A single band does not always represent single bacterial strains in denaturing gradient gel electrophoresis analysis

DNA in a denaturing gradient gel electrophoresis (DGGE) band that could not be sequenced after recovery from the gel was cloned into a TA cloning vector and a library was constructed and then 13 clones randomly picked up from the library was sequenced. Although the excised DNA from the DGGE gel showed a single band, the library consisted of several different sequences phylogenetically. This phenomenon was also observed in several other DGGE bands. Therefore, this suggests that a single DGGE band does not always represent a single bacterial strain and a new bias for quantitative analyses based on band intensities has been identified.     

Evaluation of PCR primers for denaturing gradient gel electrophoresis analysis of fungal communities in compost

AIMS: Three previously published fungal specific PCR primer sets, referred to as the NS, EF and NL primer sets, were evaluated for use in compost microbial community analysis by PCR and denaturing gradient gel electrophoresis (DGGE). METHODS AND RESULTS: Primers were first evaluated based on their tolerance to PCR inhibitors. Due to its sensitivity to inhibitors, the NS primer set was determined to require a 10-fold smaller volume addition of compost DNA to PCR than the EF and NL primer sets, based on a logistic regression model for a 75% PCR success rate. Further evaluation of the EF and NL primer sets involved testing the resolution of PCR products from pure fungal cultures on DGGE. The NL primer set, which targets the more variable 28S rDNA, resulted in multiple bands for each pure culture. Thus, the EF primer set was used to monitor the microbial community during compost colonization studies, where three fungi were inoculated onto autoclaved grape pomace and rice straw compost. CONCLUSIONS: Of the three primer sets evaluated, the EF primer set was determined to be the best for PCR-DGGE of compost fungal populations; however, concerns with the EF primer set included the lack of sequence divergence in the targeted region of 18S rDNA and PCR artifacts which interfered with detection of inoculated fungi in the colonization studies. SIGNIFICANCE AND IMPACT OF THE STUDY: There are many factors related to PCR primers that need to be assessed prior to applying PCR-DGGE to fungal communities in complex environments such as compost.     

Comparison of microbial communities in four different composting processes as evaluated by denaturing gradient gel electrophoresis analysis

AIMS: We aimed to systematically understand the composting processes by a comparison of microbial communities during four full-scale composting processes. METHODS AND RESULTS: Microbial communities during the four different full-scale composting processes were analysed by denaturing gradient gel electrophoresis combined with measurement of physicochemical parameters. Two composting processes utilized sewage sludge and two utilized food-waste. Comparison of the four processes indicated that the concentration of dissolved organic carbon was higher in the food-waste-composting than in the sewage-sludge-composting processes, and microbial communities varied with composting substrate. The tendency for different microbes to appear in the composting process with different concentrations of dissolved organic carbon agreed with a previous study that showed that microbial succession occurred with a decrease in dissolved organic carbon in a laboratory-scale food-waste-composting process. CONCLUSIONS: Our results suggested that the main factor affecting microbial communities in the composting process is the concentration of dissolved organic materials. SIGNIFICANCE AND IMPACT OF THE STUDY: In addition to studying microbial communities involved in composting, this research is also the first to study composting mechanisms using molecular methods. The results of our studies may be helpful in the design and management of composting processes.     

应用PCR与温度梯度凝胶电泳分析龈上菌斑微生物群落

目的应用PCR与温度梯度凝胶电泳(PCR—TGGE)分子技术对成年健康口腔的龈上菌斑中微生物群落组成进行分析。方法8例成年个体包括4男4女,年龄19～29岁,分别采取每例个体上下颌牙周龈上菌斑样品,共18份(个体Subl间隔10天采集2次样品)。提取菌斑DNA,PCR扩增16SrDNAV3可变区,产物经TGGE后进行相似系数分析。结果同一个体的上下颌微生物群落组成相似性系数为81％～95％,而不同个体的龈上菌斑微生物群落组成相似性系数,均在60％以下。结论不同个体具有其独特的牙周微生物群落,而且在一定时期内组成稳定。     

DGGE and 16S rDNA analysis reveals a highly diverse and rapidly colonising bacterial community on different substrates in the rumen of goats

