Application of Denaturing Gradient Gel Electrophoresis (DGGE) To Study the Diversity of Marine Picoeukaryotic Assemblages and Comparison of DGGE with Other Molecular Techniques

We used denaturing gradient gel electrophoresis (DGGE) to study the diversity of picoeukaryotes in natural marine assemblages. Two eukaryote-specific primer sets targeting different regions of the 18S rRNA gene were tested. Both primer sets gave a single band when used with algal cultures and complex fingerprints when used with natural assemblages. The reproducibility of the fingerprints was estimated by quantifying the intensities of the same bands obtained in independent PCR and DGGE analyses, and the standard error of these estimates was less than 2% on average. DGGE fingerprints were then used to compare the picoeukaryotic diversity in samples obtained at different depths and on different dates from a station in the southwest Mediterranean Sea. Both primer sets revealed significant differences along the vertical profile, whereas temporal differences at the same depths were less marked. The phylogenetic composition of picoeukaryotes from one surface sample was investigated by excising and sequencing DGGE bands. The results were compared with an analysis of a clone library and a terminal restriction fragment length polymorphism fingerprint obtained from the same sample. The three PCR-based methods, performed with three different primer sets, revealed very similar assemblage compositions; the same main phylogenetic groups were present at similar relative levels. Thus, the prasinophyte group appeared to be the most abundant group in the surface Mediterranean samples as determined by our molecular analyses. DGGE bands corresponding to prasinophytes were always found in surface samples but were not present in deep samples. Other groups detected were prymnesiophytes, novel stramenopiles (distantly related to hyphochytrids or labyrinthulids), cryptophytes, dinophytes, and pelagophytes. In conclusion, the DGGE method described here provided a reasonably detailed view of marine picoeukaryotic assemblages and allowed tentative phylogenetic identification of the dominant members.     

Application of denaturing gradient gel electrophoresis (DGGE) to study the diversity of marine picoeukaryotic assemblages and comparison of DGGE with other molecular techniques.

We used denaturing gradient gel electrophoresis (DGGE) to study the diversity of picoeukaryotes in natural marine assemblages. Two eukaryote-specific primer sets targeting different regions of the 18S rRNA gene were tested. Both primer sets gave a single band when used with algal cultures and complex fingerprints when used with natural assemblages. The reproducibility of the fingerprints was estimated by quantifying the intensities of the same bands obtained in independent PCR and DGGE analyses, and the standard error of these estimates was less than 2% on average. DGGE fingerprints were then used to compare the picoeukaryotic diversity in samples obtained at different depths and on different dates from a station in the southwest Mediterranean Sea. Both primer sets revealed significant differences along the vertical profile, whereas temporal differences at the same depths were less marked. The phylogenetic composition of picoeukaryotes from one surface sample was investigated by excising and sequencing DGGE bands. The results were compared with an analysis of a clone library and a terminal restriction fragment length polymorphism fingerprint obtained from the same sample. The three PCR-based methods, performed with three different primer sets, revealed very similar assemblage compositions; the same main phylogenetic groups were present at similar relative levels. Thus, the prasinophyte group appeared to be the most abundant group in the surface Mediterranean samples as determined by our molecular analyses. DGGE bands corresponding to prasinophytes were always found in surface samples but were not present in deep samples. Other groups detected were prymnesiophytes, novel stramenopiles (distantly related to hyphochytrids or labyrinthulids), cryptophytes, dinophytes, and pelagophytes. In conclusion, the DGGE method described here provided a reasonably detailed view of marine picoeukaryotic assemblages and allowed tentative phylogenetic identification of the dominant members.     

