PCR–DGGE method for analyzing the bacterial community in a high temperature petroleum reservoir

Different PCR–denaturing gradient gel electrophoresis (DGGE) protocols were employed to investigate bacterial communities in a high temperature and water flooded petroleum reservoir in Dagang oil field, China. Bacterial universal primers sets frequently used in PCR–DGGE were evaluated. Three primers sets P1 (341F-GC and 534R), P2 (341F-GC and 907R) and P3 (1055F and 1406R-GC) showed different DGGE patterns. Good separation and quality of patterns were obtained in DGGE analysis with the set P3. A total of 12 DNA fragments were excised from the DGGE gels and their sequences were determined. Clustering analysis of the DGGE profiles showed that bacteria in this petroleum reservoir belonged to four clusters. These results indicate that the procedure of DGGE analysis with the primer P3 (1055F and 1406R-GC) is suitable for investigating microbial community in petroleum reservoirs.     

Direct comparison of single-strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) to characterize a microbial community on the basis of 16S rRNA gene fragments

Characterization of microbial communities using single-strand conformation polymorphism (SSCP) was compared with that using denaturing gradient gel electrophoresis (DGGE). This comparison was based on the V3-4 region (Escherichia coli positions: 341-806) of 16S rRNA gene of bacterial or archaeal communities obtained from a methanogenic bioreactor. Significant differences in the bacterial banding profiles were observed while attempting to detect the diversity of the community and its succession during the reactor operation. The SSCP produced a number of sharp bands and differentiated the bacterial community structures to which the DGGE gave an identical pattern. On the other hand, the SSCP and DGGE provided similar succession patterns for archaeal community.     

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     

基于不同通用引物比较分析肠道微生物群落变化

肠道微生物对于人体健康的重要作用已经得到广泛证实,目前,对肠道微生物的研究大多采用基于扩增细菌16S rRNA基因V3-V4区的高通量测序分析,对古菌的关注较少。本研究选择了一对可以同时扩增细菌和古菌16S rRNA基因的引物,通过比较人为干扰肠道微生物前后的群落变化,说明这对引物适宜分析人类肠道细菌和古菌群落变化并具有一定优越性。采集志愿者粪便样品,同时用仅能扩增细菌引物 (B引物) 和细菌古菌通用引物 (AB引物) 进行扩增和高通量测序;使用几个常用的rRNA数据库判断引物对细菌的覆盖度和对古菌的扩增能力。结果表明,AB引物在可以展示B引物扩增出的细菌群落的基础上,可以得到肠道中常见的产甲烷古菌的序列,同时也展示出人为干扰肠道微生物前后的群落结构变化。AB引物可以仅通过一次扩增和测序同时分析肠道中的细菌和古菌群落,更加全面展示肠道微生物群落结构,适用于肠道微生物相关研究。     

利用时间进程法优化活性污泥DG-DGGE图谱

目的:为了探讨电泳时间对双梯度-变性梯度凝胶电泳(DG-DGGE)分析活性污泥样品时的影响。方法:提取污泥DNA后,以通用引物338f/534r扩增16S rDNA序列,采用时间进程法优化PCR扩增产物的DG-DGGE分离效果。结果:采用不同电泳时间进行DGGE分析时,DGGE图谱存在显著的差异。16S rDNA V3区(200 bp)在凝胶梯度6%~12%,变性剂梯度30%~60%时,在200V电压下,最佳电泳时间为5h。     

福建省稻田土壤细菌群落的16S rDNA-PCR-DGGE分析

用不依赖细菌培养的16S rDNA-PCR-DGGE方法对福建省6个不同地区12个取样点的稻田土壤进行细菌群落结构分析.对12份样品直接提取其总DNA,用F341GC/R534引物扩增16SrDNA基因的V3可变区,结合DGGE(denaturing gradient gel electrophoresis)技术分析样品细菌群落组成.结果表明,福建省不同地区的稻田土壤之间细菌群落结构存在较大差异.犬体上可分为闽东、闽南、闽北、闽西4个大类.同一地区的根际土和表土样品之间也存在差异,但差异相对较低,其中龙岩根际土和表土细菌群落结构相似性最大,永泰差异性最大.回收了DGGE图谱中11个条带,测序结果经过Blast比对表明其中10个条带代表的细菌是不可培养的,显示了DGGE技术的优越性.     

