变性梯度凝胶电泳(DGGE)在微生物生态学中的应用

由于从环境样品中分离和培养细菌的困难,分子生物学方法已发展用来描述和鉴定微生物群落。近年来基于DNA方法的群落分析得到了迅速的发展,如PCR扩增技术,克隆文库法,荧光原位杂交法,限制性酶切片段长度多态性法,变性和温度梯度凝胶电泳法。DGGE已广泛用于分析自然环境中细菌、蓝细菌,古菌、微微型真核生物、真核生物和病毒群落的生物多样性。这一技术能够提供群落中优势种类信息和同时分析多个样品。具有可重复和容易操作等特点,适合于调查种群的时空变化,并且可通过对切下的带进行序列分析或与特异性探针杂交分析鉴定群落成员。DGGE分析微生物群落的一般步骤如下：一是核酸的提取,二是16S rRNA,18S rRNA或功能基因如可容性甲烷加单氧酶羟化酶基因(mmoX)和氨加单氧酶a一亚单位基因(amoA)片段的扩增,三是通过DGGE分析PCR产物。DGGE使用具有化学变性剂梯度的聚丙烯酰胺凝胶,该凝胶能够有区别的解链PCR扩增产物。由PCR产生的不同的DNA片段长度相同但核苷酸序列不同。因此不同的双链DNA片段由于沿着化学梯度的不同解链行为将在凝胶的不同位置上停止迁移。DNA解链行为的不同导致一个凝胶带图案,该图案是微生物群落中主要种类的一个轮廓。DGGE使用所有生物中保守的基因片段如细菌中的16S rRNA基因片段和真菌中的18S rRNA基因片段。然而同其他分子生物学方法一样,DGGE也有缺陷,其中之一是只能分离较小的片段,使用于系统发育分析比较和探针设计的序列信息量受到了限制。在某些情况下,由于所用基因的多拷贝导致一个种类多于一条带,因此不易鉴定群落结构到种的水平。此外,该技术具有内在的如单一细菌种类16S rDNA拷贝之间的异质性问题,可导致自然群落中微生物数量的过多估计。DGGE是分析微生物群落的一种有力的工具。不过为了减少DGGE和其它技术的缺陷,建议研究者结合DGGE和其它分子及微生物学方法以便更详细的观察微生物的群落结构和功能。     

应用DGGE研究微生物群落时的常见问题分析

变性梯度凝胶电泳(DGGE)是通过核酸片段对微生物群落进行研究,可以监测未培养细菌及其功能基因,被广泛地应用于微生物群落多样性和动态分析,并成为微生物分子生态学研究中的重要手段之一。文中论述了DGGE操作过程中遇到的常见问题,并提出了相应的解决方法。全面分析了样品预处理过程和PCR扩增效果对DGGE分析的影响,探讨了DGGE图谱的优化过程和图谱分析方法,并对DGGE的应用前景进行了综述。     

Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology

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.     

Molecular inference of dominant picocyanobacterial populations by denaturing gradient gel electrophoresis of PCR amplified 16S rRNA gene fragments

Quantitative accuracy based on the fluorescent intensity of bands in a denaturing gradient gel electrophoresis (DGGE) profile of polymerase chain reaction (PCR)‐amplified 16S rRNA gene fragments was evaluated for the molecular inference of dominant populations using a cyanobacterial primer pair in a picocyanobacterial community. A serial dilution technique of the template prior to PCR of extracted nucleic acids allowed for elimination of minor strains (less than 10% of the whole cell number) using a cell mixture of three known cultured Synechococcus species with different ratios. When the most abundant strain among the three accounted for more than 80% of the cells, the single band derived from the most abundant one was detected exclusively after the template dilution. In the case of two or three strains evenly distributed in the sample, all strains remained as bands after template dilution. The technique used in the present study was also applied to lake water samples collected from depths of 1 and 5 m on 27 August 1999. The same dominant Synechococcus population was detected in both samples. Thus, the template‐dilution technique prior to PCR is useful to determine dominant picocyanobacterial populations in the DGGE profiling.     

