Use of the T-RFLP technique to assess spatial and temporal changes in the bacterial community structure within an agricultural soil planted with transgenic and non-transgenic potato plants

The aim of this study was to examine whether the terminal restriction fragment length polymorphism (T-RFLP) analysis represents an appropriate technique for monitoring highly diverse soil bacterial communities, i.e. to assess spatial and/or temporal effects on bacterial community structure. The T-RFLP method, a recently described fingerprinting technique, is based on terminal restriction fragment length polymorphisms between distinct small-subunit rRNA gene sequence types. This technique permits an automated quantification of the fluorescence signal intensities of the individual terminal restriction fragments (T-RFs) in a given community fingerprint pattern. The indigenous bacterial communities of three soil plots located within an agricultural field of 110 m(2) were compared. The first site was planted with non-transgenic potato plants, while the other two were planted with transgenic GUS and Barnase/Barstar potato plants, respectively. Once prior to planting and three times after planting, seven parallel samples were taken from each of the three soil plots. The T-RFLP analysis resulted in very complex but highly reproducible community fingerprint patterns. The percentage abundance values of defined T-RFs were calculated for the seven parallel samples of the respective soil plot. A multivariate analysis of variance was used to test T-RFLP data sets for significant differences. The statistical treatments clearly revealed spatial and temporal effects, as well as spacextime interaction effects, on the structural composition of the bacterial communities. T-RFs which showed the highest correlations to the discriminant factors were not those T-RFs which showed the largest single variations between the seven-sample means of individual plots. In summary, the T-RFLP technique, although a polymerase chain reaction-based method, proved to be a suitable technique for monitoring highly diverse soil microbial communities for changes over space and/or time.     

Community Structure of Denitrifiers, Bacteria, and Archaea along Redox Gradients in Pacific Northwest Marine Sediments by Terminal Restriction Fragment Length Polymorphism Analysis of Amplified Nitrite Reductase (nirS) and 16S rRNA Genes

Steep vertical gradients of oxidants (O₂ and NO₃⁻) in Puget Sound and Washington continental margin sediments indicate that aerobic respiration and denitrification occur within the top few millimeters to centimeters. To systematically explore the underlying communities of denitrifiers, Bacteria, and Archaea along redox gradients at distant geographic locations, nitrite reductase (nirS) genes and bacterial and archaeal 16S rRNA genes (rDNAs) were PCR amplified and analyzed by terminal restriction fragment length polymorphism (T-RFLP) analysis. The suitablility of T-RFLP analysis for investigating communities of nirS-containing denitrifiers was established by the correspondence of dominant terminal restriction fragments (T-RFs) of nirS to computer-simulated T-RFs of nirS clones. These clones belonged to clusters II, III, and IV from the same cores and were analyzed in a previous study (G. Braker, J. Zhou, L. Wu, A. H. Devol, and J. M. Tiedje, Appl. Environ. Microbiol. 66:2096–2104, 2000). T-RFLP analysis of nirS and bacterial rDNA revealed a high level of functional and phylogenetic diversity, whereas the level of diversity of Archaea was lower. A comparison of T-RFLPs based on the presence or absence of T-RFs and correspondence analysis based on the frequencies and heights of T-RFs allowed us to group sediment samples according to the sampling location and thus clearly distinguish Puget Sound and the Washington margin populations. However, changes in community structure within sediment core sections during the transition from aerobic to anaerobic conditions were minor. Thus, within the top layers of marine sediments, redox gradients seem to result from the differential metabolic activities of populations of similar communities, probably through mixing by marine invertebrates rather than from the development of distinct communities.     

