Web-Based Phylogenetic Assignment Tool for Analysis of Terminal Restriction Fragment Length Polymorphism Profiles of Microbial Communities

Culture-independent DNA fingerprints are commonly used to assess the diversity of a microbial community. However, relating species composition to community profiles produced by community fingerprint methods is not straightforward. Terminal restriction fragment length polymorphism (T-RFLP) is a community fingerprint method in which phylogenetic assignments may be inferred from the terminal restriction fragment (T-RF) sizes through the use of web-based resources that predict T-RF sizes for known bacteria. The process quickly becomes computationally intensive due to the need to analyze profiles produced by multiple restriction digests and the complexity of profiles generated by natural microbial communities. A web-based tool is described here that rapidly generates phylogenetic assignments from submitted community T-RFLP profiles based on a database of fragments produced by known 16S rRNA gene sequences. Users have the option of submitting a customized database generated from unpublished sequences or from a gene other than the 16S rRNA gene. This phylogenetic assignment tool allows users to employ T-RFLP to simultaneously analyze microbial community diversity and species composition. An analysis of the variability of bacterial species composition throughout the water column in a humic lake was carried out to demonstrate the functionality of the phylogenetic assignment tool. This method was validated by comparing the results generated by this program with results from a 16S rRNA gene clone library.     

Web-based phylogenetic assignment tool for analysis of terminal restriction fragment length polymorphism profiles of microbial communities

Culture-independent DNA fingerprints are commonly used to assess the diversity of a microbial community. However, relating species composition to community profiles produced by community fingerprint methods is not straightforward. Terminal restriction fragment length polymorphism (T-RFLP) is a community fingerprint method in which phylogenetic assignments may be inferred from the terminal restriction fragment (T-RF) sizes through the use of web-based resources that predict T-RF sizes for known bacteria. The process quickly becomes computationally intensive due to the need to analyze profiles produced by multiple restriction digests and the complexity of profiles generated by natural microbial communities. A web-based tool is described here that rapidly generates phylogenetic assignments from submitted community T-RFLP profiles based on a database of fragments produced by known 16S rRNA gene sequences. Users have the option of submitting a customized database generated from unpublished sequences or from a gene other than the 16S rRNA gene. This phylogenetic assignment tool allows users to employ T-RFLP to simultaneously analyze microbial community diversity and species composition. An analysis of the variability of bacterial species composition throughout the water column in a humic lake was carried out to demonstrate the functionality of the phylogenetic assignment tool. This method was validated by comparing the results generated by this program with results from a 16S rRNA gene clone library.     

Analysis of the microbial community structure in a membrane bioreactor during initial stages of filtration

Membrane biofouling was investigated during the early stages of filtration in a laboratory-scale membrane bioreactor operated on molasses wastewater. The bacterial diversity and composition of the membrane biofilm and activated sludge were analyzed using terminal restriction fragment length polymorphism coupled with 16S rRNA clone library construction and sequencing. The amount of extracellular polymeric substances produced by bacteria was investigated using spectroscopic methods. The results reveal that the bacterial community of activated sludge differs significantly from that of the membrane biofilm, especially at the initial phase. Phylogenetic analysis based on 16S rRNA gene sequences identified 25 pioneer OTUs responsible for membrane surface colonization. Also, the relationship between the identified bacterial strains and the system specifications was explored.     

Nitrospira community composition in nitrifying reactors operated with two different dissolved oxygen levels

Nitrospira is a dominant member of nitrite-oxidizing bacteria (NOB) in nitrifying bioreactors as well as in natural habitats. In this study, Nitrospira NOB were investigated in the two nitrifying reactors operated with high and low dissolved oxygen (DO) concentrations for a period of 300 days. Phylogenetic and terminal restriction fragment length polymorphism analyses based on 16S rRNA gene sequences revealed that the Nitrospira community compositions of the two reactors during the early period related to group 1 and half of the Nitrospira community composition shifted to group 2 in the high-DO reactor after day 179, although there was no significant change in the low-DO reactor. These results suggested that DO was an important factor affecting Nitrospira community compositions in the nitrifying reactors.     

