Phylogenetic specificity and reproducibility and new method for analysis of terminal restriction fragment profiles of 16S rRNA genes from bacterial communities

Terminal restriction fragment (TRF) analysis of 16S rRNA genes is an increasingly popular method for rapid comparison of microbial communities, but analysis of the data is still in a developmental stage. We assessed the phylogenetic resolution and reproducibility of TRF profiles in order to evaluate the limitations of the method, and we developed an essential analysis technique to improve the interpretation of TRF data. The theoretical phylogenetic resolution of TRF profiles was determined based on the specificity of TRFs predicted from 3,908 16S rRNA gene sequences. With sequences from the Proteobacteria or gram-positive division, as much as 73% of the TRFs were phylogenetically specific (representing strains from at most two genera). However, the fraction decreased when sequences from the two divisions were combined. The data show that phylogenetic inference will be most effective if TRF profiles represent only a single bacterial division or smaller group. The analytical precision of the TRF method was assessed by comparing nine replicate profiles of a single soil DNA sample. Despite meticulous care in producing the replicates, numerous small, irreproducible peaks were observed. As many as 85% of the 169 distinct TRFs found among the profiles were irreproducible (i.e., not present in all nine replicates). Substantial variation also occurred in the height of synonymous peaks. To make comparisons of microbial communities more reliable, we developed an analytical procedure that reduces variation and extracts a reproducible subset of data from replicate TRF profiles. The procedure can also be used with other DNA fingerprinting techniques for microbial communities or microbial genomes.     

Phylogenetic Specificity and Reproducibility and New Method for Analysis of Terminal Restriction Fragment Profiles of 16S rRNA Genes from Bacterial Communities

Terminal restriction fragment (TRF) analysis of 16S rRNA genes is an increasingly popular method for rapid comparison of microbial communities, but analysis of the data is still in a developmental stage. We assessed the phylogenetic resolution and reproducibility of TRF profiles in order to evaluate the limitations of the method, and we developed an essential analysis technique to improve the interpretation of TRF data. The theoretical phylogenetic resolution of TRF profiles was determined based on the specificity of TRFs predicted from 3,908 16S rRNA gene sequences. With sequences from the Proteobacteria or gram-positive division, as much as 73% of the TRFs were phylogenetically specific (representing strains from at most two genera). However, the fraction decreased when sequences from the two divisions were combined. The data show that phylogenetic inference will be most effective if TRF profiles represent only a single bacterial division or smaller group. The analytical precision of the TRF method was assessed by comparing nine replicate profiles of a single soil DNA sample. Despite meticulous care in producing the replicates, numerous small, irreproducible peaks were observed. As many as 85% of the 169 distinct TRFs found among the profiles were irreproducible (i.e., not present in all nine replicates). Substantial variation also occurred in the height of synonymous peaks. To make comparisons of microbial communities more reliable, we developed an analytical procedure that reduces variation and extracts a reproducible subset of data from replicate TRF profiles. The procedure can also be used with other DNA fingerprinting techniques for microbial communities or microbial genomes.     

有机污染物对水体真细菌群落结构的影响

为了揭示有机污染物对环境真细菌组成和多样性的影响,应用末端限制性片段长度多态性(tRFLP)和16S rDNA文库技术并结合水质分析方法,比较分析了松花江流域内受不同程度有机污染的4个水体及其沉积物中真细菌的群落结构。tRFLP分析表明各水体及底泥均呈现较为复杂的群落结构模式,不同底泥群落形成的末端限制性片段(TRF)图谱具有很高的相似性,但随着污染程度的加强,部分类群明显富集,而且在水样组和泥样组内,群落结构的相似性同水质相似性是一致的,主成分分析(PCA)显示水样和泥样中的真细菌TRF形成不同的群。16SrDNA文库分析表明松花江哈尔滨段底泥中真细菌分布于10个门,Proteobacteria门占优势,达群落总数的21.92%(β-Proteobacteria亚门占10.96%),而有机染污物严重超标的生活污水排污道底泥中的微生物多样性较低,分布于7个门,Proteobacteria门为优势群,占群落的47.37%(α-Proteobacteria亚门占21.05%,δ/ε-Proteobacteria亚门占15.79%)。该研究表明向水体中长期排放高浓度有机物能使系统中微生物群落多样性降低,与污染物降解相关的功能微生物类群明显富集。     