Nonradioactive method to study genetic profiles of natural bacterial communities by PCR-single-strand-conformation polymorphism.

We describe a new method for studying the structure and diversity of bacterial communities in the natural ecosystem. Our approach is based on single-strand-conformation polymorphism (SSCP) analysis of PCR products of 16S rRNA genes from complex bacterial populations. A pair of eubacterial universal primers for amplification of the variable V3 region were designed from the 16S rRNA sequences of 1,262 bacterial strains. The PCR conditions were optimized by using genomic DNAs from five gram-positive and seven gram-negative strains. The SSCP analysis of the PCR products demonstrated that a bacterial strain generated its characteristic band pattern and that other strains generated other band patterns, so that the relative diversity in bacterial communities could be measured. In addition, this method was sensitive enough to detect a bacterial population that made up less than 1.5% of a bacterial community. The distinctive differences between bacterial populations were observed in an oligotrophic lake and a eutrophic pond in a field study. The method presented here, using combined PCR amplification and SSCP pattern analyses of 16S rRNA genes, provides a useful tool to study bacterial community structures in various ecosystems.     

Analyses of soil fungal communities in adjacent natural forest and hoop pine plantation ecosystems of subtropical Australia using molecular approaches based on 18S rRNA genes

Soil fungal communities were studied using 18S rDNA-based molecular techniques. Soil DNA was analyzed using temperature gradient gel electrophoresis (TGGE), single-stranded conformational polymorphism (SSCP), cloning and sequencing methods, following community DNA extraction and polymerase chain reaction (PCR). The extracted community DNA was successfully amplified using the primer pair of EF4f-Fung5r which produced ca. 550bp 18S rDNA fragments. TGGE screening of the PCR products showed some differences in band position and intensity between two soil samples in adjacent natural forest (YNF) and hoop pine plantation (YHP) ecosystems at Yarraman in subtropical Australia. TGGE and SSCP could be used for screening PCR products. However, care must be exercised when interpreting the TGGE and SSCP results with respect to microbial diversity, because one band may not necessarily represent one species. It is recommended that the PCR products should be purified before TGGE or SSCP screening. SSCP screening of the clone sequences revealed differences among the clones. Sequence and phylogenetic analyses revealed that all obtained clones were affiliated to the kingdom Fungi, including three phyla, i.e., Zygomycota, Ascomycota and Basidiomycota. Our results suggested that community DNA extraction, PCR, cloning, SSCP screening of clones, sequencing of selected clones and phylogentic analyses could be a good strategy in investigation of soil fungal community and diversity.     

A New Approach To Utilize PCR–Single-Strand-Conformation Polymorphism for 16S rRNA Gene-Based Microbial Community Analysis

Single-strand-conformation polymorphism (SSCP) of DNA, a method widely used in mutation analysis, was adapted to the analysis and differentiation of cultivated pure-culture soil microorganisms and noncultivated rhizosphere microbial communities. A fragment (approximately 400 bp) of the bacterial 16S rRNA gene (V-4 and V-5 regions) was amplified by PCR with universal primers, with one primer phosphorylated at the 5′ end. The phosphorylated strands of the PCR products were selectively digested with lambda exonuclease, and the remaining strands were separated by electrophoresis with an MDE polyacrylamide gel, a matrix specifically optimized for SSCP purposes. By this means, reannealing and heteroduplex formation of DNA strands during electrophoresis could be excluded, and the number of bands per organism was reduced. PCR products from 10 of 11 different bacterial type strains tested could be differentiated from each other. With template mixtures consisting of pure-culture DNAs from 5 and 10 bacterial strains, most of the single strains could be detected from such model communities after PCR and SSCP analyses. Purified bands amplified from pure cultures and model communities extracted from gels could be reamplified by PCR, but by this process, additional products were also generated, as detected by further SSCP analysis. Profiles generated with DNAs of rhizosphere bacterial communities, directly extracted from two different plant species grown in the same field site, could be clearly distinguished. This study demonstrates the potential of the selected PCR–single-stranded DNA approach for microbial community analysis.     

Distribution of bacterioplankton in meromictic Lake Saelenvannet, as determined by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA.

