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Critical Reviews in Microbiology

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Molecular Techniques for Determining Microbial Diversity and Community Structure in Natural Environments
J. Theron a; T. E. Cloete a a Department of Microbiology and Plant Pathology, University of Pretoria, 0002 Pretoria, South Africa. Online Publication Date: 01 January 2000

To cite this Article Theron, J. and Cloete, T. E.(2000)'Molecular Techniques for Determining Microbial Diversity and Community

Structure in Natural Environments',Critical Reviews in Microbiology,26:1,37 57

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

Molecular Techniques for Determining Microbial Diversity and Community Structure in Natural Environments
J. 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 community is fundamental to the understanding of the structure and function of an ecosystem. The simple morphology 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 obstacles 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 community 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 sequencing, polymeric chain reaction, nucleic acid hybridization, polymorphism-based assays.

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I. INTRODUCTION Historically, it has been very difficult to determine the diversity of microorganisms that constitute natural and artificial ecosystems. Unlike most eukaryotes, few bacteria can be recognized on the basis of morphological features. The classic approach to enumerate bacteria in environmental samples has been culture-dependent techniques combined with a simultaneous or subsequent differentiation of the isolates based on batteries of physiological and biochemical tests. However, culture-dependent methods do not accu1040-841X/00/$.50 2000 by CRC Press LLC

rately reflect the actual bacterial community structure, but rather the selectivity of growth media for certain bacteria. Furthermore, (1) all techniques rely on cultivation and are time consuming and expensive as are the physiological and biochemical differentiation tests; (2) after many generations necessary to form plate colonies, the organism may deviate from its physiology, and possibly even from genotypic mix, of the population in nature; (3) only a minor fraction (0.1 to 10%) of the bacteria can be cultivated using standard techniques; and (4) it offers a very limited insight into the spatial distribution of the microorganisms.14

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Fluorescent antibody techniques offered major advances in aut-ecological studies of microorganisms.59 These methods allowed direct identification and enumeration of individual bacteria in environmental samples without requirement of prior growth in culture media. The technique involves the use of specific antibodies raised against surface markers of defined pure cultures that are either labeled directly with fluorescent dye molecules or via a fluorescent secondary antibody. Although this approach has yielded important insights into the spatial distribution of microorganisms, it has the following limitations:10,11 (1) expression of the antigen may be influenced by environmental factors; (2) false-positive and false-negative results may be obtained due to cross-reactivity or lack of reaction; (3) nonspecific binding of antibodies may result in high levels of background fluorescence; and (4) production of specific antibodies requires a pure culture of the organism of interest. Subsequently, various recombinant DNA techniques for the identification of bacteria have been developed that are independent of cultivation methods. Pace et al.1 and Olsen et al.10 outlined an approach based on identification of microorganisms via 16S rRNA gene sequences determined directly from the biomass without cultivation. The rRNA molecules comprise highly conserved sequence domains interspersed with more variable regions.12,13 Consequently, comparative analyses of rRNA sequences can identify so-called signature sequence motifs on various taxonomic levels that are targets for an evolutionary-based identification.4,1416 Advantages using the rRNAs include: (1) their occurrence in high copy numbers of usually more than 1000 in any living cell; (2) their sequences can be retrieved from the samples of interest without prior cultivation; and (3) 16S rRNA sequences have been determined for a large fraction of bacterial species.4,10,18 The use of recombinant DNA techniques have overcome many of the stumbling blocks of cultivation. It provides ways of charac-

terizing mixed microbial communities on a molecular basis in a relatively unbiased way, thereby enhancing the science and practice of microbial ecology. In this article, we describe recombinant DNA technologies applied to the analysis of mixed microbial populations. The various approaches and tools used in these analysis are outlined in Figure 1.

II. MOLECULAR TECHNIQUES A. 16S rDNA Sequencing Approach Initially, the analysis of the diversity of natural microbial populations relied on direct extraction, purification, and sequencing of 5S rRNA molecules from environmental samples.1921 Although these studies yielded interesting insights, the information content of the 120 nucleotides long 5S rRNA is relatively small, and its paucity of independently varying nucleotide positions limits its usefulness to less complex ecosystems. An average bacterial 16S rRNA molecule has a length of approximately 1500 nucleotides and thus contains considerably more information for reliable analyses than the 5S rRNA molecule. Consequently, the use of the larger rRNA molecules for studies in microbial ecology was suggested.10 In addition, the development of robust DNA cloning techniques and the polymerase chain reaction (PCR) have facilitated higherresolution analyses of more complex communities using 16S rRNA sequence analysis.

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1. Construction of 16S rRNA Gene Clone Libraries

The starting point for the recovery of rRNA sequence information from nucleic acid extracted from environmental samples is the efficient extraction of nucleic acids of sufficient quality for use in subsequent procedures. Various methods are available for the extraction and purification of nucleic acids from a

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FIGURE 1.

Commonly used molecular approaches in microbial ecology.

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wide range of environmental samples. These are usually based on chemical and/or physical disruption of cells combined with treatments to remove contaminating materials, such as humic acids and metals that can inhibit the efficiency of subsequent enzymatic reactions. Any one of three basic approaches can then be used to obtain rRNA gene clones from the total community nucleic acids. Initial approaches relied on shotgun cloning of the size-fractionated community DNA into bacteriophage lambda,1,10,22 after which the library is screened for the presence of rRNA genes. This is a laborious procedure, as rRNA genes will only represent a small fraction (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 other than those encoding rRNA. A second approach is to use the enzyme reverse transcriptase and universal or group-specific primers to synthesize single-stranded DNA complementary to rRNA in the community.23,24 A PCR step with rDNA-specific primers following reverse transcription may also be used to synthesize double-stranded rDNA for cloning.11 Starting the sequence retrieval from rRNA when compared with DNA has the advantage that due to the smaller size of rRNA more rigorous nucleic acid extraction techniques can be applied.25 Furthermore, the resulting community profile will offer some reflection of the most metabolically active organisms, because cells that produce more RNA will be better represented in the clone library than metabolically inactive cells.26 The simplest and currently the most widely adopted method to obtain 16S rRNA genes from the environment is through the use of PCR. rRNA genes can be amplified directly from the total community DNA using rRNAspecific primers and then cloned using standard methods.27 By taking advantage of the highly conserved nature of rRNA, universal primers capable of annealing to rRNA genes from all three domains (Archaea, Bacteria,

Eukarya) or primers designed to amplify rRNA genes from a particular group of organisms can be used.3,17,28 Following PCR amplification, the amplified products can be cloned. Commercially available kits exploit the fact that PCR-amplified products have an overhanging 3 deoxyadenosine residue at each end when certain DNA polymerases are used. This allows cloning of the product into a sequencing-ready vector containing a complementary deoxythymidine overhang, in many cases without requiring the product to be purified or further modified. Alternatively, the PCR products can be cloned by filling overhanging residues followed by blunt-end ligation procedures.29

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2. Screening of Clone Libraries for rRNA Genes

Contrary to shotgun gene clone libraries, the majority of clones in PCR-based gene libraries will contain rRNA sequences. To avoid unnecessary sequence determinations, redundant clones in both types of libraries need to be eliminated using the following strategies: (1) single-nucleotide sequencing;24,30 (2) dot or colony hybridization using species or group-specific probes;3,10,31,32 (3) restriction fragment length polymorphisms (RFLP) of purified plasmid DNA;31,33 and (4) colony PCR (using sequencing primers with priming sites that flank the insert DNA).34

3. Sequencing
The rapid screening and analysis of large gene clone libraries have been facilitated by automated DNA sequence systems.35 Complete sequencing of the cloned rRNA genes is facilitated by the presence of conserved sequence domains.36 Because of their length (15 to 20 nucleotides) and their universality, oligonucleotide primers can be designed that permit sequencing of the complete rRNA gene.10,20,37

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Once a sequence database has been generated from a clone library, the sequences can be compared to each other as well as previously published sequences or databases38,39 to indicate the kinds of microorganisms present in the sample. Therefore, the technique provides a list of 16S rRNA sequences as surrogate identification of individuals from the sample.