基于T-RFLP方法的连栽杉木根际土壤细菌群落变化研究

运用末端限制性片段长度多态性技术(T-RFLP),研究福建省尤溪国有林场的多代连栽杉木根际土壤细菌群落变化特征。结果表明:杉木多代连栽后根际微生态失衡,表现为碳循环菌和氮循环菌等有益菌数量减少,病原菌数量增加。在不同代数杉木土壤中,4种内切酶消化后产生的末端限制性片段(T-RFs)数量具有显著差异,第一代杉木(first Chinese fir rotation plantation,FCP)最高,第二代杉木(second Chinese fir rotation plantation,SCP)次之,第三代杉木(third Chinese fir rotation plantation,TCP)最少。土壤理化性质结果表明连栽导致土壤养分逐代降低,除土壤碳氮比与细菌群落多样性呈显著负相关外,土壤养分含量指标与细菌群落多样性呈显著正相关。T-RFLP实验共鉴定出细菌门类12门,其中厚壁菌门、蓝藻门、放线菌门和变形菌门是主要优势菌群。随栽植代数的增加,变形菌门细菌比例递增,而厚壁菌门细菌比例递减。土壤细菌群落多样性指数,表现为随栽植代数增长而减少,呈现FCPSCPTCP趋势,与T-RFs片段数量变化趋势基本一致,表明多代连栽降低了杉木根际土壤细菌群落多样性。依据功能性不同,可将鉴定出的细菌划分为5类:碳循环功能菌、硫循环功能菌、氮循环功能菌、纤维降解菌及病原菌。连栽导致碳循环菌和氮循环菌相对含量降低,硫循环菌和纤维降解菌相对含量提高,病原菌相对含量随栽植代数增加而上升。     

Effect of Freeze-Thaw Cycles on Bacterial Communities of Arctic Tundra Soil

The effect of freeze-thaw (FT) cycles on Arctic tundra soil bacterial community was studied in laboratory microcosms. FT-induced changes to the bacterial community were followed over a 60-day period by terminal restriction fragment length polymorphism (T-RFLP) profiles of amplified 16S rRNA genes and reverse transcribed 16S rRNA. The main phylotypes of the active, RNA-derived bacterial community were identified using clone analysis. Non-metric multidimensional scaling ordination of the T-RFLP profiles indicated some shifts in the bacterial communities after three to five FT cycles at −2, −5, and −10°C as analyzed both from the DNA and rRNA. The dominating T-RFLP peaks remained the same, however, and only slight variation was generally detected in the relative abundance of the main T-RF sizes of either DNA or rRNA. T-RFLP analysis coupled to clone analysis of reverse transcribed 16S rRNA indicated that the initial soil was dominated by members of Bacteroidetes, Acidobacteria, Alpha-, Beta-, and Gammaproteobacteria. The most notable change in the rRNA-derived bacterial community was a decrease in the relative abundance of a Betaproteobacteria-related phylotype after the FT cycles. This phylotype decreased, however, also in the control soil incubated at constant +5°C suggesting that the decrease was not directly related to FT sensitivity. The results indicate that FT caused only minor changes in the bacterial community structure.     

持续干旱对樱桃根际土壤细菌数量及结构多样性影响

以1年生吉塞拉实生容器苗为试材,采用绿色荧光蛋白基因标记技术,研究了干旱胁迫(连续干旱0、7、14、21、28 d和35 d)对樱桃根际促生细菌YT3的标记菌YT3-gfp数量的影响,同时结合平板计数法和末端限制性片段长度多态性分析(terminal restriction fragment length polymorphism,T-RFLP)技术,研究了干旱对樱桃土壤中的微生物数量及细菌群落结构多样性影响。结果表明:樱桃根际土壤中的YT3-gfp数量是非根际土壤中的8.75—28.77倍,随着持续干旱强度的增加,YT3-gfp的数量先增加后减小。干旱对根际土壤中YT3-gfp数量的影响大于对非根际土壤的影响,分别在持续干旱至第21天和28天时,YT3-gfp的数量达到最大值。随着持续干旱强度的增加,根际土壤中细菌和放线菌数量先增加后减小,而真菌的数量一直减少。此外,持续干旱至第21天或28天时,樱桃根际土壤具有最高的丰富度指数、多样性指数和最低的优势度指数。基于T-RFLP的主成分分析结果显示持续干旱14—35 d时,其细菌群落结构成为一个相对独立的群,群落结构趋于多样性;而持续干旱7 d和42 d构成另外两个相对独立的群,群落结构趋于简单。以上分析可知,干旱对土壤微生物影响显著,一定强度的干旱可提高细菌和放线菌数量,提高细菌群落结构多样性,适当干旱对维持根际土壤细菌群落结构多样性是有益的。     

