PCR-DGGE Fingerprinting Research Articles

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.

Characterizing a stable methane-utilizing mixed culture used in the synthesis of a high-quality biopolymer in an open system

To characterize a methane-utilizing poly-beta-hydroxybutyrate (PHB)-producing microbial community. Three different approaches based on microbiology, analytical chemistry and molecular biology were used to determine the composition of the mixed culture. The dominant species, Methylocystis sp. GB25, represents more than 86% of the total biomass. Seven accompanying bacterial species are present in the mixed culture of which two are methylotrophic bacteria and five are utilizers of complex carbon sources. Both these groups were found to be present at the same ratio with respect to each other. Results of fatty acid analysis and PCR-DGGE fingerprints reflect the stability of the mixed-culture composition in the open system during multiple continuous growth and polymer formation processes throughout a period of 29 months. The consistently high quality of the accumulated polymer further corroborates this finding. The methane-utilizing mixed culture has the potential of self-regulation resulting in a stable composition even under non-aseptic conditions. Avoiding the necessity of sterile conditions, as demonstrated in this paper, is an important step towards the development of a viable large-scale process for the production of PHB using cheap substrates like methane from natural or renewable sources. This is the first report characterizing a bacterial mixed culture being used for the biotechnological production of a high-value product in an open system.

Soil fumigation and compost amendment alter soil microbial community composition but do not improve tree growth or yield in an apple replant site

Apple replant disease (ARD) is a disease complex that reduces survival, growth and yield of replanted trees, and is often encountered in establishing new orchards on old sites. Methyl bromide (MB) has been the fumigant used most widely to control ARD, but alternatives to MB and cultural methods of control are needed. In this experiment, we evaluated the response of soil microbial communities and tree growth and yield to three pre-plant soil treatments (compost amendment, soil treatment with a broad-spectrum fumigant, and untreated controls), and use of five clonal rootstock genotypes (M.7, M.26, CG.6210, G.30 and G.16), in an apple replant site in Ithaca, New York. Polymerase chain reaction (PCR)—denaturing gradient gel electrophoresis (DGGE) analysis was used to assess changes in the community composition of bacteria and fungi in the bulk soil 8, 10, 18 and 22 months after trees were replanted. PCR-DGGE was also used to compare the community composition of bacteria, fungi and pseudomonads in untreated rhizosphere soil of the five rootstock genotypes 31 months after planting. Tree caliper and extension growth were measured annually in November from 2002 to 2004. Apple yield data were recorded in 2004, the first fruiting year after planting. Trees on CG.6210 rootstocks had the most growth and highest yield, while trees on M.26 rootstocks had the least growth and lowest yield. Tree growth and yield were not affected by pre-plant soil treatment except for lateral extension growth, which was longer in trees growing in compost-treated soil in 2003 as compared to those in the fumigation treatment. Bulk soil bacterial PCR-DGGE fingerprints differed strongly among the different soil treatments 1 year after their application, with the fingerprints derived from each pre-plant soil treatment clustering separately in a hierarchical cluster analysis. However, the differences in bacterial communities between the soil treatments diminished during the second year after planting. Soil fungal communities converged more rapidly than bacterial communities, with no discernable pattern related to pre-plant soil treatments 10 months after replanting. Changes in bulk soil bacterial and fungal communities in response to soil treatments had no obvious correlation with tree performance. On the other hand, rootstock genotypes modified their rhizosphere environments which differed significantly in their bacterial, pseudomonad, fungal and oomycete communities. Cluster analysis of PCR-DGGE fingerprints of fungal and pseudomonad rhizosphere community DNA revealed two distinct clusters. For both analyses, soil sampled from the rhizosphere of the two higher yielding rootstock genotypes clustered together, while the lower yielding rootstock genotypes also clustered together. These results suggest that the fungal and pseudomonad communities that have developed in the rhizosphere of the different rootstock genotypes may be one factor influencing tree growth and yield at this apple replant site.

