Amplified fragment length polymorphism (AFLP): a review of the procedure and its applications

Amplified fragment length polymorphism (AFLP) is a novel molecular fingerprinting technique that can be applied to DNAs of any source or complexity. Total genomic DNA is digested using two restriction enzymes. Double-stranded nucleotide adapters are ligated to the DNA fragments to serve as primer binding sites for PCR amplification. Primers complementary to the adapter and restriction site sequence, with additional nucleotides at the 3′-end, are used as selective agents to amplify a subset of ligated fragments. Polymorphisms are identified by the presence or absence of DNA fragments following analysis on polyacrylamide gels. This technique has been extensively used with plant DNA for the development of high-resolution genetic maps and for the positional cloning of genes of interest. However, its application is rapidly expanding in bacteria and higher eukaryotes for determining genetic relationships and for epidemiological typing. This review describes the AFLP procedure, and recent, novel applications in the molecular fingerprinting of DNA from both eukaryotic and prokaryotic organisms. Received 19 December 1997/ Accepted in revised form 3 June 1998     

Adaptation of acidogenic sludge to increasing glycerol concentrations for biohydrogen production

Hydrogen is a promising alternative as an energetic carrier and its production by dark fermentation from wastewater has been recently proposed, with special attention to crude glycerol as potential substrate. In this study, two different feeding strategies were evaluated for replacing the glucose substrate by glycerol substrate: a one-step strategy (glucose was replaced abruptly by glycerol) and a step-by-step strategy (progressive decrease of glucose concentration and increase of glycerol concentration from 0 to 5 g L^?1), in a continuous stirred tank reactor (12 h of hydraulic retention time (HRT), pH 5.5, 35 °C). While the one-step strategy led to biomass washout and unsuccessful H₂ production, the step-by-step strategy was efficient for biomass adaptation, reaching acceptable hydrogen yields (0.4?±?0.1 mol_H2?mol^?1 _{glycerol consumed}) around 33 % of the theoretical yield independently of the glycerol concentration. Microbial community structure was investigated by single-strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) fingerprinting techniques, targeting either the total community (16S ribosomal RNA (rRNA) gene) or the functional Clostridium population involved in H₂ production (hydA gene), as well as by 454 pyrosequencing of the total community. Multivariate analysis of fingerprinting and pyrosequencing results revealed the influence of the feeding strategy on the bacterial community structure and suggested the progressive structural adaptation of the community to increasing glycerol concentrations, through the emergence and selection of specific species, highly correlated to environmental parameters. Particularly, this work highlighted an interesting shift of dominant community members (putatively responsible of hydrogen production in the continuous stirred tank reactor (CSTR)) according to the gradient of glycerol proportion in the feed, from the family Veillonellaceae to the genera Prevotella and Clostridium sp., putatively responsible of hydrogen production in the CSTR.     

Resource availability influences the diversity of a functional group of heterotrophic soil bacteria

Resource availability is a key factor regulating biodiversity and ecosystem functioning, but the relationship between resource availability and diversity has only been rarely investigated in microbial communities. The aim of this study was to determine how diversity and community structure of a functional group of soil bacteria are influenced by resource concentration. To achieve this, we used soil microcosms to investigate degradation of benzoate, which served as a model compound, by soil bacterial communities. Microcosms were supplied with ¹³C-labelled benzoate at four concentrations and RNA-stable isotope probing followed by molecular fingerprinting analysis of 16S rRNA genes was employed to identify bacteria able to assimilate benzoate at different concentrations. The composition of the benzoate degrader community differed at different concentrations and there was a significant decrease in taxa evenness at the highest substrate concentration. Active organisms could be grouped into generalists, occurring at all substrate concentrations, specialists, active at one particular benzoate concentration only, and taxa that were active at either the two lowest or two highest concentrations. The study comprises the first explicit demonstration that resource availability has an effect on the diversity of a functional group of heterotrophic soil bacteria.     

Determination of relative rate constants for in vitro RNA processing reactions by internal competition

Studies of RNA recognition and catalysis typically involve measurement of rate constants for reactions of individual RNA sequence variants by fitting changes in substrate or product concentration to exponential or linear functions. A complementary approach is determination of relative rate constants by internal competition, which involves quantifying the time-dependent changes in substrate or product ratios in reactions containing multiple substrates. Here, we review approaches for determining relative rate constants by analysis of both substrate and product ratios and illustrate their application using the in vitro processing of precursor transfer RNA (tRNA) by ribonuclease P as a model system. The presence of inactive substrate populations is a common complicating factor in analysis of reactions involving RNA substrates, and approaches for quantitative correction of observed rate constants for these effects are illustrated. These results, together with recent applications in the literature, indicate that internal competition offers an alternate method for analyzing RNA processing kinetics using standard molecular biology methods that directly quantifies substrate specificity and may be extended to a range of applications.     

