Terminal Restriction Fragment Length Polymorphism Fingerprints Research Articles

Determination of the discriminant score of intestinal microbiota as a biomarker of disease activity in patients with ulcerative colitis

BackgroundIn recent years, the gut microbiota has been found to provide an important link to the development of inflammatory bowel diseases (IBD) like ulcerative colitis (UC). Accordingly, inter-individual variation in the gut microbial community may be linked to inter-individual variation in the risk of IBD or other diseases. Further, the Terminal Restriction Fragment Length Polymorphism (T-RFLP) is a molecular biology technique for profiling bacterial species in faecal samples. This study was to evaluate a biomarker based on intestinal microbiota.MethodsThe study subjects were 69 patients with UC together with 80 relatives as controls. Twenty-three patients had active UC (group I) and 46 had quiescent UC (group II). The later included 17 patients with mild inflammation in the large intestine (group IIa), 29 without inflammation (group IIb). The patients’ relatives were consanguineous (group III, n = 47), and non-consanguineous (group IV, n = 33). Faecal samples were obtained from all subjects for the investigation of intestinal microbiota by applying the T-RFLP method. The Discriminant analysis of operational-taxonomic-unit (OTU) on T-RFLP fingerprints was performed. The Canonical Discriminant Function Coefficient (Df) for each OTU was calculated. The individual OTUs were multiplied by the Df value, and the sum was termed the Discriminant Score (Ds).ResultsThe Ds decreased thus: group I > group IIa > group IIb > group III > group IV. Significant difference was calculated for group I vs group IV (P < 0.01), group I vs group IIb (P < 0.05), group I vs group III (P < 0.01), group IIa vs IV (P < 0.01), group IIb vs group IV (P < 0.01), group III vs group IV (P < 0.01), indicating a strong association between gut microbial species and the development of UC.ConclusionsIn this study, the Ds related to UC, or otherwise absence of UC in the five groups. Potentially, Ds may become a clinically relevant biomarker of disease activity in UC. To our knowledge, this is the first application of the Ds to the study of microbiota in UC patients, consanguineous and non-consanguineous relatives.Trial registrationClinical trial No: UMIN 000004123.

An Improved Protocol for Extraction of Metagenomic DNA from High Humus, Alkaline and Saline Soil of Chinampa for T-RFLP Fingerprinting Analysis

Aims: The principle aim of this study was to obtain high quality metagenomic DNA from the high humus-containing, alkaline soils of the chinampas, an artificial sustainable agroecosystem. Study Design: Different protocols reported previously were tested and were modified to extract the metagenomic DNA. Quality of the DNA samples was evaluated by amplification of 16S rRNA gene with PCR and T-RFLP (Terminal Restriction Fragment Length Polymorphism) analysis. Place and Duration of Study: This study was performed in Department of Microbiology, Escuela Nacional de Ciencias Biologicas, Instituto Politecnico Nacional during 2011-2012. Methodology: Four soil samples were collected from two chinampas at the depth of 0-30 Original Research Article British Microbiology Research Journal, 4(7): 821-830, 2014 822 cm and 30-60cm. A protocol started with repeated prewashing before the direct cell lysis with lysozyme followed by SDS treatments, frozen and melting cycling was developed which combined the DNA isolation and purification procedures. The 16SrRNA genes were amplified from the extracted metagenomic DNAs and were used for T-RFLP fingerprinting analysis. Results: The 16SrRNA genes were amplified from all the DNA extracts corresponding to the four soil samples and were successfully used in the T-RFLP analysis, which generated 25 to 109T-RFs in the four soil samples digested separately with the restriction endonucleases HaeIII, HhaI and MspI. Conclusion: The protocol developed in the present study could generate high molecular weight and high quality metagenomic DNA from soils with high content of humic materials, for which the other reported protocols were not functioned. This soil harboured very diverse and unique bacterial communities belonging to at least nine phyla that might contribute to the high soil fertility.