Stability in a Denitrifying Fluidized Bed Reactor

This study evaluates changes in the microbial community structure and function of a pilot-scale denitrifying fluidized bed reactor during periods of constant operating conditions and periods of perturbation. The perturbations consisted of a shutdown period without feed, two disturbances in which biofilms were mechanically sheared from carrier particles, and a twofold step increase in feed nitrate concentration. In the absence of perturbations, nitrate removal was stable and consistently greater than 99%. The structure and dynamics of the microbial community were studied using cloning and sequencing techniques and terminal restriction fragment length polymorphism (T-RFLP) of the SSU rRNA gene. Under unperturbed operating conditions, stable function was accompanied by high constancy and low variability of community structure with the majority of terminal restriction fragments (T-RFs) appearing throughout operation at consistent relative abundances. Several of the consistently present T-RFs correlated with clone sequences closely related to Acidovorax (98% similarity), Dechloromonas (99% similarity), and Zoogloea (98% similarity), genera recently identified by molecular analyses of similar systems. Significant changes in community structure and function were not observed after the shutdown period. In contrast, following the increase in loading rate and the mechanical disturbances, new T-RFs appeared. After both mechanical disturbances, function and community structure recovered. However, function was much more resilient than community structure. The similarity of response to the mechanical disturbances despite differences in community structure and operating conditions suggests that flexible community structure and potentially the activity of minor members under nonperturbation conditions promotes system recovery.     

Changes in Bacterial Community Structure in the Colon of Pigs Fed Different Experimental Diets and after Infection with Brachyspira hyodysenteriae

Bacterial communities in the large intestines of pigs were compared using terminal restriction fragment length polymorphism (T-RFLP) analysis targeting the 16S ribosomal DNA. The pigs were fed different experimental diets based on either modified standard feed or cooked rice supplemented with dietary fibers. After feeding of the animals with the experimental diets for 2 weeks, differences in the bacterial community structure in the spiral colon were detected in the form of different profiles of terminal restriction fragments (T-RFs). Some of the T-RFs were universally distributed, i.e., they were found in all samples, while others varied in distribution and were related to specific diets. The reproducibility of the T-RFLP profiles between individual animals within the diet groups was high. In the control group, the profiles remained unchanged throughout the experiment and were similar between two independent but identical experiments. When the animals were experimentally infected with Brachyspira hyodysenteriae, causing swine dysentery, many of the T-RFs fluctuated, suggesting a destabilization of the microbial community.     

Characterization of depth-related population variation in microbial communities of a coastal marine sediment using 16S rDNA-based approaches and quinone profiling

Depth-related changes in whole-community structure were evaluated in a coastal marine sediment using a molecular fingerprinting method, terminal restriction fragment length polymorphism (T-RFLP) analysis, and a chemotaxonomic technique (quinone profiling). Dendrograms derived from both T-RFLP analysis and quinone profiling indicated a significant variation in microbial community structure between the 0-2 cm layer and deeper layers. This corresponded to the dramatic change in the redox potential, acid-volatile sulphide-sulphur and bacterial numbers observed at 0-2 cm and 2-4 cm depths. A significant change in the number of terminal restriction fragments (T-RFs) was also detected at this transition depth. However, the change in major T-RFs with depth was not seen in electropherograms. The population changes were primarily variations in minor ribotypes. Most quinone homologues were detected at all depths, although the quinone composition changed with depth. Therefore, quinone profiling also suggested that the depth-related variation was primarily attributable to minor bacterial groups rather than change in the major population structure. 16S rDNA clone library analysis revealed that clones belonging to the genera Vibrio and Serratia predominated as major bacterial groups at all depths. Our data suggested that the sediment community might result from sedimentation effects of sinking particles. Overall, our results demonstrated that the combined methods of T-RFLP analysis and quinone profiling were effective for assessing depth-related microbial populations.     

