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PubMed1996

A computer-simulated restriction fragment length polymorphism analysis of bacterial small-subunit rRNA genes: efficacy of selected tetrameric restriction enzymes for studies of microbial diversity in nature.

An assessment of 10 tetrameric restriction enzymes (TREs) was conducted by using a computer-simulated restriction fragment length polymorphism (RFLP) analysis for over 100 proximally and distally related bacterial small-subunit (SSU) rRNA gene sequences. Screening SSU rDNA clone libraries with TREs has become an effective strategy because of logistic simplicity, commercial availability, and economy. However, the rationale for selecting the type and number of TREs has not been systematically evaluated. Our objective was to identify the optimal combination of TREs for RFLP screening of cloned SSU rRNA genes from undefined bacterial clone libraries. After computer-simulated TRE digestion, the resultant fragments were categorized on the basis of the frequency of different restriction fragment size classes. Three groups of distribution patterns for the TREs were determined and further examined via graphical exploratory data analysis. The RFLP size-frequency distribution data for each group of enzymes were then used to infer phylogenetic relationships via the neighbor-joining method. The resulting bootstrap values and the correct placement of node bifurcations were used as additional criteria to evaluate the efficacy of the selected TREs. These RFLP data were compared with known phylogenetic relationships based on SSU rRNA sequence analysis as defined by the Ribosomal Database Project. A heuristic approach testing random combinations of TREs showed that three or more TRE combinations detected > 99% of the operational taxonomic units (OTUs) within the model data set. OTUs that remained undetected after three TRE treatments had a median sequence similarity of 96.1%. Of the 10 restriction enzymes examined, HhaI, RsaI, and BstUI (group 3) were the most efficacious at detecting and differentiating bacterial SSU rRNA genes on the basis of their ability to correctly classify OTUs. Group 3 TREs are therefore recommended for screening in studies using bacterial SSU rRNA genes as descriptors of in situ microbial diversity.

Computer Simulation↗

PubMed2003

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).

Bacteria↗

PubMed1997

A molecular view of microbial diversity and the biosphere.

Over three decades of molecular-phylogenetic studies, researchers have compiled an increasingly robust map of evolutionary diversification showing that the main diversity of life is microbial, distributed among three primary relatedness groups or domains: Archaea, Bacteria, and Eucarya. The general properties of representatives of the three domains indicate that the earliest life was based on inorganic nutrition and that photosynthesis and use of organic compounds for carbon and energy metabolism came comparatively later. The application of molecular-phylogenetic methods to study natural microbial ecosystems without the traditional requirement for cultivation has resulted in the discovery of many unexpected evolutionary lineages; members of some of these lineages are only distantly related to known organisms but are sufficiently abundant that they are likely to have impact on the chemistry of the biosphere.

Archaea↗

PubMed2005

Microbial diversity of benthic mats along a tidal desiccation gradient.

We investigated the influence of desiccation frequency, indicated by tidal position, on microbial community structure, diversity and richness of microbial mats. We independently characterized cyanobacterial, bacterial and archaeal communities, and their spatial variability for two distinct microbial mat systems: subtidal hypersaline mats and intertidal sand flat mats. Community fingerprints based on 16S rDNA were obtained via denaturing gradient gel electrophoresis using polymerase chain reaction primers specific for each group. Fingerprints for all three groups were consistently similar [> or =85% according to Weighted Pair Group with Arithmetic Mean (WPGMA) analysis] along a 1-km-long transect in subtidal mats. Here, pair-wise comparison analysis yielded minimal variation in diversity and richness for all groups. Fingerprints of three sites along an intertidal transect were heterogenous (> or =32% similarity according to WPGMA analysis) with clear shifts in community structure in all three microbial groups. Here, all groups exhibited statistically significant decreases in richness and diversity with tidal height (as desiccation frequency increases). Regression analysis yielded a strong correlation between diversity or richness estimates and position along the tidal gradient, for both Archaea and Bacteria, with Cyanobacteria exhibiting a weaker correlation. These results suggest that desiccation frequency can shape the structure of microbial mat communities, with Archea being least tolerant and Cyanobacteria most tolerant.

