Application of denaturing gradien...
Antonie van Leeuwenhoek 73: 127���141, 1998. 127 c 1998 Kluwer Academic Publishers. Printed in the Netherlands. Mini Review Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology Gerard Muyzer1 & Kornelia Smalla2 1 Max-Planck-Institute for Marine Microbiology, Celsiusstra��e 1, D-28359 Bremen, Germany 2 Biologische Bundesanstalt f�� ur Land- und Forstwirtschaft, Messeweg 11/12, D-38104 Braunschweig, Germany ( author for correspondence) Received 24 January 1997 accepted 14 October 1997 Key words: DGGE, genetic fingerprinting, microbial ecology, molecular microbial ecology, PCR, rRNA, TGGE Abstract Here, the state of the art of the application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology will be presented. Furthermore, the potentials and limitations of these techniques will be discussed, and it will be indicated why their use in ecological studies has become so important. Abbreviations: ARDRA ��� amplified ribosomal DNA restriction analysis DGGE ��� denaturing gradient gel elec- trophoresis DMSO ��� dimethylsulfoxide PEG ��� polyethylene glycol PCR ��� polymerase chain reaction RAPD ��� randomly amplified polymorphic DNA rDNA ��� ribosomal DNA RDP ��� Ribosomal Database Project RFLP ��� restriction fragment length polymorphism rRNA ��� ribosomal RNA SSCP ��� single strand conformation polymor- phism SSU ��� small-subunit TGGE ��� temperature gradient gel electrophoresis Introduction It is now well recognised among microbiologists that only a small fraction of all bacteria have been iso- lated and characterised (Wayne et al., 1987 Ward et al., 1992). Comparison of the percentage of cul- turable bacteria with total cell counts from different habitats showed enormous discrepancies (summarised by Amann et al., 1995). One of the reasons for this difference might be the interdependency of different organisms upon each other, the most obvious example being the endosymbiotic bacteria in specific worms and molluscs (e.g., Fisher, 1990) another reason is certainly the lack of knowledge of the real conditions under which most of the bacteria are growing in their natural environment. So, to obtain a better understand- ing of the role of microbial diversity in the maintenance of ecosystems, other approaches, which complement the traditional microbiological procedures are needed. The application of molecular biological techniques to detect and identify microorganisms by certain molec- ular markers, such as 16S rRNA or its encoding gene (���the rRNA approach��� Olsen et al., 1986 Amann et al., 1995), is now more and more frequently used to explore the microbial diversity and to analyse the struc- ture of microbial communities (e.g., Muyzer and Ram- sing, 1995, and references therein). The application of these techniques in microbial ecological studies has even become a discipline on its own, i.e. molecular microbial ecology (Akkermans et al., 1995). So far, most results with the molecular approach have been obtained by cloning of 16S rDNA fragments obtained either after reverse transcription of rRNA (e.g., Ward et al., 1990 Weller et al., 1991), or after enzymatic amplification of DNA extracted from different habi- tats, such as sediments (e.g., Gray & Herwig, 1996), soil (e.g., Liesack & Stackebrandt, 1992 Borneman et al., 1996), hot springs (e.g., Barns et al., 1994), and seawater (e.g., Giovannoni et al., 1990 Fuhrman et al. 1993). The results of these studies have shown
128 the enormous wealth of microbial diversity, and at the same time the limitations of traditional cultivation tech- niques to retrieve this diversity. However, although successful, these studies have only focused on the exploration of microbial diver- sity, they have not given any information on the complex dynamics which microbial communities can undergo by diel and seasonal fluctuations or after environmental perturbations. As microbial ecology is the study of interactions among microorganisms and between microorganisms and their environment, microbial ecosystems have to be studied over longer time periods. For this purpose the cloning approach is not useful, because it is time-consuming and labour intensive, and hence impractical for multiple sample analysis. A better approach to investigate population shifts is the use of taxon-specific probes in dot-blot hybridisation of extracted rRNA (e.g., Stahl