Spatial differences in bacterioplankton composition along the Catalan coast (NW Mediterranean) assessed by molecular fingerprinting

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments was used to compare surface bacterioplankton assemblages along the Catalan coast (NW Mediterranean). Samples from three coastal stations were compared with samples taken inside the Barcelona harbour and open sea samples taken during a cruise. The bacterial assemblage of each sample showed a characteristic and reproducible DGGE fingerprint. Between 17 and 35 bands were detected in each sample, and about 40% of the bands accounted for more than 80% of the band intensity in each sample. The presence of bands as well as their relative intensity was used to compare bacterial assemblages. Clear differences between the harbour samples and the coastal samples were evident during all periods. Marked temporal changes in the bacterial assemblages were detectable for the coastal sites, suggesting seasonal succession of coastal bacterioplankton. During each season, two stations presented a very similar bacterial composition (Barcelona and Masnou) whereas bacterial assemblages in Blanes were slightly different. These differences were consistent with the different hydrography of the area. Diversity indices calculated from DGGE fingerprints were relatively similar for all samples analysed, even though harbour samples were expected to present lower diversity values.     

Response of bacterioplankton community structures to hydrological conditions and anthropogenic pollution in contrasting subtropical environments

Bacterioplankton community structures under contrasting subtropical marine environments (Hong Kong waters) were analyzed using 16S rRNA gene denaturing gradient gel electrophoresis (DGGE) and subsequent sequencing of predominant bands for samples collected bimonthly from 2004 to 2006 at five stations. Generally, bacterial abundance was significantly higher in the summer than in the winter. The general seasonal variations of the bacterial community structure, as indicated by cluster analysis of the DGGE pattern, were best correlated with temperature at most stations, except for the station close to a sewage discharge outfall, which was best explained by pollution-indicating parameters (e.g. biochemical oxygen demand). Anthropogenic pollutions appear to have affected the presence and the intensity of DGGE bands at the stations receiving discharge of primarily treated sewage. The relative abundance of major bacterial species, calculated by the relative intensity of DGGE bands after PCR amplification, also indicated the effects of hydrological or seasonal variations and sewage discharges. For the first time, a systematic molecular fingerprinting analysis of the bacterioplankton community composition was carried out along the environmental and pollution gradient in a subtropical marine environment, and it suggests that hydrological conditions and anthropogenic pollutions altered the total bacterial community as well as the dominant bacterial groups.     

Genetic Diversity of Plankton Community as Depicted by PCR-DGGE Fingerprinting and its Relation to Morphological Composition and Environmental Factors in Lake Donghu

To collect information about the genetic diversity of the plankton community and to study how plankton respond to environmental conditions, plankton samples were collected from five stations representing different trophic levels in a shallow, eutrophic lake (Lake Donghu), and investigated by PCR-DGGE fingerprinting. A total of 100 bands (61 of 16S rDNA bands and 39 of 18S rDNA bands) were detected. The DGGE bands unique to any single station accounted for 38% of the total bands, whereas common bands detected at all five stations accounted for only 11%. Using UPGMA clustering and MDS ordination of DGGE fingerprints, stations I and II were found to initially group together into one cluster, which was later joined by station V. Stations III and IV were isolated into two separate groups of one station each. Some differences in grouping relationships were found when analysis was completed on the basis of chemical characteristics and morphological composition, with zooplankton composition showing the greatest variability. However, the most similar stations (I and II) were always initially grouped into one cluster. Moreover, stations that exhibited the same or similar trophic level (stations III and IV), but different concentrations of heavy metals, were further differentiated by the DGGE method. Results of the present study indicated that PCR-DGGE fingerprinting was more sensitive than the traditional methods, as other studies suggested. Additionally, PCR-DGGE appears to be more appropriate for diversity characterization of the plankton community, as it is more canonical, systematic, and effective. Most importantly, fingerprinting results are more convenient for the comparative analyses between different studies. Therefore, the use of the described fingerprinting analysis may provide an operable and sensitive biomonitoring approach to identify critical, and potentially negative, stress within an aquatic ecosystem.     

RISA、DGGE和2D-PAGE技术对大豆根际土壤细菌多样性分析的比较

采用基于PCR扩增的核糖体间隔区分析(RISA)、变性梯度凝胶电泳(DGGE)和双向电泳(2D-PAGE)3种分子生态学技术对大豆根际土壤细菌多样性比对分析.结果表明:2D-PAGE技术得到的土壤细菌多样性(基因点)最丰富,其次为DGGE技术(基因片段),RISA技术(基因片段)最低.RISA技术得到的条带数最少,但结...     