TRFLP analysis reveals that fungi rather than bacteria are associated with premature yeast flocculation in brewing

Premature yeast flocculation (PYF) is a sporadic fermentation problem in the brewing industry that results in incomplete yeast utilization of fermentable sugars in wort. Culture-independent, PCR-based fingerprinting techniques were applied in this study to identify the associations between the occurrence of the PYF problem during brewery fermentation with barley malt-associated microbial communities (both bacteria and fungi). Striking differences in the microbial DNA fingerprint patterns for fungi between PYF positive (PYF +ve) and negative (PYF ?ve) barley malts were observed using the terminal restriction fragment length polymorphism (TRFLP) technique. The presence of terminal restriction fragments (TRFs) of 360–460 bp size range, for fungal HaeIII restriction enzyme-derived TRFLP profiles appeared to vary substantially between PYF +ve and PYF ?ve samples. The source of the barley malt did not influence the fungal taxa implicated in PYF. TRFLP analysis indicates bacterial taxa are unlikely to be important in causing PYF. Virtual digestion of fungal sequences tentatively linked HaeIII TRFs in the 360–460 bp size range to a diverse range of yeast/yeast-like species. Findings from this study suggest that direct monitoring of barley malt samples using molecular methods could potentially be an efficient and viable alternative for monitoring PYF during brewery fermentations.     

Modified T-RFLP methods for taxonomic interpretation of T-RF

Terminal restriction fragment length polymorphism (TRFLP) is a method that has been frequently used to survey the microbial diversity of environmental samples and to monitor changes in microbial communities. T-RFLP is a highly sensitive and reproducible procedure that combines a PCR with a labeled primer, restriction digestion of the amplified DNA, and separation of the terminal restriction fragment (T-RF). The reliable identification of T-RF requires the information of nucleotide sequences as well as the size of T-RF. However, it is difficult to obtain the information of nucleotide sequences because the T-RFs are fragmented and lack a priming site of 3'-end for efficient cloning and sequence analysis. Here, we improved on the T-RFLP method in order to analyze the nucleotide sequences of the distinct TRFs. The first method is to selectively amplify the portion of T-RF ligated with specific oligonucleotide adapters. In the second method, the termini of T-RFs were tailed with deoxynucleotides using terminal deoxynucleotidyl transferase (TdT) and amplified by a second round of PCR. The major T-RFs generated from reference strains and from T-RFLP profiles of activated sludge samples were efficiently isolated and identified by using two modified T-RFLP methods. These methods are less time consuming and labor-intensive when compared with other methods. The T-RFLP method using TdT has the advantages of being a simple process and having no limit of restriction enzymes. Our results suggest that these methods could be useful tools for the taxonomic interpretation of T-RFs.     

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.     

Interpreting ecological diversity indices applied to terminal restriction fragment length polymorphism data: insights from simulated microbial communities