et al., 1988 Raskin et al., 1995) or in whole cell hybridisation (for an overview, see Amann et al., 1995, and refer- ences therein). These studies however only focus on particular microorganisms for which probes have been developed. Therefore, to study the complex structures of microbial communities and their dynamics other molecular biological techniques are needed. Genetic fingerprinting techniques Genetic fingerprinting techniques provide a pattern or profile of the genetic diversity in a microbial commu- nity. One of the fingerprinting techniques that has been used in microbial ecology for more than a decade is the electrophoretic separation in high resolution polyacry- lamide gels of low molecular weight rRNA molecules (5S rRNA and tRNA) extracted from natural samples (e.g., Hofle, �� 1988 1990). Recently, another genetic fingerprinting technique, denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal DNA fragments has been introduced into microbial ecology (Muyzer et al., 1993). Within a short period of time this method has attracted the attention of many environmental micro- biologists, and the technique is now used in many lab- oratories. In this paper we describe the theoretical and prac- tical aspects of DGGE and the related technique called temperature gradient gel electrophoresis (TGGE) and their application to the analysis of microbial commu- nities. Furthermore, we will discuss the potentials and limitations of these approaches for studies in microbial ecology. Theoretical and practical aspects of DGGE and TGGE In DGGE (Fischer & Lerman, 1979, 1983 Myers et al., 1987) as well as in TGGE (Rosenbaum and Riesner, 1987 Riesner et al., 1991) DNA fragments of the same length but with different sequences can be separated. Separation is based on the decreased electrophoretic mobility of a partially melted double-stranded DNA molecule in polyacrylamide gels containing a linear gradient of DNA denaturants (a mixture of urea and formamide) or a linear temperature gradient. The melt- ing of DNA fragments proceeds in discrete so-called melting domains: stretches of base-pairs with an iden- tical melting temperature. Once a domain with the lowest melting temperature reaches its melting tem- perature (Tm) at a particular position in the denaturing or temperature gradient gel, a transition of a helical to a partially melted molecule occurs, and migration of the molecule will practically halt. Sequence variation within such domains causes the melting temperatures to differ, and molecules with different sequences will stop migrating at different positions in the gel. By using DGGE or TGGE, 50% of the sequence variants can be detected in DNA fragments up to 500 bp (Myers et al., 1985). This percentage can be increased to nearly 100% by the attachment of a GC- rich sequence, a so-called GC-clamp, to one side of the DNA fragment (Myers et al., 1985 Sheffield et al., 1989). A sequence of guanines (G) and cytosines (C) is added to the 50 -end of one of the PCR primers,coampli- fied and thus introduced into the amplified DNA frag- ments (Sheffield et al., 1989 Sheffield et al., 1992). The GC-rich sequence acts as a high melting domain preventing the two DNA strands from complete disso- ciation into single strands. The length of the GC-clamp can vary between 30 and 50 nucleotides (see Table 2 in Muyzer et al., 1997). As an alternative to GC-clamps, chemical clamps have been used (Fuhr, �� 1996). One of the PCR primers is labelled at its 50 -end with a photoac- tivatable compound, such as psoralene, which interca- lates between the base plates of both DNA strands and will covalently link them together after UV irradiation. The use of a so-called ChemiClamp has the advantage that both primers have a similar length, but also has disadvantages. Firstly, DGGE bands with this clamp cannot be reamplified directly, because of the covalent