Bacterial community structure in Apis florea larvae analyzed by denaturing gradient gel electrophoresis and 16S rRNA gene sequencing

This study characterizes the colonization and composition of bacterial flora in dwarf Asian honeybee (Apis florea) larvae and compares bacterial diversity and distribution among different sampling locations. A. florea larvae were collected from 3 locations in Chiang Mai province, Thailand. Bacterial DNA was extracted from each larva using the phenol–chloroform method. Denaturing gradient gel electrophoresis was performed, and the dominant bands were excised from the gels, cloned, and sequenced for bacterial species identification. The result revealed similarities of bacterial community profiles in each individual colony, but differences between colonies from the same and different locations. A. florea larvae harbor bacteria belonging to 2 phyla (Firmicutes and Proteobacteria), 5 classes (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacilli, and Clostridia), 6 genera (Clostridium, Gilliamella, Melissococcus, Lactobacillus, Saccharibacter, and Snodgrassella), and an unknown genus from uncultured bacterial species. The classes with the highest abundance of bacteria were Alphaproteobacteria (34%), Bacilli (25%), Betaproteobacteria (11%), Gammaproteobacteria (10%), and Clostridia (8%), respectively. Similarly, uncultured bacterial species were identified (12%). Environmental bacterial species, such as Saccharibacter floricola, were also found. This is the first study in which sequences closely related to Melissococcus plutonius, the causal pathogen responsible for European foulbrood, have been identified in Thai A. florea larvae.     

Two-step denaturing gradient gel electrophoresis (2S-DGGE), a gel-based strategy to capture full-length 16S rRNA gene sequences

Obtaining full-length 16S rRNA gene sequences is important for generating accurate taxonomy assignments of bacteria, which normally is realized via clone library construction. However, the application of clone library has been hindered due to its limitations in sample throughput and in capturing minor populations (<1?% of total microorganisms). To overcome these limitations, a new strategy, two-step denaturing gradient gel electrophoresis (2S-DGGE), is developed to obtain full-length 16S rRNA gene sequences. 2S-DGGE can compare microbial communities based on its first-round DGGE profiles and generate partial 16S rRNA gene sequences (8-534?bp, Escherichia coli numbering). Then, strain-specific primers can be designed based on sequence information of bacteria of interest to PCR amplify their remaining 16S rRNA gene sequences (515-1541?bps, E. coli numbering). The second-round DGGE can confirm DNA sequence purity of these PCR products. Finally, the full-length 16S rRNA gene sequences can be obtained through combining the two partial DNA sequences. By employing 2S-DGGE, taxonomies of a group of dehalogenating bacteria have been assigned based on their full-length 16S rRNA gene sequences, several of which existed in dehalogenating microcosms as minor populations. In all, 2S-DGGE can be utilized as a medium throughput method for taxonomic identification of interested/minor populations from single or multiple microbial consortia.     

Screening of alpha-1-antitrypsin gene by denaturing gradient gel electrophoresis (DGGE)

Alpha-1-antitrypsin (AAT) is a serine protease inhibitor whose deficiency could cause emphysema and liver disease and, as recently described, could be a risk factor for lung cancer development. Alpha-1-antitrypsin inhibits a variety of proteases but its primary target is neutrophil elastase, an extracellular endopeptidase capable of degrading most protein components of the extracellular matrix. Inhibition of neutrophil elastase by AAT has an important role in maintaining the integrity of connective tissue. The gene encoding for AAT spans over 12.2 kb, consists of seven exons and is highly polymorphic. Therefore several methods for mutation screening of alpha-1-antitrypsin gene have been developed. Method described here is based on denaturing gradient gel electrophoresis (DGGE). This method is highly efficient and reliable and allows rapid analysis of entire coding region of alpha-1-antitrypsin gene, including splice junction sites. Previously described DGGE based analysis of AAT gene included overnight electrophoresis of individually amplified fragments. The optimization of the method described in this paper is directed towards the shortening of the duration of electrophoresis and amplification of fragments in multiplex reaction in order to make the analysis less time-consuming and therefore more efficient.     