Ecological diversity indices are frequently applied to molecular profiling methods, such as terminal restriction fragment length polymorphism (T-RFLP), in order to compare diversity among microbial communities. We performed simulations to determine whether diversity indices calculated from T-RFLP profiles could reflect the true diversity of the underlying communities despite potential analytical artifacts. These include multiple taxa generating the same terminal restriction fragment (TRF) and rare TRFs being excluded by a relative abundance (fluorescence) threshold. True community diversity was simulated using the lognormal species abundance distribution. Simulated T-RFLP profiles were generated by assigning each species a TRF size based on an empirical or modeled TRF size distribution. With a typical threshold (1%), the only consistently useful relationship was between Smith and Wilson evenness applied to T-RFLP data (TRF-E(var)) and true Shannon diversity (H'), with correlations between 0.71 and 0.81. TRF-H' and true H' were well correlated in the simulations using the lowest number of species, but this correlation declined substantially in simulations using greater numbers of species, to the point where TRF-H' cannot be considered a useful statistic. The relationships between TRF diversity indices and true indices were sensitive to the relative abundance threshold, with greatly improved correlations observed using a 0.1% threshold, which was investigated for comparative purposes but is not possible to consistently achieve with current technology. In general, the use of diversity indices on T-RFLP data provides inaccurate estimates of true diversity in microbial communities (with the possible exception of TRF-E(var)). We suggest that, where significant differences in T-RFLP diversity indices were found in previous work, these should be reinterpreted as a reflection of differences in community composition rather than a true difference in community diversity.     

Terminal Restriction Fragment Length Polymorphism Data Analysis for Quantitative Comparison of Microbial Communities

Terminal restriction fragment length polymorphism (T-RFLP) is a culture-independent method of obtaining a genetic fingerprint of the composition of a microbial community. Comparisons of the utility of different methods of (i) including peaks, (ii) computing the difference (or distance) between profiles, and (iii) performing statistical analysis were made by using replicated profiles of eubacterial communities. These samples included soil collected from three regions of the United States, soil fractions derived from three agronomic field treatments, soil samples taken from within one meter of each other in an alfalfa field, and replicate laboratory bioreactors. Cluster analysis by Ward's method and by the unweighted-pair group method using arithmetic averages (UPGMA) were compared. Ward's method was more effective at differentiating major groups within sets of profiles; UPGMA had a slightly reduced error rate in clustering of replicate profiles and was more sensitive to outliers. Most replicate profiles were clustered together when relative peak height or Hellinger-transformed peak height was used, in contrast to raw peak height. Redundancy analysis was more effective than cluster analysis at detecting differences between similar samples. Redundancy analysis using Hellinger distance was more sensitive than that using Euclidean distance between relative peak height profiles. Analysis of Jaccard distance between profiles, which considers only the presence or absence of a terminal restriction fragment, was the most sensitive in redundancy analysis, and was equally sensitive in cluster analysis, if all profiles had cumulative peak heights greater than 10,000 fluorescence units. It is concluded that T-RFLP is a sensitive method of differentiating between microbial communities when the optimal statistical method is used for the situation at hand. It is recommended that hypothesis testing be performed by redundancy analysis of Hellinger-transformed data and that exploratory data analysis be performed by cluster analysis using Ward's method to find natural groups or by UPGMA to identify potential outliers. Analyses can also be based on Jaccard distance if all profiles have cumulative peak heights greater than 10,000 fluorescence units.     

Terminal restriction fragment length polymorphism data analysis for quantitative comparison of microbial communities

Terminal restriction fragment length polymorphism (T-RFLP) is a culture-independent method of obtaining a genetic fingerprint of the composition of a microbial community. Comparisons of the utility of different methods of (i) including peaks, (ii) computing the difference (or distance) between profiles, and (iii) performing statistical analysis were made by using replicated profiles of eubacterial communities. These samples included soil collected from three regions of the United States, soil fractions derived from three agronomic field treatments, soil samples taken from within one meter of each other in an alfalfa field, and replicate laboratory bioreactors. Cluster analysis by Ward's method and by the unweighted-pair group method using arithmetic averages (UPGMA) were compared. Ward's method was more effective at differentiating major groups within sets of profiles; UPGMA had a slightly reduced error rate in clustering of replicate profiles and was more sensitive to outliers. Most replicate profiles were clustered together when relative peak height or Hellinger-transformed peak height was used, in contrast to raw peak height. Redundancy analysis was more effective than cluster analysis at detecting differences between similar samples. Redundancy analysis using Hellinger distance was more sensitive than that using Euclidean distance between relative peak height profiles. Analysis of Jaccard distance between profiles, which considers only the presence or absence of a terminal restriction fragment, was the most sensitive in redundancy analysis, and was equally sensitive in cluster analysis, if all profiles had cumulative peak heights greater than 10,000 fluorescence units. It is concluded that T-RFLP is a sensitive method of differentiating between microbial communities when the optimal statistical method is used for the situation at hand. It is recommended that hypothesis testing be performed by redundancy analysis of Hellinger-transformed data and that exploratory data analysis be performed by cluster analysis using Ward's method to find natural groups or by UPGMA to identify potential outliers. Analyses can also be based on Jaccard distance if all profiles have cumulative peak heights greater than 10,000 fluorescence units.     