B. PCR and Related Techniques The expanding rRNA sequence database contains several thousand sequences. By comparative sequence analysis of the more variable regions of the 16S rRNA molecule, it is possible to design oligonucleotides of varying phylogenetic resolution. These can be used in the detection and enumeration of specific groups of bacteria by PCR40 or combined with the use of specific oligonucleotide probes.41 PCR provides a way of amplifying nucleic acid sequences that might be in low abundance in a complex mixture of whole cell extracts. A thermostable DNA polymerase, often the Taq enzyme from Thermus aquaticus, is used to amplify the specific DNA sequence of interest. The target sequence is defined by two sequence-specific oligonucleotide primers that flank the target sequence and that anneal to the complementary strands of the target sequence. During the PCR process, repetitive cycles of DNA denaturation, annealing of the oligonucleotide primers to the target DNA, and extension of the primers across the target sequence results in increasingly greater quantities of target sequence.

rRNA operons in order to detect specific organisms or groups of organisms in environmental samples.4245 Although the method is both specific and sensitive, such standard PCR reactions are not quantitative. To obtain quantitative data from PCR-based analyses, statistical methods based on most probable number (MPN) estimations have been used.44,46,47 In MPN-PCR, DNA extracts are diluted before PCR amplification and limits are set on the number of genes in the sample by reference to known control dilutions. Another way to quantify PCR-amplified products for comparison is to include an internal control in the PCR reaction.4850 Here, a known amount of target DNA is added to a PCR reaction containing DNA from the mixed microbial population. The known target DNA is complementary to the same primers and thus competes with the target sequences in the extracted DNA sample. By preparing a dilution series of the known and unknown DNA species, it is possible to quantify the amount of product produced from the complementary gene in the extracted DNA. The known DNA target can be generated by cloning the gene of interest or purifying the PCR-amplified product, after which a deletion is introduced to give a differently sized PCR product.41 There exist many variations of the PCR technique. The sensitivity and specificity of the PCR may be improved by adopting a nested approach. The initial amplification is carried out with a pair of primers that are not organism specific, whereafter a second round of amplification is conducted on the product using primers specific for an organism with target sites internal to the first primer pair.51,52

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1. PCR with Specific Primers

The presence of universally conserved 5 and 3 sequences37 allows both the recovery of rRNA sequences as cDNA and amplification of nearly complete 16S rRNA genes extracted from natural samples. Specific primers have been used to amplify fragments of C. Nucleic Acid Hybridization Techniques The easiest way of detecting specific nucleic acid sequences is through direct hybridization of 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 nylon or nitrocellulose membrane. Bacterial colonies can also be replica-plated from agar plates to membranes and their nucleic acids exposed in situ following lysis for subsequent hybridization. Probes may be used to detect genes in the bacterial genome (Southern blots) or to detect mRNA or rRNA (Northern blots).41 For the in situ identification of individual whole cells, it is necessary to make the cells permeable to oligonucleotide probes hybridizing with rRNA.17 These hybridization techniques rely on the specific binding of nucleic acid probes to complementary DNA or RNA (target nucleic acid). These probes are single strands of nucleic acid with the potential of carrying detectable marker molecules highly specific to complementary target sequences, even if these sequences account for only a small fraction of the target nucleic acid. Either DNA or RNA can serve as a nucleic acid probe, but for a number of reasons (e.g., ease of synthesis and stability), most studies have employed DNA probes.2

D. GENOMIC DNA HYBRIDIZATION TECHNIQUES Nucleic acid hybridization methods such as DNA reassociation and reciprocal hybridization of community DNA provides limited information of specific sequence content. Furthermore, the extent to which these methods can be used to estimate species diversity and individual population identity varies with the method and community.

The principle of DNA renaturation kinetics is that the rate of hybridization is proportional to the concentration of complementary DNA sequences and inversely proportional to the total length of different sequences in the sample.2,56 Thus, as microbial community diversity (heterogenecity) increases (e.g., there are a greater number of unique genomes), the rate of reassociation of DNA extracted from the community decreases.57 Experimentally, DNA reassociation is measured over time and the fraction of reassociated DNA (C/Co) is expressed as a function of Cot, where Co is the initial molar concentration of nucleotides in single-stranded DNA and t is the time in seconds. The plot of this relationship is referred to as a Cot curve. The reaction rate constant, k, can be expressed as 1/Cot1/2, where t1/2 denotes the time in seconds required for 50% reassociation. Under defined conditions, most importantly temperature and monovalent ion concentration, Cot1/2 is proportional to the complexity (e.g., number of unique genomes) of the DNA.58 The conclusion from DNA reassociation studies is generally that natural microbial ecosystems are very complex.2,54 DNA reassociation can provide a useful measure of community structure.57 However, one concern in the interpretation of DNA reassociation estimates is a reduction in the rate of reassociation resulting from impurities in the DNA sample. It is also important to evaluate changes in reassociation kinetics that might result from the use of different extraction and purification techniques.

1. DNA Reassociation
DNA reassociation kinetics have been used to investigate genomic sequence complexity53 and to assess the diversity of natural microbial communities.54,55 In these analyses, community DNA isolated from the environment is denatured and then allowed to reanneal.

2. Reciprocal Hybridization of Community DNA

Reciprocal hybridization of total community DNA59,60 provides an approach to determine whether two samples share the same kinds of organisms, regardless of exactly their species. This is done by cross-hybridization of the community DNA extracted from the two samples,

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based on the idea that significant cross-hybridization of pure culture DNA occurs only between identical or very closely related species.59 The prokaryotic DNA is extracted from each sample, radioactively labeled by standard techniques and then hybridized under stringent conditions to DNA on filters. Radioactive probes from each are hybridized to filters containing DNA spots from the same sample (control) as well as the test sample. The result (measured radioactivity bound to the test spot) is expressed as a percentage of the self-hybridization result and is referred to as the similarity between the samples. However, interpretation is complicated by uncertainties about relative population abundances and hybridization kinetics as well as possible differences in labeling efficiencies of DNAs comprising different DNA pools. Nevertheless, the method provides a relatively clear assessment of community relationship when the communities are nearly identical (high reciprocal hybridization similarity) or distinct (low reciprocal hybridization). Intermediate cross-hybridization values may, however, be more difficult to interpret.57,61

E. Quantitative DNA Hybridization Qualitative and quantitative estimates of community structure can be made using tailormade oligonucleotide probes that bind to targets with a wide range of specificities from domain to strain. Possibly the most important hybridization techniques for microbial ecology, because they are the most direct, are quantitative dot-blot hybridizations of extracted nucleic acids,25 and in situ hybridization of fixed environmental samples.4,18

latter are used more frequently because they are short enough to allow for single mismatch discrimination of target nucleic acids and large quantities of oligonucleotides can be rapidly and inexpensively produced. During oligonucleotide synthesis a variety of marker or linker sequences can be introduced to the 5 end of the oligonucleotide. The principal conjugate fluorochromes are derivatives of fluorescein or rhodamine, although labels such as digoxigenin and biotin have also been used.4,11,62 The design of rRNA-targeted group- or species-specific probes should be based on a good rRNA sequence database and be performed in a computer-assisted way using the appropriate software. A resource for such rRNA studies is the Ribosomal Database Project,38,39 which provides aligned rRNA sequences as well as a variety of other services such as probe analysis and sequence similarity analysis. The organisms encompassed by a probe varies according to the region of the 16S rRNA selected as the hybridization target. Species-specific probes complement the most variable regions, while more general probes target more conserved regions of the molecule.3,25,63 The principal steps involved in the design of probes are (1) the alignment of rRNA gene sequences; (2) the identification of sequence idiosyncrasies; (3) the synthesis and labeling of complementary nucleic acid probes; and (4) the experimental evaluation and optimization of the probe specificities and assay sensitivities using cultured reference strains.16,17,64

2. Quantitative Dot-Blot Hybridization

Although the comparative analysis of sequences retrieved from an environmental sample yields information on the identity or relatedness of new sequences compared with the available sequences, it gives a minimal estimate of the microbial diversity in the examined sample. Clone or sequence abundances may also be a biased measure of organ-

1. Oligonucleotide Probes
According to their length, DNA probes can be grouped as either polynucleotide probes (more than 50 nucleotides) or as oligonucleotide probes (less than 20 nucleotides).18 The