Dominant bacterioplankton populations in the meromictic Lake Suigetsu as determined by denaturing gradient gel electrophoresis of 16S rRNA gene fragments

Lake Suigetsu is a typical meromictic lake in Japan characterized by a permanent chemocline at a depth of between 3 and 8 m separating the oxic freshwater mixolimnion from anoxic saline sulfidogenic monimolimnion. Dominant bacterioplankton populations in Lake Suigetsu were investigated using PCR-denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. The bacterial population was vertically stratified, and temporal shifts in the microbial communities were observed in both the oxic and anoxic layers of Lake Suigetsu during the sampling period. Several dominant DGGE bands were excised and sequenced. In the chemocline, green sulfur bacteria phylogenetically related to the genera Prosthecochloris, Pelodyctyon, and Chlorobium within the phylum Chlorobi were dominant; the colorless sulfur bacteria closely related to the genus Thiomicrospira were detected. These sulfur bacterial groups appear to be important in the biogeochemical cycling of sulfur and/or carbon in Lake Suigetsu. Bacterial sequences affiliated with the Bacteroidetes phylum were frequent among the dominant fragments in the DGGE profiles throughout the water column. Populations possessing a fermentative metabolism exist in Bacteroidetes, suggesting they may contribute to the degradation of organic matter in the anoxic environment of Lake Suigetsu.     

Application of single-strand conformation polymorphism and denaturing gradient gel electrophoresis for fla sequence typing of Campylobacter jejuni

Campylobacter jejuni is a frequent cause of bacterial gastroenteritis in humans all over the world. Several molecular typing methods are used to study the epidemiology of Campylobacter spp. infections. The aim of the present study was to investigate the application of single-strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) analysis as rapid primary subtyping methods for C. jejuni. A variable fragment from the 3' end of the flaA to the 3' end of the intergenic region, separating the flaA and flaB genes, was subjected to SSCP and DGGE analysis. A total of 48 clinical C. jejuni isolates, 49 C. jejuni strains isolated from poultry, 2 strains isolated from ducks and 1 strain isolated from a pheasant were assigned to 24 distinct SSCP patterns. Sequence analysis of the respective DNA fragments revealed that every different fla sequence type could be distinguished by SSCP. DGGE proved to be equally discriminatory. Both methods can be applied as primary subtyping methods, because pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP) analysis further differentiated isolates belonging to the same fla sequence types.     

Digitization of DGGE (denaturing gradient gel electrophoresis) profile and cluster analysis of microbial communities

Denaturing gradient gel electrophoresis (DGGE) of 16S rDNA profiles were objectively digitized using an image analyzer; the individual microbial species in a community can thus be precisely quantified. The similarity between various microbial communities was compared to the digitized DGGE profiles using the cluster analyses technique. The microbial community in a biofilm was considerably different from that in suspended sludge obtained from the same system.     

Identification of bacteria in a biodegraded wall painting by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA.

Medieval wall paintings are often affected by biodecay. An inventory of the existing microorganisms associated with the damage to the paintings is not yet an integral part of the restoration process. This stems from the lack of effective means for such a stocktaking. Nevertheless, fungi and bacteria cause severe damage through mechanical processes from growth into the painting and its grounding and through their metabolism. Detailed information on the bacterial colonization of ancient wall paintings is essential for the protection of the paintings. We used a molecular approach based on the detection and identification of DNA sequences encoding rRNA (rDNA) to identify bacteria present on an ancient wall painting without prior cultivation of the organisms, since it has been shown that most of these bacteria cannot be cultivated under laboratory conditions. To trace the noncultivated fraction of bacteria, total DNA from a biodegraded wall painting sample from a 13th century fresco was extracted and 194-bp fragments of the 16S rDNA were amplified with eubacterial primers. The 16S rDNA fragments of uniform length obtained from the different bacterial species were separated according to their sequence differences by denaturing gradient gel electrophoresis (DGGE). By sequencing excised and reamplified individual DNA bands, we characterized the phylogenetic affiliation of the corresponding bacteria. Using this approach, we identified members or close relatives of the genera Halomonas, Clostridium, and Frankia. To our knowledge, these groups of bacteria have not yet been isolated and implicated by conventional microbiological techniques as contributing to the biodegradation of wall paintings.     