Long-term monitoring of the succession of a microbial community in activated sludge from a circulation flush toilet as a closed system

The microbial diversity and community succession of a circulation flush toilet were investigated by terminal restriction fragment length polymorphism and cloning analyses. Clonal libraries of 16S rRNA gene on day 3 and day 127 were constructed. On day 3, 102 clones were sequenced; Proteobacteria and Bacteroidetes accounted for 27% and 45%, respectively. On day 127, Proteobacteria had increased to 43% and Bacteroidetes had decreased to 26% of a total of 100 clones. Terminal restriction fragment length polymorphism peaks were identified by in silico analysis of clone libraries. The relative abundances of Nitrosomonas increased from 1% to 6% with commencement of nitrification and denitrification. Similarly, the relative abundance of terminal restriction fragments generated from Xanthomonas increased from 3% to 10%. Therefore, these bacteria could play a prominent role in this process. To reveal the relationship between stability of the microbial community and performance of the system, microbial community succession was visualized by multidimensional scaling analysis. The microbial community structure changed markedly, particularly during the start-up period of the system. The plots then became stable after the start of nitrification and denitrification. This result suggests that the succession of microbial community structure had a correlation with the performance of the system.     

Anaerobic microbial communities in Lake Pavin, a unique meromictic lake in France

The Bacteria and Archaea from the meromictic Lake Pavin were analyzed in samples collected along a vertical profile in the anoxic monimolimnion and were compared to those in samples from the oxic mixolimnion. Nine targeted 16S rRNA oligonucleotide probes were used to assess the distribution of Bacteria and Archaea and to investigate the in situ occurrence of sulfate-reducing bacteria and methane-producing Archaea involved in the terminal steps of the anaerobic degradation of organic material. The diversity of the complex microbial communities was assessed from the 16S rRNA polymorphisms present in terminal restriction fragment (TRF) depth patterns. The densities of the microbial community increased in the anoxic layer, and Archaea detected with probe ARCH915 represented the largest microbial group in the water column, with a mean Archaea/Eubacteria ratio of 1.5. Terminal restriction fragment length polymorphism (TRFLP) analysis revealed an elevated archaeal and bacterial phylotype richness in anoxic bottom-water samples. The structure of the Archaea community remained rather homogeneous, while TRFLP patterns for the eubacterial community revealed a heterogeneous distribution of eubacterial TRFs.     

Microbial Community Assembly and Succession on Lake Sturgeon Egg Surfaces as a Function of Simulated Spawning Stream Flow Rate

We investigated microbial succession on lake sturgeon (Acipenser fulvescens) egg surfaces over the course of their incubation period as a function of simulated stream flow rate. The primary objective was to characterize the microbial community assembly during succession and to examine how simulated stream flow rate affect the successional process. Sturgeon eggs were reared under three flow regimes; high (0.55 m/s), low (0.18 m/s), and variable (0.35 and 0.11 m/s alternating 12 h intervals). Eggs were collected from each flow regime at different egg developmental stages. Microbial community DNA was extracted from egg surface and the communities were examined using 16S rRNA gene-based terminal restriction fragment length polymorphism and 454 pyrosequencing. Analysis of these datasets using principal component analysis revealed that microbial communities were clustered by egg developmental stages (early, middle, and late) regardless of flow regimes. 454 pyrosequencing data suggested that 90–98 % of the microbial communities were composed of the phyla Proteobacteria and Bacteroidetes throughout succession. β-Protebacteria was more dominant in the early stage, Bacteroidetes became more dominant in the middle stage, and α-Proteobacteria became dominant in the late stage. A total of 360 genera and 5,826 OTUs at 97 % similarity cutoff were associated with the eggs. Midway through egg development, the egg-associated communities of the low flow regime had a higher diversity than those communities developed under high or variable flow regimes. Results show that microbial community turnover occurred during embryogenesis, and stream flow rate influenced the microbial succession processes on the sturgeon egg surfaces.     