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ism abundance due to variations in extraction and cloning efficiencies.4,22 Furthermore, it is possible that a sequence may have originated from the pool of free DNA present in the ecosystem or from contamination and not from cells in the habitat. Thus, the abundance or relative abundance of a certain rRNA or rDNA in the extracted nucleic acid pool can be determined more reliably by dot-blot hybridization or of a certain clone in the library by colony hybridization. For quantitative dot-blot hybridization25,65 the samples of interest are treated to maximize cell lysis and release of nucleic acids. Quantification of a certain 16S rRNA compared with total 16S rRNA can then be obtained by dot-blot hybridization of a directly isolated nucleic acid mixture with universal and specific oligonucleotide probes. The relative abundance is calculated by dividing the amount of specific probe bound to a given sample by the amount of hybridized universal probe. The relative abundance of a given rRNA might reflect changes in the abundance of the cell population of interest or in the cellular rRNA content. For several organisms, a direct linear relationship has been shown between growth rate and cellular ribosome content.4,66 The relative abundance of a rRNA thus can be interpreted as the relative importance of a defined species in terms of actual metabolic activity or potential metabolic activity, but cannot be directly translated into cell numbers.18

3. Whole Cell In Situ Hybridization

Analysis on a single cell level can provide a more detailed picture than dot-blot hybridization. Not only can the cell morphology of an uncultured microbe be determined, but also their spatial distribution in situ. Early applications of in situ nucleic acid hybridization relied on the use of isotopically labeled oligonucleotides that bound to the rRNAs of intact, fixed cells, and following autoradiog-

raphy organisms could be phylogenetically identified.15 However, microautoradiography of such in situ hybridization probes require long exposure times to photographic emulsions. Moreover, the useful resolution is no less than about 1 m, because of the scatter of radioactive disintegrations. The use of fluorescent dyelabeled rRNA-targeted oligonucleotide probes allows the detection of individual cells,67 and this approach has been used to analyze many different ecosystems.4,18 rRNA sequences and thereby phylogenetic affiliations were assigned to individual cells of hitherto uncultured endosymbionts of protozoa68,69 and of magnetotactic bacteria.70,71 Other applications encompassed enumeration and spatial distribution of bacterial populations in activated sludge7278 and in biofilms.11,7983 During whole cell in situ hybridization the morphology of the cells in the sample has to be stabilized in order to maintain morphological integrity of the cells under harsh hybridization conditions. The cell walls and membranes have to be permeabilized to allow free penetration of fluorescent oligonucleotides to the intracellular rRNA. This can be achieved with fixatives such as aldehydes and/or alcohols.4,67,70 The cells are either attached to gelatin-coated microscope slides or hybridized in suspension and immersed in hybridization solution containing a fluorescently labeled oligonucleotide. Following incubation at the hybridization temperature for one to several hours, to allow the probe to bind to complementary rRNA sequences, washing steps are used to remove unbound or part of the nonspecifically bound fluorescent probe, and the sample is then viewed by epifluorescence microscopy. Probes will only bind correctly under defined hybridization conditions, and the optimization of hybridization and washing conditions is as important as the probe design.16,63 Several probes labeled with spectrally different fluorochromes can be simultaneously used on one sample,18,84 while counterstaining of the fixed cells with DAPI (4,6-diamidino-2-phenylindole) allows total counts to be made.85

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A direct correlation between the growth rates of bacterial cells, the average ribosome contents and the probe-conferred fluorescence has been reported.67,86 This is used to estimate the growth rates of individual cells in situ.87,88 Often, it is also important to obtain information about how the functional components of an ecological system relates to the organization of the system. In communities that have an inherent architecture, such as biofilms and flocs, the question of where various organisms are located is of interest. These determinations are difficult to make with conventional epifluorescence microscopy. By coupling in situ hybridization with fluorescently labeled rRNA-targeted oligonucleotide probes with confocal scanning laser microscopy,89 it is possible to place the labeled microbes in a three-dimensional reconstruction of the intact microbial community.78,83,9093 Fluorescently labeled probes have also been used in combination with flow cytometry for rapid, automated identification and counting or collection of microorganisms in hybridized cell suspensions.9496

ments following separation by gel electrophoresis. Although the detection of small differences in specific DNA sequences can give important information about community structure and the diversity of microbes, they provide little or no direct information of specific microbial population identity.

1. Amplified Ribosomal DNA Restriction Analysis (ARDRA)

The amplified portions of 16S rRNA genes from a mixed microbial population might be of similar sizes with a particular set of oligonucleotide primers, but have small differences at the nucleotide level. One way of detecting these differences is to restrict the PCR-amplified product with restriction endonucleases and examine the pattern of restriction fragments.31,101104 The analytical power of this procedure derives from the fractionation step. The separation of DNA fragments derived from different populations requires that they differ in sequence at the restriction endonuclease sites, or differ in length of DNA flanked by common restriction sites. The separated DNA fragments may also be transferred to filters for hybridization with probes specific for an organism of interest, that is, are subjected to Southern transfer.105 By design, the probe for each organism will hybridize to a fragment of a different size and thus can be used to resolve different environmental populations.106

F. Polymorphism-Based Procedures The different polymorphism-based procedures that have been applied to microbial ecological systems are generally coupled to a PCR reaction. The techniques of amplified ribosomal DNA restriction analysis (ARDRA) and denaturing gradient gel electrophoresis (DGGE) is specifically addressed. Two other protocols use a single primer to amplify fragments with PCR before examination on agarose gels or restriction. PCR amplification of repetitive extragenic palindromic sequences (REP-PCR) takes advantage of repetitive sequences found in the microbial genome.97,98 Randomly amplified polymorphic DNA (RAPD) or arbitrarily primed PCR99,100 use primers that are not specific for a particular gene to amplify fragments. These methods generate a fingerprint that is represented by a banding pattern of nucleic acid frag-

2. Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing gradient gel electrophoresis (DGGE)107 is a method by which fragments of DNA of identical or near identical length but different in sequence composition can be resolved electrophoretically. DGGE has been extended to the analysis of PCR-amplified 16S rRNA genes from environmental samples.108111 In DGGE analysis, separation is based on changes in electrophoretic mobility

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of DNA fragments migrating in a vertical polyacrylamide gel containing a linearly increasing concentration of DNA denaturants (formamide and/or urea). As the DNA fragments are subjected to electrophoresis, partial melting of the double-stranded DNA occurs in discrete regions, the so-called melting domains, at a denaturant concentration specific for the nucleotide sequence of the DNA. The migration of the fragment therefore is severely retarded. Sequence variation within such domains alters their melting behavior, and sequence variants of the different amplification products stop migrating at different positions in the denaturing gradient.112,113 Although DGGE analysis of PCR-amplified 16S rDNA fragments provide a rapid method to characterize community population structure, more specific information of population composition can be obtained by secondary analysis of the DGGE banding pattern. Individual bands (fragments) may be excised from the gel, subjected to a second round of PCR amplification and sequenced.111,114 Alternatively, the DNA can be transferred to nylon membranes and then challenged with group- and species-specific oligonucleotide probes to identify specific populations within the microbial community.108,115 As DGGE is relatively rapid to perform and many samples can be electrophoresed simultaneously, the method is particularly useful when examining time series and population dynamics. Once the identity of an organism associated with any particular band has been determined, fluctuations in individual components of a microbial population, due to environmental perturbations, can be rapidly assessed.34

III. LIMITATIONS OF MOLECULAR METHODS A. Extraction of Nucleic Acids A major limitation of the described nucleicbased methodologies, with the exception of

whole cell in situ hybridization, is the quantitative recovery of nucleic acids from the environment. Two aspects of nucleic acid recovery deserve special mention.57 The first aspect relates to the recovery efficiency of the extraction method. For example, if the total amount of nucleic acids present in a sample is unknown, then it is difficult to assess the recovery efficiency by any extraction method. This is compounded by the fact that spores are more resistant to cell lysis than vegetative cells, and Gram-negative cells are more susceptible to cell lysis than Gram-positive cells. It is also possible that the same lysis technique may give different results with different types of sample (e.g., water, sediment, or soil), and recovery might be reduced by degradation or adsorption of nucleic acids to matrix material (e.g., clays). The degree of cell lysis therefore should be determined independently. Microscopic enumeration of the cells in an environmental sample before and after lysis treatments can yield a reasonable indication of the efficiency of cell lysis.116 A second consideration is representative nucleic acid recovery. For example, a population resistant to breakage would be fractionally underrepresented, while microorganisms that are easily lysed would be overrepresented.57 It has been noted that small cells (0.3 to 1.2 m) in a soil sample were more resistant to lysis than larger cells and even bacterial endospores.117 This may influence the recovery of sequences from environmental samples where many cells are likely to be small due to a state of starvation. Starting 16S rRNA sequence retrieval from RNA compared with DNA has the advantage that it is more resistant to mechanical breakage so that more vigorous nucleic acid extraction techniques can be applied.25 However, the RNA is subject to degradation during the following extraction as a consequence of endogenous nucleases or nuclease contamination. One consequence of partial degradation of the sample is variable destruction of different primer or probe target sites.118 However, sample integrity may be evaluated by acry-

