Amplified Ribosomal DNA Restriction Analysis Patterns Research Articles

Phylogeny based on 16S rDNA and nifH sequences of Ralstonia taiwanensis strains isolated from nitrogen-fixing nodules of Mimosa pudica, in India.

Bacterial symbionts present in the indeterminate-type nitrogen (N)-fixing nodules of Mimosa pudica grown in North and South India showed maximum similarity to Ralstonia taiwanensis on the basis of carbon-source utilization patterns and 16S rDNA sequence. Isolates from the nodules of M. pudica from North India and South India showed identical ARDRA (Amplified Ribosomal DNA Restriction Analysis) patterns with Sau3AI and RsaI, but AluI revealed dimorphy between the North Indian and South Indian isolates. Alignment of 16S rDNA sequences revealed similarity of North Indian isolates with an R. taiwanensis strain isolated from M. pudica in Taiwan, whereas South Indian isolates showed closer relatedness with the isolates from Mimosa diplotricha. Alignment of nifH sequences from both North Indian and South Indian isolates with that of the related isolates revealed their closer affinity to alpha-rhizobia, suggesting that nif genes in the beta-rhizobia might have been acquired from alpha-rhizobia via lateral transfer during co-occupancy of nodules by alpha-rhizobia and progenitors of R. taiwanensis, members of the beta-subclass of Proteobacteria. Immunological cross-reaction of the bacteroid preparation of M. pudica nodules showed strong a positive signal with anti-dinitrogenase reductase antibody, whereas a weak positive cross-reaction was observed with free-living R. taiwanensis grown microaerobically in minimal medium with and without NH4Cl. In spite of the expression of dinitrogenase reductase under free-living conditions, acetylene reduction was not observed under N-free conditions even after prolonged incubation.

Relationship between spatial and genetic distance in Agrobacterium spp. in 1 cubic centimeter of soil.

The spatial and genetic unit of bacterial population structure is the clone. Surprisingly, very little is known about the spread of a clone (spatial distance between clonally related bacteria) and the relationship between spatial distance and genetic distance, especially at very short scale (microhabitat scale), where cell division takes place. Agrobacterium spp. Biovar 1 was chosen because it is a soil bacterial taxon easy to isolate. A total of 865 microsamples 500 microm in diameter were sampled with spatial coordinates in 1 cm(3) of undisturbed soil. The 55 isolates obtained yielded 42 ribotypes, covering three genomic species based on amplified ribosomal DNA restriction analysis (ARDRA) of the intergenic spacer 16S-23S, seven of which contained two to six isolates. These clonemates (identical ARDRA patterns) could be found in the same microsample or 1 cm apart. The genetic diversity did not change with distance, indicating the same habitat variability across the cube. The mixing of ribotypes, as assessed by the spatial position of clonemates, corresponded to an overlapping of clones. Although the population probably was in a recession stage in the cube (10(3) agrobacteria g(-1)), a high genetic diversity was maintained. In two independent microsamples (500 microm in diameter) at the invasion stage, the average genetic diversity was at the same level as in the cube. Quantification of the microdiversity landscape will help to estimate the probability of encounter between bacteria under realistic natural conditions and to set appropriate sampling strategies for population genetic analysis.

High bacterial diversity of a waste gas-degrading community in an industrial biofilter as shown by a 16S rDNA clone library.

The bacterial diversity of an industrial biofilter used for waste gas abatement in an animal-rendering plant was investigated. A 16S rDNA clone library was generated and 444 clones were screened using computer-aided amplified ribosomal DNA restriction analysis (ARDRA). Of the screened clones, 60.8% showed unique ARDRA patterns and the remaining 174 clones were clustered into 65 groups. Almost full-length 16S rDNA sequences of 106 clones were determined and 90.5% of the clones were affiliated with the two phyla Proteobacteria and Bacteroidetes. Alpha-, Beta-, and Gammaproteobacteria accounted for 22.1, 17.6 and 18.6% respectively. Minor portions were affiliated with the Actinobacteria (2.0%), Firmicutes and Verrucomicrobia (both 1.0%), and the Deltaproteobacteria and Thermomicrobia (each 0.5%). Only six out of the 106 16S rDNA sequences exhibited similarities of more than 97% to classified bacterial species indicating that a substantial fraction of the clone sequences were derived from unknown taxa. It was also evaluated whether a database containing 281 computer-simulated bacterial rDNA fragment patterns generated from published reference sequences can be used for identification purposes. The data analysis demonstrated that this was possible only for a small number of clones, which were closely related to described bacterial strains. Rarefaction analysis of ARDRA clusters demonstrated that the 444 clones screened are insufficient to describe the entire diversity of the clone library.