Biases for detecting arbuscular mycorrhizal fungal mixture by terminal restriction fragment length polymorphism (T-RFLP)

Terminal restriction fragment length polymorphism (T-RFLP) analysis of amplified ribosomal RNA genes is used for profiling microbial communities and sometimes for species richness and relative abundance estimation in environmental samples. However, the T-RFLP fingerprint may be subject to biases during the procedure, influencing the detection of real community structures in the environment. To investigate possible sources of T-RFLP bias, 18S rRNA gene clones derived from two arbuscular mycorrhizal fungal sequences were combined in simple pairwise mixes to assess the effects of polymerase chain reaction cycle number, plant genomic DNA purification method and varying template ratio on the template-to-product ratio as measured by relative T-RF peak area. Varying cycle numbers indicated that amplification was still in the exponential phase at the cycle numbers lower than 18, so these small cycle numbers were used for the comparison of template-to-product quantities. Relative abundance estimated from T-RF peak ratios varied with different purification procedures, but the best results, closest to input ratios, were obtained by using phenol-chloroform purification. The presence of an excess of unpurified non-target plant genomic DNA generated a bias towards lower or overestimation of relative abundance. We conclude that a low number of amplification cycles and stringent DNA purification are necessary for accurate mixed sample analysis by T-RFLP.

Secondary succession of bacterial communities and co-occurrence of phylotypes in oil-polluted Populus rhizosphere

Oil pollution can be considered as a driver for bacterial secondary succession. A greenhouse study was conducted using forest mineral soil contaminated by heavy fuel oil and planted with hybrid aspen. The aim was to compare the bacterial succession in oil- and plant-amended ecosystems. Samples were taken weekly from bulk and rhizosphere soil. The oil degrading bacteria were enumerated by most-probable number assay and the bacterial community structures were determined by terminal restriction fragment length polymorphism (T-RFLP) of the 16S rRNA and extradiol dioxygenase genes. Hierarchical clustering of phylotypes coupled with heatmap analysis of T-RFLP fingerprints revealed patterns of co-occurrence of bacterial groups. Co-occurring OTUs present only in the oil treatments were observed from both 16S rRNA and extradiol dioxygenase gene heatmaps, which represent the bacteria adapted to oil pollution. A rhizosphere effect was detected in oil-contaminated soil with elevated bacterial 16S rRNA gene diversity and MPN counts. The forest soil was dominated by two phyla, Acidobacteria and Proteobacteria, while Betaproteobacteria were clearly activated by addition of oil. Directional succession of bacterial communities was unique to oil treatments, shown by the 16S rRNA gene community structure. These results advance our understanding of secondary succession of bacterial communities in bio- and rhizoremediation.

Testing the Limits of 454 Pyrotag Sequencing: Reproducibility, Quantitative Assessment and Comparison to T-RFLP Fingerprinting of Aquifer Microbes

The characterization of microbial community structure via 16S rRNA gene profiling has been greatly advanced in recent years by the introduction of amplicon pyrosequencing. The possibility of barcoding gives the opportunity to massively screen multiple samples from environmental or clinical sources for community details. However, an on-going debate questions the reproducibility and semi-quantitative rigour of pyrotag sequencing, similar to the early days of community fingerprinting. In this study we demonstrate the reproducibility of bacterial 454 pyrotag sequencing over biological and technical replicates of aquifer sediment bacterial communities. Moreover, we explore the potential of recovering specific template ratios via quantitatively defined template spiking to environmental DNA. We sequenced pyrotag libraries of triplicate sediment samples taken in annual sampling campaigns at a tar oil contaminated aquifer in Düsseldorf, Germany. The abundance of dominating lineages was highly reproducible with a maximal standard deviation of ∼4% read abundance across biological, and ∼2% across technical replicates. Our workflow also allows for the linking of read abundances within defined assembled pyrotag contigs to that of specific ‘in vivo’ fingerprinting signatures. Thus we demonstrate that both terminal restriction fragment length polymorphism (T-RFLP) analysis and pyrotag sequencing are capable of recovering highly comparable community structure. Overall diversity was roughly double in amplicon sequencing. Pyrotag libraries were also capable of linearly recovering increasing ratios (up to 20%) of 16S rRNA gene amendments from a pure culture of Aliivibrio fisheri spiked to sediment DNA. Our study demonstrates that 454 pyrotag sequencing is a robust and reproducible method, capable of reliably recovering template abundances and overall community structure within natural microbial communities.

