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.     

Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients.

A group-specific primer, F243 (positions 226 to 243, Escherichia coli numbering), was developed by comparison of sequences of genes encoding 16S rRNA (16S rDNA) for the detection of actinomycetes in the environment with PCR and temperature or denaturing gradient gel electrophoresis (TGGE or DGGE, respectively). The specificity of the forward primer in combination with different reverse ones was tested with genomic DNA from a variety of bacterial strains. Most actinomycetes investigated could be separated by TGGE and DGGE, with both techniques giving similar results. Two strategies were employed to study natural microbial communities. First, we used the selective amplification of actinomycete sequences (E. coli positions 226 to 528) for direct analysis of the products in denaturing gradients. Second, a nested PCR providing actinomycete-specific fragments (E. coli positions 226 to 1401) was used which served as template for a PCR when conserved primers were used. The products (E. coli positions 968 to 1401) of this indirect approach were then separated by use of gradient gels. Both approaches allowed detection of actinomycete communities in soil. The second strategy allowed the estimation of the relative abundance of actinomycetes within the bacterial community. Mixtures of PCR-derived 16S rDNA fragments were used as model communities consisting of five actinomycetes and five other bacterial species. Actinomycete products were obtained over a 100-fold dilution range of the actinomycete DNA in the model community by specific PCR; detection of the diluted actinomycete DNA was not possible when conserved primers were used. The methods tested for detection were applied to monitor actinomycete community changes in potato rhizosphere and to investigate actinomycete diversity in different soils.     

Separation of fluorescence-labelled terminal restriction fragment DNA on a two-dimensional gel (T-RFs-2D) - an efficient approach for microbial consortium characterization

Fingerprinting techniques provide access to understanding the ecology of uncultured microbial consortia. However, the application of current techniques such as terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) has been hindered due to their limitations in characterizing complex microbial communities. This is due to that different populations possibly share the same terminal restriction fragments (T-RFs) and DNA fragments may co-migrate on DGGE gels. To overcome these limitations, a new approach was developed to separate terminal restriction fragments (T-RFs) of 16S rRNA genes on a two-dimensional gel (T-RFs-2D). T-RFs-2D involves restriction digestion of terminal fluorescence-labelled PCR amplified 16S rRNA gene products and their high-resolution separation via a two-dimensional (2D) gel electrophoresis based on the T-RF fragment size (1(st) D) and its sequence composition on the denaturing gradient gel (2(nd) D). The sequence information of interested T-RFs on 2D gels can be obtained through serial poly(A) tailing reaction, PCR amplification and subsequent DNA sequencing. By employing the T-RFs-2D method, bacteria with MspI digested T-RF size of 436 (±1) bp and 514 (±1) bp were identified to be a Lysobacter sp. and a Dehalococcoides sp. in a polychlorinated biphenyl (PCB) dechlorinating culture. With the high resolution of 2D separation, T-RFs-2D separated 63 DNA fragments in a complex river-sediment microbial community, while traditional DGGE detected only 41 DNA fragments in the same sample. In all, T-RFs-2D has its advantage in obtaining sequence information of interested T-RFs and also in characterization of complex microbial communities.     

Increase of inheritable polymorphisms of arbitrary primed PCR products on DGGE gels

Summary Single 10-mer primers of arbitrary sequence were used to amplify, by polymerase chain reaction (PCR), random genomic regions of two closely related hexaploid wheat cultivars. Polymorphic bands between the two genomes were visualized on both agarose and denaturing gradient gel electrophoresis (DGGE) gels. There was approximately a four-fold increase in inheritable polymorphisms when the PCR fragments were separated on DGGE gels.     

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。     

Recruitment of Antarctic marine eukaryotes onto artificial surfaces

Activities related to Antarctic research stations have caused significant local impacts on the marine environment, potentially affecting the recruitment of benthic invertebrates. Herein, we report the community structure of recruiting marine eukaryotes onto artificial substrata using molecular techniques. Slides were deployed at three sites adjacent to McMurdo Station, Scott Base, and Cape Armitage in McMurdo Sound. Denaturing gradient gel electrophoresis (DGGE) analysis revealed complex and diverse eukaryotic communities had established on artificial surfaces deployed at a range of site and depth regimes after 12 months. Analysis of similarity results detected significantly greater variability in community profiles among sites than within sites. The nonmetric multidimensional scaling plot constructed from DGGE banding patterns revealed different benthic communities had established at 12 and 18 m depths. Despite this, the variation in community composition was greater among sites than between depths, especially at Cape Armitage and Scott Base. Sequence analysis of excised DGGE bands revealed a predominance of arthropod and dinoflagellate sequences at Cape Armitage. In contrast, a wide diversity of phyla including cnidaria, bryozoa, protozoa, dinoflagellates, arthropods, platyhelminths, and annelids were present adjacent to the two research stations. The abundance of diatoms detected in Cape Armitage benthic assemblages exceeded the abundance of diatoms from McMurdo Station and Scott Base by almost two orders of magnitude. The discovery that distinct eukaryotic communities recruit at different sites and depths is probably due to complex interactions between multiple factors including water quality, larval supply, and light. The detection of sessile phyla on slides at each of the sites indicates that the pollution profiles present at each site is not an impediment to successful recruitment of these species.     

