Horizontal heterogeneity of denitrifying bacterial communities in marine sediments by terminal restriction fragment length polymorphism analysis

Although it is widely believed that horizontal patchiness exists in microbial sediment communities, determining the extent of variability or the particular members of the bacterial community which account for the observed differences among sites at various scales has not been routinely demonstrated. In this study, horizontal heterogeneity was examined in time and space for denitrifying bacteria in continental shelf sediments off Tuckerton, N.J., at the Rutgers University Long-Term Ecosystem Observatory (LEO-15). Characterization of the denitrifying community was done using PCR amplification of the nitrous oxide reductase (nosZ) gene combined with terminal restriction fragment length polymorphism analysis. Spatial scales from centimeters to kilometers were examined, while temporal variation was assayed over the course of 1995 to 1996. Sorenson's indices (pairwise similarity values) were calculated to permit comparison between samples. The similarities of benthic denitrifiers ranged from 0.80 to 0.85 for centimeter scale comparisons, from 0.52 to 0.79 for meter level comparisons, and from 0.23 to 0.53 for kilometer scale comparisons. Sorenson's indices for temporal comparisons varied from 0.12 to 0.74. A cluster analysis of the similarity values indicated that the composition of the denitrifier assemblages varied most significantly at the kilometer scale and between seasons at individual stations. Specific nosZ genes were identified which varied at centimeter, meter, or kilometer scales and may be associated with variability in meio- or macrofaunal abundance (centimeter scale), bottom topography (meter scale), or sediment characteristics (kilometer scale).     

Monitoring of phytoplankton community structure using terminal restriction fragment length polymorphism (T-RFLP)

To establish molecular monitoring for the phytoplankton community in aquatic ecosystems, we analysed the terminal restriction fragment length polymorphism (T-RFLP) of small subunit ribosomal RNA gene (18S rDNA) sequences of nuclear genomes from the algal strains of culture collections and environmental samples of two freshwater reservoirs (Sangcheon reservoir and Seoho reservoir, Korea). Terminal restriction fragment (T-RF) length database was also constructed from twelve strains of algal culture collections to annotate and identify the phytoplankton species from T-RFLP profiles. Algal species in reservoirs were identified and monitored through the colony sequencing and T-RF length patterns of 18S rRNA. In this study, 41 unique clones were identified from two reservoirs including Chlorophyta, Cryptophyta, and Alveolata. In the case of Cryptomonas sp., we found significant linear relationships between T-RF peak areas and biovolumes by cell counting. Our results suggest that T-RFLP analysis can be a fast and quantitative monitoring tool for species changes in phytoplankton communities.     

Effect of hydrogen on soil bacterial community structure in two soils as determined by terminal restriction fragment length polymorphism

The metabolism of hydrogen evolved from HUP^? legume nodules can alter bacterial community structures in the rhizosphere. Our earlier experiments demonstrated increased hydrogen uptake and appearance of white spots within bacterial colonies in H₂-treated soil. We were also able to isolate hydrogen-oxidizing bacteria from soil samples exposed to hydrogen, but not from samples exposed to air. To further understand the effect of hydrogen metabolism on soil microbial communities, in this study 16S rRNA terminal restriction fragment (TRF) profiles of different soil samples exposed to hydrogen gas under laboratory, greenhouse, and field conditions were analyzed. Relationships between soil bacterial community structures from hydrogen-treated soil samples and controls, illustrated by UPGMA (unpaired group mathematical averages) dendrograms, indicated a significant contribution of hydrogen metabolism to the variation in bacterial community. The intensity variation of TRF peaks includes both hydrogen-utilizing bacteria, whose growth were stimulated by hydrogen exposure, and other bacterial species whose growth was inhibited. Comparison of TRF profiles between laboratory and greenhouse samples showed that T-RFLP is a useful technique in the detection of root-related effects on soil bacterial community structure.     

Formation of pseudo-terminal restriction fragments,a PCR-related bias affecting terminal restriction fragment length polymorphism analysis of microbial community structure

Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology. In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency. A total of 50% of 109 bacterial and 78% of 68 archaeal clones from the guts of cetoniid beetle larvae, using MspI and AluI as restriction enzymes, respectively, were affected by the presence of these additional T-RFs. These peaks were called "pseudo-T-RFs" since they can be detected as terminal fluorescently labeled fragments in T-RFLP analysis but do not represent the primary terminal restriction site as indicated by sequence data analysis. Pseudo-T-RFs were also identified in T-RFLP profiles of pure culture and environmental DNA extracts. Digestion of amplicons with the single-strand-specific mung bean nuclease prior to T-RFLP analysis completely eliminated pseudo-T-RFs. This clearly indicates that single-stranded amplicons are the reason for the formation of pseudo-T-RFs, most probably because single-stranded restriction sites cannot be cleaved by restriction enzymes. The strong dependence of pseudo-T-RF formation on the number of cycles used in PCR indicates that (partly) single-stranded amplicons can be formed during amplification of 16S rRNA genes. In a model, we explain how transiently formed secondary structures of single-stranded amplicons may render single-stranded amplicons accessible to restriction enzymes. The occurrence of pseudo-T-RFs has consequences for the interpretation of T-RFLP profiles from environmental samples, since pseudo-T-RFs may lead to an overestimation of microbial diversity. Therefore, it is advisable to establish 16S rRNA gene sequence clone libraries in parallel with T-RFLP analysis from the same sample and to check clones for their in vitro digestion T-RF pattern to facilitate the detection of pseudo-T-RFs.     

New threshold and confidence estimates for terminal restriction fragment length polymorphism analysis of complex bacterial communities

Terminal restriction fragment length polymorphism (T-RFLP) analysis has the potential to be useful for comparisons of complex bacterial communities, especially to detect changes in community structure in response to different variables. To do this successfully, systematic variations have to be detected above method-associated noise, by standardizing data sets and assigning confidence estimates to relationships detected. We investigated the use of different standardizing methods in T-RFLP analysis of PCR-amplified 16S rRNA genes to elucidate the similarities between the bacterial communities in 17 soil and sediment samples. We developed a robust method for standardizing data sets that appeared to allow detection of similarities between complex bacterial communities. We term this the variable percentage threshold method. We found that making conclusions about the similarities of complex bacterial communities from T-RFLP profiles generated by a single restriction enzyme (RE) may lead to erroneous conclusions. Instead, the use of multiple REs, each individually, to generate multiple data sets allowed us to determine a confidence estimate for groupings of apparently similar communities and at the same time minimized the effects of RE selection. In conjunction with the variable percentage threshold method, this allowed us to make confident conclusions about the similarities of the complex bacterial communities in the 17 different samples.     

Diversity analysis of bacterial community compositions in sediments of urban lakes by terminal restriction fragment length polymorphism (T-RFLP)

Bacteria are crucial components in lake sediments and play important role in various environmental processes. Urban lakes in the densely populated cities are often small, shallow, highly artificial and hypereutrophic compared to rural and natural lakes and have been overlooked for a long time. In the present study, bacterial community compositions in surface sediments of three urban lakes (Lake Mochou, Lake Qianhu and Lake Zixia) in Nanjing City, China, were investigated using the terminal restriction fragment length polymorphism (T-RFLP) of PCR-amplified 16S rRNA gene and clone libraries. Remarkable differences in the T-RFLP patterns were observed in different lakes or different sampling stations of the same lake. Canonical correspondence analysis indicated that total nitrogen (TN) had significant effects on bacterial community structure in the lake sediments. Chloroflexi were the most dominant bacterial group in the clone library from Lake Mochou (21.7?% of the total clones) which was partly associated with its higher TN and organic matters concentrations. However, Bacteroidetes appeared to be dominated colonizers in the sediments of Lake Zixia (20.4?% of the total clones). Our study gives a comprehensive insight into the structure of bacterial community of urban lake sediments, indicating that the environmental factors played a key role in influencing the bacterial community composition in the freshwater ecosystems.     

Use of primer selection and restriction enzymes to assess bacterial community diversity in an agricultural soil used for potato production via terminal restriction fragment length polymorphism

The disparity of secondary metabolites in Penicillium chrysogenum between two scales of penicillin G fermentation (50 L as pilot process and 150,000 L as industrial one) was investigated by ion-pair reversed-phase liquid chromatography tandemed with hybrid quadrupole time-of-flight mass spectrometry. In industrial process, the pools of intracellular L-α-aminoadipyl-L-cysteinyl-D-valine (LLD-ACV) and isopenicillin N (IPN) were remarkably less than that in the pilot one, which indicated that the productivity of penicillin G might be higher in the large scale of fermentation. This conclusion was supported by the higher intracellular penicillin G concentration as well as its higher yield per unit biomass in industrial cultivation. The different changing tendencies of IPN, 6-aminopenicillanic acid and 6-oxopiperide-2-carboxylic acid between two processes also suggested the same conclusion. The higher content of intracellular LLD-ACV in pilot process lead to a similarly higher concentration of bis-δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine, which had an inhibitory effect on ACV synthetase and also subdued the activity of IPN synthetase. The interconversion of secondary metabolites and the influence they put on enzymes would intensify the discrepancy between two fermentations more largely. These findings provided new insight into the changes and regulation of secondary metabolites in P. chrysogenum under different fermentation sizes.     