The community structure of bacterioplankton in meromictic Lake Saelenvannet was examined by PCR amplification of the V3 region of 16S rRNA from microbial communities recovered from various depths in the water column. Two different primer sets were used, one for amplification of DNA from the domain Bacteria and another specific for DNA from the domain Archaea. Amplified DNA fragments were resolved by denaturing gradient gel electrophoresis (DGGE), and the resulting profiles were reproducible and specific for the communities from different depths. Bacterial diversity estimated from the number and intensity of specific fragments in DGGE profiles decreased with depth. The reverse was true for the Archaea, with the diversity increasing with depth. Hybridization of DGGE profiles with oligonucleotide probes specific for phylogenetic groups of microorganisms showed the presence of both sulfate-reducing bacteria and methanogens throughout the water column, but they appeared to be most abundant below the chemocline. Several dominant fragments in the DGGE profiles were excised and sequenced. Among the dominant populations were representatives related to Chlorobium phaeovibrioides, chloroplasts from eukaryotic algae, and unidentified Archaea.     

Design and Evaluation of PCR Primers for Analysis of Bacterial Populations in Wine by Denaturing Gradient Gel Electrophoresis

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal DNA (rDNA) is routinely used to compare levels of diversity of microbial communities and to monitor population dynamics. While using PCR-DGGE to examine the bacteria in wine fermentations, we noted that several commonly used PCR primers for amplifying bacterial 16S rDNA also coamplified yeast, fungal, or plant DNA present in samples. Unfortunately, amplification of nonbacterial DNA can result in a masking of bacterial populations in DGGE profiles. To surmount this problem, we developed two new primer sets for specific amplification of bacterial 16S rDNA in wine fermentation samples without amplification of eukaryotic DNA. One primer set, termed WLAB1 and WLAB2, amplified lactic acid bacteria, while another, termed WBAC1 and WBAC2, amplified both lactic acid bacterial and acetic acid bacterial populations found in wine. Primer specificity and efficacy were examined with DNA isolated from numerous bacterial, yeast, and fungal species commonly found in wine and must samples. Importantly, both primer sets effectively distinguished bacterial species in wine containing mixtures of yeast and bacteria.     

Design and evaluation of PCR primers for analysis of bacterial populations in wine by denaturing gradient gel electrophoresis

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal DNA (rDNA) is routinely used to compare levels of diversity of microbial communities and to monitor population dynamics. While using PCR-DGGE to examine the bacteria in wine fermentations, we noted that several commonly used PCR primers for amplifying bacterial 16S rDNA also coamplified yeast, fungal, or plant DNA present in samples. Unfortunately, amplification of nonbacterial DNA can result in a masking of bacterial populations in DGGE profiles. To surmount this problem, we developed two new primer sets for specific amplification of bacterial 16S rDNA in wine fermentation samples without amplification of eukaryotic DNA. One primer set, termed WLAB1 and WLAB2, amplified lactic acid bacteria, while another, termed WBAC1 and WBAC2, amplified both lactic acid bacterial and acetic acid bacterial populations found in wine. Primer specificity and efficacy were examined with DNA isolated from numerous bacterial, yeast, and fungal species commonly found in wine and must samples. Importantly, both primer sets effectively distinguished bacterial species in wine containing mixtures of yeast and bacteria.     

Influence of Commonly Used Primer Systems on Automated Ribosomal Intergenic Spacer Analysis of Bacterial Communities in Environmental Samples

Due to the high diversity of bacteria in many ecosystems, their slow generation times, specific but mostly unknown nutrient requirements and syntrophic interactions, isolation based approaches in microbial ecology mostly fail to describe microbial community structure. Thus, cultivation independent techniques, which rely on directly extracted nucleic acids from the environment, are a well-used alternative. For example, bacterial automated ribosomal intergenic spacer analysis (B-ARISA) is one of the widely used methods for fingerprinting bacterial communities after PCR-based amplification of selected regions of the operon coding for rRNA genes using community DNA. However, B-ARISA alone does not provide any taxonomic information and the results may be severely biased in relation to the primer set selection. Furthermore, amplified DNA stemming from mitochondrial or chloroplast templates might strongly bias the obtained fingerprints. In this study, we determined the applicability of three different B-ARISA primer sets to the study of bacterial communities. The results from in silico analysis harnessing publicly available sequence databases showed that all three primer sets tested are specific to bacteria but only two primers sets assure high bacterial taxa coverage (1406f/23Sr and ITSF/ITSReub). Considering the study of bacteria in a plant interface, the primer set ITSF/ITSReub was found to amplify (in silico) sequences of some important crop species such as Sorghum bicolor and Zea mays. Bacterial genera and plant species potentially amplified by different primer sets are given. These data were confirmed when DNA extracted from soil and plant samples were analyzed. The presented information could be useful when interpreting existing B-ARISA results and planning B-ARISA experiments, especially when plant DNA can be expected.     