Quantitative molecular assay for fingerprinting microbial communities of wastewater and estrogen-degrading consortia

A quantitative fingerprinting method, called the real-time terminal restriction fragment length polymorphism (real-time-t-RFLP) assay, was developed for simultaneous determination of microbial diversity and abundance within a complex community. The real-time-t-RFLP assay was developed by incorporating the quantitative feature of real-time PCR and the fingerprinting feature of t-RFLP analysis. The assay was validated by using a model microbial community containing three pure strains, an Escherichia coli strain (gram negative), a Pseudomonas fluorescens strain (gram negative), and a Bacillus thuringiensis strain (gram positive). Subsequently, the real-time-t-RFLP assay was applied to and proven to be useful for environmental samples; the richness and abundance of species in microbial communities (expressed as the number of 16S rRNA gene copies of each ribotype per milliliter) of wastewater and estrogen-degrading consortia (enriched with 17alpha-estradiol, 17beta-estradiol, or estrone) were successfully characterized. The results of this study strongly suggested that the real-time-t-RFLP assay can be a powerful molecular tool for gaining insight into microbial communities in various engineered systems and natural habitats.     

Characterisation of micromonosporae from aquatic environments using molecular taxonomic methods

Large numbers of strains assigned to the genus Micromonospora on the basis of typical colonial and pigmentation features were isolated from diverse aquatic sediments using a standard selective isolation procedure. Two hundred and six isolates and eight representatives of the genus Micromonospora were assigned to 24 multimembered groups based on a numerical analysis of banding patterns generated using BOX and ERIC primers. Representatives of multimembered groups encompassing isolated micromonosporae were the subject of 16S rRNA gene sequencing analyses. Good congruence was found between the molecular fingerprinting and 16S rRNA sequence data indicating that the groups based upon the former are taxonomically meaningful. Nearly all of the isolates that were chosen for the 16S rRNA gene sequencing analyses showed that the ecosystems studied are a rich source of novel micromonosporae. These findings have implications for high throughput screening for novel micromonosporae as BOX and ERIC fingerprinting, which is rapid and reproducible, can be applied as a robust dereplication procedure to indicate which environmental isolates have been cultured previously.     

Virion RNA species of the arenaviruses Pichinde, Tacaribe, and Tamiami.

The principal RNA species isolated from labeled preparations of the arenavirus Pichinde usually include a large viral RNA species L (apparent molecular weight = 3.2 X 10(6)), and a smaller viral RNA species S (apparent molecular weight = 1.6 X 10(6)). In addition, either little or considerable quantities of 28S rRNA as well as 18S rRNA can also be obtained in virus extracts, depending on the virus stock and growth conditions used to generate virus preparations. Similar RNA species have been identified in RNA extracted from Tacaribe and Tamiami arenavirus preparations. Oligonucleotide fingerprint analyses have confirmed the host ribosomal origin of the 28S and 18S species. Such analyses have also indicated that the Pichinde viral L and S RNA species each contain unique nucleotide sequences. Viral RNA preparations isolated by conventional phenol-sodium dodecyl sulfate extraction often have much of their L and S RNA species in the form of aggregates as visualized by either electron microscopy or oligonucleotide fingerprinting of material recovered from the top of gels (run by using undenatured RNA preparations). Circular and linear RNA forms have also been seen in electron micrographs of undenatured RNA preparations, although denatured viral RNA preparations have yielded mostly linear RNA species with few RNA aggregates or circular forms.     

Selection of international molecular standards for DNA fingerprinting of <Emphasis Type="Italic">Theobroma cacao</Emphasis>

A collaborative international program was initiated to identify and describe the genetic diversity of living germplasm collections of Theobroma cacao genotypes that are maintained in several international collections scattered throughout tropical cacao growing countries of the world. Simple sequence repeat (SSR) DNA analysis was identified as the most appropriate molecular tool for DNA fingerprinting these collections during an international forum representing academic, government and industry scientists in the cacao community. Twenty-five SSR primers, which had been previously described, were evaluated as potential candidates to define an efficient, standardized, molecular fingerprinting protocol for T. cacao accessions. These primers have been evaluated for reliability, widespread distribution across the cacao genome, number of alleles produced by the SSR primers in cacao and their ability to discriminate between cacao accessions. Approximately 690 cacao accessions were used to evaluate the utility of these SSR primers as international molecular standards, and a small number of test samples of T. cacao were sent to two other independent laboratories for verification. DNA fragments were selectively amplified by PCR, using the SSR primers labeled with fluorescent dyes, and separated by capillary electrophoresis. Based on this study, the 15 SSR primers that had the highest reproducibility and consistency within a common genotype, while allowing the differentiation of separate divergent genotypes, were selected as international molecular standards for DNA fingerprinting of T. cacao.     