Seasonality in bacterial diversity in north-west Mediterranean coastal waters: assessment through clone libraries, fingerprinting and FISH

We combined denaturing gradient gel electrophoresis (DGGE), catalysed reporter deposition-FISH (CARD-FISH) and clone libraries to investigate the seasonality of the bacterial assemblage composition in north-west Mediterranean coastal waters. DGGE analysis indicated that bacterial diversity changed gradually throughout the year, although with a clear distinction of the summer period. Alphaproteobacteria were the dominant group on an annual basis [29% of the DAPI (4',6-diamidino-2-phenylindole) counts by CARD-FISH, and 70% of the bacterial clones]. The SAR11 clade was most abundant during spring and summer (>20% of DAPI counts), while the Roseobacter clade was abundant primarily in winter and spring (up to 7% of DAPI counts). The phylum Bacteroidetes constituted the second most important group and was quantitatively uniform throughout the year (average 11% of the DAPI counts). Gammaproteobacteria showed a peak during summer (8% of DAPI counts), when most of them belonged to the NOR5 cluster. Clone libraries and CARD-FISH showed reasonable agreement in the quantitative proportions of Bacteroidetes and Gammaproteobacteria, but Alphaproteobacteria were overrepresented in clone libraries. Sequencing of the most predominant DGGE bands failed to detect the SAR11 group despite their high abundance. The combination of the three molecular approaches allowed a comprehensive assessment of seasonal changes in bacterial diversity.     

Phylogenetic composition of bacterioplankton assemblages from the Arctic Ocean

We analyzed the phylogenetic composition of bacterioplankton assemblages in 11 Arctic Ocean samples collected over three seasons (winter-spring 1995, summer 1996, and summer-fall 1997) by sequencing cloned fragments of 16S rRNA genes. The sequencing effort was directed by denaturing gradient gel electrophoresis (DGGE) screening of samples and the clone libraries. Sequences of 88 clones fell into seven major lineages of the domain Bacteria: alpha(36%)-, gamma(32%)-, delta(14%)-, and epsilon(1%)-Proteobacteria; Cytophaga-Flexibacter-Bacteroides spp. (9%); Verrucomicrobium spp. (6%); and green nonsulfur bacteria (2%). A total of 34% of the cloned sequences (excluding clones in the SAR11 and Roseobacter groups) had sequence similarities that were <94% compared to previously reported sequences, indicating the presence of novel sequences. DGGE fingerprints of the selected samples showed that most of the bands were common to all samples in all three seasons. However, additional bands representing sequences related to Cytophaga and Polaribacter species were found in samples collected during the summer and fall. Of the clones in a library generated from one sample collected in spring of 1995, 50% were the same and were most closely affiliated (99% similarity) with Alteromonas macleodii, while 50% of the clones in another sample were most closely affiliated (90 to 96% similarity) with Oceanospirillum sp. The majority of the cloned sequences were most closely related to uncultured, environmental sequences. Prominent among these were members of the SAR11 group. Differences between mixed-layer and halocline samples were apparent in DGGE fingerprints and clone libraries. Sequences related to alpha-Proteobacteria (dominated by SAR11) were abundant (52%) in samples from the mixed layer, while sequences related to gamma-proteobacteria were more abundant (44%) in halocline samples. Two bands corresponding to sequences related to SAR307 (common in deep water) and the high-G+C gram-positive bacteria were characteristic of the halocline samples.     