Effect of long‐term mineral fertilisation on soil microbial abundance,community structure and diversity in a Typic Hapludoll under intensive farming systems

Fertiliser application can not only influence plant communities, but also the soil microbial community dynamics, and consequently soil quality. Specifically, mineral fertilisation can directly or indirectly affect soil chemical properties, microbial abundance and, the structure and diversity of soil microbial communities. We investigated the impact of six different mineral fertiliser regimes in a maize/soybean rotation system: control (CK, without fertilisation), PS (application of phosphorus plus sulphur), NS (application of nitrogen plus S), NP (application of N plus P), NPS (application of N, P plus S) and NPSm (application of N, P, S plus micronutrients). Soil samples were collected at the physiological maturity stage of maize and soybean in March of 2013 and 2014, respectively. Overall, mineral fertilisation resulted in significantly decreased soil pH and increased total organic carbon compared with the control (CK). The analysis of terminal restriction fragment length polymorphism (T‐RFLP) revealed that mineral fertilisers caused a shift in the composition of both bacterial and fungal communities. In 2013, the highest value of Shannon diversity of bacterial terminal restriction fragments (TRFs) was found in control soils. In 2014, NPSm treated soils showed the lowest values of diversity for both bacterial and fungal TRFs. In both crop growing seasons, the analysis of phospholipid fatty acid (PLFA) detected the lowest value of total microbial biomass under CK. As PLFA analysis can be used to evaluate total microbial community, this result suggests that fertilisation increased total microbial biomass. When the bacterial and fungal abundance were examined using real time polymerase chain reaction, the results revealed that mineral fertilisation led to decreased bacterial abundance (16S rRNA), while fungal abundance (18S rRNA) was found to be increased in both crop growing seasons. Our results show that mineral fertiliser application has a significant impact on soil properties, bacterial and fungal abundance and microbial diversity. However, further studies are needed to better understand the mechanisms involved in the changes to microbial communities as a consequence of mineral fertilisation.     

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.     

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.     

Reliability for detecting composition and changes of microbial communities by T-RFLP genetic profiling

Terminal restriction fragment length polymorphism (T-RFLP) analysis is commonly used for profiling microbial communities in various environments. However, it may suffer from biases during the analytic process. This study addressed the potential of T-RFLP profiles (1) to reflect real community structures and diversities, as well as (2) to reliably detect changing components of microbial community structures. For this purpose, defined artificial communities of 30 SSU rRNA gene clones, derived from nine bacterial phyla, were used. PCR amplification efficiency was one primary bias with a maximum variability factor of 3.5 among clones. PCR downstream analyses such as enzymatic restriction and capillary electrophoresis introduced a maximum bias factor of 4 to terminal restriction fragment (T-RF) signal intensities, resulting in a total maximum bias factor of 14 in the final T-RFLP profiles. In addition, the quotient between amplification efficiency and T-RF size allowed predicting T-RF abundances in the profiles with high accuracy. Although these biases impaired detection of real community structures, the relative changes in structures and diversities were reliably reflected in the T-RFLP profiles. These data support the suitability of T-RFLP profiling for monitoring effects on microbial communities.     