In the rumen, plant particles are colonised and degraded by the rumen micro-organisms. Although numerous important findings about fibre-associated bacterial community were obtained using traditional or molecular techniques, little information is available on the dynamics of bacteria associated with feed particles during incubation in the rumen. In the present study, ryegrass leaf, ryegrass stem and rice straw, representing different carbohydrate compositions, were used as substrates and placed in the rumen of goats by using nylon bags, and PCR/DGGE (denaturing gradient gel electrophoresis) with subsequent sequence analysis were used to monitor the dynamics of and identify bacteria associated with the substrates during 24 h of incubation. DGGE results showed that substrate samples collected from 10 min to 6 h had similar DGGE patterns, with up to 24 predominant bands to each sample, including 14 common bands to all samples, suggesting a rapid and stable colonisation by a highly diverse bacterial community. Substrate samples collected at 12 and 24 h showed similar DGGE patterns but had great difference in DGGE patterns from those collected at 10 min to 6 h, suggesting an apparent shift in bacterial community. Sequence analysis indicated that most substrate-associated bacteria were closely related to fibrolytic bacteria. In conclusion, a highly diverse and similar rumen bacterial community could immediately colonise to different substrates and remained stable during the initial 6 h of incubation, but experienced a marked change after 12 h of incubation. Italian ryegrass leaf, Italian ryegrass stem and rice straw were colonised with a similar bacterial community.     

Characterisation of the bacterial community associated with early stages of Great Scallop (Pecten maximus), using denaturing gradient gel electrophoresis (DGGE)

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA was used to characterise and compare bacterial communities associated with scallop larvae (Pecten maximus), in different production units in a shellfish hatchery. Water and larvae samples were collected from three different aquaculture systems; stagnant, flow-through and a flow- through system with seawater treated with ozone. Samples were also collected from different algal cultures, inlet tanks and water pipes leading to the different aquaculture systems. Clear differences were seen between the bacterial community associated with the larvae and in the water from the different aquaculture systems. However, there were high similarities in the community composition between different water samples and between larvae samples collected at different time periods, indicating a high stability in the bacterial communities. Fifty three percent of the sequences from these samples were similar to 16S rRNA gene sequences of members of the gamma-subclass of the Proteobacteria. The different algal cultures had different bacterial communities, however 73 percent of the sequences were similar to 16S rRNA gene sequences of members of the alpha-subclass of the Proteobacteria. Differences in the DGGE profiles were also seen between the samples taken from the inlet tanks and water pipes, indicating a change in the bacterial community composition as the water passed through the pipes. To our knowledge this is the first study investigating bacterial communities associated with Great Scallop larvae in different aquaculture systems including noncultured components.     

应用变性梯度凝胶电泳和16S rDNA序列分析对kefir粒中细菌多样性的研究

以开菲尔(Kefir)粒为材料,经过DNA抽提和16S rDNA V3区PCR扩增,扩增产物经变性梯度凝胶电泳(DGGE)分离并切割电泳条带进行序列测定,并与现有的数据库进行了比较,对Kefir粒的细菌多样性进行分析。结果表明,DGGE图谱中可检测到的8条带的16S rDNA基因序列中有7个基因序列与GenBank数据库登录的相关序列的相似性大于98%,余下的1个基因序列的相似性也大于96%。相似性大于98%的7个克隆中,有3个属于鞘氨醇杆菌属(Sphingobacterium),2个属于乳杆菌属(Lactobacillus),其它2个分别属于肠杆菌属(Enterobacter)和不动杆菌属(Acinetobacter)。首次报道了鞘氨醇杆菌作为优势菌群存在开菲尔Kefir粒中。     

Denaturing gradient gel electrophoresis (DGGE) analysis of bacterial community composition in deep-sea sediments of the South China Sea

The South China Sea, which is one of the largest marginal seas in the world, is predicted to have suitable accumulation conditions and exporting prospects for natural gas hydrate. The aim of this study was to explore the bacterial community composition of deep-sea sediments from such an ecosystem. DNA was extracted by five different methods and used as templates for PCR amplification of the V3 regions of the 16S rRNA gene. Denaturing gradient gel electrophoresis (DGGE) was used to separate the amplified products and analyse the 16S rRNA gene diversity of sediment samples. The results of DGGE indicated that the bacterial community composition is influenced by DNA extraction methods. Sequencing dominant bands demonstrated that the major phylogenetic groups identified by DGGE belong to Proteobacteria, Bacteroidetes, gram-positive bacteria and Archaea. Integrating different DNA extraction procedures are needed to analyse the actual bacterial diversity from environment when the amplification of 16S rRNA gene and construction of representative clone library were adopted.     