Seasonal Fluctuations of Bacterial Community Diversity in Agricultural Soil and Experimental Validation by Laboratory Disturbance Experiments

Natural fluctuations in soil microbial communities are poorly documented because of the inherent difficulty to perform a simultaneous analysis of the relative abundances of multiple populations over a long time period. Yet, it is important to understand the magnitudes of community composition variability as a function of natural influences (e.g., temperature, plant growth, or rainfall) because this forms the reference or baseline against which external disturbances (e.g., anthropogenic emissions) can be judged. Second, definition of baseline fluctuations in complex microbial communities may help to understand at which point the systems become unbalanced and cannot return to their original composition. In this paper, we examined the seasonal fluctuations in the bacterial community of an agricultural soil used for regular plant crop production by using terminal restriction fragment length polymorphism profiling (T-RFLP) of the amplified 16S ribosomal ribonucleic acid (rRNA) gene diversity. Cluster and statistical analysis of T-RFLP data showed that soil bacterial communities fluctuated very little during the seasons (similarity indices between 0.835 and 0.997) with insignificant variations in 16S rRNA gene richness and diversity indices. Despite overall insignificant fluctuations, between 8 and 30% of all terminal restriction fragments changed their relative intensity in a significant manner among consecutive time samples. To determine the magnitude of community variations induced by external factors, soil samples were subjected to either inoculation with a pure bacterial culture, addition of the herbicide mecoprop, or addition of nutrients. All treatments resulted in statistically measurable changes of T-RFLP profiles of the communities. Addition of nutrients or bacteria plus mecoprop resulted in bacteria composition, which did not return to the original profile within 14 days. We propose that at less than 70% similarity in T-RFLP, the bacterial communities risk to drift apart to inherently different states.     

Post-amplification Klenow fragment treatment alleviates PCR bias caused by partially single-stranded amplicons

Partially single-stranded amplicons, formed during PCR amplification of single and mixed templates, are a potential source of bias in genetic diversity studies. The analysis of 16S rRNA gene diversity in mixed template samples by the fingerprinting technique terminal restriction fragment length polymorphism (T-RFLP) analysis can be biased by the occurrence of pseudo-T-RFs, i.e., restriction fragments occurring in addition to the expected terminal restriction fragments of single amplicons. This bias originates from PCR products, which are single-stranded at their terminal restriction site. Here we show that treatment of PCR amplicons with Klenow fragment prior to restriction digest and T-RFLP analysis minimized effectively the occurrence of pseudo-T-RFs. Klenow fragment activity filled in bases into the partially single-stranded amplicons and thereby restored the affected amplicons to complete double strands. Our method allowed to improve the assessment of genetic diversity and gene ratios from T-RFLP analysis of an original environmental sample. Since partially single-stranded amplicons might influence many PCR-based techniques, post-amplification treatment with Klenow fragment may be useful for a wide range of applications, which assess the composition of amplicon pools, e.g., the analysis of marker gene diversity in mixed template samples by fingerprinting techniques or the analysis of sequence diversity by cloning.     

Analysis of the endophytic actinobacterial population in the roots of wheat (Triticum aestivum L.) by terminal restriction fragment length polymorphism and sequencing of 16S rRNA clones

The endophytic actinobacterial population in the roots of wheat grown in three different soils obtained from the southeast part of South Australia was investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis of the amplified 16S rRNA genes. A new, validated approach was applied to the T-RFLP analysis in order to estimate, to the genus level, the actinobacterial population that was identified. Actinobacterium-biased primers were used together with three restriction enzymes to obtain terminal restriction fragments (TRFs). The TRFs were matched to bacterial genera by the T-RFLP Analysis Program, and the data were analyzed to validate and semiquantify the genera present within the plant roots. The highest diversity and level of endophytic colonization were found in the roots of wheat grown in a dark loam from Swedes Flat, and the lowest were found in water-repellent sand from Western Flat. This molecular approach detected a greater diversity of actinobacteria than did previous culture-dependent methods, with the predominant genera being Mycobacterium (21.02%) in Swedes Flat, Streptomyces (14.35%) in Red Loam, and Kitasatospora (15.02%) in Western Flat. This study indicates that the soil that supported a higher number of indigenous organisms resulted in wheat roots with higher actinobacterial diversity and levels of colonization within the plant tissue. Sequencing of 16S rRNA clones, obtained using the same actinobacterium-biased PCR primers that were used in the T-RFLP analysis, confirmed the presence of the actinobacterial diversity and identified a number of Mycobacterium and Streptomyces species.     