Chitinase genes revealed and compared in bacterial isolates, DNA extracts and a metagenomic library from a phytopathogen-suppressive soil

Soil that is suppressive to disease caused by fungal pathogens is an interesting source to target for novel chitinases that might be contributing towards disease suppression. In this study, we screened for chitinase genes, in a phytopathogen-suppressive soil in three ways: (1) from a metagenomic library constructed from microbial cells extracted from soil, (2) from directly extracted DNA and (3) from bacterial isolates with antifungal and chitinase activities. Terminal restriction fragment length polymorphism (T-RFLP) of chitinase genes revealed differences in amplified chitinase genes from the metagenomic library and the directly extracted DNA, but approximately 40% of the identified chitinase terminal restriction fragments (TRFs) were found in both sources. All of the chitinase TRFs from the isolates were matched to TRFs in the directly extracted DNA and the metagenomic library. The most abundant chitinase TRF in the soil DNA and the metagenomic library corresponded to the TRF¹⁰³ of the isolate Streptomyces mutomycini and/or Streptomyces clavifer . There were good matches between T-RFLP profiles of chitinase gene fragments obtained from different sources of DNA. However, there were also differences in both the chitinase and the 16S rRNA gene T-RFLP patterns depending on the source of DNA, emphasizing the lack of complete coverage of the gene diversity by any of the approaches used.     

Interpreting Ecological Diversity Indices Applied to Terminal Restriction Fragment Length Polymorphism Data: Insights from Simulated Microbial Communities

Ecological diversity indices are frequently applied to molecular profiling methods, such as terminal restriction fragment length polymorphism (T-RFLP), in order to compare diversity among microbial communities. We performed simulations to determine whether diversity indices calculated from T-RFLP profiles could reflect the true diversity of the underlying communities despite potential analytical artifacts. These include multiple taxa generating the same terminal restriction fragment (TRF) and rare TRFs being excluded by a relative abundance (fluorescence) threshold. True community diversity was simulated using the lognormal species abundance distribution. Simulated T-RFLP profiles were generated by assigning each species a TRF size based on an empirical or modeled TRF size distribution. With a typical threshold (1%), the only consistently useful relationship was between Smith and Wilson evenness applied to T-RFLP data (TRF-E_var) and true Shannon diversity (H′), with correlations between 0.71 and 0.81. TRF-H′ and true H′ were well correlated in the simulations using the lowest number of species, but this correlation declined substantially in simulations using greater numbers of species, to the point where TRF-H′ cannot be considered a useful statistic. The relationships between TRF diversity indices and true indices were sensitive to the relative abundance threshold, with greatly improved correlations observed using a 0.1% threshold, which was investigated for comparative purposes but is not possible to consistently achieve with current technology. In general, the use of diversity indices on T-RFLP data provides inaccurate estimates of true diversity in microbial communities (with the possible exception of TRF-E_var). We suggest that, where significant differences in T-RFLP diversity indices were found in previous work, these should be reinterpreted as a reflection of differences in community composition rather than a true difference in community diversity.     

Soil bacterial diversity in a loblolly pine plantation: influence of ectomycorrhizas and fertilization

We studied the effect of ectomycorrhizas and fertilization on soil microbial communities associated with roots of 10-year-old loblolly pine. Ectomycorrhizas were identified using a combination of community terminal restriction fragment profiling and matching of individual terminal restriction fragments to those produced from ectomycorrhizal clones and sequences recovered from roots and sporocarps. Differences between bacterial communities were initially determined using cluster analysis on community terminal restriction fragment profiles and through subsequent recovery of 16S rDNA clones. Analysis of bacterial clones revealed that terminal restriction fragment length was often shared between taxonomically dissimilar bacterial types. Consequently, we could not reliably infer the identity of peaks in the bacterial community profile with some exceptions, notably chloroplast rDNA that generated an approximate peak size of 80.2 bp. Fertilization increased the frequency of a Piloderma-like ectomycorrhiza. However, we did not detect clear effects of fertilization or the presence of viable ectomycorrhizas on bacterial communities. Bacterial communities seemed to be determined largely by the carbon and nitrogen content of soil. These results suggest that important soil microbial groups respond differently to soil conditions and management practices, with ectomycorrhizal communities reflecting past nutrient conditions and bacterial communities reflecting current environmental conditions of soil microsites.     