Microbial biogeography along an estuarine salinity gradient: combined influences of bacterial growth and residence time

Shifts in bacterioplankton community composition along the salinity gradient of the Parker River estuary and Plum Island Sound, in northeastern Massachusetts, were related to residence time and bacterial community doubling time in spring, summer, and fall seasons. Bacterial community composition was characterized with denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA. Average community doubling time was calculated from bacterial production ([(14)C]leucine incorporation) and bacterial abundance (direct counts). Freshwater and marine populations advected into the estuary represented a large fraction of the bacterioplankton community in all seasons. However, a unique estuarine community formed at intermediate salinities in summer and fall, when average doubling time was much shorter than water residence time, but not in spring, when doubling time was similar to residence time. Sequencing of DNA in DGGE bands demonstrated that most bands represented single phylotypes and that matching bands from different samples represented identical phylotypes. Most river and coastal ocean bacterioplankton were members of common freshwater and marine phylogenetic clusters within the phyla Proteobacteria, Bacteroidetes, and ACTINOBACTERIA: Estuarine bacterioplankton also belonged to these phyla but were related to clones and isolates from several different environments, including marine water columns, freshwater sediments, and soil.     

Characterization of Protistan Assemblages in the Ross Sea, Antarctica, by Denaturing Gradient Gel Electrophoresis

The diversity of protistan assemblages has traditionally been studied using microscopy and morphological characterization, but these methods are often inadequate for ecological studies of these communities because most small protists inherently lack adequate taxonomic characters to facilitate their identification at the species level and many protistan species also do not preserve well. We have therefore used a culture-independent approach (denaturing gradient gel electrophoresis [DGGE]) to obtain an assessment of the genetic composition and distribution of protists within different microhabitats (seawater, meltwater or slush on sea-ice floes, and ice) of the Ross Sea, Antarctica. Samples of the same type (e.g., water) shared more of the same bands than samples of different types (e.g., ice versus water), despite being collected from different sites. These findings imply that samples from the same environment have a similar protistan species composition and that the type of microenvironment significantly influences the protistan species composition of these Antarctic assemblages. It should be noted that a large number of bands among the samples within each microhabitat were distinct, indicating the potential presence of significant genetic diversity within each microenvironment. Sequence analysis of selected DGGE bands revealed sequences that represent diatoms, dinoflagellates, ciliates, flagellates, and several unidentified eukaryotes.     

Genetic Diversity of Eukaryotic Plankton Assemblages in Eastern Tibetan Lakes Differing by their Salinity and Altitude

Eukaryotic plankton assemblages in 11 high-mountain lakes located at altitudes of 2,817 to 5,134 m and over a total area of ca. one million square kilometers on the Eastern Tibet Plateau, spanning a salinity gradient from 0.2 (freshwater) to 187.1 g l⁻¹ (hypersaline), were investigated by cultivation independent methods. Two 18S rRNA gene-based fingerprint approaches, i.e., the terminal restriction fragment length polymorphism and denaturing gradient gel electrophoresis (DGGE) with subsequent band sequencing were applied. Samples of the same lake type (e.g., freshwater) generally shared more of the same bands or T-RFs than samples of different types (e.g., freshwater versus saline). However, a certain number of bands or T-RFs among the samples within each lake were distinct, indicating the potential presence of significant genetic diversity within each lake. PCA indicated that the most significant environmental gradient among the investigated lakes was salinity. The observed molecular profiles could be further explained (17–24%) by ion percentage of chloride, carbonate and bicarbonate, and sulfate, which were also covaried with change of altitude and latitude. Sequence analysis of selected major DGGE bands revealed many sequences (largely protist) that are not related to any known cultures but to uncultured eukaryotic picoplankton and unidentified eukaryotes. One fourth of the retrieved sequences showed ≤97% similarity to the closest sequences in the GenBank. Sequences related to well-known heterotrophic nanoflagellates were not retrieved from the DGGE gels. Several groups of eukaryotic plankton, which were found worldwide and detected in low land lakes, were also detected in habitats located above 4,400 m, suggesting a cosmopolitan distribution of these phylotypes. Collectively, our study suggests that there was a high beta-diversity of eukaryotic plankton assemblages in the investigated Tibetan lakes shaped by multiple geographic and environmental factors.     