Response of Estuarine Biofilm Microbial Community Development to Changes in Dissolved Oxygen and Nutrient Concentrations

The information content and responsiveness of microbial biofilm community structure, as an integrative indicator of water quality, was assessed against short-term changes in oxygen and nutrient loading in an open-water estuarine setting. Biofilms were grown for 7-day periods on artificial substrates in the Pensacola Bay estuary, Florida, in the vicinity of a wastewater treatment plant (WWTP) outfall and a nearby reference site. Substrates were deployed floating at the surface and near the benthos in 5.4 m of water. Three sampling events covered a 1-month period coincident with declining seasonal WWTP flow and increasing dissolved oxygen (DO) levels in the bottom waters. Biomass accumulation in benthic biofilms appeared to be controlled by oxygen rather than nutrients. The overriding effect of DO was also seen in DNA fingerprints of community structure by terminal restriction fragment length polymorphism (T-RFLP) of amplified 16S rRNA genes. Ribotype diversity in benthic biofilms at both sites dramatically increased during the transition from hypoxic to normoxic. Terminal restriction fragment length polymorphism patterns showed pronounced differences between benthic and surface biofilm communities from the same site in terms of signal type, strength, and diversity, but minor differences between sites. Sequencing of 16S rRNA gene clone libraries from benthic biofilms at the WWTP site suggested that low DO levels favored sulfate-reducing prokaryotes (SRP), which decreased with rising oxygen levels and increasing overall diversity. A 91-bp ribotype in the CfoI-restricted 16S rRNA gene T-RFLP profiles, indicative of SRP, tracked the decrease in relative SRP abundance over time.     

Soil parent material is a key determinant of the bacterial community structure in arable soils

The bacterial community composition in soil and rhizosphere taken from arable field sites, differing in soil parent material and soil texture, was analyzed using terminal restriction fragment length polymorphism (T-RFLP) of 16S rRNA genes. Nine sandy to silty soils from North-East Germany could clearly be distinguished from each other, with a relatively low heterogeneity in the community structure within the field replicates. There was a relationship between the soil parent material, i.e. different glacial and aeolian sediments, and the clustering of the profiles from different sites. A site-specific grouping of T-RFLP profiles was also found for the rhizosphere samples of the same field sites that were planted with potatoes. The branching of the rhizosphere profiles corresponded partly with the soil parent material, whereas the effect of the plant genotype was negligible. Selected terminal restriction fragments differing in their relative abundance within the nine soils were analyzed based on the cloning of the 16S rRNA genes of one soil sample. A high phylogenetic diversity observed to include Acidobacteria, Betaproteobacteria, Bacteroidetes, Verrucomicrobia, and Gemmatimonadetes. The assignment of three out of the seven selected terminal restriction fragments to members of Acidobacteria suggested that this group seems to participate frequently in the shifting of community structures that result from soil property changes.     

Bacterial community dynamics during the ensilage of wilted grass

Aims:  Grass silage is the product formed by a natural lactic acid bacterial fermentation when grass is stored under anaerobic conditions, and represents an important ruminant feedstuff on farms during winter. Of the two commonly employed methods of ensiling forage, baled silage composition frequently differs from that of comparable precision-chop silage reflecting a different ensiling environment. The aim of this study was to investigate the dynamics of the silage fermentation in wilted grass and between ensiling systems.
Methods and Results:  Fermentation dynamics were examined using traditional methods of silage analyses, including microbial enumeration and analysis of fermentation products, and culture-independent terminal restriction fragment length polymorphism (T-RFLP). A successful fermentation was achieved in both systems, with the fermentation (increase in lactic acid bacteria and lactic acid concentration, decrease in pH) proceeding rapidly once the herbage was ensiled.
Conclusions:  Under controlled conditions, little difference in silage quality and microbial composition were observed between ensiling systems and this was further reflected in the T-RFLP community analysis.
Significance and Impact of the Study:  T-RFLP proved a potentially useful tool to study the ensilage process and could provide valid support to traditional methods, or a viable alternative to these methods, for investigating the dynamics of the bacterial community over the course of the fermentation.     