Denaturing gradient gel electrophoresis analyses of the vertical distribution and diversity of Vibrio spp. populations in the Cariaco Basin

The Cariaco system is the second largest permanently anoxic marine water body in the world. Its water column is characterized by a pronounced vertical layering of microbial communities. The goal of our study was to investigate the vertical distribution and diversity of Vibrio spp. present in the Cariaco Basin waters using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments. Representatives of the Vibrio genus were detected by nested and direct PCR in seawater at 10 depths. Sequence analyses of 55 DGGE bands revealed that only 11 different operational taxonomic units (OTU) are identified as Vibrio species. Between one and five OTUs were detected at each depth and the most common OTUs were OTU 1 and OTU 2, which phylogenetically clustered with Vibrio chagasii and Vibrio fortis, respectively. OTUs 3 and 4 were only found in the anoxic zone and were identified as Vibrio orientalis and Vibrio neptunius, respectively. Several Vibrio species detected are potentially pathogenic to human, prawns and corals such as Vibrio parahaemolyticus, Vibrio fischeri and Vibrio shilonii. In the Cariaco Basin, different Vibrio species were found to be specific to specific depths strata, suggesting that this genus is a natural component of the microbial communities in this marine redox environment.     

Polymerase chain reaction and denaturing gradient gel electrophoresis monitoring of fecal bifidobacterium populations in a prebiotic and probiotic feeding trial

A culture-independent approach based on genus-specific PCR and denaturing gradient gel electrophoresis (DGGE) was used to monitor qualitative changes in fecal bifidobacterial communities in a human feeding trial. DNA was extracted directly from feces and bifidobacterial 16S rDNA sequences were amplified using genus-specific PCR. The PCR fragments were subsequently separated in a sequence-specific manner by DGGE in order to obtain a profile of bifidobacterial fragments. The DGGE profiles revealed that in general, administration for two weeks of galactooligosaccharide and/or Bifidobacterium lactis Bb-12 (8 g and 3 x 10(10) cfu per day, respectively) did not affect the qualitative composition of the indigenous Bifidobacterium population, while B. lactis Bb-12 transiently colonised the gut.     

Impact of protozoan grazing on bacterial community structure in soil microcosms

The influence of grazing by a mixed assemblage of soil protozoa (seven flagellates and one amoeba) on bacterial community structure was studied in soil microcosms amended with a particulate resource (sterile wheat roots) or a soluble resource (a solution of various organic compounds). Sterilized soil was reinoculated with mixed soil bacteria (obtained by filtering and dilution) or with bacteria and protozoa. Denaturing gradient gel electrophoresis (DGGE) of PCR amplifications of 16S rRNA gene fragments, as well as community level physiological profiling (Biolog plates), suggested that the mixed protozoan community had significant effects on the bacterial community structure. Excising and sequencing of bands from the DGGE gels indicated that high-G+C gram-positive bacteria closely related to Arthrobacter spp. were favored by grazing, whereas the excised bands that decreased in intensity were related to gram-negative bacteria. The percentages of intensity found in bands related to high G+C gram positives increased from 4.5 and 12.6% in the ungrazed microcosms amended with roots and nutrient solution, respectively, to 19.3 and 32.9% in the grazed microcosms. Protozoa reduced the average bacterial cell size in microcosms amended with nutrient solution but not in the treatment amended with roots. Hence, size-selective feeding may explain some but not all of the changes in bacterial community structure. Five different protozoan isolates (Acanthamoeba sp., two species of Cercomonas, Thaumatomonas sp., and Spumella sp.) had different effects on the bacterial communities. This suggests that the composition of protozoan communities is important for the effect of protozoan grazing on bacterial communities.     

Development of a polymerase chain reaction-based denaturing gradient gel electrophoresis technique to study nematode species biodiversity using the 18s rDNA gene

A polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) technique was developed to evaluate nematode biodiversity. Amplified fragments (approximately 630 bp) of nematode 18S rDNA could be separated by DGGE at 60 °C for 5.5 h using a gradient of 30–55% denaturant. No DNA from the nematodes’ food source was observed on the gels. The sensitivity of the system was tested using DNA from six species of Steinernema. The system clearly separated PCR fragments from all species except S. tami and S. carpocapsae, which had similar melting behaviour despite being widely separated on the phylogenetic tree.     