RRNA-based profiling of bacteria in the axilla of healthy males suggests right-left asymmetry in bacterial activity

The activity of the human armpit microbiota triggers the formation of body odor. We used differential 16S rRNA gene (rDNA)- and rRNA-based terminal-restriction fragment length polymorphism fingerprinting in combination with cloning and sequencing to identify active members of the human armpit microbiota. DNA and RNA were isolated from skin scrub samples taken from both armpits of 10 preconditioned, healthy males. The fingerprint profiles indicated pronounced similarities between the armpit microbiota in the right and the left axillae of an individual test person, but larger differences between the axilla microbiota of different individuals. Using 16S rDNA and rRNA sequence data, the majority of peaks in the armpit profiles were assigned to bacteria affiliated with well-known genera of skin bacteria. The relative abundances of all groups were similar among the rDNA and rRNA samples, suggesting that all groups of armpit bacteria were active. Surprisingly, the relative abundance of sequences affiliated with Peptoniphilus sp. was by far and with statistical significance the highest in the rRNA samples of the right armpits. Thus, bacteria affiliated with Peptoniphilus sp. might have been particularly active in the right axillae of the test persons, possibly owing to the handedness of the test persons, which might cause different environmental conditions in the right axillae.

The influence of the cage system and colonisation of Salmonella Enteritidis on the microbial gut flora of laying hens studied by T-RFLP and 454 pyrosequencing

BackgroundIn the EU conventional cages for laying hens are forbidden beginning in January 2012, however concerns about a higher transmission rate of Salmonella in alternative cages systems have been raised. The extent to which cage systems may affect the intestinal microbiota of laying hens is not known, and different microbiota may demonstrate different resistance towards colonization with Salmonella. To investigate this, ileal and caecal samples from two experimental studies where laying hens were inoculated with Salmonella Enteritidis and housed in different systems (conventional cage, furnished cage or aviary), were compared using Terminal Restriction Fragment Length Polymorphism (T-RFLP). The distribution of genera in the microbiota in caecum was furthermore described by next generation sequencing of 16S rDNA libraries.ResultsHens in the same cage type developed similar T-RFLP fingerprints of the ileal and caecal microbiota, and these could be separated from layers in the other cages types. No significant difference in the fingerprint profiles could be observed between Salmonella positive and negative samples from same cage. By deep sequencing of 16S rDNA libraries from caecum, 197 different Operational Taxonomic Units (OTU) were identified, and 195 and 196 OTU respectively, were found in hens in aviary and furnished cages, but only 178 OTU of these were recovered from conventional cages. The ratio between the dominating phyla or families and genera in the microbiota remained fairly constant throughout the study. Faecalibacterium and Butyricimonas were the most prevalent genera found in the caecal microbiota of layers irrespective of the cage type.ConclusionsHens confined in the same cage group tend to develop similar microbiota in their ileum and caecum possibly due to isolation, while differences in the microbiota between cages may be caused by environmental or individual bird factors. Although the cages type had influence on composition of the microbiota in the layers by promoting higher diversity in furnished and aviary systems, we did not observe differences in colonization and excretion pattern of Salmonella from these groups. We suggest, that differences in group size and exposure to a more faecally contaminated environment in the alternative systems may explain the observed differences in diversity of the caecal microbiota.

Community analysis of ammonia-oxidizing bacteria in activated sludge of eight wastewater treatment systems

We investigated the communities of ammonia-oxidizing bacteria (AOB) in activated sludge collected from eight wastewater treatment systems using polymerase chain reaction (PCR) followed by terminal restriction fragment length polymorphism (T-RFLP), cloning, and sequencing of the α-subunit of the ammonia monooxygenase gene ( amoA). The T-RFLP fingerprint analyses showed that different wastewater treatment systems harbored distinct AOB communities. However, there was no remarkable difference among the AOB T-RFLP profiles from different parts of the same system. The T-RFLP fingerprints showed that a full-scale wastewater treatment plant (WWTP) contained a larger number of dominant AOB species than a pilot-scale reactor. The source of influent affected the AOB community, and the WWTPs treating domestic wastewater contained a higher AOB diversity than those receiving mixed domestic and industrial wastewater. However, the AOB community structure was little affected by the treatment process in this study. Phylogenetic analysis of the cloned amoA genes clearly indicated that all the dominant AOB in the systems was closely related to Nitrosomonas spp. not to Nitrosospira spp. Members of the Nitrosomonas oligotropha and Nitrosomonas communis clusters were found in all samples, while members of Nitrosomonas europaea cluster occurred in some systems.