Assessment of microbial diversity in four southwestern United States soils by 16S rRNA gene terminal restriction fragment analysis

The ability of terminal restriction fragment (T-RFLP or TRF) profiles of 16S rRNA genes to provide useful information about the relative diversity of complex microbial communities was investigated by comparison with other methods. Four soil communities representing two pinyon rhizosphere and two between-tree (interspace) soil environments were compared by analysis of 16S rRNA gene clone libraries and culture collections (Dunbar et al., Appl. Environ. Microbiol. 65:1662-1669, 1998) and by analysis of 16S rDNA TRF profiles of community DNA. The TRF method was able to differentiate the four communities in a manner consistent with previous comparisons of the communities by analysis of 16S rDNA clone libraries. TRF profiles were not useful for calculating and comparing traditional community richness or evenness values among the four soil environments. Statistics calculated from RsaI, HhaI, HaeIII, and MspI profiles of each community were inconsistent, and the combined data were not significantly different between samples. The detection sensitivity of the method was tested. In standard PCRs, a seeded population comprising 0.1 to 1% of the total community could be detected. The combined results demonstrate that TRF analysis is an excellent method for rapidly comparing the relationships between bacterial communities in environmental samples. However, for highly complex communities, the method appears unable to provide classical measures of relative community diversity.     

Analysis of the Endophytic Actinobacterial Population in the Roots of Wheat (Triticum aestivum L.) by Terminal Restriction Fragment Length Polymorphism and Sequencing of 16S rRNA Clones

The endophytic actinobacterial population in the roots of wheat grown in three different soils obtained from the southeast part of South Australia was investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis of the amplified 16S rRNA genes. A new, validated approach was applied to the T-RFLP analysis in order to estimate, to the genus level, the actinobacterial population that was identified. Actinobacterium-biased primers were used together with three restriction enzymes to obtain terminal restriction fragments (TRFs). The TRFs were matched to bacterial genera by the T-RFLP Analysis Program, and the data were analyzed to validate and semiquantify the genera present within the plant roots. The highest diversity and level of endophytic colonization were found in the roots of wheat grown in a dark loam from Swedes Flat, and the lowest were found in water-repellent sand from Western Flat. This molecular approach detected a greater diversity of actinobacteria than did previous culture-dependent methods, with the predominant genera being Mycobacterium (21.02%) in Swedes Flat, Streptomyces (14.35%) in Red Loam, and Kitasatospora (15.02%) in Western Flat. This study indicates that the soil that supported a higher number of indigenous organisms resulted in wheat roots with higher actinobacterial diversity and levels of colonization within the plant tissue. Sequencing of 16S rRNA clones, obtained using the same actinobacterium-biased PCR primers that were used in the T-RFLP analysis, confirmed the presence of the actinobacterial diversity and identified a number of Mycobacterium and Streptomyces species.     

Diversity and persistence of arbuscular mycorrhizas in a low-Arctic meadow habitat

Little is known about the ecology and diversity of arbuscular mycorrhizal (AM) fungi in Arctic ecosystems. Here, the diversity and composition of the AM fungal community and its response to host plant community composition were studied in a low-Arctic meadow habitat. The natural vegetation in two low-Arctic meadow sites was manipulated. Plots with natural vegetation, monoculture and no vegetation were established. Seeds of Solidago virgaurea were sown into the plots and the AM fungal community in the seedling roots was analysed using the terminal restriction fragment length polymorphism (T-RFLP) method. The vegetation manipulation treatments affected the community composition but not the diversity of AM fungi found in S. virgaurea roots. The diversity of AM fungi was higher in S. virgaurea roots in the site with naturally higher plant species diversity. These results show that AM fungi in low-Arctic meadows are able to survive for a period of 2 yr without a host plant. This ability buffers the AM fungal community against short-term changes in host plant community composition and diversity.     

Chilling and cultivar type affect the diversity of bacterial endophytes colonizing sweet pepper (Capsicum anuum L.)