基于PCR-DGGE基因指纹的对虾体内优势细菌组成分析

采用不依赖分离培养的16S rDNA的PCR-DGGE基因指纹技术对刀额新对虾与中国对虾的鳃部与肠道优势细菌种群组成进行比较分析。研究发现:对虾鳃部与肠道存在着丰富多样的细菌;根据DGGE指纹图的聚类分析发现不同对虾及同一种对虾的鳃部与肠道内的细菌组成差异性非常大;同时也发现不同对虾体内有相同的细菌存在。首次尝试建立基于16S rDNA的PCR-DGGE基因指纹的对虾体内细菌组成揭示方法,对于今后建立对虾与养殖水体微生物和相关疾病的关系具有重要意义。     

Dereplication for biotechnology screening: PyMS analysis and PCR–RFLP–SSCP (PRS) profiling of 16S rRNA genes of marine and terrestrial actinomycetes

The search for exploitable biology is a major task for biotechnology-based industries. In this context, discrimination between previously tested or recovered micro-organisms (dereplication) is imperative, in order to reduce screening costs by sorting large collections of isolates, which are then subjected to further detailed evaluation. Pyrolysis mass spectrometry (PyMS) is a whole-cell fingerprinting technique that enables the rapid and reproducible sorting of micro-organisms, uses small samples and has the advantage of being fully automated. In this study, we compare chemometric fingerprinting with a ribotyping fingerprinting method, in order to investigate the extent to which pyrogroups formed by PyMS analysis relate to genetic diversity, using polymerase chain reaction-restriction fragment length polymorphism-single-strand conformational polymorphism (PRS). A mixture of environmental strains of mycolic acid containing actinomycetes was used to mimic the selection of colonies from primary isolation plates. The congruence found between the clusters defined by the chemometric and molecular fingerprinting techniques was very high and demonstrated the effectiveness of PyMS as a rapid sorting and dereplicating procedure for putatively novel strains, criteria that are critical for biotechnological screens. Moreover, PyMS analysis revealed significant variation within pyrogroups that contained strains with the same genotypic (PRS) characteristics, thus emphasising its discriminatory capacity at the infraspecies level.     

Determination of the primary structure of oligodeoxyribonucleotides containing P-S-C(5') bonds

Analogues of oligodeoxynucleotides with P-S-C(5') bonds, which, due to their unusual substrate properties, may find interesting applications in molecular biology, can not be structurally analysed by the Maxam-Gilbert or Sanger (fingerprinting) methods. We, therefore, devised a modification of the fingerprinting technique making possible the sequence determination of these analogues.     

Development and application of an enzymatic and cell flotation treatment for the recovery of viable microbial cells from environmental matrices such as anaerobic sludge

Efficient dissociation of microorganisms from their aggregate matrix is required to study the microorganisms without interaction with their native environment (e.g., biofilms, flocs, granules, etc.) and to assess their community composition through the application of molecular or microscopy techniques. To this end, we combined enzymatic treatments and a cell extraction by density gradient to efficiently recover anaerobic microorganisms from urban wastewater treatment plant sludge. The enzymes employed (amylase, cellulase, DNase, and pectinase) as a pretreatment softly disintegrated the extrapolymeric substances (EPS) interlocked with the microorganisms. The potential damaging effects of the applied procedure on bacterial and archaeal communities were assessed by studying the variations in density (using quantitative PCR), diversity (using capillary electrophoresis single-strand conformation polymorphism fingerprinting [CE-SSCP]), and activity (using a standard anaerobic activity test) of the extracted microorganisms. The protocol preserved the general capacity of the microbial community to produce methane under anaerobic conditions and its diversity; particularly the archaeal community was not affected in terms of either density or structure. This cell extraction procedure from the matrix materials offers interesting perspectives for metabolic, microscopic, and molecular assays of microbial communities present in complex matrices constituted by bioaggregates or biofilms.     