The structure of the bacterial and archaeal community in a biogas digester as revealed by denaturing gradient gel electrophoresis and 16S rDNA sequencing analysis

Aims:  To identify the bacterial and archaeal composition in a mesophilic biogas digester treating pig manure and to compare the consistency of two 16S rDNA-based methods to investigate the microbial structure.
Methods and results:  Sixty-nine bacterial operational taxonomic units (OTU) and 25 archaeal OTU were identified by sequencing two 16S rDNA clone libraries. Most bacterial OTU were identified as phyla of Firmicutes (47·2% of total clones), Bacteroides (35·4%) and Spirochaetes (13·2%). Methanoculleus bourgensis (29·0%), Methanosarcina barkeri (27·4%) and Methanospirillum hungatei (10·8%) were the dominant methanogens. Only 9% of bacterial and 20% of archaeal OTU matched cultured isolates at a similarity index of ≥97%. About 78% of the dominant bacterial (with abundance >3%) and 83% of archaeal OTU were recovered from the denaturing gradient gel electrophoresis (DGGE) bands of V3 regions in 16S rDNAs.
Conclusions:  In the digester, most bacterial and archaeal species were uncultured; bacteria belonging to Firmicutes , Bacteroides and Spirochaetes seem to take charge of cellulolysis, proteolysis, acidogenesis, sulfur-reducing and homoacetogenesis; the most methanogens were typical hydrogenotrophic or hydrogenotrophic/aceticlastic; DGGE profiles reflected the dominant microbiota.
Significance and Impact of the Study:  This study gave a first insight of the overall microbial structure in a rural biogas digester and also indicated DGGE was useful in displaying its dominant microbiota.     

Characterization of benthic bacterial assemblages in a polluted stream using denaturing gradient gel electrophoresis

To study differences in bacterial assemblages among sites with different environmental conditions, sediment samples were collected from three sites along a South Carolina (U.S.A.) coastalplain stream with varying levels of anthropogenic perturbation. The objective of this study was to compare the bacterial assemblages among these sites to detect possible impacts from the disturbance. To accomplish this comparison, DNA was extracted from the samples and subjected to the polymerase chain reaction using primers designed to amplify bacterial 16S rRNA genes. Relative measures of bacterial genetic diversity, assessed using denaturing gradient gel electrophoresis (DGGE), revealed greater numbers of unique sequences at the disturbed sites. The number of bands, which is analogous to species richness, did not vary predictably among sites. Similarity indices revealed a high level of similarity among replicate samples from each site and low similarity between samples from different sites. This study demonstrated that bacterial assemblages differed among sites and that the presence or absence of certain species, represented by unique DGGE bands, differed among sites; unique bands were most commonly encountered at the disturbed sites. Based on the evidence gathered, we conclude that benthic bacterial assemblages vary longitudinally and that anthropogenic disturbance may alter the bacterial component of streams.     

茶园土壤微生物群落基因多样性

应用PCR技术,直接从土壤中抽提总DNA,扩增16S rDNA V3 片段,采用变性梯度凝胶电泳(DGGE)分析16S rDNA V3 片段的多态性,研究了杭州西湖梅家坞不同植茶年龄(8、50和90年)、不同利用方式(茶园、荒地和林地)的土壤微生物群落基因多样性.结果表明：不同植茶年龄和不同土地利用方式影响土壤微生物群落的基因多样性.荒地、茶园和林地土壤微生物群落基因多样性指数明显不同(P<0.05),其排列顺序为荒地>茶园>林地.不同植茶年龄的土壤中,50年茶园土壤的微生物群落基因多样性指数、微生物量碳和基础呼吸明显高于8年和90年茶园土壤(P<0.05).     