Fidelity of select restriction endonucleases in determining microbial diversity by terminal-restriction fragment length polymorphism

An evaluation of 18 DNA restriction endonucleases for use in terminal-restriction fragment length polymorphism (T-RFLP) analysis was performed by using richness and density indices in conjunction with computer simulations for 4,603 bacterial small-subunit rRNA gene sequences. T-RFLP analysis has become a commonly used method for screening environmental samples for precursory identification and community comparison studies due to its precision and high-throughput capability. The accuracy of T-RFLP analysis for describing a community has not yet been thoroughly evaluated. In this study, we attempted to classify restriction endonucleases based upon the ability to resolve unique terminal-restriction fragments (T-RFs) or operational taxonomic units (OTUs) from a database of gene sequences. Furthermore, we assessed the predictive accuracy of T-RFLP at fixed values of community richness (n = 1, 5, 10, 50, and 100). Classification of restriction endonuclease fidelity was performed by measuring richness and density for the entire database of T-RFs. Further analysis of T-RFLP accuracy for determining richness was performed by iterative, random sampling from the derived database of T-RFs. It became apparent that two constraints were influential for measuring the fidelity of a given restriction endonuclease: (i) the ability to resolve unique sequence variants and (ii) the number of unique T-RFs that fell within a measurable size range. The latter constraint was found to be more significant for estimating restriction endonuclease fidelity. Of the 18 restriction endonucleases examined, BstUI, DdeI, Sau96I, and MspI had the highest frequency of resolving single populations in model communities. All restriction endonucleases used in this study detected < or =70% of the OTUs at richness values greater than 50 OTUs per modeled community. Based on the results of our in silico experiments, the most efficacious uses of T-RFLP for microbial diversity studies are those that address situations where there is low to intermediate species richness (e.g., colonization, early successional stages, biofilm formation).     

The Diversity of Bacterial Communities Associated with Atlantic Cod <Emphasis Type="Italic">Gadus morhua</Emphasis>

The spatial and temporal changes in the bacterial communities associated with the Atlantic cod Gadus morhua were investigated using terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S recombinant DNA (rDNA). Epidermal mucous was sampled from 366 cod caught in three harvest locations (Baltic, Icelandic, and North Seas) over three seasons (spring 2002, autumn 2002, and spring 2003), and an automated method for the high-throughput processing of environmental samples was developed using a Qiagen BioRobot. The analysis revealed that a diverse consortium of bacteria were found on fish; γ-proteobacteria and Cytophaga–Flavobacter–Bacteroides (CFB) species were dominant. T-RFLP peak profiles suggested that operational taxonomic units (OTUs) related to Photobacterium sp., Psychrobacter sp., and Bacteroides sp. were common to all sites in all three seasons, but there were intersite variations in community composition. Cod caught from different seas had distinct reproducible bacterial assemblages. Whereas communities from fish caught in the Baltic and Icelandic Seas were relatively stable over the three seasons, those from fish from the North Sea changed significantly over time.     

Critical evaluation of solid waste sample processing for DNA-based microbial community analysis

Landfills represent a unique microbial ecosystem and play a significant role in global biogeochemical processes. The study of complex ecosystems such as landfills using DNA-based techniques can be advantageous since they allow for analysis of uncultured organisms and offer higher resolution in measuring demographic and metabolic (functional) diversity. However, sample acquisition and processing from refuse is challenging due to material heterogeneity. Decomposed refuse was used to evaluate the effect of seven sample processing methods on Bacteria and Archaea community structure using T-RFLP. Bias was assessed using measured richness and by comparing community structure using multi-dimensional scaling (MDS). Generally, direct methods were found to be most biased while indirect methods (i.e., removal of cellular material from the refuse matrix before DNA extraction) were least biased. An indirect method using PO₄ buffer gave consistently high bacterial and archaeal richness and also resulted in 28 and 34% recovery of R. albus and M. formicicum spiked into refuse, respectively. However, the highest recovery of less abundant T-RFs was achieved using multiple processing methods. Results indicate differences in measured T-RF diversity from studies of landfill ecosystems could be caused by methodological (i.e., processing method) variation rather than refuse heterogeneity or true divergence in community structure.     