129 bond, and secondly, irradiation of the PCR products with UV might damage the amplified DNA causing multiple bands or even a smear in the DGGE analysis (Cariello et al., 1988 Fuhr, �� 1996). DNA bands in DGGE and TGGE profiles can be visualised using ethidium bromide. Recently, SYBR Green I was introduced as an alternative to ethidi- um bromide (Muyzer et al., 1997). The advantage of SYBR Green I is the lack of background staining, which makes it possible to observe less dominant DNA fragments. A more sensitive detection method is silver staining (Felske et al., 1996). However, silver staining also stains single stranded DNA, and silver stained gels cannot be used for subsequent hybridization analysis (Heuer & Smalla, 1997). Prior to DGGE or TGGE analysis of DNA frag- ments it is necessary to determine the melting behav- iour of the DNA fragments. Furthermore, to obtain the best separation of different DNA fragments, it is necessary to optimise the gradient and the duration of electrophoresis. The melting behaviour of DNA fragments, as well as the optimal gradient can be determined experimen- tally with perpendicular gradient gels. Perpendicular gels have an increasing gradient of denaturants or tem- perature from left to right, perpendicular to the direc- tion of electrophoresis. The sample is applied across the entire width of the gel and electrophoresed for about 3 hours at 200 Volts. After staining the gel with ethidium bromide and UV-transillumination, the elec- trophoretic pattern will appear as a sigmoid-shaped curve. DNA molecules at the left side of the gel, where the concentration of denaturants or the temperature is low, will migrate as double-stranded DNA. At the other side of the gel, where the concentration of denat- urants or temperature is high, the molecules melt into branched molecules as soon as they enter the gel and therefore halt. At intermediate concentrations of denat- urants, the molecules have different degrees of melting, and concomitantly different mobilities. A steep transi- tion in mobility occurs at the denaturant concentration corresponding to the melting temperature of the lowest melting domain of the fragment. Perpendicular gels are used to determine the melting behaviour of the DNA fragments. In addition, from these gels the optimal gradient can be determined for multi-lane analysis in parallel gels. The optimal time of electrophoresis is determined by parallel gradient electrophoresis. Parallel gradient gels have an increasing gradient of denaturants or tem- perature from top-to-bottom, parallel to the direction of electrophoresis. They are used for analysing multiple samples on the same gel. Before analysing samples on a parallel gel, the duration of electrophoresis must be determined to obtain maximum resolution between the different DNA fragments. For this purpose, individu- al samples are loaded onto the parallel gel at constant time intervals, a so-called time travel experiment. DGGE equipment can be obtained from differ- ent commercial companies, such as Bio-Rad (Her- cules, USA), INGENY (Leiden, The Netherlands), and C.B.S. Scientific Co., Inc. (Del Mar, USA). TGGE equipment originally sold by Diagen GmbH (Ger- many), can now be purchased from Biometra (Ger- many) Applications of DGGE and TGGE in microbial ecology Studying community complexity DGGE of PCR-amplified 16S rDNA fragments was first used to profile community complexity of a micro- bial mat and bacterial biofilms (Muyzer et al., 1993). For this purpose bacterial genomic DNA was extracted from natural samples, and segments of the 16S rRNA genes were amplified in the polymerase chain reaction (PCR Saiki et al., 1988). This resulted in a mixture of PCR products obtained from the different bacte- ria present in the sample. The individual PCR products were subsequently separated by DGGE. The result was a pattern of bands, for which the number of bands cor- responded to the number of predominant members in the microbial communities. To obtain more detailed information about some of the community members, DGGE profiles were blotted onto nylon membranes and hybridised with a radioactively-labelled oligonu- cleotide probe specific for sulfate-reducing bacteria (Amann et al., 1992). In a subsequent study, Muyzer and de Waal (1994) were able to identify community members by sequencing of DNA eluted from excised DGGE bands. Figure 1 gives a flow chart of the differ- ent steps in this strategy. Muyzer et al. (1995) used DGGE analysis of PCR- amplified rDNA fragments to provide information on the genetic diversity of microbial communities found around hydrothermal vents. Denaturing gradient gel electrophoresis of DNA fragments obtained after enzy- matic amplification of the 16S rDNA using genom- ic DNA extracted from 2 different hydrothermal vent samples and bacterial primers, showed only 1 band for