Archaea↗

PubMed2003

Microbial diversity of marine sponges.

The recent application of molecular microbial ecology tools to sponge-microbe associations has revealed a glimpse into the biodiversity of these microbial communities, that is considered just 'the tip of the iceberg'. This chapter provides an overview over these new findings with regard to identity, diversity and distribution patterns of sponge-associated microbial consortia. The sponges Aplysina aerophoba (Verongida), Rhopaloeides odorabile (Dicytoceratida) and Theonella swinhoei (Lithistida) were chosen as model systems for this review because they have been subject to both, cultivation-dependent and cultivation-independent approaches. A discussion of the microbial assemblages of Halichondriapanicea is presented in the accompanying chapter by Imhoff and Stöhr. Considering that a large fraction of sponge-associated microbes is not yet amenable to cultivation, an emphasis has been placed on the techniques centering around the 16S rRNA gene. A section has been included that covers the potential of sponge microbial communities for drug discovery. Finally, a 'sponge-microbe interaction model' is presented that summarizes our current understanding of the processes that might have shaped the community structure of the microbial assemblages within sponges.

Animals↗

PubMed2001

Biolog for the determination of microbial diversity in activated sludge systems.

In this study, different carbon source profiles were generated by inoculating Biolog GN microwell plates, with different dilutions of microbial communities from a number of activated sludge systems. This led to the successful generation of patterns reflecting diversity and evenness in the different systems. The high number of substrates utilized at the lower dilutions (10(-1) and 10(-2)) indicated a high microbial diversity in the community, but not necessarily evenness of each species. Evenness of each species was reflected upon further dilution. Our results indicated differences in the microbial community composition amongst some of the activated sludge systems studied. These differences were not specifically related to phosphate removing and non phosphate removing systems.

Bacteria↗

PubMed2002

Microbial diversity and complexity in hypersaline environments: a preliminary assessment.

The microbial communities in solar salterns and a soda lake have been characterized using two techniques: BIOLOG, to estimate the metabolic potential, and amplicon length heterogeneity analysis, to estimate the molecular diversity of these communities. Both techniques demonstrated that the halophilic Bacteria and halophilic Archaea populations in the Eilat, Israel saltern are dynamic communities with extensive metabolic potentials and changing community structures. Halophilic Bacteria were detected in Mono Lake and the lower salinity ponds at the Shark Bay saltern in Western Australia, except when the crystallizer samples were stressed by exposure to Acid Green Dye #9899. At Shark Bay, halophilic Archaea were found only in the crystallizer samples. These data confirm both the metabolic diversity and the phylogenetic complexity of the microbial communities and assert the need to develop more versatile media for the cultivation of the diversity of bacteria in hypersaline environments.

Archaea↗

PubMed2005

Natural microbial diversity in superficial sediments of Milazzo Harbor (Sicily) and community successions during microcosm enrichment with various hydrocarbons.

Hydrocarbon-contaminated superficial sediments collected from the Harbor of Milazzo (Tirrenean Sea, northern Sicily), a zone strongly affected by anthropogenic activities, were examined for in situ biodegradative capacities. A culture-independent molecular phylogenetic approach was used to study the influence of hydrocarbon and nutrient addition on the activity and diversity of the indigenous microbiota during a microcosm evaluation. The autochthonous microbial community in non-polluted sediments was represented by eubacterial phylotypes grouped within Proteobacteria, CFB and Firmicutes. The archaeal domain was represented by members of Marine Group I of Crenarchaeota. The majority of recovered sequences was affiliated with heterotrophic genera Clostridium and Vibrio, typical members of eutrophic coastal environments. Amendments of hydrocarbons and mineral nutrients to microcosms dramatically changed the initial diversity of the microbial community. Only bacterial phylotypes affiliated with Proteobacteria and CFB division were detected. The decrease in diversity observed in several microcosms could be explained by the strong selection for microorganisms belonging to group of marine hydrocarbonoclastic gamma-Proteobacteria, namely Alcanivorax, Cycloclasticus, Marinobacter, Marinobacterium/Neptunomonas and Thalassolituus. This study demonstrated that nutrient amendment to hydrocarbon-contaminated superficial sediments enhanced the indigenous microbial biodegradation activity and that highly specialized marine hydrocarbonoclastic bacteria, representing a minor fraction in the natural microbial community, play an important role in the biodegradation of petroleum hydrocarbons accidentally entering the coastal environment.

