lysis tech-nique may give different results with differ-ent types of sample (e.g., water, sediment, orsoil), and recovery might be reduced by deg-

radation or adsorption of nucleic acids to ma-trix material (e.g., clays). The degree of celllysis therefore should be determined indepen-dently. Microscopic enumeration of the cellsin an environmental sample before and afterlysis treatments can yield a reasonable indica-tion of the efficiency of cell lysis.116 A secondconsideration is representative nucleic acidrecovery. For example, a population resistantto breakage would be fractionally underrep-resented, while microorganisms that are eas-ily lysed would be overrepresented.57 It hasbeen noted that small cells (0.3 to 1.2 m) ina soil sample were more resistant to lysis thanlarger cells and even bacterial endospores.117

This may influence the recovery of sequencesfrom environmental samples where many cellsare likely to be small due to a state of starvation.

Starting 16S rRNA sequence retrieval fromRNA compared with DNA has the advantagethat it is more resistant to mechanical break-age so that more vigorous nucleic acid extrac-tion techniques can be applied.25 However,

the RNA is subject to degradation during thefollowing extraction as a consequence of en-dogenous nucleases or nuclease contamina-tion. One consequence of partial degradationof the sample is variable destruction of differ-ent primer or probe target sites.118 However,sample integrity may be evaluated by acry-


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lamide gel electrophoresis to demonstrate re-covery of high-molecular-weight species.

B. PCR and Cloning

Although the analysis of a microbial com-munity by PCR and cloning provides a conve-nient and rapid alternative to shotgun cloning,there are several factors that could skew diver-sity estimates.119 PCR reactions are very sensi-tive to reaction conditions and even duplicatesmight not give quantitatively identical results.41

Due to the sensitivity and specificity of thePCR reactions, minor contamination can leadto false-positive signals and false-negative am-plifications are often seen.120 It has been sug-

gested that templates with a high % G + Ccontent are discriminated against due to lowefficiency of strand separation during thedenaturation step of the PCR reaction,121 andsequences that are more abundant than otherless abundant sequences may be preferentiallyamplified.3 Certain sequences may also be dis-criminated slightly against others due to selec-tive priming or higher-order structure elementsthat could result in huge differences after multi-ple cycles. Amplification of rRNA sequencesby using general primers in some cases ex-clude important environmental populations.122

The work of Suzuki and Giovannoni123 indi-cated that the amount and kind of backgroundDNA in a sample can affect the results throughcompetition. Furthermore, depending on whetherthe same batch of PCR product was cloned us-ing either blunt-end or sticky-end cloning pro-cedures, different results may be obtained.121

Depending on the quality of the DNA usedfor PCR, in vitro recombinants of two or morewild- type rRNA genes, so-called chimeric

sequences, can be formed at frequencies ofseveral percent.124,125 One study demonstratedthat 30% of products generated during co-amplification of similar templates were chi-meric.126 Although computer programs havebeen developed to help identify chimeric se-quences,125 the programs may indicate the

presence of chimeric sequences even whennone exist and the programs have difficultiesin identifying chimeras when the two sequencesfrom which the chimera is formed show greaterthan 85% homology.

Furthermore, depending on the particular

thermostable DNA polymerase used, the fidel-ity of the PCR may vary.34 Although carefulanalysis of secondary interactions should iden-tify discrepencies due to misincorporation ofnucleotides during PCR, there is a danger thatthe presence of novel taxa may be assumedas a result of infidelity in DNA replication.

Many prokaryotes have several rRNA op-erons127 and even though the rRNA codingregions are usually highly similar, they arenot necessarily identical.128 The archaeonHa-

loarcula marismortui has interoperon differ-ences of up to 5% between the two 16S rRNAgene sequences.129 The cloning step in rRNAanalysis separates not only the rRNA genesof different organisms but also the differentgenes. Thus, slightly different gene fragmentscould originate from one strain and would notindicate the presence of closely related or-ganisms.130 This is of concern when makingconclusions about biodiversity from data ob-tained with rRNA gene clone libraries.

C. Whole Cell In SituHybridization

The methodological constraints regardingwhole cell hybridization can be divided intofour main categories: cell permeability prob-lems, target site accessibility, target specific-ity, and sensitivity. Permeability of fixed cellsmay be affected by their state of growth. Forexample, alterations in the cell wall structureof dormant cells (e.g., spores) increase their

resistance to adverse environmental condi-tions.131 Poor permeability of fixed cells tothe oligonucleotide probe could result in weakfluorescence intensity. Simple fixation meth-ods using alcohols, formaldehyde, or para-formaldehyde tend to permeabilize 70 to 90%of microscopically visible cells.4,34 However,


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the cell permeability may be enhanced by addi-tional treatment with enzymes,132 solvents,133

or acid.134

If a cell has been permeabilized, the ac-cessibility of the target sequence in the rRNAmay determine whether probe hybridization

to rRNA will occur within the cell. Hybrid-ization of the probe to the target rRNA maybe prevented due to highly stable secondarystructure elements of the rRNA itself or dueto strong interactions of the rRNA with ribo-somal proteins.17,18 This problem can be de-tected by a strong hybridization signal beingobtained with a universal probe that is knownto target an accessible site on the rRNA mol-ecule. Poor accessibility of the target site canthen be indicated by the absence of a hybrid-

ization signal in the same cell(s) using a dif-ferent probe.4,34

Generally, probes containing a single la-beled molecule give a strong signal only ifcells are metabolically active and contain largenumbers of ribosomes and target rRNA. Con-versely, low cellular ribosome contents mayresult in weak fluorescence intensity. This isa significant concern in slowly growing natu-ral populations.86,135 Various approaches havebeen adopted in order to improve the sensi-tivity. These have included the use of multi-ple probes labeled with a single fluor;84,136 orlabeled with multiple fluors86,137 and enzyme-linked probes or detection systems11,62,138,139

that allow signal amplification. The latter in-direct approach not only has the potential forsignal amplification, but may also be used innatural samples showing high levels of au-tofluorescence.17,140

