Terminal Restriction Fragment Length Polymorphism Approach Research Articles

Botanical microbiomes on the cheap: Inexpensive molecular fingerprinting methods to study plant-associated communities of bacteria and fungi.

High‐throughput sequencing technologies have revolutionized the study of plant‐associated microbial populations, but they are relatively expensive. Molecular fingerprinting techniques are more affordable, yet yield considerably less information about the microbial community. Does this mean they are no longer useful for plant microbiome research? In this paper, we review the past 10 years of studies on plant‐associated microbiomes using molecular fingerprinting methodologies, including single‐strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), amplicon length heterogeneity PCR (LH‐PCR), ribosomal intergenic spacer analysis (RISA) and automated ribosomal intergenic spacer analysis (ARISA), and terminal restriction fragment length polymorphism (TRFLP). We also present data juxtaposing results from TRFLP methods with those generated using Illumina sequencing in the comparison of rhizobacterial populations of Brazilian maize and fungal surveys in Canadian tomato roots. In both cases, the TRFLP approach yielded the desired results at a level of resolution comparable to that of the MiSeq method, but at a fraction of the cost. Community fingerprinting methods (especially TRFLP) remain relevant for the identification of dominant microbes in a population, the observation of shifts in plant microbiome community diversity, and for screening samples before their use in more sensitive and expensive approaches.

Long-term monitoring reveals stable and remarkably similar microbial communities in parallel full-scale biogas reactors digesting energy crops.

Biogas is an important renewable energy carrier. It is a product of stepwise anaerobic degradation of organic materials by highly diverse microbial communities forming complex interlinking metabolic networks. Knowledge about the microbial background of long-term stable process performance in full-scale reactors is crucial for rationally improving the efficiency and reliability of biogas plants. To generate such knowledge, in the present study three parallel mesophilic full-scale reactors fed exclusively with energy crops were sampled weekly over one year. Physicochemical process parameters were determined and the microbial communities were analysed by terminal restriction fragment length polymorphism (T-RFLP) fingerprinting and 454-amplicon sequencing. For investigating the methanogenic community, a high-resolution T-RFLP approach based on the mcrA gene was developed by selecting restriction enzymes with improved taxonomic resolution compared to previous studies. Interestingly, no Methanosarcina-related generalists, but rather specialized hydrogenotrophic and acetoclastic methanogenic taxa were detected. In general, the microbial communities in the non-connected reactors were remarkably stable and highly similar indicating that identical environmental and process parameters resulted in identical microbial assemblages and dynamics. Practical implications such as flexible operation schemes comprising controlled variations of process parameters for an efficient microbial resource management under fluctuating process conditions are discussed.

A time-course analysis of four full-scale anaerobic digesters in relation to the dynamics of change of their microbial communities

This study describes the microbial community richness, -dynamics, and -organization of four full-scale anaerobic digesters during a time-course study of 45 days. The microbial community was analyzed using a Bacteria- and Archaea-targeting 16S rRNA gene-based Terminal-Restriction Fragment Length Polymorphism approach. Clustering analysis separated meso- and thermophilic reactors for both archaeal and bacterial communities. Regardless of the operating temperature, each installation possessed a distinct community profile. For both microbial domains, about 8 dominant terminal-restriction fragments could be observed, with a minimum of 4 and a maximum of 14. The bacterial community organization (a coefficient which describes the specific degree of evenness) showed a factor 2 more variation in the mesophilic reactors, compared with the thermophilic ones. The archaeal community structure of the mesophilic UASB reactor was found to be more stable. The community composition was highly dynamic for Bacteria and Archaea, with a rate of change between 20-50% per 15 days. This study illustrated that microbial communities in full-scale anaerobic digesters are unique to the installation and that community properties are dynamic. Converging complex microbial processes such as anaerobic digestion which rely on a multitude of microbial teams apparently can be highly dynamic.

Novel lineages of Prochlorococcus thrive within the oxygen minimum zone of the eastern tropical South Pacific

The eastern tropical Pacific Ocean holds two of the main oceanic oxygen minimum zones of the global ocean. The presence of an oxygen-depleted layer at intermediate depths, which also impinges on the seafloor and in some cases the euphotic zone, plays a significant role in structuring both pelagic and benthic communities, and also in the vertical partitioning of microbial assemblages. Here, we assessed the genetic diversity and distribution of natural populations of the cyanobacteria Prochlorococcus and Synechococcus within oxic and suboxic waters of the eastern tropical Pacific using cloning and sequencing, and terminal restriction fragment length polymorphism (T-RFLP) analyses applied to the 16S-23S rRNA internal transcribed spacer region. With the T-RFLP approach we could discriminate 19 cyanobacterial clades, of which 18 were present in the study region. Synechococcus was more abundant in the surface oxic waters of the eastern South Pacific, while Prochlorococcus dominated the subsurface low-oxygen waters. Two of the dominant clades in the oxygen-deficient waters belong to novel and yet uncultivated lineages of low-light adapted Prochlorococcus.

Development of a cultivation‐independent approach for the study of genetic diversity of Sinorhizobium meliloti populations

The development of a species-specific marker for the analysis of the genetic polymorphism of the nitrogen-fixing symbiotic bacterium Sinorhizobium meliloti directly from environmental DNA is reported. The marker is based on terminal-restriction fragment length polymorphism (T-RFLP) methodology targeting specifically the 16S-23S Ribosomal Intergenic Spacer of S. meliloti. Species-specificity and polymorphism of the marker were tested on DNA extracted from soil samples and from a collection of 130 S. meliloti bacterial isolates. These primers and the T-RFLP approach proved useful for the detection and analysis of polymorphism of S. meliloti populations.

Assessing the relationship between patch type and soil mites: A molecular approach

An urgent need exists for indicators of soil health and patch functionality in extensive rangelands that can be measured efficiently and at low cost. Soil mites are candidate indicators, but their identification and handling is so specialised and time-consuming that their inclusion in routine monitoring is unlikely. The aim of this study was to measure the relationship between patch type and mite assemblages using a conventional approach. An additional aim was to determine if a molecular approach traditionally used for soil microbes could be adapted for soil mites to overcome some of the bottlenecks associated with soil fauna diversity assessment. Soil mite species abundance and diversity were measured using conventional ecological methods in soil from patches with perennial grass and litter cover (PGL), and compared to soil from bare patches with annual grasses and/or litter cover (BAL). Soil mite assemblages were also assessed using a molecular method called terminal-restriction fragment length polymorphism (T-RFLP) analysis. The conventional data showed a relationship between patch type and mite assemblage. The Prostigmata and Oribatida were well represented in the PGL sites, particularly the Aphelacaridae (Oribatida). For T-RFLP analysis, the mite community was represented by a series of DNA fragment lengths that reflected mite sequence diversity. The T-RFLP data showed a distinct difference in the mite assemblage between the patch types. Where possible, T-RFLP peaks were matched to mite families using a reference 18S rDNA database, and the Aphelacaridae prevalent in the conventional samples at PGL sites were identified, as were prostigmatids and oribatids. We identified limits to the T-RFLP approach and this included an inability to distinguish some species whose DNA sequences were similar. Despite these limitations, the data still showed a clear difference between sites, and the molecular taxonomic inferences also compared well with the conventional ecological data. The results from this study indicated that the T-RFLP approach was effective in measuring mite assemblages in this system. The power of this technique lies in the fact that species diversity and abundance data can be obtained quickly because of the time taken to process hundreds of samples, from soil DNA extraction to data output on the gene analyser, can be as little as 4 days.