T-RFPred: a nucleotide sequence size prediction tool for microbial community description based on terminal-restriction fragment length polymorphism chromatograms

BackgroundTerminal-Restriction Fragment Length Polymorphism (T-RFLP) is a technique used to analyze complex microbial communities. It allows for the quantification of unique or numerically dominant phylotypes in amplicon pools and it has been used primarily for comparisons between different communities. T-RFPred, Terminal-Restriction Fragment Prediction, was developed to identify and assign taxonomic information to chromatogram peaks of a T-RFLP fingerprint for a more comprehensive description of microbial communities. The program estimates the expected fragment size of representative 16S rRNA gene sequences (either from a complementary clone library or from public databases) for a given primer and restriction enzyme(s) and provides candidate taxonomic assignments.ResultsTo show the accuracy of the program, T-RFLP profiles of a marine bacterial community were described using artificial bacterioplankton clone libraries of sequences obtained from public databases. For all valid chromatogram peaks, a phylogenetic group could be assigned.ConclusionsT-RFPred offers enhanced functionality of T-RFLP profile analysis over current available programs. In particular, it circumvents the need for full-length 16S rRNA gene sequences during taxonomic assignments of T-RF peaks. Thus, large 16S rRNA gene datasets from environmental studies, including metagenomes, or public databases can be used as the reference set. Furthermore, T-RFPred is useful in experimental design for the selection of primers as well as the type and number of restriction enzymes that will yield informative chromatograms from natural microbial communities.

Microbial community structure changes during oyster mushroom substrate preparation

Although oyster mushroom (Pleurotus spp.) is a valuable food, cultivated worldwide on an industrial scale, still very little is known about the microbial dynamics during oyster mushroom substrate preparation. Therefore, the characterization of the microbial dynamics by chemical and biological tools was the objective of this study. During substrate preparation, enzymatic digestibility of the substrate improved by 77%, whereas the cellulose and hemicellulose to lignin ratios decreased by 9% and 19%, respectively. Fluorescein diacetate hydrolysis reached its minimum value at the temperature maximum of the process during the composting phase and exceeded the initial level at the end of the process. Fungal species played part in the initial mesophilic phase of the substrate preparation process, but they disappeared after pasteurization in tunnels at constant elevated temperatures. Changes in the microbiota showed a marked bacterial community succession during substrate preparation investigated by 16S ribosomal deoxyribonucleic acid-based terminal restriction fragment length polymorphism (T-RFLP). Mature samples represented the least variance, which indicated the effect of the standardized preparation protocol. The relation between mushroom yield and the bacterial community T-RFLP fingerprints was investigated, but the uniformity of mushroom yields did not support any significant correlation.

Zeta-Proteobacteria Dominate the Colonization and Formation of Microbial Mats in Low-Temperature Hydrothermal Vents at Loihi Seamount, Hawaii

In situ colonization experiments were performed to study the pioneer populations of bacteria at Loihi Seamount, Hawaii. Over a ten-year sampling period, 41 microbial growth chambers (MGCs) were deployed and recovered in Pele's Pit and the surrounding area after short-term (4–10 days) and long-term (∼1–6 years) incubations in the flow of hydrothermal effluent. Terminal-restriction fragment length polymorphism (T-RFLP) analysis of the small subunit rRNA gene (SSU rDNA) revealed that the short-term MGC communities exhibited a low number of represented populations when compared to the long-term MGC communities and naturally occurring microbial mats. Cluster analysis of T-RFLP fingerprints showed the short-term MGC communities all had similar richness but were separated into three distinct groups with different arrays of colonizing populations. Clone library analysis showed that cooler vents (T ave = 40°C) were primarily colonized by Mariprofundus ferrooxydans, a neutrophilic Fe-oxidizing ζ −Proteobacteria while warmer vents (T ave = 71°C) were colonized by Sulfurimonas spp. and other sulfur-cycling members of the ϵ −Proteobacteria. Vents with an intermediate temperature (T ave = 51°C) were colonized by representatives of both ζ -Proteobacteria and ϵ -Proteobacteria. Long-term MGC communities did not cluster with any of the short-term communities and exhibited higher richness, indicating a greater number of bacterial populations were able to colonize and grow in the long-term growth chambers.