Diversity of 16S rDNA and naphthalene dioxygenase genes from coal-tar-waste-contaminated aquifer waters.

Microbial diversity in four wells along a groundwater flowpath in a coal-tar-waste-contaminated aquifer was examined using RFLP analysis of both 16S rDNA and naphthalene dioxygenase (NDO) genes. Amplified ribosomal DNA restriction analysis (ARDRA) relied upon eubacteria-specific primers to generate four clone libraries. From each library, 100 clones were randomly picked for analysis. Sixty percent of 400 clones contained unique ARDRA patterns. Diversity indices calculated for each community were high (Shannon-Weaver, H = 3.53 to 3.69). Clones representing ARDRA patterns found in the highest abundance were sequenced (31 total). Sequences related to aerobic bacteria (e.g., Nitrospira, Methylomonas, and Gallionella) predominated among those retrieved from the uncontaminated area of the site, whereas sequences related to facultatively aerobic and anaerobic bacteria (e.g. Azoarcus, Syntrophus, and Desulfotomaculum) predominated among those retrieved from contaminated areas of the site. Using NDO-specific primers and low-stringency PCR conditions, variability in RFLP patterns was only detected in community-derived DNA (3 of 4 wells) and not in 5 newly isolated naphthalene-degrading pure cultures. The ARDRA patterns of the pure culture isolates were not found in the clone libraries. Polymorphisms in community 16S rDNA and NDO genes found in well-water microorganisms reflected distinctive geochemical conditions across the site. Sequences related to sulfate-reducing bacteria were found in groundwater that contained sulfide, while sequences related to Gallionella, Syntrophus, and nitrate-reducing aromatic hydrocarbon-degrading bacteria were found in groundwater that contained ferrous iron, methane, and naphthalene, respectively.

Molecular discrimination of lactobacilli used as starter and probiotic cultures by amplified ribosomal DNA restriction analysis.

Lactic acid bacteria such as Lactobacillus helveticus, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. lactis, L. delbrueckii subsp. bulgaricus, L. acidophilus, and L. casei related taxa which are widely used as starter or probiotic cultures can be identified by amplified ribosomal DNA restriction analysis (ARDRA). The genetic discrimination of the related species belonging to these groups was first obtained by PCR amplifications by using group-specific or species-specific 16S rDNA primers. The numerical analysis of the ARDRA patterns obtained by using CfoI, HinfI, Tru9I, and ScrFI was an efficient typing tool for identification of species of the L. acidophilus and L. casei complex. ARDRA by using CfoI was a reliable method for differentiation of L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. Finally, strains ATCC 393 and ATCC 15820 exhibited unique ARDRA patterns with CfoI and Tru9I restriction enzymes as compared with the other strains of L. casei, L. paracasei, and L. rhamnosus.

Random Amplified Ribosomal DNA Restriction Analysis for Rapid Identification of Thermophilic Actinomycete-like Bacteria Involved in Hypersensitivity Pneumonitis