A climate chamber experiment was conducted to assay the effect of low temperatures (chilling) on the diversity of bacteria colonizing the endospheres of two thermophilic sweet pepper (Capsicum anuum L.) cultivars, Milder Spiral and Ziegenhorn Bello. Structural diversity was analyzed by 16S rRNA-based terminal restriction fragment length polymorphism (T-RFLP) analysis and by the generation of 16S rRNA gene libraries to determine dominant community members in T-RFLP profiles. Cultivable community members colonizing lines Milder Spiral and Ziegenhorn Bello were identified by 16S rRNA gene analysis. T-RFLP profiles and 16S rRNA gene libraries revealed a high heterogeneity of community composition due to chilling and suggested further the existence of cultivar-specific communities. The majority of isolates obtained from the cultivar Milder Spiral were assigned as high-G+C Gram-positive bacteria (Microbacterium sp., Micrococcus sp., Rhodococcus sp.) and Firmicutes (Staphylococcus sp.). Of the isolated endophytes obtained from cultivar Zeigenhorn Bello, 93% were affiliated with Staphylococcus aureus and Bacillus sp. (Firmicutes). The experimental set-up was suited to demonstrate that chilling and cultivar type can influence the diversity of bacterial endophytes colonizing sweet pepper. We propose additional chilling experiments to investigate the effect of chilling on functional, plant-beneficial abilities of bacterial endophytes associated with low-temperature-sensitive crops, such as sweet pepper.     

Bacterial community composition of a wastewater treatment system reliant on N2 fixation

The temporal stability and change of the dominant phylogenetic groups of the domain bacteria were studied in a model plant-based industrial wastewater treatment system showing high levels of organic carbon removal supported by high levels of N₂ fixation. Community profiles were obtained through terminal restriction fragment length polymorphism analysis and cloning of 16S rRNA amplicons followed by sequencing. Bacterial community profiles showed that ten common terminal restriction fragments made up approximately 50% of the measured bacterial community. As much as 42% of the measured bacterial community could be monitored by using quantitative PCR and primers that targeted three dominant operational taxonomic units. Despite changes in wastewater composition and dissolved oxygen levels, the bacterial community composition appeared stable and was dominated by α-Proteobacteria and β-Proteobacteria, with a lesser amount of the highly diverse bacterial phylum Bacteroidetes. A short period of considerable change in the bacterial community composition did not appear to affect treatment performance indicating functional redundancy in this treatment system.     

Genetic characterization of microbial communities living at the surface of building stones

Aims:  The aim of the present study was to reveal the microbial genetic diversity of epilithic biofilms using a DNA-based procedure.
Methods and Results:  A DNA extraction protocol was first selected to obtain PCR-amplifiable metagenomic DNA from a limestone biofilm. Extracted DNA was used to amplify either 16S rRNA genes or ITS regions from prokaryotic and eukaryotic genomes, respectively. Amplified DNAs were subsequently cloned, amplified by colony PCR and screened by restriction analysis [restriction analyses of amplified ribosomal DNA (ARDRA)] for DNA sequencing. Phylogenetic analysis using 16S rDNA sequences showed that predominating bacteria were Alphaproteobacteria belonging to the genera Sphingomonas , Erythrobacter , Porphyrobacter , Rhodopila and Jannashia ; Cyanobacteria and Actinobacteria were also identified. Analysis of ITS rDNA sequences revealed the presence of algae of the Chlorophyceae family and fungi related either to Rhinocladiella or to a melanized ascomycete. Statistical analysis showed that the specific richness evidenced was representative of the original sampled biofilm.
Conclusions:  The molecular methodology developed here constitutes a valuable tool to investigate the genetic diversity of microbial biofilms from building stone.
Significance and Impact of the Study:  The easy-to-run molecular method described here has practical importance to establish microbiological diagnosis and to define strategies for protection and restoration of stone surfaces.     

Terminal restriction fragment length polymorphism analysis program, a web-based research tool for microbial community analysis

Rapid analysis of microbial communities has proven to be a difficult task. This is due, in part, to both the tremendous diversity of the microbial world and the high complexity of many microbial communities. Several techniques for community analysis have emerged over the past decade, and most take advantage of the molecular phylogeny derived from 16S rRNA comparative sequence analysis. We describe a web-based research tool located at the Ribosomal Database Project web site (http://www.cme.msu.edu/RDP/html/analyses. html) that facilitates microbial community analysis using terminal restriction fragment length polymorphism of 16S ribosomal DNA. The analysis function (designated TAP T-RFLP) permits the user to perform in silico restriction digestions of the entire 16S sequence database and derive terminal restriction fragment sizes, measured in base pairs, from the 5' terminus of the user-specified primer to the 3' terminus of the restriction endonuclease target site. The output can be sorted and viewed either phylogenetically or by size. It is anticipated that the site will guide experimental design as well as provide insight into interpreting results of community analysis with terminal restriction fragment length polymorphisms.