Terminal-restriction fragment length polymorphism analysis of biphenyl dioxygenase genes from a polychlorinated biphenyl-polluted soil

Polychlorinated biphenyls (PCBs) are ubiquitous persistent organic pollutants that can be co-metabolically biotransformed by biphenyl-utilizing bacteria. In this study, terminal- restriction fragment length polymorphism (T-RFLP) was applied to the substrate specificity-determining region of the 2,3-biphenyl dioxygenase encoding genes of a microbial community found in a PCB-polluted soil. Notably, both the total biphenyl/PCB-utilizing community and its members actively expressing the 2,3-biphenyl dioxygenase gene were analyzed. T-RFLP fingerprinting along with gene library construction allowed us not only to detect biphenyl dioxygenases related to the well characterized catabolic patterns of Pseudomonas pseudoalcaligenes KF707 and Burkholderia xenovorans LB400, but also to reveal novel environmental enzyme classes displaying amino acid substitutions that may be related to broader specificity and improved catalytic properties. Furthermore, space and time of sampling along with bioavailability conditions of different PCBs were considered possible sources of profile variability.

Plant-by-Plant Variations of Bacterial Communities Associated with Leaves of the Nickel Hyperaccumulator Alyssum bertolonii Desv.

Bacteria associated with tissues of metal-hyperaccumulating plants are of great interest due to the multiple roles they may play with respect to plant growth and resistance to heavy metals. The variability of bacterial communities associated with plant tissues of three populations of Alyssum bertolonii, a Ni hyperaccumulator endemic of serpentine outcrops of Central Italy, was investigated. Terminal-restriction fragment length polymorphism (T-RFLP) analysis of bacterial 16S rRNA genes was applied to DNA extracted from leaf tissues of 30 individual plants from three geographically separated serpentine outcrops. Moreover, T-RFLP fingerprinting was also performed on DNA extracted from the same soils from which the plants were collected. Fifty-nine unique terminal-restriction fragments (TRFs) were identified, with more than half of the taxonomically interpreted TRFs assigned to Alpha- and Gamma-Proteobacteria and Clostridia. Data were then used to define the extent of variation of bacterial communities due to single plants or to plant populations. Results indicated a very high plant-by-plant variation of leaf-associated community (more than 93% of total variance observed). However, a core (numerically small) of plant-specific TRFs was found. This work demonstrates that plant-associated bacterial communities represent a large reservoir of biodiversity and that the high variability existing between plants, even from the same population, should be taken into account in future studies on association between bacteria and metal-hyperaccumulating plants.

Linking phylogenetic identities of bacteria to starch fermentation in an in vitro model of the large intestine by RNA‐based stable isotope probing

Carbohydrates, including starches, are an important energy source for humans, and are known for their interactions with the microbiota in the digestive tract. Largely, those interactions are thought to promote human health. Using 16S ribosomal RNA (rRNA)-based stable isotope probing (SIP), we identified starch-fermenting bacteria under human colon-like conditions. To the microbiota of the TIM-2 in vitro model of the human colon 7.4 g l(-1) of [U-(13)C]-starch was added. RNA extracted from lumen samples after 0 (control), 2, 4 and 8 h was subjected to density-gradient ultracentrifugation. Terminal-restriction fragment length polymorphism (T-RFLP) fingerprinting and phylogenetic analyses of the labelled and unlabelled 16S rRNA suggested populations related to Ruminococcus bromii, Prevotella spp. and Eubacterium rectale to be involved in starch metabolism. Additionally, 16S rRNA related to that of Bifidobacterium adolescentis was abundant in all analysed fractions. While this might be due to the enrichment of high-GC RNA in high-density fractions, it could also indicate an active role in starch fermentation. Comparison of the T-RFLP fingerprints of experiments performed with labelled and unlabelled starch revealed Ruminococcus bromii as the primary degrader in starch fermentation in the studied model, as it was found to solely predominate in the labelled fractions. LC-MS analyses of the lumen and dialysate samples showed that, for both experiments, starch fermentation primarily yielded acetate, butyrate and propionate. Integration of molecular and metabolite data suggests metabolic cross-feeding in the system, where populations related to Ruminococcus bromii are the primary starch degrader, while those related to Prevotella spp., Bifidobacterium adolescentis and Eubacterium rectale might be further involved in the trophic chain.