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

B. PCR and Cloning Although the analysis of a microbial community by PCR and cloning provides a convenient and rapid alternative to shotgun cloning, there are several factors that could skew diversity estimates.119 PCR reactions are very sensitive to reaction conditions and even duplicates might not give quantitatively identical results.41 Due to the sensitivity and specificity of the PCR reactions, minor contamination can lead to false-positive signals and false-negative amplifications are often seen.120 It has been suggested that templates with a high % G + C content are discriminated against due to low efficiency of strand separation during the denaturation step of the PCR reaction,121 and sequences that are more abundant than other less abundant sequences may be preferentially amplified.3 Certain sequences may also be discriminated slightly against others due to selective priming or higher-order structure elements that could result in huge differences after multiple cycles. Amplification of rRNA sequences by using general primers in some cases exclude important environmental populations.122 The work of Suzuki and Giovannoni123 indicated that the amount and kind of background DNA in a sample can affect the results through competition. Furthermore, depending on whether the same batch of PCR product was cloned using either blunt-end or sticky-end cloning procedures, different results may be obtained.121 Depending on the quality of the DNA used for PCR, in vitro recombinants of two or more wild- type rRNA genes, so-called chimeric sequences, can be formed at frequencies of several percent.124,125 One study demonstrated that 30% of products generated during coamplification of similar templates were chimeric.126 Although computer programs have been developed to help identify chimeric sequences,125 the programs may indicate the

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presence of chimeric sequences even when none exist and the programs have difficulties in identifying chimeras when the two sequences from which the chimera is formed show greater than 85% homology. Furthermore, depending on the particular thermostable DNA polymerase used, the fidelity of the PCR may vary.34 Although careful analysis of secondary interactions should identify discrepencies due to misincorporation of nucleotides during PCR, there is a danger that the presence of novel taxa may be assumed as a result of infidelity in DNA replication. Many prokaryotes have several rRNA operons127 and even though the rRNA coding regions are usually highly similar, they are not necessarily identical.128 The archaeon Haloarcula marismortui has interoperon differences of up to 5% between the two 16S rRNA gene sequences.129 The cloning step in rRNA analysis separates not only the rRNA genes of different organisms but also the different genes. Thus, slightly different gene fragments could originate from one strain and would not indicate the presence of closely related organisms.130 This is of concern when making conclusions about biodiversity from data obtained with rRNA gene clone libraries.

C. Whole Cell In Situ Hybridization The methodological constraints regarding whole cell hybridization can be divided into four main categories: cell permeability problems, target site accessibility, target specificity, and sensitivity. Permeability of fixed cells may be affected by their state of growth. For example, alterations in the cell wall structure of dormant cells (e.g., spores) increase their resistance to adverse environmental conditions.131 Poor permeability of fixed cells to the oligonucleotide probe could result in weak fluorescence intensity. Simple fixation methods using alcohols, formaldehyde, or paraformaldehyde tend to permeabilize 70 to 90% of microscopically visible cells.4,34 However,

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the cell permeability may be enhanced by additional treatment with enzymes,132 solvents,133 or acid.134 If a cell has been permeabilized, the accessibility of the target sequence in the rRNA may determine whether probe hybridization to rRNA will occur within the cell. Hybridization of the probe to the target rRNA may be prevented due to highly stable secondary structure elements of the rRNA itself or due to strong interactions of the rRNA with ribosomal proteins.17,18 This problem can be detected by a strong hybridization signal being obtained with a universal probe that is known to target an accessible site on the rRNA molecule. Poor accessibility of the target site can then be indicated by the absence of a hybridization signal in the same cell(s) using a different probe.4,34 Generally, probes containing a single labeled molecule give a strong signal only if cells are metabolically active and contain large numbers of ribosomes and target rRNA. Conversely, low cellular ribosome contents may result in weak fluorescence intensity. This is a significant concern in slowly growing natural populations.86,135 Various approaches have been adopted in order to improve the sensitivity. These have included the use of multiple probes labeled with a single fluor;84,136 or labeled with multiple fluors86,137 and enzymelinked probes or detection systems11,62,138,139 that allow signal amplification. The latter indirect approach not only has the potential for signal amplification, but may also be used in natural samples showing high levels of autofluorescence.17,140 Any thorough identification method has to include positive and negative controls.11,17 False-positive results may either be caused by cells emitting autofluorescence after excitation or by nonspecific binding of the probe to nontarget cells. Samples therefore should be checked for autofluorescence before hybridization and a negative control with a fluorescent oligonucleotide not complementary to rRNA has to be applied to check for se-

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quence-independent nonspecific binding. Such nonspecific binding may be due to interaction of the dye compound of the probe with hydrophobic cell components. Failures to detect cells containing target sequences (false-negatives) may originate from cells with either low cellular ribosome content or limited permeability of the cell periphery for the fluorescent probe.16 With the rapidly expanding database of 16S rRNA sequences, the problem of probe specificity has become more apparent and the design of diagnostic probes is becoming increasingly difficult.34 These problems are also applicable to PCR and other oligonucleotide-dependent techniques. The problem of probe specificity may be overcome by using multiple specific oligonucleotide probes targeting different sites on the rRNA molecule and labeled with different fluorochromes.4 While a single oligonucleotide target sequence may be found in a number of related taxa, the probability that target sites for three designed oligonucleotides are found in a nontarget organism is, however, much reduced.

D. Denaturing Gradient Gel Electrophoresis (DGGE) In addition to concerns raised earlier regarding the representative PCR amplification of individual populations within the target collection and the formation of chimeric sequences between populations,122,124 there are other limitations associated with the technique. Available technology does not allow for the separation of multiple bands amplified from a highly diverse bacterial community,57,107 but resolution may be improved by using a narrower range of denaturants or two-dimensional electrophoresis.107 The information obtained from sequencing of individual bands is limited, because only fragments of up to approximately 500 bp can be well separated. Another concern associated with the technique is the assignment of particular bands to individual

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populations, particularly where multiple bands occur. Individual organisms could potentially contribute to multiple bands on a DGGE gel because the sequences between rRNA operons of an individual organism can vary significantly.57,129,141

IV. CONCLUSIONS AND FUTURE PROSPECTS Classic methods for the detection and identification of microorganisms require their cultivation in pure cultures. However, often only a minor fraction of the bacteria actually present in a natural sample can be grown under standard conditions, and the inevitable selectivity of cultivation techniques hardly reflects the true microbial community composition.4 The ability to characterize microbes rapidly in mixed cultures by a molecular approach provides a way of monitoring community structure and biodiversity in natural and engineered ecosystems. Due to functional constraints, the rRNA genes are highly conserved and therefore have been used extensively in these analyses. Organisms catalogued by sequence will, however, permit assessments of diversity only. The challenge of the described molecular technologies therefore is the assignment of function to groups of microorganisms in their particular ecosystem. Integrated studies combining molecular measures of species composition and the abundance of important microbial groups with measurement of particular processes and environmental parameters are being more widely adopted. Such studies have the potential to relate community structure to function and activity in complex microbial communities. For example, community structure and function have been analyzed through a combination of whole cell in situ hybridization and microsensors. rRNA-based localizations of ammonia- and nitrite-oxidizing bacteria were done on 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 demonstrated on a microscopic scale. The distribution of sulfate-reducing and methanogenic bacteria was also determined in a similar manner, with respect to activity.65 Such studies will be essential in the future if we are to reap the maximum rewards from nucleic acid-based studies of microbial ecology. Moreover, many microorganisms have been isolated that are capable of remarkable enzymatic transformations. Subsequently, molecular methods can be used to obtain essentially any gene encoding potentially useful products directly from the environment, without the need to cultivate the organism. The specific identity of the organism that contributes a useful gene would usually be unimportant and, in any case, could be obtained using the sequence of the gene and methods such as those described. This type of approach to the natural microbial world is being investigated by biotechnological companies, and the door thus has been thrown open for the enhancement and practice of environmental biotechnology. Nevertheless, the role of classic microbial ecology should not be underestimated. Molecular studies complemented by appropriate culture-based investigations will assist in identifying organisms that are truly representative of those important in nature. It is only by selecting a range of appropriate tools in a complementary fashion that some of the mysteries of microbial ecology can be unlocked and the wealth of novel biodiversity presented by natural microbial communities can be harvested.