Differentiation of bacterial strains by thermal gradient gel electrophoresis using non-GC-clamped PCR primers for the 16S-23S rDNA intergenic spacer region

The method for DNA fingerprinting of the 16S-23S rDNA intergenic spacer region was modified to increase resolution of bacterial strains by thermal gradient gel electrophoresis (TGGE) analysis. By utilizing the high melting temperature region of the tRNA gene located in the middle of the 16S-23S rDNA intergenic spacer region as an internal clamp for TGGE, multiple melting domain problems were solved. PCR primers lacking a stretch of GC-rich sequences (GC-clamp) amplified the intergenic spacer region more efficiently than GC-clamped primers. Therefore, PCR artifacts were avoided by using low, 17-cycle, PCR. The method was successfully applied to diverse bacterial species for strain differentiation by TGGE without requiring a special PCR primer set.     

Phylogenetic analysis of bacterial communities associated with larvae of the Atlantic halibut propose succession from a uniform normal flora

Halibut, the largest of all flatfishes is a valuable species with a great potential for aquaculture. Bacteria play an important role in regulating the health of the early life stages. The present article is the first broad-range molecular analysis of bacterial communities in larvae of the Atlantic halibut (Hippoglossus hippoglossus). DNA was extracted from larvae, water and silo biofilm from hatcheries in Norway, Scotland, Iceland and Canada. Eubacterial 16S rRNA gene fragments were amplified by polymerase chain reaction (PCR) with broad-range primers. Sequences spanning the hyper variable V3 region representing individual bacterial species were separated into community profiles by denaturing gradient gel electrophoresis (DGGE). The profiles revealed simple communities after hatching and bacterial succession following growth. Sequencing and phylogenetic analysis of excised DGGE bands suggested aerobic heterotrophs related to groups of Pseudomonas, Janthinobacterium and possibly Marinomonas to be the primary colonisers of the larvae. After onset of feeding, fermentative species (Vibrio) were detected as well. Comparative analysis of bacterial communities from different geographical regions indicated that larvae of the Atlantic halibut possess a distinct and specific normal flora.     

Bacterial and archaeal assemblages in sediments of a large shallow freshwater lake, Lake Taihu, as revealed by denaturing gradient gel electrophoresis

Aims:  To explore the association of microbial community structure with the development of eutrophication in a large shallow freshwater lake, Lake Taihu.
Methods and Results:  The bacterial and archaeal assemblages in sediments of different lake areas were analysed using denaturing gradient gel electrophoresis (DGGE) of amplified 16S rDNA fragments. The bacterial DGGE profiles showed that eutrophied sites, grass-bottom areas and relatively clean sites with a eutrophic (albeit dredged) site are three respective clusters. Fifty-one dominant bacterial DGGE bands were detected and 92 corresponding clones were sequenced, most of which were affiliated with bacterial phylotypes commonly found in freshwater ecosystems. Actinobacteria were detected in the centre of the lake and not at eutrophied sites whereas the opposite was found with respect to Verrucomicrobiales . Twenty-five dominant archaeal DGGE bands were detected and 31 corresponding clones were sequenced, most of which were affiliated with freshwater archaeal phylotypes.
Conclusions:  The bacterial community structures in the sediments of different areas with similar water quality and situation tend to be similar in Taihu Lake.
Significance and Impact of the Study:  This study may expand our knowledge on the relationship between the overall microbial assemblages and the development of eutrophication in the shallow freshwater lake.     

Denaturing gradient gel electrophoresis (DGGE) screening of clones prior to sequencing

Whilst cloning and sequencing techniques are used ubiquitously for the identification of novel DNA sequences, the necessity to determine a consensus sequence means that this can be both labour‐intensive and costly. Here we describe a rapid and cost effective method of using denaturing gradient gel electrophoresis (DGGE) for the analysis of large numbers of clones prior to sequencing. This procedure allows for the selection of specific clones, eliminates the need to sequence multiple copies of the same clone, and reduces the likelihood of sequencing recombinant PCR product.     