凝胶条带内DNA回收测序评估DGGE可靠性

为了评估DGGE的可靠性,对DGGE条带中回收的DNA片段进行了测序比较分析,并引入了DGGE可靠性指数的概念评价其可靠性。结果显示同一条DGGE条带回收的DNA来自同一属的概率为64.7%,相同位置的DGGE条带可以被认为是同一OTU;不同的DGGE条带回收到类似的DNA序列（16S rDNA V3区差异小于4 bp）的概率为10.5%;DGGE可靠性指数为74.8%。以上结果表明尽管DGGE技术与理论预期存在一定的差距,但是DGGE技术基本能够反映微生物群落的多样性。       

Characterization of protistan assemblages in the Ross Sea, Antarctica, by denaturing gradient gel electrophoresis

The diversity of protistan assemblages has traditionally been studied using microscopy and morphological characterization, but these methods are often inadequate for ecological studies of these communities because most small protists inherently lack adequate taxonomic characters to facilitate their identification at the species level and many protistan species also do not preserve well. We have therefore used a culture-independent approach (denaturing gradient gel electrophoresis [DGGE]) to obtain an assessment of the genetic composition and distribution of protists within different microhabitats (seawater, meltwater or slush on sea-ice floes, and ice) of the Ross Sea, Antarctica. Samples of the same type (e.g., water) shared more of the same bands than samples of different types (e.g., ice versus water), despite being collected from different sites. These findings imply that samples from the same environment have a similar protistan species composition and that the type of microenvironment significantly influences the protistan species composition of these Antarctic assemblages. It should be noted that a large number of bands among the samples within each microhabitat were distinct, indicating the potential presence of significant genetic diversity within each microenvironment. Sequence analysis of selected DGGE bands revealed sequences that represent diatoms, dinoflagellates, ciliates, flagellates, and several unidentified eukaryotes.     

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.     

Diversity of an ectomycorrhizal fungal community studied by a root tip and total soil DNA approach

Molecular methods based on soil DNA extracts are increasingly being used to study the fungal diversity of ectomycorrhizal (EM) fungal communities in soil. Contrary to EM root tip identification, the use of molecular methods enables identification of extramatrical mycelia in soil. To compare fungal diversity as determined by root tip identification and mycelial identification, six soil samples were analysed. Root tips were extracted from the six samples and after amplification, the basidiomycete diversity on the root tips was analysed by denaturing gradient gel electrophoresis (DGGE). The soil from the six samples was sieved, total soil DNA was extracted and after amplification, the basidiomycete diversity in the soil fractions was analysed by DGGE. Fourteen different bands were excised from the DGGE gel and sequenced; fungal taxon names could be assigned to eight bands. Out of a total of 14 fungal taxa detected in soil, 11 fungal taxa were found on root tips, of which seven were EM fungal taxa. To examine whether the sieving treatment would affect EM species diversity, two different sieve mesh sizes were used and in addition, the organic soil fraction was analysed separately. DGGE analysis showed no differences in banding pattern for the different soil fractions. The organic fraction gave the highest DGGE band intensities. This work demonstrates that there is a high correspondence between basidiomycete diversity detected by molecular analysis of root tips and soil samples, irrespective of the soil fraction being analysed.     

Microbial Biogeography along an Estuarine Salinity Gradient: Combined Influences of Bacterial Growth and Residence Time

Shifts in bacterioplankton community composition along the salinity gradient of the Parker River estuary and Plum Island Sound, in northeastern Massachusetts, were related to residence time and bacterial community doubling time in spring, summer, and fall seasons. Bacterial community composition was characterized with denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA. Average community doubling time was calculated from bacterial production ([¹⁴C]leucine incorporation) and bacterial abundance (direct counts). Freshwater and marine populations advected into the estuary represented a large fraction of the bacterioplankton community in all seasons. However, a unique estuarine community formed at intermediate salinities in summer and fall, when average doubling time was much shorter than water residence time, but not in spring, when doubling time was similar to residence time. Sequencing of DNA in DGGE bands demonstrated that most bands represented single phylotypes and that matching bands from different samples represented identical phylotypes. Most river and coastal ocean bacterioplankton were members of common freshwater and marine phylogenetic clusters within the phyla Proteobacteria, Bacteroidetes, and Actinobacteria. Estuarine bacterioplankton also belonged to these phyla but were related to clones and isolates from several different environments, including marine water columns, freshwater sediments, and soil.     