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REFERENCES
1. Pace, N. R., Stahl, D. A., Lane, D. J., and Olsen, G. J., The analysis of natural microbial populations by ribosomal RNA sequences, Adv. Microb. Ecol., 9, 1, 1986.

49

2. Holben, W. E. and Tiedje, J. M., Applications of nucleic acid hybridization in microbial ecology, Ecology, 69, 561, 1988. 3. Ward, D. M., Bateson, M. M., Weller, R., and Ruff-Roberts, A. L., Ribosomal RNA analysis of microorganisms as they occur in nature, Adv. Microb. Ecol., 12, 219, 1992. 4. Amann, R. I., Ludwig, W., and Schleifer, K-H., Phylogenetic identification and in situ detection of individual microbial cells without cultivation, Microbiol. Rev., 59, 143, 1995. 5. Bohlool, B. B. and Schmidt, E. L., The immunofluorescence approach in microbial ecology, Adv. Microbiol. Ecol., 4, 203, 1980. 6. Cloete, T. E. and Steyn, P. L., A combined membrane filter immunofluorescent technique for the in situ identification and enumeration of Acinetobacter in activated sludge, Wat. Res., 22, 961, 1988. 7. Macario, A. J. L., Conway de Macario, E., Ney, U., Schoberth, S. M., and Sahm, H., Shifts in methanogenic subpopulations measured with antibody probes in a fixed-bed loop anaerobic bioreactor treating sulfite evaporator condensate, Appl. Environ. Microbiol., 55, 1996, 1989. 8. Desmonts, C., Minet, J., Colwell, R., and Cormier, M., Fluorescent-antibody method useful for detecting viable but nonculturable Salmonella spp. in chlorinated wastewater, Appl. Environ. Microbiol., 56, 1448, 1990. 9. Witzel, K-P., Approaches to bacterial population dynamics, in Aquatic Microbial Ecology: Biochemical and Molecular Approaches, Overbeck J. and Chrst, R. J., Eds., SpringerVerlag, New York, 1990, 96. 10. Olsen, G. J., Lane, D. J., Giovannoni, S. J., Pace, N. R., and Stahl, D. A., Microbial ecology and evolution: a ribosomal RNA approach, Annu. Rev. Microbiol., 40, 337, 1986. 11. Amann, R., Ludwig, W., and Schleifer, KH., Identification and in situ detection of individual bacterial cells, FEMS Microbiol. Lett., 100, 45, 1992. 12. Gutell, R. R., Larsen, N., and Woese, C. R., Lessons from an evolving rRNA: 16S and 23S rRNA structures from a comparative perspective, Microbiol. Rev., 58, 10, 1994.

13. Van de Peer, Y., Chapelle, S., and De Wachter, R., A quantitative map of nucleotide substitution rates in bacterial rRNA, Nucl. Acids Res., 24, 3381, 1996. 14. Woese, C. R., Bacterial evolution, Microbiol. Rev., 51, 221, 1987. 15. Giovannoni, S. J., DeLong, E. F., Olsen, G. J., and Pace, N. R., Phylogenetic group-specific oligodeoxynucleotide probes for identification of single microbial cells, J. Bacteriol., 170, 720, 1988. 16. Manz, W., Amann, R., Ludwig, W., Wagner, M., and Schleifer, K-H., Phylogenetic oligodeoxynucleotide probes for the major subclasses of proteobacteria: problems and solutions, System. Appl. Microbiol., 15, 593, 1992. 17. Amann, R. I., In situ identification of microorganisms by whole cell hybridization with rRNA-targeted nucleic acid probes, in Molecular Microbial Ecology Manual, Akkermans, A. D. L., van Elsas, J. D., and de Bruijn, F. J., Eds., Kluwer Academic Publishers, Dordrecht, 1995, 1. 18. Amann, R., Glckner, F-O., and Neef, A., Modern methods in subsurface microbiology: in situ identification of microorganisms with nucleic acid probes, FEMS Microbiol. Rev., 20, 191, 1997. 19. Stahl, D. A., Lane, D. J., Olsen, G. J., and Pace, N. R., Analysis of hydrothermal ventassociated symbionts by ribosomal RNA sequences, Science, 224, 409, 1984. 20. Lane, D. J., Pace, B., Olsen, G. J., Stahl, D. A., Sogin, M. L., and Pace, N. R., Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses, Proc. Natl. Acad. Sci. U.S.A., 82, 6955, 1985. 21. Stahl, D. A., Lane, D. J., Olsen, G. J., and Pace, N. R., Characterization of a Yellowstone hot spring microbial community by 5S ribosomal RNA sequences, Appl. Environ. Microbiol., 49, 1379, 1985. 22. Schmidt, T. M., DeLong, E. F., and Pace, N. R., Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing, J. Bacteriol., 173, 4371, 1991. 23. Weller, R. and Ward, D. M., Selective recovery of 16S rRNA sequences from natural

Downloaded By: [ENEA] At: 14:48 13 November 2008

50

microbial communities in the form of cDNA, Appl. Environ. Microbiol., 55, 1818, 1989. 24. Ward, D. M., Weller, R., and Bateson, M. M., 16S rRNA sequences reveal numerous uncultured microorganisms in a natural community, Nature, 345, 63, 1990. 25. Stahl, D. A., Flesher, B., Mansfield, H., and Montgomery, L., Use of phylogenetically based hybridization probes for studies of ruminal microbial ecology, Appl. Environ. Microbiol., 54, 1079, 1988. 26. Hugenholtz, P. and Pace, N. R., Identifying microbial diversity in the natural environment: a molecular phylogenetic approach, Tibtech, 14, 190, 1996. 27. Giovannoni, S. J., Britschgi, T. B., Moyer, C. L., and Field, K. G., Genetic diversity in Sargasso Sea bacterioplankton, Nature, 345, 60, 1990. 28. Amann, R. I., Stromley, J., Devereux, R., Key, R., and Stahl, D. A., Molecular and microscopic identification of sulfate reducing bacteria in multispecies biofilms, Appl. Environ. Microbiol., 58, 614, 1992. 29. Sambrook, J., Fritsch, E. F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989. 30. Reysenbach, A. L., Wickham, G. S., and Pace, N. R., Phylogenetic analysis of the hyperthermophilic pink filament community in Octopus Spring, Yellowstone National Park, Appl. Environ. Microbiol., 60, 2113, 1994. 31. Britschgi, T. B. and Giovannoni, S. J., Phylogenetic analysis of a natural marine bacterioplankton population by rRNA gene cloning and sequencing, Appl. Environ. Microbiol., 57, 1707, 1991. 32. Liesack, W. and Stackebrandt, E., Occurrence of novel groups of the domain Bacteria as revealed by analysis of genetic material isolated from an Australian terrestrial environment, J. Bacteriol., 174, 5072, 1992. 33. DeLong, E. F., Franks, D. G., and Alldredge, A. L., Phylogenetic diversity of aggregateattached vs. free-living marine bacterial assemblages, Limnol. Oceanogr., 38, 924, 1993.

34. Head, I. M., Saunders, J. R., and Pickup, R. W., Microbial evolution, diversity and ecology: A decade of ribosomal RNA analysis of uncultivated microorganisms, Microb. Ecol., 35, 1, 1998. 35. Vainio, E. J., Moilanen, A., Koivula, T. T., Bamford, D. H., and Hantula, J., Comparison of partial 16S rRNA gene sequences obtained from activated sludge bacteria, Appl. Microbiol. Biotechnol., 48, 73, 1997. 36. Gutell, R. R., Weiser, B., Woese, C. R., and Noller, H. F., Comparative anatomy of 16Slike ribosomal RNA, Prog. Nucleic Acid Res. Mol. Biol., 32, 155, 1985. 37. Edwards, U., Rogall, T., Blcker, H., Emde, M., and Bttger, E. C., Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA, Nucl. Acids Res., 17, 7843, 1989. 38. Maidak, B. L., Larsen, N., McCaughey, M. J., Overbeek, R., Olsen, G. J., Fogel, K., Blandy, J., and Woese, C. R., The Ribosomal Database Project, Nucl. Acids Res., 22, 3485, 1994. 39. Maidak, B. L., Olsen, G. J., Larsen, N., Overbeek, R., McCaughey, M. J., and Woese, C. R., The RDP (Ribosomal Database Project), Nucl. Acids Res., 25, 109, 1997. 40. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Ehrlich, H. A., Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase, Science, 239, 487, 1988. 41. Schneegurt, M. A. and Kulpa, C. F., The application of molecular techniques in environmental biotechnology for monitoring microbial systems, Biotechnol. Appl. Biochem., 27, 73, 1998. 42. Hiorns, W. D., Hastings, R. C., Head, I. M., McCarthy, A. J., Saunders, J. R., Pickup, R. W., and Hall, G. H., Amplification of 16S ribosomal RNA genes of autotrophic ammonia-oxidizing bacteria demonstrates the ubiquity of nitrosospiras in the environment, Microbiology, 141, 2793, 1995. 43. Holmes, A. J., Owens, N. J. P., and Murrell, J. C., Detection of novel marine methanotrophs using phylogenetic and functional gene