Activity and diversity of methanotrophs in the soil-water interface and rhizospheric soil from a flooded temperate rice field

To combine molecular and cultivation techniques to characterize the methanotrophic community in the soil-water interface (SWI) and rhizospheric soil from flooded rice fields in Uruguay, a temperate region in South America. A novel type I, related to the genus Methylococcus, and three type II methanotrophs were isolated from the highest positive dilution steps from the most probable number (MPN) counts. Potential methane oxidation activities measured in slurried samples were higher in the rhizospheric soil compared to the SWI and were stimulated by N-fertilization. PmoA (particulate methane monooxygenase) clone libraries were constructed for both rice microsites. SWI clones clustered in six groups related to cultivated and uncultivated members from different ecosystems of the genera Methylobacter, Methylomonas, Methylococcus and a novel type I sublineage while cultivation and T-RFLP (terminal restriction fragment length polymorphism) analysis confirmed the presence of type II methanotrophs. Cultivation techniques, cloning analysis and T-RFLP fingerprinting of the pmoA gene revealed a diverse methanotrophic community in the rice rhizospheric soil and SWI. This study reports, for the first time, the analysis of the methanotrophic diversity in rice SWI and this diversity may be exploited in reducing methane emissions.

DGGE and T-RFLP Analysis of Bacterial Succession during Mushroom Compost Production and Sequence-aided T-RFLP Profile of Mature Compost

The amount of button mushroom (Agaricus bisporus) harvested from compost is largely affected by the microbial processes taking place during composting and the microbes inhabiting the mature compost. In this study, the microbial changes during the stages of this specific composting process were monitored, and the dominant bacteria of the mature compost were identified to reveal the microbiological background of the favorable properties of the heat-treated phase II mushroom compost. 16S ribosomal deoxyribonucleic acid (rDNA)-based denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) molecular fingerprinting methods were used to track the succession of microbial communities in summer and winter composting cycles. DNA from individual DGGE bands were reamplified and subjected to sequence analysis. Principal component analysis of fingerprints of the composting processes showed intensive changes in bacterial community during the 22-day procedure. Peak temperature samples grouped together and were dominated by Thermus thermophilus. Mature compost patterns were almost identical by both methods (DGGE, T-RFLP). To get an in-depth analysis of the mature compost bacterial community, the sequence data from cultivation of the bacteria and cloning of environmental 16S rDNA were uniquely coupled with the output of the environmental T-RFLP fingerprints (sequence-aided T-RFLP). This method revealed the dominance of a supposedly cellulose-degrading consortium composed of phylotypes related to Pseudoxanthomonas, Thermobifida, and Thermomonospora.

High aromatic ring-cleavage diversity in birch rhizosphere: PAH treatment-specific changes of I.E.3 group extradiol dioxygenases and 16S rRNA bacterial communities in soil

Genes encoding key enzymes of catabolic pathways can be targeted by DNA fingerprinting to explore genetic degradation potential in pristine and polluted soils. We performed a greenhouse microcosm experiment to elucidate structural and functional bacterial diversity in polyaromatic hydrocarbon (PAH)-polluted soil and to test the suitability of birch (Betula pendula) for remediation. Degradation of PAHs was analysed by high-performance liquid chromatography, DNA isolated from soil amplified and fingerprinted by restriction fragment length polymorphism (RFLP) and terminal restriction fragment length polymorphism (T-RFLP). Bacterial 16S rRNA T-RFLP fingerprinting revealed a high structural bacterial diversity in soil where PAH amendment altered the general community structure as well as the rhizosphere community. Birch augmented extradiol dioxygenase diversity in rhizosphere showing a rhizosphere effect, and further pyrene was more efficiently degraded in planted pots. Degraders of aromatic compounds upon PAH amendment were shown by the changed extradiol ring-cleavage community structure in soil. The RFLP analysis grouped extradiol dioxygenase marker genes into 17 distinct operational taxonomic units displaying novel phylogenetic clusters of ring-cleavage dioxygenases representing putative catabolic pathways, and the peptide sequences contained conserved amino-acid signatures of extradiol dioxygenases. A branch of major environmental TS cluster was identified as being related to Parvibaculum lavantivorans ring-cleavage dioxygenase. The described structural and functional diversity demonstrated a complex interplay of bacteria in PAH pollution. The findings improve our understanding of rhizoremediation and unveil the extent of uncharacterized enzymes and may benefit bioremediation research by facilitating the development of molecular tools to detect and monitor populations involved in degradative processes.