Residual polymerase activity-induced bias in terminal restriction fragment length polymorphism analysis

Residual activity of polymerase chain reaction DNA polymerases in restriction analyses strongly affected genetic profiling based on terminal restriction fragment length polymorphisms. Artificial fragment sizes produced as a result of 5'-overhang restriction site fill-in and addition of a terminal A may bias genetic profiling and genotyping of microbial communities. Efficient removal of polymerases retained original fragment sizes and significantly reduced this profiling bias in soil bacterial communities.     

Evaluation of terminal-restriction fragment length polymorphism analysis in contrasting marine environments

Terminal-restriction fragment length polymorphism (T-RFLP) analysis is widely used in microbial ecology studies. In the present study, T-RFLP analysis of PCR products digested by five restriction enzymes (AluI, HaeIII, MspI, Sau3AI and TaqI) was applied for 20 samples from three contrasting coastal environments to assess the biases associated with the choice of enzyme digestion and T-RF analysis. The five enzyme digestions produced highly variable species richness (in terms of number of T-RFs). Analysis of peak areas with a threshold of 0.5% of the total peak area, which recovered 92-96% of the total peak area, revealed different diversity indexes from the five enzyme digestions. Multidimensional scaling, based on matrices that were generated by scoring peak presence/absence and area, revealed similar bacterial community structure patterns among the 20 samples, regardless of the choice of restriction enzymes. Our results strongly argue that the choice of different digestion enzymes in the T-RFLP technique generated valid and consistent bacterial community structures but highly variable species richness and diversity indices. The biases associated with the choice of digestion enzymes needs to be evaluated carefully or at least to be addressed when using T-RFLP analysis.     

Application of a new PCR primer for terminal restriction fragment length polymorphism analysis of the bacterial communities in plant roots

Contamination with plastid small subunit (SSU) rDNA is a major drawback when analyzing the bacterial communities of plant roots using culture-independent methods. In this study, a polymerase chain reaction (PCR) primer, 783r, was designed and tested to specifically amplify the SSU rDNA of various bacterial species without amplifying the SSU rDNA of plant plastids. To confirm how useful the community analysis of rhizobacteria is using 783r, the terminal restriction fragment length polymorphism (T-RFLP) method was performed with wheat (Triticum aestivum) and spinach (Spinacea oleracea) root samples. Using the standard T-RFLP method, a large T-RF peak of plant plastid SSU rDNA interfered with the bacterial community analysis. In contrast, the T-RFLP method using the 783r primer was able to detect the bacterial DNA while directly eliminating the influence of the plant-derived DNA extracted from the plant roots. Primer 783r might, therefore, be a useful PCR primer for the culture-independent analysis of bacterial communities in plant roots using SSU rDNA.     

Determining a minimum detection threshold in terminal restriction fragment length polymorphism analysis

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a common technique used to characterize soil microbial diversity. The fidelity of this technique in accurately reporting diversity has not been thoroughly evaluated. Here we determine if rare fungal species can be reliably detected by T-RFLP analysis. Spores from three arbuscular mycorrhizal fungal species were each mixed at a range of concentrations (1%, 10%, 50%, and 100%) with Glomus irregulare to establish a minimum detection threshold. T-RFLP analysis was capable of detecting diagnostic peaks of rare taxa at concentrations as low as 1%. The relative proportion of the target taxa in the sample and DNA concentration influenced peak detection reliability. However, low concentrations produced small, inconsistent electropherogram peaks contributing to difficulty in differentiating true peaks from signal noise. The results of this experiment suggest T-RFLP is a reproducible and high fidelity procedure, which requires careful data interpretation in order to accurately characterize sample diversity.     

Comparison of two fingerprinting techniques, terminal restriction fragment length polymorphism and automated ribosomal intergenic spacer analysis, for determination of bacterial diversity in aquatic environments

We investigated bacterial diversity in different aquatic environments (including marine and lagoon sediments, coastal seawater, and groundwater), and we compared two fingerprinting techniques (terminal restriction fragment length polymorphism [T-RFLP] and automated ribosomal intergenic spacer analysis [ARISA]) which are currently utilized for estimating richness and community composition. Bacterial diversity ranged from 27 to 99 phylotypes (on average, 56) using the T-RFLP approach and from 62 to 101 genotypes (on average, 81) when the same samples were analyzed using ARISA. The total diversity encountered in all matrices analyzed was 144 phylotypes for T-RFLP and 200 genotypes for ARISA. Although the two techniques provided similar results in the analysis of community structure, bacterial richness and diversity estimates were significantly higher using ARISA. These findings suggest that ARISA is more effective than T-RFLP in detecting the presence of bacterial taxa accounting for <5% of total amplified product. ARISA enabled also distinction among aquatic bacterial isolates of Pseudomonas spp. which were indistinguishable using T-RFLP analysis. Overall, the results of this study show that ARISA is more accurate than T-RFLP analysis on the 16S rRNA gene for estimating the biodiversity of aquatic bacterial assemblages.     