Downloaded By: [ENEA] At: 14:48 13 November 2008

51

probes after methane enrichment, Microbiology, 141, 1947, 1995. 44. Pizarro, J., Jedlicki, E., Orellana, O., Romero, J., and Espejo, R. T., Bacterial populations in samples of bioleached copper ore as revealed by analysis of DNA obtained before and after cultivation, Appl. Environ. Microbiol., 62, 1323, 1996. 45. Zhou, J., Palumbo, A. V., and Tiedje, J. M., Sensitive detection of a novel class of toluenedegrading denitrifiers, Azoarcus tolulyticus, with small-subunit rRNA primers and probes, Appl. Environ. Microbiol., 63, 2384, 1997. 46. Degrange, V. and Bardin, R., Detection and counting of Nitrobacter populations in soil by PCR, Appl. Environ. Microbiol., 61, 2093, 1995. 47. Wand, H., Laht, T., Peters, M. M., Becker, P. M., Stottmeister, U., and Heinaru, A., Monitoring of biodegradative Pseudomonas putida strains in aquatic environments using molecular techniques, Microb. Ecol., 33, 124, 1997. 48. Diviacco, S., Norio, P., Zentilin, L., Menzo, S., Clementi, M., Biamonti, G., Riva, S., Falaschi, A., and Giacca, M., A novel procedure for quantitative polymerase chain reaction by coamplification of competitive templates, Gene, 122, 313, 1992. 49. Leser, T. D., Boye, M., and Hendriksen, N. B., Survival and activity of Pseudomonas sp. strain B13 (FR1) in a marine microcosm determined by quantitative PCR and an rRNA-targeting probe and its effect on the indigenous bacterioplankton, Appl. Environ. Microbiol., 61, 1201, 1995. 50. Wikstrm, P., Wiklund, A., Andersson, AC., and Forsman, M., DNA recovery and PCR quantification of catechol 2,3dioxygenase genes from different soil types, J. Biotechnol., 52, 107, 1996. 51. el Fantroussi, S., Mahillon, J., Naveau, H., and Agathos, S. N., Introduction of anaerobic dechlorinating bacteria into soil slurry microcosms and nested-PCR monitoring, Appl. Environ. Microbiol., 63, 806, 1997. 52. Lvesque, M-J., La Boissire, S., Thomas, J. C., Beaudet, R., and Villemur, R., Rapid method for detecting Desulfitobacterium frappieri strain PCP-1 in soil by the polymerase

chain reaction, Appl. Microbiol. Biotechnol., 47, 719, 1997. 53. Britten, R. J. and Kohne, D. E., Repeated sequences in DNA, Science, 161, 529, 1968. 54. Torsvik, V., Goksyr, J., and Daae, F. L., High diversity in DNA of soil bacteria, Appl. Environ. Microbiol., 56, 782, 1990. 55. Torsvik, V., Salte, K., Srheim, R., and Goksyr, J., Comparison of phenotypic diversity and DNA heterogeneity in a population of soil bacteria, Appl. Environ. Microbiol., 56, 776, 1990. 56. Chelm, B. K., DNA-DNA renaturation kinetics, in Methods in Chloroplast Molecular Biology, Edelman, M., Hallick, R. B. and Chua, H-H., Eds., Elsevier Biomedical, New York, 1982, 301. 57. Stahl, D. A., Molecular approaches for the measurement of density, diversity, and phylogeny, in Manual of Environmental Microbiology, Hurst, C. J, Knudsen, G. R., McInerney, M. J., Stetzenbach, L. D., and Walter, M. V., Eds., American Society for Microbiology, Washington, DC, 1997, 102. 58. Britten, R. J., Graham, D. E., and Neufeld, B. R., Analysis of repeating DNA sequences by reassociation, Methods Enzymol., 29, 363, 1974. 59. Lee, S. and Fuhrman, J. A., DNA hybridization to compare species compositions of natural bacterioplankton assemblages, Appl. Environ. Microbiol., 56, 739, 1990. 60. Lee, S. and Fuhrman, J. A., Spatial and temporal variation of natural bacterioplankton assemblages studied by total genomic DNA cross-hybridization, Limnol. Oceanogr., 36, 1277, 1991. 61. Fuhrman, J. A., Lee, S. H., Masuchi, Y., Davis, A. A., and Wilcox, R. M., Characterization of marine prokaryotic communities via DNA and RNA, Microb. Ecol., 28, 133, 1994. 62. Zarda, B., Amann, R., Wallner, G., and Schleifer, K-H., Identification of single bacterial cells using digoxigenin-labeled, rRNAtargeted oligonucleotides, J. Gen. Microbiol., 137, 2823, 1991. 63. Stahl, D. A. and Amann, R., Development and application of nucleic acid probes in bacterial systematics, in Sequencing and Hybrid-

Downloaded By: [ENEA] At: 14:48 13 November 2008

52

ization Techniques in Bacterial Systematics, Stackebrandt, E. and Goodfellow, M., Eds., John Wiley and Sons, Chichester, 1991, 205. 64. Devereux, R., Kane, M. D., Winfrey, J. and Stahl, D. A., Genus- and group-specific hybridization probes for determinative and environmental studies of sulfate-reducing bacteria, Syst. Appl. Microbiol., 15, 601, 1992. 65. Raskin, L., Poulsen, L. K., Noguera, D. R., Rittmann, B. E., and Stahl, D. A., Quantification of methanogenic groups in anaerobic biological reactors using oligonucleotide probe hybridization, Appl. Environ. Microbiol., 60, 1241, 1994. 66. Schaechter, M. O., Maaloe, O., and Kjeldgaard, N. O., Dependency on medium and temperature of cell size and chemical composition during balanced growth of Salmonella typhimurium, J. Gen. Microbiol., 19, 952, 1958. 67. DeLong, E. F., Wickham, G. S., and Pace, N. R., Phylogenetic stains: ribosomal RNAbased probes for the identification of single microbial cells, Science, 243, 1360, 1989. 68. Embley, T. M., Finlay, B. J., and Brown S., RNA sequence analysis shows that the symbionts in the ciliate Metopus contortus are polymorphs of a single methanogen species, FEMS Microbiol. Lett., 97, 57, 1992. 69. Springer, N., Ludwig, W., Amann, R., Schmidt, H. J., Gortz, H-D., and Schleifer, K-H., Occurrence of fragmented 16S rRNA in an obligate bacterial endosymbiont of Paramecium caudatum, Proc. Natl. Acad. Sci. U.S.A., 90, 9892, 1993. 70. Spring, S., Amann, R., Ludwig, W., Schleifer, K-H., and Petersen, N., Phylogenetic diversity and identification of nonculturable magnetotactic bacteria, Syst. Appl. Microbiol., 15, 116, 1992. 71. Spring, S., Amann, R., Ludwig, W, Schleifer, K-H., van Gemerden, H., and Petersen, N., Dominating role of an unusual magnetotactic bacterium in the microaerophilic zone of a freshwater sediment, Appl. Environ. Microbiol., 59, 2397, 1993. 72. Wagner, M., Amann, R., Lemmer, H., and Schleifer, K-H., Probing activated sludge with oligonucleotides specific for proteobacteria:

inadequacy of culture-dependent methods for describing microbial community structure, Appl. Environ. Microbiol., 59, 1520, 1993. 73. Wagner, M., Amann, R., Kmpfer, P., Assmus, B., Hartmann, A., Hutzler, P., Springer, N., and Schleifer, K-H., Identification and in situ detection of Gram-negative filamentous bacteria in activated sludge, Syst. Appl. Microbiol., 17, 405, 1994. 74. Wagner, M., Erhardt, D., Manz, W., Amann, R., Lemmer, N., Wedi, D., and Schleifer, K-H., Development of an rRNA-targeted oligonucleotyide probe specific for the genus Acinetobacter, its application for in situ monitoring in activated sludge, Appl. Environ. Microbiol., 60, 792, 1994. 75. Snaidr, J., Amann, R., Huber, I., Ludwig, W., and Schleifer K-H., Phylogenetic analysis and in situ identification of bacteria in activated sludge, Appl. Environ. Microbiol., 63, 2884, 1997. 76. Juretschko, S., Timmermann, G., Schmid, M., Schleifer, K-H., Pommerening-Roser, A., Koops, H. P., and Wagner, M., Combined molecular and conventional analyses of nitrifying bacterium diversity in activated sludge: Nitrosococcus mobilis and Nitrospiralike bacteria as dominant populations, Appl. Environ. Microbiol., 64, 3042, 1998. 77. Schuppler, M., Wagner, M., Schon, G., and Gobel, U.B., In situ identification of nocardioform actinomycetes in activated sludge using fluorescent rRNA-targeted oligonucleotide probes, Microbiology, 144, 249, 1998. 78. Sekiguchi, Y., Kamagata, Y., Nakamura, K., Ohashi, A., and Harada, H., Fluorescence in situ hybridization using 16S rRNA-targeted oligonucleotides reveals localization of methanogens and selected uncultured bacteria in mesophilic and thermophilic sludge granules, Appl. Environ. Microbiol., 65, 1280, 1999. 79. Manz, W., Szewzyk, U., Eriksson, P., Amann, R., Schleifer, K-H., and Stenstrm, T-A., In situ identification of bacteria in drinking water and adjoining biofilms by hybridization with 16S and 23S rRNA-directed fluorescent oligonucleotide probes, Appl. Environ. Microbiol., 59, 2293, 1993. 80. Ramsing, N. B., Khl, M., and Jrgensen, B. B., Distribution of sulphate-reducing bac-

Downloaded By: [ENEA] At: 14:48 13 November 2008

53

teria, O2 and H2S in photosynthetic biofilms determined by oligonucleotide probes and microelectrodes, Appl. Environ. Microbiol., 59, 3820, 1993. 81. Szewzyk, U., Manz, W., Amann, R., Schleifer, K-H., and Stenstrm, T-A., Growth and in situ detection of a pathogenic Escherichia coli in biofilms of a heterotrophic water bacterium by use of 16S- and 23SrRNA-directed fluorescent oligonucleotide probes, FEMS Microbiol. Ecol., 13, 169, 1994. 82. Kalmbach, S., Manz, W., and Szewzyk, U., Isolation of new bacterial species from drinking water biofilms and proof of their in situ dominance with highly specific 16S rRNA probes, Appl. Environ. Microbiol., 63, 4164, 1997. 83. Manz, W., Wendt-Potthoff, K., Neu, T. R., Szewzyk, U., and Lawrence, J. R., Phylogenetic composition, spatial structure, and dynamics of lotic bacterial biofilms investigated by fluorescent in situ hybridization and confocal laser scanning microscopy, Microb. Ecol., 37, 225, 1999. 84. Lee, S., Malone, C., and Kemp, P. F., Use of multiple 16S RNA-targeted fluorescent probes to increase signal strength and measure cellular RNA from natural planktonic bacteria, Marine Ecol. Prog. Ser., 101, 193, 1993. 85. Hicks, R., Amann, R. I., and Stahl, D. A., Dual staining of natural bacterioplankton with 4,6-diamidino-2-phenylindole and fluorescent oligonucleotide probes targeting kingdom level 16S rRNA sequences, Appl. Environ. Microbiol., 58, 2158, 1992. 86. Wallner, G., Amann, R., and Beisker, W., Optimizing fluorescent in situ hybridization of suspended cells with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms, Cytometry, 14, 136, 1993. 87. Poulsen, L. K., Ballard, G., and Stahl, D. A., Use of rRNA fluorescent in situ hybridization for measuring the activity of single cells in young and established biofilms, Appl. Environ. Microbiol., 59, 1354, 1993. 88. Mller, S., Kristensen, C. S., Poulsen, L. K., Carstensen, J. M., and Molin, S., Bacterial growth on surfaces: Automated image analysis for quantification of growth rate-related

parameters, Appl. Environ. Microbiol., 61, 741, 1995. 89. Caldwell, D. E., Korber, D. R., and Lawrence, J. R., Confocal laser microscopy and digital image analysis in microbial ecology, Adv. Microb. Ecol., 12, 1, 1992. 90. Lawrence, J. R., Korber, D. R., Hoyle, B. D., Costerton, J. W., and Caldwell, D. E., Optical sectioning of microbial biofilms, J. Bacteriol., 173, 6558, 1991. 91. Embley, T. M. and Finlay, B. J., The use of small subunit rRNA sequences to unravel the relationships between anaerobic ciliates and their methanogen symbionts, Microbiol., 140, 225, 1994. 92. Mller, S., Pedersen, A. R., Poulsen, L. K., Arvin, E. and Molin, S., Activity and threedimensional distribution of toluene-degrading Pseudomonas putida in a multispecies biofilm assessed by quantitative in situ hybridization and scanning confocal laser microscopy, Appl. Environ. Microbiology, 62, 4632, 1996. 93. Schramm, A., Larsen, L. H., Revsbech, N. P., Ramsing, N. B., Amann, R., and Schleifer, K-H., Structure and function of a nitrifying biofilm as determined by in situ hybridization and the use of microelectrodes, Appl. Environ. Microbiol., 62, 4641, 1996. 94. Amann, R. I., Binder, B. J., Olson, R. J., Chisholm, S. W., Devereux, R., and Stahl, D. A., Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations, Appl. Environ. Microbiol., 56, 1919, 1990. 95. Wallner, G., Steinmentz, I., Bitter-Suermann, D., and Amann. R., Combination of rRNAtargeted hybridization probes and immunoprobes for the identification of bacteria by flow cytometry, System. Appl. Microbiol., 19, 569, 1996. 96. Wallner, G., Fuchs, B., Spring, S., Beisker, W., and Amann, R., Flow sorting of microorganisms for molecular analysis, Appl. Environ. Microbiol., 63, 4223, 1997. 97. Fries, M. R., Hopkins, G. D., McCarty P. L., Forney, L. J., and Tiedje, J. M., Microbial succession during a field evaluation of phenol and toluene as the primary substrates for

Downloaded By: [ENEA] At: 14:48 13 November 2008

54

trichloroethene cometabolism, Appl. Environ. Microbiol., 63, 1515, 1997. 98. Fries, M. R., Zhou, J., Chee-Sanford, J., and Tiedje, J. M., Isolation, characterization, and distribution of denitrifying toluene degraders from a variety of habitats, Appl. Environ. Microbiol., 60, 2802, 1994. 99. Hadrys, H., Balick, M., and Schierwater, B., Applications of random amplified polymorphic DNA (RAPD) in molecular ecology, Mol. Ecol., 1, 55, 1992. 100. Breen, A., Rope, A. F., Taylor, D., Loper, J. C., and Sferra, P. R., Application of DNA amplification fingerprinting (DAF) to mixed culture bioreactors. J. Ind. Microbiol., 14, 10, 1995. 101. Porteous, L. A., Armstrong, J. L., Seidler, R. J., and Watrud, L. S., An effective method to extract DNA from environmental samples for polymerase chain reaction amplification and DNA fingerprinting, Curr. Microbiol., 29, 301, 1994. 102. Moyer, C. L., Dobbs, F. C., and Karl, D. M., Estimation of diversity and community structure through restriction fragment length polymorphism distribution analysis of bacterial 16S rRNA genes from a microbial mat at an active, hydrothermal vent system, Loihi Seamount, Hawaii, Appl. Environ. Microbiol., 60, 871, 1994. 103. Smit, E., Leeflang, P., and Wemars, K., Detection of shifts in microbial community structure and diversity in soil caused by copper contamination using amplified ribosomal DNA restriction analysis, FEMS Microbiol. Ecol., 23, 249, 1997. 104. Massol-Deya, A., Weller, R., Rios-Hernandez, L., Zhou, J-Z., Hickey, R. F., and Tiedje, J. M., Succession and convergence of biofilm communities in fixed-film reactors treating aromatic hydrocarbons in groundwater, Appl. Environ. Microbiol., 63, 270, 1997. 105. Southern, E. M., Detection of specific sequences among DNA fragments separated by gel electrophoresis, J. Mol. Biol., 98, 503, 1975. 106. Lovell, C. R. and Hui, Y., Design and testing of a functional group-specific probe for the