Quantitative Molecular Assay for Fingerprinting Microbial Communities of Wastewater and Estrogen-Degrading Consortia

A quantitative fingerprinting method, called the real-time terminal restriction fragment length polymorphism (real-time-t-RFLP) assay, was developed for simultaneous determination of microbial diversity and abundance within a complex community. The real-time-t-RFLP assay was developed by incorporating the quantitative feature of real-time PCR and the fingerprinting feature of t-RFLP analysis. The assay was validated by using a model microbial community containing three pure strains, an Escherichia coli strain (gram negative), a Pseudomonas fluorescens strain (gram negative), and a Bacillus thuringiensis strain (gram positive). Subsequently, the real-time-t-RFLP assay was applied to and proven to be useful for environmental samples; the richness and abundance of species in microbial communities (expressed as the number of 16S rRNA gene copies of each ribotype per milliliter) of wastewater and estrogen-degrading consortia (enriched with 17α-estradiol, 17β-estradiol, or estrone) were successfully characterized. The results of this study strongly suggested that the real-time-t-RFLP assay can be a powerful molecular tool for gaining insight into microbial communities in various engineered systems and natural habitats.     

iFoldRNA: three-dimensional RNA structure prediction and folding

Three-dimensional RNA structure prediction and folding is of significant interest in the biological research community. Here, we present iFoldRNA, a novel web-based methodology for RNA structure prediction with near atomic resolution accuracy and analysis of RNA folding thermodynamics. iFoldRNA rapidly explores RNA conformations using discrete molecular dynamics simulations of input RNA sequences. Starting from simplified linear-chain conformations, RNA molecules (<50 nt) fold to native-like structures within half an hour of simulation, facilitating rapid RNA structure prediction. All-atom reconstruction of energetically stable conformations generates iFoldRNA predicted RNA structures. The predicted RNA structures are within 2-5 A root mean squre deviations (RMSDs) from corresponding experimentally derived structures. RNA folding parameters including specific heat, contact maps, simulation trajectories, gyration radii, RMSDs from native state, fraction of native-like contacts are accessible from iFoldRNA. We expect iFoldRNA will serve as a useful resource for RNA structure prediction and folding thermodynamic analyses. AVAILABILITY: http://iFoldRNA.dokhlab.org.     

Specific detection, isolation, and characterization of selected, previously uncultured members of the freshwater bacterioplankton community

High-throughput cultivation was combined with rapid and group-specific phylogenetic fingerprinting in order to recover representatives of three freshwater bacterioplankton communities. A total of 570 bacterial cultures were obtained by employing the most probable number and MicroDrop techniques. The majority of the cultured bacteria were closely related to previously uncultured bacteria and grouped with the alpha-Proteobacteria, beta-Proteobacteria, Actinobacteria, Firmicutes, or Flavobacteria-Cytophaga lineage. Correspondingly, the natural bacterioplankton community was analyzed by high-resolution phylogenetic fingerprinting of these five bacterial lineages. 16S rRNA gene fragments were generated for each lineage and subsequently separated by denaturing gradient gel electrophoresis. By the combination of five group-specific PCR protocols, the total number of 16S rRNA gene fingerprints generated from the natural communities was increased sixfold compared to conventional (eubacterial) fingerprinting. Four of the environmental alpha-Proteobacteria 16S rRNA gene sequences obtained from the natural community were found to be identical to those of bacterial isolates. One of these phylotypes was detected in 14 different cultures and hence represented the most frequently cultured bacterium. Three of these 14 strains were characterized in detail. Their complete 16S rRNA gene sequences showed only 93% similarity to that of Sandaracinobacter sibiricus, the closest relative described so far. The novel phylotype of bacterium is a strict aerobe capable of using numerous organic carbon substrates and contains bacteriochlorophyll a bound to two different photosynthetic light-harvesting complexes. Dot blot hybridization revealed that the strains occur in lakes of different trophic status and constitute up to 2% of the microbial community.     

Application of polymerase chain reaction with arbitrary primer (AP-PCR) to strain identification of Porphyromonas (Bacteroides) gingivalis

Several molecular methods are currently available for identification and discrimination of bacterial strains within the same species, which vary in efficiency and required labour. Here we applied a novel method for fingerprinting genomes, called arbitrarily primed PCR (AP-PCR), to the delineation of strains within the species Porphyromonas gingivalis. Using a single primer on a set of nine strains, nine simple distinct banding patterns, indicative of genetic polymorphism, were observed. Common amplicons and amplicons shared by only some strains were also observed, the latter suggesting that AP-PCR can be used to generate polymorphic markers. Genomic fingerprinting obtained by AP-PCR was independent of the quality of DNA. Assays performed directly using whole cells as a source of DNA template indicated that AP-PCR from colony is a quick, simple and accurate procedure.