Hypersensitivity pneumonitis (HP) is a pulmonary disease characterised by inflammation that can be caused by, amongst other substances, a subset of 4 thermophilic mycelial bacteria: Saccharopolyspora rectivirgula, Saccharomonospora viridis, Thermoactinomyces sacchari, and Thermoactinomyces vulgaris. Air sampling analyses in highly contaminated environments are often performed to evaluate exposure to these species which are difficult and fastidious to identify by conventional techniques. The aim of this study was to use amplified ribosomal DNA restriction analysis (ARDRA) to develop a method of identification for those thermophilic organisms that would be more rapid and simple. Strains of these 4 species were obtained from the American type culture collection (ATCC) and were characterized using biochemical tests and ARDRA patterns obtained on their partial-lenght amplified 16S rDNAs. To validate this approach, ARDRA with two restriction enzymes, TaqI and HhaI, was applied to 49 thermophilic actinomycete-like strains from environmental samples (sawmills). The results obtained show that combining some cultural characteristics and biochemical tests, such as xanthine or hypoxanthine decomposition, growth in the presence of NaCl, lysozyme or novobiocin, and spore resistance over 100 degrees C provide a rough identification and selection of the genera of interest. Consequently, target species could be confirmed by digestion of partial-lenght 16S rDNA with the use of Taql and HhaI restriction enzymes that gave specific restriction patterns. ARDRA analyses on the 49 environmental actinomycete-like organisms revealed the presence of 8 Saccharopolyspora rectivirgula, 2 Saccharomonospora viridis, and 15 Thermoactinomyces vulgaris strains, the other strains had restriction patterns different than those of the species of interest. Results of the present study will be applicable to other potential HP environments such as dairy barns, peat bogs and compost plants.

Culturability and In situ abundance of pelagic bacteria from the North Sea.

The culturability of abundant members of the domain Bacteria in North Sea bacterioplankton was investigated by a combination of various cultivation strategies and cultivation-independent 16S rRNA-based techniques. We retrieved 16S rRNA gene (rDNA) clones from environmental DNAs and determined the in situ abundance of different groups and genera by fluorescence in situ hybridization (FISH). A culture collection of 145 strains was established by plating on oligotrophic medium. Isolates were screened by FISH, amplified ribosomal DNA restriction analysis (ARDRA), and sequencing of representative 16S rDNAs. The majority of isolates were members of the genera Pseudoalteromonas, Alteromonas, and Vibrio. Despite being readily culturable, they constituted only a minor fraction of the bacterioplankton community. They were not detected in the 16S rDNA library, and FISH indicated rare (<1% of total cell counts) occurrence as large, rRNA-rich, particle-associated bacteria. Conversely, abundant members of the Cytophaga-Flavobacteria and gamma proteobacterial SAR86 clusters, identified by FISH as 17 to 30% and up to 10% of total cells in the North Sea bacterioplankton, respectively, were cultured rarely or not at all. Whereas SAR86-affiliated clones dominated the 16S rDNA library (44 of 53 clones), no clone affiliated to the Cytophaga-Flavobacterum cluster was retrieved. The only readily culturable abundant group of marine bacteria was related to the genus Roseobacter. The group made up 10% of the total cells in the summer, and the corresponding sequences were also present in our clone library. Rarefaction analysis of the ARDRA patterns of all of the isolates suggested that the total culturable diversity by our method was high and still not covered by the numbers of isolated strains but was almost saturated for the gamma proteobacteria. This predicts a limit to the isolation of unculturable marine bacteria, particularly the gamma-proteobacterial SAR86 cluster, as long as no new techniques for isolation are available and thus contrasts with more optimistic accounts of the culturability of marine bacterioplankton.

Assessment of microbial community structure changes by amplified ribosomal DNA restriction analysis (ARDRA).

Amplified ribosomal DNA restriction analysis (ARDRA) is a simple method based on restriction endonuclease digestion of the amplified bacterial 16S rDNA. In this study we have evaluated the suitability of this method to detect differences in activated sludge bacterial communities fed on domestic or industrial wastewater, and subject to different operational conditions. The ability of ARDRA to detect these differences has been tested in modified Ludzack-Ettinger (MLE) configurations. Samples from three activated sludge wastewater treatment plants (WWTPs) with the MLE configuration were collected for both oxic and anoxic reactors, and ARDRA patterns using double enzyme digestions AluI+MspI were obtained. A matrix of Dice similarity coefficients was calculated and used to compare these restriction patterns. Differences in the community structure due to influent characteristics and temperature could be observed, but not between the oxic and anoxic reactors of each of the three MLE configurations. Other possible applications of ARDRA for detecting and monitoring changes in activated sludge systems are also discussed.

How stable is stable? Function versus community composition.