Monitoring of microbial community structure and succession in the biohydrogen production reactor by denaturing gradient gel electrophoresis (DGGE)

To study the structure of microbial communities in the biological hydrogen production reactor and determine the ecological function of hydrogen producing bacteria, anaerobic sludge was obtained from the continuous stirred tank reactor (CSTR) in different periods of time, and the diversity and dynamics of microbial communities were investigated by denaturing gradient gel electrophoresis (DGGE). The results of DGGE demonstrated that an obvious shift of microbial population happened from the beginning of star-up to the 28th day, and the ethanol type fermentation was established. After 28 days the structure of microbial community became stable, and the climax community was formed. Comparative analysis of 16S rDNA sequences from reamplifying and sequencing the prominent bands indicated that the dominant population belonged to low G+C Gram-positive bacteria (Clostridium sp. andEthanologenbacterium sp.), β-proteobacteria (Acidovorax sp.), γ-proteobacteria (Kluyvera sp.), Bacteroides (uncultured bacterium SJA-168), and Spirochaetes (uncultured eubacterium E1-K13), respectively. The hydrogen production rate increased obviously with the increase ofEthanologenbacterium sp.,Clostridium sp. and uncultured Spirochaetes after 21 days, meanwhile the succession of ethanol type fermentation was formed. Throughout the succession the microbial diversity increased however it decreased after 21 days. Some types ofClostridium sp.Acidovorax sp.,Kluyvera sp., and Bacteroides were dominant populations during all periods of time. These special populations were essential for the construction of climax community. Hydrogen production efficiency was dependent on both hydrogen producing bacteria and other populations. It implied that the cometabolism of microbial community played a great role of biohydrogen production in the reactors.     

Monitoring of microbial community structure and succession in the biohydrogen production reactor by denaturing gradient gel electrophoresis (DGGE)

Biohydrogen production has been concerned ex-tremely as a new technology of energy resource pro-duction by many scientists[1—4]. Enhancement of hy-drogen production efficiency and cutting down the operating cost are very important problems, which are the limiting factors for the industrialization of hydro-gen production process. The fermentation hydrogen production technology offers a new method to resolve these difficulties[5—8]. Compared with photosynthetic hydrogen production possesses, f…     

Analyses of microbial activity in biomass-recycle reactors using denaturing gradient gel electrophoresis of 16S rDNA and 16S rRNA PCR products

The relationship between mixed microbial community structure and physiology when grown under substrate-limited conditions was investigated using continuous-flow bioreactors with 100% biomass recycle. Community structure was analyzed by denaturing gradient gel electrophoresis (DGGE) of the PCR and RT-PCR amplified V3 region of 16S rDNA and 16S rRNA templates, respectively. Comparisons were made of communities exposed to different types of transient conditions (e.g., long- and short-term starvation, increasing nutrients). With progressively more stringent substrate limitation over time, the specific content of community RNA declined by more than 10-fold and closely followed the decline in specific growth rate. In contrast, the DNA content was variable (up to 3-fold differences) and did not follow the same trend. Cluster analysis of the presence or absence of individual bands indicated that the fingerprints generated by the two templates were different, and community response was first observed in the rRNA fraction. However, both the rDNA and rRNA fingerprints provided a picture of temporal population dynamics. Dice similarity coefficients gave a quantitative measure of the differences and changes between the communities. In comparison, standard cultivation techniques yielded only a quarter of the phylotypes detected by DGGE, but included the most dominant population based on rRNA. Nucleotide-sequence analyses of the almost complete 16S rRNA genes of these isolates place them in the same group of organisms that is typically cultivated from environmental samples: alpha, beta, and gamma Proteobacteria and the high GC and the low GC Gram-positive divisions.     

Analysis of bacterial communities on historical glass by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA.

The present study describes the analysis of bacterial communities on historical window glass by denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA fragments. So far, only a few studies have been published in which the microflora and the corrosion mechanisms of glass surfaces have been investigated. Some microorganisms, especially fungi, have been isolated from different glass samples in the past. However, our results demonstrate that bacterial communities on biodeteriorated glass surfaces are much more complex than previously believed. In addition, bacteria were identified, which have never been isolated from glass samples before.     