Any thorough identification method hasto include positive and negative controls.11,17

False-positive results may either be caused

by cells emitting autofluorescence after exci-tation or by nonspecific binding of the probeto nontarget cells. Samples therefore shouldbe checked for autofluorescence before hy-bridization and a negative control with a fluo-rescent oligonucleotide not complementaryto rRNA has to be applied to check for se-

quence-independent nonspecific binding. Suchnonspecific binding may be due to interactionof the dye compound of the probe with hydro-phobic cell components. Failures to detect cellscontaining target sequences (false-negatives)may originate from cells with either low cel-

lular ribosome content or limited permeabil-ity of the cell periphery for the fluorescentprobe.16

With the rapidly expanding database of16S rRNA sequences, the problem of probespecificity has become more apparent andthe design of diagnostic probes is becomingincreasingly difficult.34 These problems arealso applicable to PCR and other oligonucle-otide-dependent techniques. The problem ofprobe specificity may be overcome by using

multiple specific oligonucleotide probes tar-geting different sites on the rRNA moleculeand labeled with different fluorochromes.4

While a single oligonucleotide target sequencemay be found in a number of related taxa,the probability that target sites for three de-signed oligonucleotides are found in a non-target organism is, however, much reduced.

D. Denaturing Gradient GelElectrophoresis (DGGE)

In addition to concerns raised earlier re-garding the representative PCR amplificationof individual populations within the targetcollection and the formation of chimeric se-quences between populations,122,124 there areother limitations associated with the technique.Available technology does not allow for theseparation of multiple bands amplified froma highly diverse bacterial community,57,107 butresolution may be improved by using a nar-

rower range of denaturants or two-dimensionalelectrophoresis.107 The information obtainedfrom sequencing of individual bands is limit-ed, because only fragments of up to approxi-mately 500 bp can be well separated. Anotherconcern associated with the technique is theassignment of particular bands to individual


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populations, particularly where multiple bandsoccur. Individual organisms could potentiallycontribute to multiple bands on a DGGE gelbecause the sequences between rRNA oper-ons of an individual organism can vary sig-nificantly.57,129,141

IV. CONCLUSIONS AND FUTUREPROSPECTS

Classic methods for the detection and iden-tification of microorganisms require their cul-tivation in pure cultures. However, often onlya minor fraction of the bacteria actually pres-ent in a natural sample can be grown under

standard conditions, and the inevitable selec-tivity of cultivation techniques hardly reflectsthe true microbial community composition.4

The ability to characterize microbes rapidlyin mixed cultures by a molecular approachprovides a way of monitoring community struc-ture and biodiversity in natural and engineeredecosystems. Due to functional constraints, therRNA genes are highly conserved and there-fore have been used extensively in these analy-ses. Organisms catalogued by sequence will,however, permit assessments of diversityonly. The challenge of the described molecu-lar technologies therefore is the assignment offunction to groups of microorganisms in theirparticular ecosystem.

Integrated studies combining molecularmeasures of species composition and the abun-dance of important microbial groups withmeasurement of particular processes and en-vironmental parameters are being more widelyadopted. Such studies have the potential to re-late community structure to function and ac-

tivity in complex microbial communities. Forexample, community structure and functionhave been analyzed through a combination ofwhole cell in situ hybridization and micro-sensors. rRNA-based localizations of ammo-nia- and nitrite-oxidizing bacteria were doneon a nitrifying biofilm following microelec-

trode measurements for O2, N2O and NO2/

NO3.93,115 A good correlation of community

structure and function could be demonstratedon a microscopic scale. The distribution ofsulfate-reducing and methanogenic bacteriawas also determined in a similar manner,

with respect to activity.65 Such studies willbe essential in the future if we are to reap themaximum rewards from nucleic acid-basedstudies of microbial ecology. Moreover, manymicroorganisms have been isolated that arecapable of remarkable enzymatic transforma-tions. Subsequently, molecular methods canbe used to obtain essentially any gene encod-ing potentially useful products directly fromthe environment, without the need to cultivatethe organism. The specific identity of the or-

ganism that contributes a useful gene wouldusually be unimportant and, in any case, couldbe obtained using the sequence of the geneand methods such as those described. Thistype of approach to the natural microbial worldis being investigated by biotechnological com-panies, and the door thus has been thrownopen for the enhancement and practice ofenvironmental biotechnology.

Nevertheless, the role of classic microbialecology should not be underestimated. Mo-lecular studies complemented by appropri-ate culture-based investigations will assist inidentifying organisms that are truly represen-tative of those important in nature. It is onlyby selecting a range of appropriate tools in acomplementary fashion that some of the mys-teries of microbial ecology can be unlockedand the wealth of novel biodiversity present-ed by natural microbial communities can beharvested.

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