Spatiotemporal variability in archaeal communities of tropical coastal waters

Spatiotemporal variations in the archaeal community structure from four locations along the central west coast of India were analyzed by denaturing gradient gel electrophoresis (DGGE) profiling and sequencing of prominent bands on the DGGE gels. A total of 36 water samples were collected during pre-monsoon, post-monsoon and monsoon seasons from three depths (surface, mid-depth , and close to bottom ～20 m). The community DNA extracts from these samples were subjected to DGGE analysis. The results of this study clearly indicate a significant difference in the community structure between sampling locations and seasons. Location-wise variation was more pronounced during pre-monsoon and post-monsoon seasons. During monsoon, however, the depth-wise variation was pronounced, suggesting monsoon influence on archaeal community structure. Sequencing of DGGE bands suggested the presence of marine group I and II archaea. Canonical correspondence analysis indicated that nitrate, nitrite and chlorophyll a were the significant explanatory variables responsible for > 60 % variation in archaeal community in these coastal waters.     

Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis Monitoring of Fecal Bifidobacterium Populations in a Prebiotic and Probiotic Feeding Trial

A culture-independent approach based on genus-specific PCR and denaturing gradient gel electrophoresis (DGGE) was used to monitor qualitative changes in fecal bifidobacterial communities in a human feeding trial. DNA was extracted directly from feces and bifidobacterial 16S rDNA sequences were amplified using genus-specific PCR. The PCR fragments were subsequently separated in a sequence-specific manner by DGGE in order to obtain a profile of bifidobacterial fragments. The DGGE profiles revealed that in general, administration for two weeks of galactooligosaccharide and/or Bifidobacterium lactis Bb-12 (8 g and 3 × 10¹⁰ cfu per day, respectively) did not affect the qualitative composition of the indigenous Bifidobacterium population, while B. lactis Bb-12 transiently colonised the gut.     

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.     

Seasonal variations in virus-host populations in Norwegian coastal waters: focusing on the cyanophage community infecting marine Synechococcus spp

Viruses are ubiquitous components of the marine ecosystem. In the current study we investigated seasonal variations in the viral community in Norwegian coastal waters by pulsed-field gel electrophoresis (PFGE). The results demonstrated that the viral community was diverse, displaying dynamic seasonal variation, and that viral populations of 29 different sizes in the range from 26 to 500 kb were present. Virus populations from 260 to 500 kb and dominating autotrophic pico- and nanoeukaryotes showed similar dynamic variations. Using flow cytometry and real-time PCR, we focused in particular on one host-virus system: Synechococcus spp. and cyanophages. The two groups covaried throughout the year and were found in the highest amounts in fall with concentrations of 7.3 x 10(4) Synechococcus cells ml(-1) and 7.2 x 10(3) cyanophage ml(-1). By using primers targeting the g20 gene in PCRs on DNA extracted from PFGE bands, we demonstrated that cyanophages were found in a genomic size range of 26 to 380 kb. The genetic richness of the cyanophage community, determined by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified g20 gene fragments, revealed seasonal shifts in the populations, with one community dominating in spring and summer and a different one dominating in fall. Phylogenetic analysis of the sequences originating from PFGE and DGGE bands grouped the sequences into three groups, all with homology to cyanomyoviruses present in cultures. Our results show that the cyanophage community in Norwegian coastal waters is dynamic and genetically diverse and has a surprisingly wide genomic size range.     

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.     

Molecular diversity analysis of rumen methanogenic Archaea from goat in eastern China by DGGE methods using different primer pairs

Aims:  To screen a pair of primers suitable for denaturing gradient gel electrophoretic (DGGE) analysis of ruminal methanogenic Archaea and to detect the archaeal communities in the rumen of goat.
Methods and Results:  Nine primer pairs for 16S rDNA of methanogenic Archaea , including six for directed polymerase chain reaction (PCR) and three for nested PCR were first evaluated by PCR amplification of the total DNA from rumen fluids and bacteria. The DGGE analysis of rumen fluids was then conducted with three primer sets (344fGC/915r, 1106fGC/1378r and 519f/915rGC) of the nine pairs tested. Good separation and quality of patterns were obtained in DGGE analysis with primer pairs 1106fGC/1378r and 519f/915rGC. A total of 40 DNA fragments were excised from the DGGE gels and their sequences were determined. All fragments belonged to methanogenic Archaea while primer pair 519f/915rGC had better amplification ranges than the other two primer pairs.
Conclusions:  The procedure of DGGE analysis with primer pair 519f/915rGC was more suitable for investigating methanogenic archaeal community in the rumen. The dominant methanogenic Archaea in the rumen of goat was Methanobrevibacter sp. and an unidentified methanogenic Archaea .
Significance and Impact of the Study:  One pair of primers suitable for DGGE analysis of ruminal methanogenic Archaea was obtained and the molecular diversity of ruminal methanogenic Archaea in goat was investigated by PCR-DGGE.     