Effect of primers hybridizing to different evolutionarily conserved regions of the small-subunit rRNA gene in PCR-based microbial community analyses and genetic profiling

Genetic profiling techniques of microbial communities based on PCR-amplified signature genes, such as denaturing gradient gel electrophoresis or single-strand-conformation polymorphism (SSCP) analysis, are normally done with PCR products of less than 500-bp. The most common target for diversity analysis, the small-subunit rRNA genes, however, are larger, and thus, only partial sequences can be analyzed. Here, we compared the results obtained by PCR targeting different variable (V) regions (V2 and V3, V4 and V5, and V6 to V8) of the bacterial 16S rRNA gene with primers hybridizing to evolutionarily conserved flanking regions. SSCP analysis of single-stranded PCR products generated from 13 different bacterial species showed fewer bands with products containing V4-V5 (average, 1.7 bands per organism) than with V2-V3 (2.2 bands) and V6-V8 (2.3 bands). We found that the additional bands (>1 per organism) were caused by intraspecies operon heterogeneities or by more than one conformation of the same sequence. Community profiles, generated by PCR-SSCP from bacterial-cell consortia extracted from rhizospheres of field-grown maize (Zea mays), were analyzed by cloning and sequencing of the dominant bands. A total of 48 sequences could be attributed to 34 different strains from 10 taxonomical groups. Independent of the primer pairs, we found proteobacteria (alpha, beta, and gamma subgroups) and members of the genus Paenibacillus (low G+C gram-positive) to be the dominant organisms. Other groups, however, were only detected with single primer pairs. This study gives an example of how much the selection of different variable regions combined with different specificities of the flanking "universal" primers can affect a PCR-based microbial community analysis.     

Conversion of AFLP markers to sequence-specific PCR markers in barley and wheat

 Conversion of amplified fragment length polymorphisms (AFLPs) to sequence-specific PCR primers would be useful for many genetic-linkage applications. We examined 21 wheat nullitetrasomic stocks and five wheat-barley addition lines using 12 and 14 AFLP primer combinations, respectively. On average, 36.8% of the scored AFLP fragments in the wheat nullitetrasomic stocks and 22.3% in the wheat-barley addition lines could be mapped to specific chromosomes, providing approximately 461 chromosome-specific AFLP markers in the wheat nullitetrasomic stocks and 174 in the wheat-barley addition lines. Ten AFLP fragments specific to barley chromosomes and 16 AFLP fragments specific to wheat 3BS and 4BS chromosome arms were isolated from the polyacrylamide gels, re-amplified, cloned and sequenced. Primer sets were designed from these sequences. Amplification of wheat and barley genomic DNA using the barley derived primers revealed that three primer sets amplified DNA from the expected chromosome, five amplified fragments from all barley chromosomes but not from wheat, one amplified a similar-sized fragment from multiple barley chromosomes and from wheat, and one gave no amplification. Amplification of wheat genomic DNA using the wheat-derived primer sets revealed that three primer sets amplified a fragment from the expected chromosome, 11 primer sets amplified a similar-sized fragment from multiple chromosomes, and two gave no amplification. These experiments indicate that polymorphisms identified by AFLP are often not transferable to more sequence-specific PCR applications. Received: 30 June 1998 / Accepted: 26 October 1998     

Effect of Primers Hybridizing to Different Evolutionarily Conserved Regions of the Small-Subunit rRNA Gene in PCR-Based Microbial Community Analyses and Genetic Profiling