Denaturing gradient gel electrophoresis analysis of bacterial communities associated with failed endodontic treatment

A great deal of evidence indicates that persistent infections of the root canal of human teeth play an important role in the failure of the root canal treatment. The present study was undertaken to apply the PCR-DGGE fingerprinting approach to examine the structure of the bacterial population infecting previously treated root canals of humans associated with persistent periradicular lesions. Samples were taken from 14 filled root canals, DNA was extracted, and part of the 16S rDNA of all bacteria was amplified by PCR and separated by DGGE, generating banding patterns representative of the community structure. Species-specific PCR for the detection of Enterococcus faecalis was also performed. The mean number of bands detected in the 16S rDNA community profiles was about 6, ranging from 1 to 26 bands. Each sample showed a unique structure of the microbial community. The species-specific PCR assay revealed the presence of E. faecalis in 10 of 14 samples, but DGGE analysis revealed it was not the dominant species. Results revealed that the intraradicular bacterial community associated with failed endodontic treatment significantly varied in composition from teeth to teeth. Persistent intraradicular infections were present in all root-filled teeth.

PCR-DGGE fingerprints of microbial succession during a manufacture of traditional water buffalo mozzarella cheese.

To monitor the process and the starter effectiveness recording a series of fingerprints of the microbial diversity occurring at different steps of mozzarella cheese manufacture and to investigate the involvement of the natural starter to the achievement of the final product. Samples of raw milk, natural whey culture (NWC) used as starter, curd after ripening and final product were collected during a mozzarella cheese manufacture. Total microbial DNA was directly extracted from the dairy samples as well as bulk colonies collected from the plates of appropriate culture media generally used for viable counts of mesophilic and thermophilic lactic acid bacteria (LAB) and used in polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) experiments. The analysis of the DGGE profiles showed a strong influence of the microflora of the NWC on the whole process because after the starter addition, the profile of all the dairy samples was identical to the one shown by the NWC. Simple indexes were calculated for the DGGE profiles to have an objective estimation of biodiversity and of technological importance of specific groups of organisms. LAB grown on Man Rogosa Sharp (MRS) and Rogosa agar at 30 degrees C showed high viable counts and the highest diversity in species indicating their importance in the cheese making, which had not been considered so far. Moreover, the NWC profiles were shown to be the most similar to the curd profile suggesting to be effective in manufacture. The PCR-DGGE analysis showed that in premium quality manufacture the NWC used as starter had a strong influence on the microflora responsible for process development. The molecular approach appeared to be valid as a tool to control process development, starter effectiveness and product identity as well as to rank cheese quality.

Stability of the bacterial communities supported by a seven-stage biological process treating pharmaceutical wastewater as revealed by PCR-DGGE

The stabilities of the bacterial community structures supported by seven full-scale biological reactors treating pharmaceutical wastewater were investigated by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) amplified 16S rRNA gene fragments. Effluent quality from this treatment process was consistently high with respect to BOD 5 (<30 mg l −1), soluble COD (<500 mg l −1), and total ammonia (< 5 mg l −1 as N) concentrations. Long-term community structure stability was studied by comparing the similarity of PCR-DGGE fingerprints from samples collected 87 days apart between which the influent wastewater characteristics were relatively stable. The Dice index ( C s) of similarity was moderately high for the first four reactors ( C s=0.61–0.77) and very high for the last three reactors ( C s=0.89–0.91). Short-term community structure stability was studied by comparing PCR-DGGE fingerprints from samples collected 15 days apart between which the influent wastewater characteristics changed significantly, while the effluent quality remained consistently high. The bacterial community composition of each of the seven bioreactors showed a moderate community shift ( C s=0.70–0.76). Short-term variability in influent wastewater composition, therefore, affected a greater community shift than did long-term operation treating a wastewater of relatively consistent composition. These results indicate that functionally stable wastewater treatment bioreactors have stable microbial community structures under normal operating conditions but are able to adapt in response to perturbations to sustain high effluent quality.