Communities of nirS-type denitrifiers in the water column of the oxygen minimum zone in the eastern South Pacific

The major sites of water column denitrification in the ocean are oxygen minimum zones (OMZ), such as one in the eastern South Pacific (ESP). To understand the structure of denitrifying communities in the OMZ off Chile, denitrifier communities at two sites in the Chilean OMZ (Antofagasta and Iquique) and at different water depths were explored by terminal restriction fragment length polymorphism analysis and cloning of polymerase chain reaction (PCR)-amplified nirS genes. NirS is a functional marker gene for denitrification encoding cytochrome cd1-containing nitrite reductase, which catalyses the reduction of nitrite to nitric oxide, the key step in denitrification. Major differences were found between communities from the two geographic locations. Shifts in community structure occurred along a biogeochemical gradient at Antofagasta. Canonical correspondence analysis indicated that O2, NO3-, NO2- and depth were important environmental factors governing these communities along the biogeochemical gradient in the water column. Phylogenetic analysis grouped the majority of clones from the ESP in distinct clusters of genes from presumably novel and yet uncultivated denitrifers. These nirS clusters were distantly related to those found in the water column of the Arabian Sea but the phylogenetic distance was even higher compared with environmental sequences from marine sediments or any other habitat. This finding suggests similar environmental conditions trigger the development of denitrifiers with related nirS genotypes despite large geographic distances.     

Growth of phototrophic biofilms from limestone monuments under laboratory conditions

In the current study, five phototrophic biofilms from different Southern Europe limestone monuments were characterised by molecular techniques and cultivated under laboratory conditions. Phototrophic biofilms were collected from Orologio Tower in Martano (Italy), Santa Clara-a-Velha Monastery and Ajuda National Palace, both in Portugal, and Seville and Granada Cathedrals from Spain. The biofilms were grown under laboratory conditions and periodically sampled in order to monitor their evolution over a three-month period. Prokaryotic communities from natural samples and cultivated biofilms were monitored using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments in conjunction with clone sequencing and phylogenetic analysis. DNA-based molecular analysis of 16S rRNA gene fragments from the natural green biofilms revealed complex and different communities composition with respect to phototrophic microorganisms. The biofilms from Orologio Tower (Martano, Italy) and Santa Clara-a-Velha Monastery (Coimbra, Portugal) were dominated by the microalga Chlorella. The cyanobacterium Chroococcidiopsis was the dominating genus from Ajuda National Palace biofilm (Lisbon, Portugal). The biofilms from Seville and Granada Cathedrals (Spain) were both dominated by the cyanobacterium Pleurocapsa. The DGGE analysis of the cultivated biofilms showed that the communities developed differently in terms of species establishment and community composition during the three-month incubation period. The biofilm culture from Coimbra (Portugal) showed a remarkable stability of the microbial components of the natural community in laboratory conditions. With this work, a multiple-species community assemblage was obtained for further stone colonisation experiments.     

Seasonal Variations in Virus-Host Populations in Norwegian Coastal Waters: Focusing on the Cyanophage Community Infecting Marine Synechococcus spp.

Viruses are ubiquitous components of the marine ecosystem. In the current study we investigated seasonal variations in the viral community in Norwegian coastal waters by pulsed-field gel electrophoresis (PFGE). The results demonstrated that the viral community was diverse, displaying dynamic seasonal variation, and that viral populations of 29 different sizes in the range from 26 to 500 kb were present. Virus populations from 260 to 500 kb and dominating autotrophic pico- and nanoeukaryotes showed similar dynamic variations. Using flow cytometry and real-time PCR, we focused in particular on one host-virus system: Synechococcus spp. and cyanophages. The two groups covaried throughout the year and were found in the highest amounts in fall with concentrations of 7.3 × 10⁴ Synechococcus cells ml⁻¹ and 7.2 × 10³ cyanophage ml⁻¹. By using primers targeting the g20 gene in PCRs on DNA extracted from PFGE bands, we demonstrated that cyanophages were found in a genomic size range of 26 to 380 kb. The genetic richness of the cyanophage community, determined by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified g20 gene fragments, revealed seasonal shifts in the populations, with one community dominating in spring and summer and a different one dominating in fall. Phylogenetic analysis of the sequences originating from PFGE and DGGE bands grouped the sequences into three groups, all with homology to cyanomyoviruses present in cultures. Our results show that the cyanophage community in Norwegian coastal waters is dynamic and genetically diverse and has a surprisingly wide genomic size range.