Genetic profiling techniques of microbial communities based on PCR-amplified signature genes, such as denaturing gradient gel electrophoresis or single-strand-conformation polymorphism (SSCP) analysis, are normally done with PCR products of less than 500-bp. The most common target for diversity analysis, the small-subunit rRNA genes, however, are larger, and thus, only partial sequences can be analyzed. Here, we compared the results obtained by PCR targeting different variable (V) regions (V2 and V3, V4 and V5, and V6 to V8) of the bacterial 16S rRNA gene with primers hybridizing to evolutionarily conserved flanking regions. SSCP analysis of single-stranded PCR products generated from 13 different bacterial species showed fewer bands with products containing V4-V5 (average, 1.7 bands per organism) than with V2-V3 (2.2 bands) and V6-V8 (2.3 bands). We found that the additional bands (>1 per organism) were caused by intraspecies operon heterogeneities or by more than one conformation of the same sequence. Community profiles, generated by PCR-SSCP from bacterial-cell consortia extracted from rhizospheres of field-grown maize (Zea mays), were analyzed by cloning and sequencing of the dominant bands. A total of 48 sequences could be attributed to 34 different strains from 10 taxonomical groups. Independent of the primer pairs, we found proteobacteria (α, β, and γ subgroups) and members of the genus Paenibacillus (low G+C gram-positive) to be the dominant organisms. Other groups, however, were only detected with single primer pairs. This study gives an example of how much the selection of different variable regions combined with different specificities of the flanking “universal” primers can affect a PCR-based microbial community analysis.     

Succession of microbial communities during hot composting as detected by PCR-single-strand-conformation polymorphism-based genetic profiles of small-subunit rRNA genes

A cultivation-independent technique for genetic profiling of PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR amplifications were performed with DNA directly extracted from compost samples and with primers targeting either (i) the V4-V5 region of eubacterial 16S rRNA genes, (ii) the V3 region in the 16S rRNA genes of actinomycetes, or (iii) the V8-V9 region of fungal 18S rRNA genes. Homologous PCR products were converted to single-stranded DNA molecules by exonuclease digestion and were subsequently electrophoretically separated by their single-strand-conformation polymorphism (SSCP). Genetic profiles obtained by this technique showed a succession and increasing diversity of microbial populations with all primers. A total of 19 single products were isolated from the profiles by PCR reamplification and cloning. DNA sequencing of these molecular isolates showed similarities in the range of 92.3 to 100% to known gram-positive bacteria with a low or high G+C DNA content and to the SSU rDNA of gamma-Proteobacteria. The amplified 18S rRNA gene sequences were related to the respective gene regions of Candida krusei and Candida tropicalis. Specific molecular isolates could be attributed to different composting stages. The diversity of cultivated bacteria isolated from samples taken at the end of the composting process was low. A total of 290 isolates were related to only 6 different species. Two or three of these species were also detectable in the SSCP community profiles. Our study indicates that community SSCP profiles can be highly useful for the monitoring of bacterial diversity and community successions in a biotechnologically relevant process.     

Universal primer PCR with DGGE for rapid detection of bacterial pathogens

A universal primer PCR (UPPCR) combined with denaturing gradient gel electrophoresis (DGGE) was evaluated as a method permitting the rapid detection of pathogens. The results show that this method is efficient at amplifying the conserved regions of bacterial 16S rRNA genes with universal primers and can detect causative bacterial pathogens rapidly. Six species of bacteria from fisheries (Pseudomonas fluorescens, Vibrio anguillarum, Aeromonas hydrophila, Vibrio fluvialis, Providencia rettgeri and Aeromonas sobria) were examined. Our results indicate that the approach we undertook can be adopted not only for axenic bacterial populations but also for mixed communities as well. Furthermore, we were able to achieve the rapid detection of multiple bacteria a single in sample. In addition, UPPCR-DGGE was shown to be better than previously reported UPPCR-single-stranded conformation polymorphism (SSCP)-based methods for the rapid detection of bacterial pathogens.     

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.     

链霉菌看家基因引物的设计与验证

看家基因的扩增与测序是进行多基因系统进化分析首先需要解决的问题。针对链霉菌这一群高(G C)mol%革兰氏阳性细菌,选定4个看家基因:atpD、recA、rpoB和trpB,利用NCBI数据库中已有的2个链霉菌和3个分枝杆菌的全基因组序列,以及另两个链霉菌的recA基因序列,通过软件分析设计了各基因的扩增和测序引物,并优化了扩增反应条件。从所试验的55株链霉菌中,均特异地扩增出了上述4个基因的片段,并成功进行了序列测定,验证了所设计引物的实用性。所归纳的引物设计方法可用于高(G C)mol%革兰氏阳性细菌的其它看家基因,以促进多基因系统进化研究的开展。