Structural diversity of microbial communities in arable soils of a heavily industrialised area determined by PCR-DGGE fingerprinting and FAME profiling

Microbial community structure in soil sampled from sites contaminated with different levels of heavy metals was assessed by PCR-DGGE analysis of 16S rDNA fragments and MIDI-FAME profiling of total cell fatty acids. Total community DNA was extracted from these soils by three methods to compare their usefulness for generation of representative pools of bacterial community 16S rRNA genes. Crude DNA extracts were purified and then amplified using eubacterial primers. PCR products were analysed by DGGE to obtain bacterial community patterns. Culturable fractions of fast growing bacteria separated from soil colloids by blending and differential centrifugation were also analysed by profiling of cellular fatty acids. PCR-DGGE analysis showed significant differences in microbial community structure between the soils studied, which were related to the contamination levels. Polluted soils could be characterised by a community differing in ‘richness’ and structure of dominating bacterial populations from those of a pristine soil. The differences in the bacterial community structure were still visible after 10-fold dilution of the target DNA, indicating that even less dominant populations were affected by heavy metals. However, organic matter content, soil type and crop cultivation could also affect the bacterial populations that established in these soils. The direct methods for DNA extraction from soil generated information about the microbial community composition different from that of the indirect method. The latter method was less efficient than both direct methods with respect to the generation of representative pools of bacterial community 16S rRNA genes. The structure of the culturable bacterial community was not dependent on the concentrations of heavy metals in soil, as determined by MIDI-FAME profiling. It is possible that this fraction of soil bacteria was less diverse (dominated by gram-positive bacteria) than the total community analysed at the DNA level without prior cultivation.

Bacterial phyllosphere communities of Solanum tuberosum L. and T4-lysozyme-producing transgenic variants

Bacterial communities in the phyllosphere of genetically modified potato plants carrying a T4-lysozyme gene and of control plants without the gene were compared in the greenhouse and in the field. The polyphasic investigation included characterisation of isolates by fatty acid analysis, community-level catabolic profiling in BIOLOG GN microplates and genetic community fingerprinting by denaturing gradient gel electrophoresis of 16S rDNA fragments amplified by PCR from community DNA (PCR-DGGE). The diversity and frequency distribution of species determined by fatty acid analysis of 190 isolates from a greenhouse experiment and 488 isolates from a field sampling, as well as PCR-DGGE fingerprints of phyllosphere samples from the field, indicated highly similar communities of T4-lysozyme plants and controls. In addition, effects on community-level catabolic profiles were detected neither in the greenhouse nor in the field trial. Differences in the percentages of culturable species were detectable for the T4-lysozyme plants, namely a higher percentage of isolates classified as Erwinia sp. and lower abundances of Gram-positive isolates and of Agrobacterium, but the observed effects were minor relative to the natural variability observed in several field samplings. The observed effect on Agrobacterium populations in the field was confirmed by PCR-DGGE.

Bacterial phyllosphere communities of Solanum tuberosum L. and T4-lysozyme-producing transgenic variants

Bacterial communities in the phyllosphere of genetically modified potato plants carrying a T4-lysozyme gene and of control plants without the gene were compared in the greenhouse and in the field. The polyphasic investigation included characterisation of isolates by fatty acid analysis, community-level catabolic profiling in BIOLOG GN microplates and genetic community fingerprinting by denaturing gradient gel electrophoresis of 16S rDNA fragments amplified by PCR from community DNA (PCR-DGGE). The diversity and frequency distribution of species determined by fatty acid analysis of 190 isolates from a greenhouse experiment and 488 isolates from a field sampling, as well as PCR-DGGE fingerprints of phyllosphere samples from the field, indicated highly similar communities of T4-lysozyme plants and controls. In addition, effects on community-level catabolic profiles were detected neither in the greenhouse nor in the field trial. Differences in the percentages of culturable species were detectable for the T4-lysozyme plants, namely a higher percentage of isolates classified as Erwinia sp. and lower abundances of Gram-positive isolates and of Agrobacterium, but the observed effects were minor relative to the natural variability observed in several field samplings. The observed effect on Agrobacterium populations in the field was confirmed by PCR-DGGE.