Application of new primer-enzyme combinations to terminal restriction fragment length polymorphism profiling of bacterial populations in human feces

New primer-enzyme combinations for terminal restriction fragment length polymorphism (T-RFLP) targeting of the 16S rRNA gene were constructed by using the T-RFLP analysis program (designated TAP T-RFLP) located at the Ribosomal Database Project website, and their performance was examined empirically. By using the fluorescently labeled 516f primer (Escherichia coli positions 516 to 532) and 1510r primer (positions 1510 to 1492), the 16S rRNA gene was amplified from human fecal DNA. The resulting amplified product was digested with RsaI plus BfaI or with BslI. When the T-RFLP was carried out with fecal DNAs from eight individuals, eight predominant operational taxonomic units (OTUs) were detected with RsaI and BfaI digestion and 14 predominant OTUs were detected with BslI digestion. The distribution of the OTUs was consistent with the results of the computer simulations with TAP T-RFLP. The T-RFLP analyses of the fecal DNAs from individuals gave characteristic profiles, while the variability of the T-RFLP profiles between duplicate DNA preparations from the same samples were minimal. This new T-RFLP method made it easy to predict what kind of intestinal bacterial group corresponded to each OTU on the basis of the terminal restriction fragment length compared with the conventional T-RFLP and, moreover, made it possible to identify the bacterial species that an OTU represents by cloning and sequencing.     

Use of multiplex terminal restriction fragment length polymorphism for rapid and simultaneous analysis of different components of the soil microbial community

A multiplex terminal restriction fragment length polymorphism (M-TRFLP) fingerprinting method was developed and validated for simultaneous analysis of the diversity and community structure of two or more microbial taxa (up to four taxa). The reproducibility and robustness of the method were examined using soil samples collected from different habitats. DNA was PCR amplified separately from soil samples using individual taxon-specific primers for bacteria, archaea, and fungi. The same samples were also subjected to a multiplex PCR with the primers for all three taxa. The terminal restriction fragment length polymorphism profiles generated for the two sets of PCR products were almost identical not only in terms of the presence of peaks but also in terms of the relative peak intensity. The M-TRFLP method was then used to investigate rhizosphere bacterial, fungal, and rhizobial/agrobacterial communities associated with the dwarf shrub Calluna vulgaris growing in either open moorland, a mature pine forest, or a transition zone between these two habitats containing naturally regenerating pine trees. Rhizosphere microbial communities associated with Vaccinium myrtillus collected from the native pine forest were also investigated. In this study, individual PCR products from the three taxa were also pooled before restriction digestion and fragment size analysis. The terminal restriction fragment length polymorphism profiles obtained with PCR products amplified individually and with multiplexed and pooled PCR products were found to be consistent with each other in terms of the number, position, and relative intensity of peaks. The results presented here confirm that M-TRFLP analysis is a highly reproducible and robust molecular tool for simultaneous investigation of multiple taxa, which allows more complete and higher resolution of microbial communities to be obtained more rapidly and economically.     

High-sensitivity stable-isotope probing by a quantitative terminal restriction fragment length polymorphism protocol

Stable-isotope probing (SIP) has proved a valuable cultivation-independent tool for linking specific microbial populations to selected functions in various natural and engineered systems. However, application of SIP to microbial populations with relatively minor buoyant density increases, such as populations that utilize compounds as a nitrogen source, results in reduced resolution of labeled populations. We therefore developed a tandem quantitative PCR (qPCR)-TRFLP (terminal restriction fragment length polymorphism) protocol that improves resolution of detection by quantifying specific taxonomic groups in gradient fractions. This method combines well-controlled amplification with TRFLP analysis to quantify relative taxon abundance in amplicon pools of FAM-labeled PCR products, using the intercalating dye EvaGreen to monitor amplification. Method accuracy was evaluated using mixtures of cloned 16S rRNA genes, DNA extracted from low- and high-G+C bacterial isolates (Escherichia coli, Rhodococcus, Variovorax, and Microbacterium), and DNA from soil microcosms amended with known amounts of genomic DNA from bacterial isolates. Improved resolution of minor shifts in buoyant density relative to TRFLP analysis alone was confirmed using well-controlled SIP analyses.     