Comparison of Rhizosphere Bacterial Communities in Arabidopsis thaliana Mutants for Systemic Acquired Resistance

Systemic acquired resistance (SAR) is an inducible systemic plant defense against a broad spectrum of plant pathogens, with the potential to secrete antimicrobial compounds into the soil. However, its impact on rhizosphere bacteria is not known. In this study, we examined fingerprints of bacterial communities in the rhizosphere of the model plant Arabidopsis thaliana to determine the effect of SAR on bacterial community structure and diversity. We compared Arabidopsis mutants that are constitutive and non-inducible for SAR and verified SAR activation by measuring pathogenesis-related protein activity via a β-glucoronidase (GUS) reporter construct driven by the β-1-3 glucanase promoter. We used terminal restriction fragment length polymorphism (T-RFLP) analysis of MspI- and HaeIII-digested 16S rDNA to estimate bacterial rhizosphere community diversity, with Lactobacillus sp. added as internal controls. T-RFLP analysis showed a clear rhizosphere effect on community structure, and diversity analysis of both rhizosphere and bulk soil operational taxonomic units (as defined by terminal restriction fragments) using richness, Shannon–Weiner, and Simpson’s diversity indices and evenness confirmed that the presence of Arabidopsis roots significantly altered bacterial communities. This effect of altered soil microbial community structure by plants was also seen upon multivariate cluster analysis of the terminal restriction fragments. We also found visible differences in the rhizosphere community fingerprints of different Arabidopsis SAR mutants; however, there was no clear decrease of rhizosphere diversity because of constitutive SAR expression. Our study suggests that SAR can alter rhizosphere bacterial communities, opening the door to further understanding and application of inducible plant defense as a driving force in structuring soil bacterial assemblages.     

Novel Bacterial Lineages at the (Sub)Division Level as Detected by Signature Nucleotide-Targeted Recovery of 16S rRNA Genes from Bulk Soil and Rice Roots of Flooded Rice Microcosms

Using a newly developed 16S rRNA gene (rDNA)-targeted PCR assay with proposed group specificity for planctomycetes, we examined anoxic bulk soil of flooded rice microcosms for the presence of novel planctomycete-like diversity. For comparison, oxic rice roots were included as an additional sample in this investigation. The bacterial diversity detectable by this PCR assay was assessed by using a combined approach that included terminal restriction fragment length polymorphism (T-RFLP) analysis and comparative sequence analysis of cloned 16S rDNA. T-RFLP fingerprint patterns generated from rice roots contained 12 distinct terminal restriction fragments (T-RFs). In contrast, the T-RFLP fingerprint patterns obtained from the anoxic bulk soil contained 33 distinct T-RFs, a clearly higher level of complexity. A survey of 176 bulk soil 16S rDNA clone sequences permitted correlation of 20 T-RFs with phylogenetic information. The other 13 T-RFs remained unidentified. The predominant T-RFs obtained from rice roots could be assigned to members of the genus Pirellula within the Planctomycetales, while most of the T-RFs obtained from the bulk soil corresponded to novel lines of bacterial descent. Using a level of 16S rDNA sequence dissimilarity to cultured microorganisms of approximately 20% as a threshold value, we detected 11 distinct bacterial lineages for which pure-culture representatives are not known. Four of these lineages could be assigned to the order Planctomycetales, while one lineage was affiliated with the division Verrucomicrobia and one lineage was affiliated with the spirochetes. The other five lineages either could not be assigned to any of the main lines of bacterial descent or clearly expanded the known diversity of division level lineages WS3 and OP3. Our results indicate the presence of bacterial diversity at a subdivision and/or division level that has not been detected previously by the so-called universal 16S rDNA PCR assays.     