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.     

Genetic Diversity of Bacterial Communities of Serpentine Soil and of Rhizosphere of the Nickel-Hyperaccumulator Plant <Emphasis Type="Italic">Alyssum bertolonii</Emphasis>

Serpentine soils are characterized by high levels of heavy metals (Ni, Co, Cr), and low levels of important plant nutrients (P, Ca, N). Because of these inhospitable edaphic conditions, serpentine soils are typically home to a very specialized flora including endemic species as the nickel hyperaccumulator Alyssum bertolonii. Although much is known about the serpentine flora, few researches have investigated the bacterial communities of serpentine areas. In the present study bacterial communities were sampled at various distances from A. bertolonii roots in three different serpentine areas and their genetic diversity was assessed by terminal restriction fragment length polymorphism (T-RFLP) analysis. The obtained results indicated the occurrence of a high genetic diversity and heterogeneity of the bacterial communities present in the different serpentine areas. Moreover, TRFs (terminal restriction fragments) common to all the investigated A. bertolonii rhizosphere samples were found. A new cloning strategy was applied to 27 TRFs that were sequenced and taxonomically interpreted as mainly belonging to Gram-positive and -Proteobacteria representatives. In particular, cloned TRFs which discriminated between rhizosphere and soil samples were mainly interpreted as belonging to Proteobacteria representatives.This revised version was published online in November 2004 with corrections to Volume 48.     

Explorative screening of complex microbial communities by real-time 16S rDNA restriction fragment melting curve analyses

We have developed restriction fragment melting curve analyses (RFMCA), which is a novel method for the real-time analysis of microbial communities. The major advantage of RFMCA compared to, for example, terminal restriction fragment length polymorphism (T-RFLP) or temperature/denaturing gradient gel electrophoresis (TGGE/DGGE) is that the physical separation of DNA fragments is avoided. The RFMCA detection is done by melting point analyses in closed tube systems, which enables high-throughput applications. The robustness of RFMCA was demonstrated by analyzing both mixtures of known samples and the microbial communities in the cecal content of poultry. Our conclusions are that RFMCA is robust, gives a relatively high resolution, and has the potential for high-throughput explorative screenings of microbial communities and large clone libraries.     

Haloacetic acid-degrading bacterial communities in drinking water systems as determined by cultivation and by terminal restriction fragment length polymorphism of PCR-amplified haloacid dehalogenase gene fragments

Aims: To characterize the HAA‐degrading bacteria in drinking water systems. Methods and Results: Haloacetic acid (HAA)‐degrading bacteria were analysed in drinking water systems by cultivation and by a novel application of terminal restriction fragment length polymorphism (tRFLP). Substantial similarities were observed among the tRFLP patterns of dehI and dehII gene fragments in drinking water samples obtained from three different cities (Minneapolis, MN; St Paul, MN; Bucharest, Romania) and from one biologically active granular activated carbon filter (Hershey, PA). The dominant fragment in the tRFLP profiles of dehI genes from the drinking water samples matched the pattern from an Afipia sp. that was previously isolated from drinking water. In contrast, the dominant fragment in the tRFLP profiles of dehII genes did not match any previously characterized dehII gene fragment. PCR cloning was used to characterize this gene fragment, which had <65% nucleotide sequence identity with any previously characterized dehII gene. Conclusions: Afipia spp. are an appropriate model organism for studying the biodegradation of HAAs in drinking water distribution systems as encoded by dehI genes; the organism that harbours the most prominent dehII gene in drinking water has yet to be cultivated and identified. Significance and Impact of the Study: The development of a novel application of tRFLP targeting dehI and dehII genes could be broadly useful in understanding HAA‐degrading bacteria in numerous environments.