study of natural populations of acetogenic bacteria, Appl. Environ. Microbiol., 57, 2602, 1991. 107. Fischer, S. G. and Lerman, L. S., Lengthindependent separation of DNA restriction fragments in two-dimensional gel electrophoresis, Cell, 16, 191, 1979. 108. Muyzer, G., de Waal, E. C., and Uitterrlinden, A. G., Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified gens coding for 16S rRNA, Appl. Environ. Microbiol., 59, 695, 1993. 109. Santegoeds, C. M., Nold, S. C., and Ward, D. M., Denaturing gradient gel electrophoresis used to monitor the enrichment culture of aerobic chemoorganotrophic bacteria from a hot spring cyanobacterial mat, Appl. Environ. Microbiol., 62, 3922, 1996. 110. Gillan, D. C., Speksnijder, A. G., Zwart, G., and De Ridder, C., Genetic diversity of the biofilm covering Montacuta ferruginosa (Mollusca, bivalvia) as evaluated by denaturing gradient gel electrophoresis analysis and cloning of PCR-amplified gene fragments coding for 16S rRNA, Appl. Environ. Microbiol., 64, 3464, 1998. 111. el Fantroussi, S., Verschuere, L., Verstraete, W., and Top, E., M., Effect of phenylurea herbicides on soil microbial communities estimated by analysis of 16S rRNA gene fingerprints and community-level physiological profiles, Appl. Environ. Microbiol., 65, 982, 1999. 112. Lerman, L. S., Fischer, S. G., Hurley, I., Silverstein, K., and Lumelsky, N., Sequence determined DNA separations, Annu. Rev. Biophys. Bioeng., 13, 399, 1984. 113. Myers, R. M., Maniatis, T., and Lerman, L. S., Detection and localization of single base changes by denaturing gradient gel electrophoresis, Methods Enzymol., 155, 501, 1987. 114. Ferris, M. J., Muyzer, G., and Ward, D. M., Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial community, Appl. Environ. Microbiol., 62, 340, 1996. 115. Santegoeds, C. M., Ferdelman, T. G., Muyzer, G., and de Beer, D., Structural and functional

Downloaded By: [ENEA] At: 14:48 13 November 2008

55

dynamics of sulfate-reducing populations in bacterial biofilms, Appl. Environ. Microbiol., 64, 3731, 1998. 116. Zhou, J., Bruns, M. A. and Tiedje, J. M., DNA recovery of soils of diverse composition, Appl. Environ. Microbiol., 62, 316, 1996. 117. Mor, M., Herrick, J. B., Silva, M. O., Ghiorse, W. C., and Madsen, E. J., Quantitative cell lysis of indigenous microorganisms and rapid extraction of microbial DNA from sediment, Appl. Environ. Microbiol., 60, 1572, 1994. 118. Risatti, J. B., Capman, W. C., and Stahl, D. A., Community structure of a microbial mat: the phylogenetic dimension, Proc. Natl. Acad. Sci. U.S.A., 91, 10173, 1994. 119. Farrelly, V., Rainey, F. A., and Stackebrandt, E., Effect of genome size and rrn gene copy number on PCR amplification of 16S rRNA genes from a mixture of bacterial species, Appl. Environ. Microbiol., 61, 2798, 1995. 120. Wilson, I. G., Inhibition and facilitation of nucleic acid amplification, Appl. Environ. Microbiol., 63, 3741, 1997. 121. Rainey, F. A., Ward, N., Sly, L. I., and Stackebrandt, E., Dependence on the taxon composition of clone libraries for PCR-amplified, naturally occurring 16S rDNA on the primer pair and the cloning system used, Experientia, 50, 796, 1994. 122. Reysenbach, A. L., Giver, L. J., Wickham, G. S., and Pace, N. R., Differential amplification of rRNA genes by polymerase chain reaction, Appl. Environ. Microbiol., 58, 3417, 1992. 123. Suzuki, M. T. and Giovannoni, S. J., Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR, Appl. Environ. Microbiol., 62, 625, 1996. 124. Liesack, W., Weyland, H., and Stackebrandt, E., Potential risks of gene amplification by PCR as determined by 16S rDNA analysis of a mixed culture of strict barophilic bacteria, Microb. Ecol., 21, 199, 1991. 125. Kopczynski, E. D., Bateson, M. M., and Ward, D. M., Recognition of chimeric small subunit ribosomal DNAs composed of genes from uncultivated microorganisms, Appl. Environ. Microbiol., 60, 746, 1994.

126. Wang, G. C-Y. and Wang, Y., The frequency of chimeric molecules as a consequence of PCR co-amplification of 16S rRNA genes from different bacterial species, Microbiology, 142, 1107, 1996. 127. Cole, S. T. and Girons, I. S., Bacterial genomics, FEMS Microbiol. Rev., 14, 139, 1994. 128. Dryden, S. C. and Kaplan, S., Localization and structural analysis of the ribosomal RNA operons of Rhodobacter sphaeroides, Nucleic Acids Res., 18, 7267, 1990. 129. Mylvaganam, S. and Dennis, P. P., Sequence heterogeneity between the two genes encoding 16S rRNA from the halophilic archaebacterium Haloarcula marismortui, Genetics, 130, 399, 1992. 130. Amann, R., Snaidr, J., Wagner, M., Ludwig, W., and Schleifer, K-H., In situ visualization of high genetic diversity in a natural microbial community, J. Bacteriol., 178, 3496, 1996. 131. Roszak, D. B. and Colwell, R. R., Survival strategies of bacteria in the natural environment, Microbiol. Rev., 51, 365, 1987. 132. Beimfohr, C., Krause, A., Amann, R., Ludwig, W., and Schleifer, K-H., In situ identification of lactococci, enterococci, and streptococci, Syst. Appl. Microbiol., 16, 450, 1993. 133. Hahn, D., Amann, R. I., and Zeyer, J., Detection of mRNA in Streptomyces cells by in situ hybridization with digoxigenin-labeled probes, Appl. Environ. Microbiol., 59, 2753, 1993. 134. MacNaughton, S. J., ODonnell, A. G., and Embley, T. M., Permeabilization of mycolic acid containing actinomycetes for in situ hybridization with fluorescently labeled oligonucleotide probes, Microbiology, 140, 2859, 1994. 135. Hahn, D., Amann, R. I., Ludwig, W., Akkermans, A. D. L., and Schleifer, K-H., Detection of microorganisms in soil after in situ hybridization with rRNA-targeted fluorescently labelled oligonucleotides, J. Gen. Microbiol., 138, 879, 1992. 136. Amann, R. I., Krumholz, L., and Stahl, D. A., Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and en-

Downloaded By: [ENEA] At: 14:48 13 November 2008

56

vironmental studies in microbiology, J. Bacteriol., 172, 762, 1990. 137. Trebesius, K. R., Amann, R., Ludwig, W., Mhlegger, K., and Schleifer, K-H., Identification of whole fixed bacterial cells with nonradioactive rRNA-targeted transcript probes, Appl. Environ. Microbiol., 60, 3228, 1994. 138. Lebaron, P., Catala, P., Fajon, C., Joux, F., Baudart, J., and Bernard, L., A new sensitive, whole-cell hybridization technique for detection of bacteria involving a biotinylated oligonucleotide probe targeting rRNA and Tyramide signal amplification, Appl. Environ. Microbiol., 63, 3274, 1997.

139. Schonhuber, W., Zarda, B., Eix, S., Rippka, R., Herdman, M., Ludwig, W., and Amann, R., In situ identification of cyanobacteria with horseradish peroxidase-labeled, rRNA-targeted oligonucleotide probes, Appl. Environ. Microbiol., 65, 1259, 1999. 140. Muyzer, G. and Ramsing, N. B., Molecular methods to study the organization of microbial communities, Wat. Sci. Technol., 32, 1, 1995. 141. Mevarech, M., Hirsch-Twizer, S., Goldman, S., Yakobsen, E., Eisenberg, H., and Dennis, P. P., Isolation and characterization of the rRNA gene clusters of Halobacterium marismortui, J. Bacteriol., 171, 3479, 1989.

Downloaded By: [ENEA] At: 14:48 13 November 2008

57