Variation of Bacterial Community Diversity in Rhizosphere Soil of Sole-Cropped versus Intercropped Wheat Field after Harvest

As the major crops in north China, spring crops are usually planted from April through May every spring and harvested in fall. Wheat is also a very common crop traditionally planted in fall or spring and harvested in summer year by year. This continuous cropping system exhibited the disadvantages of reducing the fertility of soil through decreasing microbial diversity. Thus, management of microbial diversity in the rhizosphere plays a vital role in sustainable crop production. In this study, ten common spring crops in north China were chosen sole-cropped and four were chosen intercropped with peanut in wheat fields after harvest. Denaturing gradient gel electrophoresis (DGGE) and DNA sequencing of one 16S rDNA fragment were used to analyze the bacterial diversity and species identification. DGGE profiles showed the bacterial community diversity in rhizosphere soil samples varied among various crops under different cropping systems, more diverse under intercropping system than under sole-cropping. Some intercropping-specific bands in DGGE profiles suggested that several bacterial species were stimulated by intercropping systems specifically. Furthermore, the identification of these dominant and functional bacteria by DNA sequencing indicated that intercropping systems are more beneficial to improve soil fertility. Compared to intercropping systems, we also observed changes in microbial community of rhizosphere soil under sole-crops. The rhizosphere bacterial community structure in spring crops showed a strong crop species-specific pattern. More importantly, Empedobacter brevis, a typical plant pathogen, was only found in the carrot rhizosphere, suggesting carrot should be sown prudently. In conclusion, our study demonstrated that crop species and cropping systems had significant effects on bacterial community diversity in the rhizosphere soils. We strongly suggest sorghum, glutinous millet and buckwheat could be taken into account as intercropping crops with peanut; while hulled oat, mung bean or foxtail millet could be considered for sowing in wheat fields after harvest in North China.     

Polycyclic Aromatic Hydrocarbon-Induced Structural Shift of Bacterial Communities in Mangrove Sediment

Mangrove sediment is well known for its susceptibility to anthropogenic pollution, including polycyclic aromatic hydrocarbons (PAHs), but knowledge of the sediment microbial community structure with regards to exposure to PAHs is limited. The study aims to assess the effects of PAHs on the bacterial community of mangrove sediment using both 16s rDNA polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and traditional enrichment methods. Both the exposure time and the PAH concentration reduced the microbial diversity, as determined by the DGGE bands. Although PAHs could act as carbon sources for microorganisms, PAHs, at a concentration as low as 20 mg l⁻¹, posed a toxic effect to the microbial community. Sequencing of DGGE bands showed that marine bacteria from the genera of Vibrio, Roseobacter, and Ferrimonas were most abundant after PAH exposure, which suggests that both marine and terrestrial bacteria coexisted in the mangrove sediment, but that the marine microbes were more difficult to isolate using the traditional culture method. DGGE determination further demonstrated that the consistency among triplicates of the enriched consortia was significantly less than that of the sediment slurries. The present study reveals that the mangrove sediment microbial structure is susceptible to PAH contamination, and complex microbial community interactions occur in mangrove sediment.     

Pulsed-field gel electrophoresis analysis of virus assemblages present in a hypersaline environment.

A method for analyzing virus assemblages in aquatic environments was developed and used for studying the highest-salinity ponds (from 13.4 to 35% salinity) from a multi-pond solar saltern in Alicante, Spain. The protocol consisted of a series of concentration and purification steps including tangential flow filtration and ultracentrifugation, followed by the preparation of total viral nucleic acids that were subsequently separated by pulsed-field gel electrophoresis. For every sample analyzed, a characteristic DNA pattern was obtained, whose complexity was related to viral diversity. The comparison of our results with a similar analysis carried out with marine virus assemblages shows that, as expected, the viral diversity corresponding to the analyzed hypersaline environment is considerably lower than that of a marine environment.