牛场肥水灌溉对土壤nirK、nirS型反硝化微生物群落结构的影响

以徐水县梁家营长期定位施肥试验田为研究对象,利用末端限制性片段长度多态性(T-RFLP)分析和克隆文库构建,研究了5种施肥处理(清水灌溉CK、无机肥灌溉CF、牛场肥水不同浓度、不同次数灌溉T4、T5和T11)下土壤中nirK、nirS型反硝化细菌群落多样性及其群落结构的演变。结果表明,不同施肥处理下nirK、nirS型反硝化细菌群落多样性无显著差异,但群落结构却有明显变化:nirK型反硝化细菌群落结构既受施肥种类又受施肥量影响,优势种群尤其对施肥种类和施肥量响应显著;nirS型反硝化细菌则主要受施肥种类影响,施肥量影响微弱。牛场肥水处理和无机肥处理分别促进和抑制不同的nirS型反硝化细菌,群落主成分受无机肥促进、牛场肥水抑制。系统发育分析结果表明,土壤中nirK型反硝化细菌主要与假单胞菌属(Pseudomonas)、产碱杆菌属(Alcaligenes)和根瘤菌属(Rhizobium)的反硝化细菌具有较近的亲缘关系;nirS型反硝化细菌主要与劳尔氏菌(Ralstonia)和红长命菌属(Rubrivivax)有较近的亲缘关系。试验土壤中反硝化微生物多与目前已报道的好氧反硝化细菌亲缘关系较近,这可能与微生物分析取自表层土有关。     

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.     

Changes in bacterial community structure in the colon of pigs fed different experimental diets and after infection with Brachyspira hyodysenteriae

Bacterial communities in the large intestines of pigs were compared using terminal restriction fragment length polymorphism (T-RFLP) analysis targeting the 16S ribosomal DNA. The pigs were fed different experimental diets based on either modified standard feed or cooked rice supplemented with dietary fibers. After feeding of the animals with the experimental diets for 2 weeks, differences in the bacterial community structure in the spiral colon were detected in the form of different profiles of terminal restriction fragments (T-RFs). Some of the T-RFs were universally distributed, i.e., they were found in all samples, while others varied in distribution and were related to specific diets. The reproducibility of the T-RFLP profiles between individual animals within the diet groups was high. In the control group, the profiles remained unchanged throughout the experiment and were similar between two independent but identical experiments. When the animals were experimentally infected with Brachyspira hyodysenteriae, causing swine dysentery, many of the T-RFs fluctuated, suggesting a destabilization of the microbial community.     

Impact of T-RFLP data analysis choices on assessments of microbial community structure and dynamics

Background

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a common DNA-fingerprinting technique used for comparisons of complex microbial communities. Although the technique is well established there is no consensus on how to treat T-RFLP data to achieve the highest possible accuracy and reproducibility. This study focused on two critical steps in the T-RFLP data treatment: the alignment of the terminal restriction fragments (T-RFs), which enables comparisons of samples, and the normalization of T-RF profiles, which adjusts for differences in signal strength, total fluorescence, between samples.

Results

Variations in the estimation of T-RF sizes were observed and these variations were found to affect the alignment of the T-RFs. A novel method was developed which improved the alignment by adjusting for systematic shifts in the T-RF size estimations between the T-RF profiles. Differences in total fluorescence were shown to be caused by differences in sample concentration and by the gel loading. Five normalization methods were evaluated and the total fluorescence normalization procedure based on peak height data was found to increase the similarity between replicate profiles the most. A high peak detection threshold, alignment correction, normalization and the use of consensus profiles instead of single profiles increased the similarity of replicate T-RF profiles, i.e. lead to an increased reproducibility. The impact of different treatment methods on the outcome of subsequent analyses of T-RFLP data was evaluated using a dataset from a longitudinal study of the bacterial community in an activated sludge wastewater treatment plant. Whether the alignment was corrected or not and if and how the T-RF profiles were normalized had a substantial impact on ordination analyses, assessments of bacterial dynamics and analyses of correlations with environmental parameters.