Comparing activated sludge fungal community population diversity using denaturing gradient gel electrophoresis and terminal restriction fragment length polymorphism

We compared the relative values of denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) for profiling fungal communities in wastewater treatment plants using both ITS and 18S rRNA gene fragments as phylogenetic markers. A similar number of fungal ribotypes was obtained with both methods for the same treatment plant when the ITS primer set was used, while a greater number of ribotypes was obtained with T-RFLP compared to DGGE with the 18S rRNA primer set. Non-metric multi-dimensional scaling of presence/absence data and analysis of similarity showed that both methods could distinguish between the different plant communities at a statistically significant level (p < 0.05), regardless of which phylogenetic marker was used. The data suggest that both methods can be used preferably together to profile activated sludge fungal communities. A comparison of profiles generated with both these phylogenetic markers based on the number of ribotypes/bands, suggests that the 18S rRNA region is more discriminatory than the ITS region. Detected differences in fungal community compositions between plants probably reflect differences in their influent compositions and operational parameters.     

Assessment of anaerobic wastewater treatment failure using terminal restriction fragment length polymorphism analysis

AIMS: The suitability of genetic fingerprinting to study the microbiological basis of anaerobic bioreactor failure is investigated. METHODS AND RESULTS: Two laboratory-scale anaerobic expanded granular sludge bed bioreactors, R1 and R2, were used for the mesophilic (37 degrees C) treatment of high-strength [10 g chemical oxygen demand (COD) l(-1)] synthetic industrial-like wastewater over a 100-day trial period. A successful start up was achieved by both bioreactors with COD removal over 90%. Both reactors were operated under identical parameters; however, increased organic loading during the trial induced a reduction in the COD removal of R1, while R2 maintained satisfactory performance (COD removal >90%) throughout the experiment. Specific methanogenic activity measurements of biomass from both reactors indicated that the main route of methane production was hydrogenotrophic methanogenesis. Terminal restriction fragment length polymorphism (TRFLP) analysis was applied to the characterization of microbial community dynamics within the system during the trial. The principal differences between the two consortia analysed included an increased abundance of Thiovulum- and Methanococcus-like organisms and uncultured Crenarchaeota in R1. CONCLUSIONS: The results indicated that there was a microbiological basis for the deviation, in terms of operational performance, of R1 and R2. SIGNIFICANCE AND IMPACT OF THE STUDY: High-throughput fingerprinting techniques, such as TRFLP, have been demonstrated as practically relevant for biomonitoring of anaerobic reactor communities.     

T-Align, a web-based tool for comparison of multiple terminal restriction fragment length polymorphism profiles

Terminal restriction fragment length polymorphism (tRFLP) is a potentially high-throughput method for the analysis of complex microbial communities. Comparison of multiple tRFLP profiles to identify shared and unique components of microbial communities however, is done manually, which is both time consuming and error prone. This paper describes a freely accessible web-based program, T-Align (http://inismor.ucd.ie/~talign/), which addresses this problem. Initially replicate profiles are compared and used to generate a single consensus profile containing only terminal restriction fragments that occur in all replicate profiles. Subsequently consensus profiles representing different communities are compared to produce a list showing whether a terminal restriction fragment (TRF) is present in a particular sample and its relative fluorescence intensity. The use of T-Align thus allows rapid comparison of numerous tRFLP profiles. T-Align is demonstrated by alignment of tRFLP profiles generated from bacterioplankton communities collected from the Irish and Celtic Seas in November 2000. Ubiquitous TRFs and site-specific TRFs were identified using T-Align.     

A Lactobacillus helveticus plasmid detects restriction fragment length polymorphism in different bacterial species

A small cryptic Lactobacillus helveticus plasmid, pLBL4, was able to reveal restriction fragment length polymorphism in different bacterial species including Lactobacillus species, Bacillus species, and Escherichia coli when used as a DNA probe. The observed polymorphism was a result of the combined hybridization of several microsatellite sequences. The 6-bp sequence (TTGTTT) was repeated 12 times, seven of which were concentrated within the region between 1791 and 1997 bp of the plasmid sequence. The polymorphic patterns generated with pLBL4 differed from those obtained with M13 DNA in the larger number of bands observed. The results presented here open the possibility of using pLBL4 as a new broad-spectrum polymorphic DNA probe for fingerprint analysis.