The microbial community dynamics of a functionally stable, well-mixed, methanogenic reactor fed with glucose were analyzed over a 605-day period. The reactor maintained constant pH and chemical oxygen demand removal during this period. Thirty-six rrn clones from each of seven sampling events were analyzed by amplified ribosomal DNA restriction analysis (ARDRA) for the Bacteria and Archaea domains and by sequence analysis of dominant members of the community. Operational taxonomic units (OTUs), distinguished as unique ARDRA patterns, showed reproducible distribution for three sample replicates. The highest diversity was observed in the Bacteria domain. The 16S ribosomal DNA Bacteria clone library contained 75 OTUs, with the dominant OTU accounting for 13% of the total clones, but just 21 Archaea OTUs were found, and the most prominent OTU represented 50% of the clones from the respective library. Succession in methanogenic populations was observed, and two periods were distinguished: in the first, Methanobacterium formicicum was dominant, and in the second, Methanosarcina mazei and a Methanobacterium bryantii-related organism were dominant. Higher variability in Bacteria populations was detected, and the temporal OTU distribution suggested a chaotic pattern. Although dominant OTUs were constantly replaced from one sampling point to the next, phylogenetic analysis indicated that inferred physiologic changes in the community were not as dramatic as were genetic changes. Seven of eight dominant OTUs during the first period clustered with the spirochete group, although a cyclic pattern of substitution occurred among members within this order. A more flexible community structure characterized the second period, since a sequential replacement of a Eubacterium-related organism by an unrelated deep-branched organism and finally by a Propionibacterium-like species was observed. Metabolic differences among the dominant fermenters detected suggest that changes in carbon and electron flow occurred during the stable performance and indicate that an extremely dynamic community can maintain a stable ecosystem function.

Detection of shifts in microbial community structure and diversity in soil caused by copper contamination using amplified ribosomal DNA restriction analysis

Amplified ribosomal DNA restriction analysis (ARDRA) was used for assessing the effect of copper contamination on the microbial community in soil. ARDRA is a DNA fingerprinting technique based on PCR amplification of 16S ribosomal DNA using primers for conserved regions, followed by restriction enzyme digestions and agarose gel electrophoresis. This results in distinguishable fingerprints for different bacterial species. Microbial community structure in soil was investigated by studying ARDRA patterns of the whole community, and by comparing ARDRA patterns of cloned 16S RNA gene sequences from soil and isolates obtained from soil by cultivation. Distinct differences in microbial community structure and a lower diversity were found in a soil contaminated with copper compared with non-contaminated soil. Diversity of ARDRA patterns from the clones was higher than that from isolates, supporting the general idea that the culturable population represents only a part of the total (amplifiable) population in soil. To compare microbial communities from various soils a method is described for the construction of a dendrogram representing the relatedness of the populations. ARDRA appears to be suitable for analysing microbial communities in soil and gives interesting information with respect to microbial ecology.

Applicability of combined amplified ribosomal DNA restriction analysis (ARDRA) patterns in bacterial phylogeny and taxonomy

A standardized method for amplified ribosomal DNA restriction analysis (ARDRA) is described. The first step involves selection of five tetracutter restriction enzymes on the basis of theoretical digestions of known 16S rDNA (rRNA) sequences. In the second step, the experimentally obtained restriction patterns are normalized and combined by means of the pattern recognition and analysis software GelCompar. Finally, numerical analysis allows the strains to be grouped according to the similarities in their combined ARDRA patterns. Results obtained with representatives of two phylogenetic lineages, the genera Alcaligenes and Bordetella and the genera Bacillus and Paenibacillus, are presented. In general, the clustering of the strains corresponded well with known species delineations and topology of phylogenetic groupings except for the discrepant position of the Bacillus lautus type strain, which can probably be explained by non-authenticity of this strain in one of the analyses. The effect of using less than five restriction enzymes on the clustering was evaluated. The frequent occurrence of interoperon variability of the 16S rRNA gene in Bacillus and Paenibacillus was also demonstrated. Because ARDRA detects interspecies and interstrain as well as interoperon variability and enables a relatively fast multiple strain analysis per taxon, this technique is appropriate to obtain indicative phylogenetic and taxonomic information. This information can be used to select strains for further detailed taxonomic studies. ARDRA fingerprinting also allows the construction of a database for indentification purposes.