Fingerprinting methods to approach multitrophic interactions among microflora and microfauna communities in soil

The soil functioning and the response of the biota to external perturbations such as organic input are based on multitrophic interactions among a wide range of organisms. However, the various components of the soil microflora and microfauna are rarely addressed in ecological studies. We have developed a molecular approach based on terminal restriction fragment length polymorphism (T-RFLP) analysis to assess the community structures of protozoa and nematodes, together with bacteria and fungi that share the same soil environment. Two soils were characterized by a specific fingerprint for each of the four groups of organisms, showing the potential of all T-RFLP procedures to differentiate the community structures. The response of the soil biota to organic inputs was addressed using T-RFLP fingerprints together with physiological profiles of bacteria communities and global microbial activities and densities. Although the impact of compost or manure on the soil biota was only slightly noticeable from the global parameters measured, it was obvious from the community level analyses. However, the different components of the soil biota were altered to various extents, depending on the group of organisms and the soil–organic matter combination. The potential of the T-RFLP strategy to analyze simultaneously different biotic groups from the same soil DNA extract will facilitate the more systematic integration of eukaryotic organisms in ecological studies to investigate multitrophic interactions among the microflora and microfauna in relation with soil processes.

Characterization of Nitrifying, Denitrifying, and Overall Bacterial Communities in Permeable Marine Sediments of the Northeastern Gulf of Mexico

Sandy or permeable sediment deposits cover the majority of the shallow ocean seafloor, and yet the associated bacterial communities remain poorly described. The objective of this study was to expand the characterization of bacterial community diversity in permeable sediment impacted by advective pore water exchange and to assess effects of spatial, temporal, hydrodynamic, and geochemical gradients. Terminal restriction fragment length polymorphism (TRFLP) was used to analyze nearly 100 sediment samples collected from two northeastern Gulf of Mexico subtidal sites that primarily differed in their hydrodynamic conditions. Communities were described across multiple taxonomic levels using universal bacterial small subunit (SSU) rRNA targets (RNA- and DNA-based) and functional markers for nitrification (amoA) and denitrification (nosZ). Clonal analysis of SSU rRNA targets identified several taxa not previously detected in sandy sediments (i.e., Acidobacteria, Actinobacteria, Chloroflexi, Cyanobacteria, and Firmicutes). Sequence diversity was high among the overall bacterial and denitrifying communities, with members of the Alphaproteobacteria predominant in both. Diversity of bacterial nitrifiers (amoA) remained comparatively low and did not covary with the other gene targets. TRFLP fingerprinting revealed changes in sequence diversity from the family to species level across sediment depth and study site. The high diversity of facultative denitrifiers was consistent with the high permeability, deeper oxygen penetration, and high rates of aerobic respiration determined in these sediments. The high relative abundance of Gammaproteobacteria in RNA clone libraries suggests that this group may be poised to respond to short-term periodic pulses of growth substrates, and this observation warrants further investigation.

Tilting at Windmills: a Response to a Recent Critique of Terminal Restriction Fragment Length Polymorphism Data

Blackwood et al. (1) assert that terminal restriction fragment length polymorphism (T-RFLP) data cannot be used to estimate the species level diversity of bacterial communities. While I do not argue with their analytical techniques, nor the interpretation of their results, their conclusion is essentially moot as T-RFLP techniques are rarely used (nor should they be used) to study the diversity of complex microbial communities at the species level. A more useful test would have been to determine if T-RFLP accurately captures bacterial diversity at coarser levels of taxonomic resolution, for which the technique is undoubtedly better suited. On the basis of their analyses, Blackwood et al. (1) argue that our work (2) should be reinterpreted. However, nowhere in our paper do we indicate that T-RFLP captures diversity at the species level, and for this reason, their analyses have no bearing on our study. My goal is not to unilaterally defend T-RFLP or related fingerprinting methods; to do so would be foolish given that such methods are rapidly being replaced by techniques that will allow us to compare a large number of communities at a high level of taxonomic resolution. Rather, my goal is to point out that we do science with the techniques we have, not the techniques we might wish to have [to paraphrase the former Secretary of Defense of the United States (“…you go to war with the Army you have. They're not the Army you might want or wish to have at a later time.” Donald H. Rumsfeld, 8 December 2004, Kuwait)]. We could spend our careers identifying every individual species in a handful of samples (currently a Sisyphean task), or we could look for patterns in microbial community composition by analyzing a relatively large number of samples at coarser scales of taxonomic resolution and moving to finer scales of resolution as the appropriate techniques become available. Although T-RFLP may not allow us to estimate diversity at very fine levels of phylogenetic resolution, such was not our goal and there is no reason to assume that the species level is the most appropriate level of taxonomic resolution for comparing levels of microbial diversity.