Conclusions

A novel method for the evaluation and correction of the alignment of T-RF profiles was shown to reduce the uncertainty and ambiguity in alignments of T-RF profiles. Large differences in the outcome of assessments of bacterial community structure and dynamics were observed between different alignment and normalization methods. The results of this study can therefore be of value when considering what methods to use in the analysis of T-RFLP data.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0360-8) contains supplementary material, which is available to authorized users.     

Elevated Atmospheric CO2 Alters Soil Microbial Communities Associated with Trembling Aspen (Populus tremuloides) Roots

Global atmospheric CO₂ levels are expected to double within the next 50 years. To assess the effects of increased atmospheric CO₂ on soil ecosystems, cloned trembling aspen (Populus tremuloides) seedlings were grown individually in 1 m³ open bottom root boxes under either elevated (720 ppm, ELEV) or ambient CO₂ (360 ppm, AMB). After 5 years, soil cores (40 cm depth) were collected from the root boxes and divided into 0–20 cm and 20–40 cm fractions. ELEV treatment resulted in significant decreases in both soil nitrate and total soil nitrogen in both the 0–20 cm and 20–40 cm soil fractions, with a 47% decrease in soil nitrate and a 50% decrease in total soil nitrogen occurring in the 0–20 cm fraction. ELEV treatment did not result in a significant change in the amount of soil microbial biomass. However, analysis of indicator phospholipid fatty acids (PLFA) indicated that ELEV treatment did result in significant increases in PLFA indicators for fungi and Gram-negative bacteria in the 0–20 cm fraction. Terminal restriction fragment length polymorphism (T-RFLP) analysis was used to analyze the composition of the soil bacterial communities (using primers targeting the 16SrRNA gene) and the soil fungal communities (using primers targeting the intergenic transcribed spacer region). T-RFLP analysis revealed shifts in both bacterial and fungal community structure, as well as increases in both bacterial and fungal species richness with ELEV treatment. These results indicated that increased atmospheric CO₂ had significant effects on both soil nutrient availability and the community composition of soil microbes associated with aspen roots.     

Nest specificity of the bacterial community in termite guts (Hodotermes mossambicus)

While recent results have provided strong evidence for the presence of a stable gut microbiota among several termite species, little is known about variations at the colony or individual level. Using a cultivation-independent approach, we investigated the structure of the bacterial community in the gut of termites from four different colonies of Hodotermes mossambicus. 16S rRNA-based terminal restriction fragment length polymorphism (T-RFLP) analysis of the bacterial gut microbiota revealed (1) a high consistency of the gut microbiota among nestmates and (2) subtle but distinct differences in community structure between individuals from different colonies. Since products of bacterial metabolism may contribute to a colony odor that can be used as discriminatory signal, the presence of a colony-specific bacterial community adds support to the hypothesis that the gut microbiota of termites is involved in nestmate recognition. Received 12 July 2005; revised 10 February and 15 March 2006; accepted 7 April 2006.     

Bacterial Community Structure of Sediments of the Bizerte Lagoon (Tunisia), a Southern Mediterranean Coastal Anthropized Lagoon

In order to estimate how pollution affects the bacterial community structure and composition of sediments, chemical and molecular approaches were combined to investigate eight stations around the Bizerte lagoon. Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified 16S rRNA genes revealed that each station was characterized by a specific bacterial community structure. The combination of this data with those of chemical analysis showed a correlation between the bacterial fingerprint and the pollutant content, principally with hydrocarbon pollution. The composition of the bacterial community of two contrasted stations related to the pollution revealed sequences affiliated to α, β, γ, δ, ε subclass of the Proteobacteria, Actinobacteria, and Acidobacteria in both stations although in different extent. Gamma and delta subclass of the Proteobacteria were dominant and represent 70% of clones in the heavy-metal-contaminated station and 47% in the polyaromatic hydrocarbon (PAH)-contaminated. Nevertheless, most of the sequences found were unaffiliated to cultured bacteria. The adaptation of the bacterial community mainly to PAH compounds demonstrated here and the fact that these bacterial communities are mainly unknown suggest that the Bizerte lagoon is an interesting environment to understand the capacity of bacteria to cope with some pollutants.