Diversity of endophytic bacterial communities in poplar grown under field conditions

Bacterial endophytes may be important for plant health and other ecologically relevant functions of poplar trees. The composition of endophytic bacteria colonizing the aerial parts of poplar was studied using a multiphasic approach. The terminal restriction fragment length polymorphism analysis of 16S rRNA genes demonstrated the impact of different hybrid poplar clones on the endophytic community structure. Detailed analysis of endophytic bacteria using cultivation methods in combination with cloning of 16S rRNA genes amplified from plant tissue revealed a high phylogenetic diversity of endophytic bacteria with a total of 53 taxa at the genus level that included Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. The community structure displayed clear differences in terms of the presence and relative proportions of bacterial taxa between the four poplar clones studied. The results showed that the genetic background of the hybrid poplar clones corresponded well with the endophytic community structure. Out of the 513 isolates and 209 clones identified, Actinobacteria, in particular the family Microbacteriaceae, made up the largest fraction of the isolates, whereas the clone library was dominated by Alpha- and Betaproteobacteria. The most abundant genera among the isolates were Pseudomonas and Curtobacterium, while Sphingomonas prevailed among the clones.     

A heteroduplex method for detection of targeted sub-populations of bacterial communities

We describe a simple method, based on heteroduplex mobility analysis of 16S rDNA fragments, for targeted detection of sub-populations of bacteria within diverse microbial communities. A small (ca. 200 bp) polymorphic fragment of the bacterial 16S rRNA gene was amplified from sample DNA using universal primers. Sample products were hybridised with a fluorescently labelled fragment amplified from a selected 'reporter' organism representing the target group. The resulting products were resolved and the labelled heteroduplex pairs detected on non-denaturing gels using automated DNA detection technology. A model, based on analysis of samples with known 16S rDNA sequences, demonstrates that heteroduplex mobility is inversely correlated with genetic distance and that beyond 26% genetic difference, heteroduplex products are not detected. The utility of the method was tested by field studies in which stream biofilms could be characterised by heteroduplex profiles generated with heterotrophic and autotrophic reporter organisms representing target groups.     

应用变性梯度凝胶电泳和16SrDNA序列分析对kefir粒中细菌多样性的研究

以开菲尔（Kefir）粒为材料,经过DNA抽提和16SrDNA V3区PCR扩增,扩增产物经变性梯度凝胶电泳（DGGE）分离并切割电泳条带进行序列测定,并与现有的数据库进行了比较,对Kefir粒的细菌多样性进行分析。结果表明,DGGE图谱中可检测到的8条带的16SrDNA基因序列中有7个基因序列与GenBank数据库登录的相关序列的相似性大于98％,余下的1个基因序列的相似性也大于96％。相似性大于98％的7个克隆中,有3个属于鞘氨醇杆菌属（Sphingobacterium）,2个属于乳杆菌属（Lactobacillus）,其它2个分别属于肠杆菌属（Errterobacter）和不动杆菌属（Acinetobacter）。首次报道了鞘氨醇杆菌作为优势菌群存在开菲尔Kefir粒中。     

对Bt蛋白敏感敏感和敏感的棉铃虫中肠细菌群落的比较

摘要：【目的】通过比较Cry1Ac蛋白抗性及敏感棉铃虫中肠细菌群落的结构组成,研究中肠微生物是否与棉铃虫Bt抗性产生有关。【方法】首先提取了棉铃虫中肠微生物基因组DNA,通过PCR扩增获得了16S rDNA全长片段及V3区。采用基于16S rDNA 的免培养技术—16S rDNA文库建立和变性梯度凝胶电泳（DGGE）研究了国内特有的Bt抗性和敏感品系棉铃虫中肠细菌群落组成,并对其进行分析和比较。【结果】16S rDNA文库测序结果表明,抗性品系与敏感品系棉铃虫中肠细菌群落特别是优势菌群非常相似,但在部分劣势菌群上存在差异。抗性品系中主要优势菌有：不可培养微生物（Uncultured bacterium）占56.4%,鹑鸡肠球菌（Enterococcus gallinarum）占17.0%,铅黄肠球菌（Enterococcus casseliflavus）占17.0%;敏感品系中主要优势菌为不可培养微生物（Uncultured bacterium）60.2%,鹑鸡肠球菌（Enterococcus gallinarum）占19.3%,铅黄肠球菌（Enterococcus casseliflavus）占14.7%。随后进行的PCR验证表明,部分有差异的劣势菌在两种品系虫体都存在。DGGE图谱分析表明,这两个品系棉铃虫中肠菌群相似性达到92.3%。【结论】敏感品系与抗性品系棉铃虫肠道菌群组成极其相似,推测抗性的产生与肠道微生物无直接关系。