Specific detection of different phylogenetic groups of chemocline bacteria based on PCR and denaturing gradient gel electrophoresis of 16S rRNA gene fragments

Specific amplification of 16S rRNA gene fragments in combination with denaturing gradient gel electrophoresis (DGGE) was used to generate fingerprints of Chromatiaceae, green sulfur bacteria, Desulfovibrionaceae, and β-Proteobacteria. Sequencing of the gene fragments confirmed that each primer pair was highly specific for the respective phylogenetic group. Applying the new primer sets, the bacterial diversity in the chemoclines of a eutrophic freshwater lake, a saline meromictic lake, and a laminated marine sediment was investigated. Compared to a conventional bacterial primer pair, a higher number of discrete DGGE bands was generated using our specific primer pairs. With one exception, all 15 bands tested yielded reliable 16S rRNA gene sequences. The highest diversity was found within the chemocline microbial community of the eutrophic freshwater lake. Sequence comparison revealed that the six sequences of Chromatiaceae and green sulfur bacteria detected in this habitat all represent distinct and previously unknown phylotypes. The lowest diversity of phylotypes was detected in the chemocline of the meromictic saline lake, which yielded only one sequence each of the Chromatiaceae, β-2-Proteobacteria, and Desulfovibrionaceae, and no sequences of green sulfur bacteria. The newly developed primer sets are useful for the detection of previously unknown phylotypes, for the comparison of the microbial diversity between different natural habitats, and especially for the rapid monitoring of enrichments of unknown bacterial species. Received: 22 January 1999 / Accepted: 28 April 1999     

Denaturing gradient electrophoresis (DGE) and single-strand conformation polymorphism (SSCP) molecular fingerprintings revisited by simulation and used as a tool to measure microbial diversity

The exact extent of microbial diversity remains unknowable. Nevertheless, fingerprinting patterns [denaturing gradient electrophoresis (DGE), single-strand conformation polymorphism (SSCP)] provide an image of a microbial ecosystem and contain diversity data. We generated numerical simulation fingerprinting patterns based on three types of distribution (uniform, geometric and lognormal) with a range of units from 10 to 500,000. First, simulated patterns containing a diversity of around 1000 units or more gave patterns similar to those obtained in experiments. Second, the number of bands or peaks saturated quickly to about 35 and were unrelated to the degree of diversity. Finally, assuming lognormal distribution, we used an estimator of diversity on in silico and experimental fingerprinting patterns. Results on in silico patterns corresponded to the simulation inputs. Diversity results in experimental patterns were in the same range as those obtained from the same DNA sample in molecular inventories. Thus, fingerprinting patterns contain extractable data about diversity although not on the basis of a number of bands or peaks, as is generally assumed to be the case.     

Molecular analysis of 16 Turkish families with DHPR deficiency using denaturing gradient gel electrophoresis (DGGE)

Dihydropteridine reductase (DHPR) catalyses the conversion of quinonoid dihydrobiopterin (qBH2) to tetrahydrobiopterin (BH4), which serves as the obligatory cofactor for the aromatic amino acid hydroxylases. DHPR deficiency, caused by mutations in the QDPR gene, results in hyperphenylalaninemia and deficiency of various neurotransmitters in the central nervous system, with severe neurological symptoms as a consequence. We have studied, at the clinical and molecular levels, 17 patients belonging to 16 Turkish families with DHPR deficiency. The patients were detected at neonatal screening for hyperphenylalaninemia or upon the development of neurological symptoms. To identify the disease causing molecular defects, we developed a sensitive screening method that rapidly scans the entire open reading frame and all splice sites of the QDPR gene. This method combines PCR amplification and "GC-clamping" of each of the seven exonic regions of QDPR, resolution of mutations by denaturing gradient gel electrophoresis (DGGE), and identification of mutations by direct sequence analysis. A total of ten different mutations were identified, of which three are known (G23D, Y150C, R221X) and the remaining are novel (G17R, G18D, W35fs, Q66R, W90X, S97fs and G149R). Six of these mutations are missense variants, two are nonsense mutations, and two are frameshift mutations. All patients had homoallelic genotypes, which allowed the establishment of genotype-phenotype associations. Our findings suggest that DGGE is a fast and efficient method for detection of mutations in the QDPR gene, which may be useful for confirmatory DNA-based diagnosis, genetic counselling and prenatal diagnosis in DHPR deficiency.