Base Sequence↗

PubMed1997

Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA.

A quantitative molecular technique was developed for rapid analysis of microbial community diversity in various environments. The technique employed PCR in which one of the two primers used was fluorescently labeled at the 5' end and was used to amplify a selected region of bacterial genes encoding 16S rRNA from total community DNA. The PCR product was digested with restriction enzymes, and the fluorescently labeled terminal restriction fragment was precisely measured by using an automated DNA sequencer. Computer-simulated analysis of terminal restriction fragment length polymorphisms (T-RFLP) for 1,002 eubacterial sequences showed that with proper selection of PCR primers and restriction enzymes, 686 sequences could be PCR amplified and classified into 233 unique terminal restriction fragment lengths or "ribotypes." Using T-RFLP, we were able to distinguish all bacterial strains in a model bacterial community, and the pattern was consistent with the predicted outcome. Analysis of complex bacterial communities with T-RFLP revealed high species diversity in activated sludge, bioreactor sludge, aquifer sand, and termite guts; as many as 72 unique ribotypes were found in these communities, with 36 ribotypes observed in the termite guts. The community T-RFLP patterns were numerically analyzed and hierarchically clustered. The pattern derived from termite guts was found to be distinctly different from the patterns derived from the other three communities. Overall, our results demonstrated that T-RFLP is a powerful tool for assessing the diversity of complex bacterial communities and for rapidly comparing the community structure and diversity of different ecosystems.

DNA, Bacterial↗

PubMed2004

The ecology and genetics of microbial diversity.

Natural communities of microbes are often diverse, a fact that is difficult to reconcile with the action of natural selection in simple, uniform environments. We suggest that this apparent paradox may be resolved by considering the origin and fate of diversity in an explicitly ecological context. Here, we review insights into the ecological and genetic causes of diversity that stem from experiments with microbial populations evolving in the defined conditions of the laboratory environment. These studies highlight the importance of environmental structure in governing the fate of diversity and shed light on the genetic mechanisms generating diversity. We conclude by emphasizing the importance of placing detailed molecular-level studies within the context of a sound ecological and evolutionary framework.

Bacteria↗

PubMed2006

Microbial diversity and succession during the manufacture and ripening of traditional, Spanish, blue-veined Cabrales cheese, as determined by PCR-DGGE.

The diversity and dynamics of the dominant microbial communities arising during the manufacture and ripening of four batches of naturally fermented Cabrales cheese were investigated by the PCR-DGGE culture-independent technique. Total microbial DNA was extracted from cheese milk, curd and cheese samples and used as template material in PCR experiments to amplify the V3 region of the bacterial 16S rRNA gene, plus the D1 region of the eukaryotic 26S rRNA gene. These regions were then analysed using DGGE. Eukaryotic and bacterial bands were identified by isolation, reamplification and sequencing. The results were compared to those obtained in a previous microbial characterization of the same four batches using classical culturing methods. Great variability was recorded between batches by the PCR-DGGE technique. This was also shown by culturing, and underlines the uniqueness of artisanal products. Lactocococcus lactis subsp. lactis was dominant from the cheese milk stage until the end of ripening, whereas populations of certain Lactobacillus species appeared during ripening. Populations of species never isolated by culturing were found to be numerous by the PCR-DGGE method, in particular Lactococcus garvieae and Lactococcus raffinolactis. Other, completely unknown lactococci were also detected. The dominant eukaryotic populations from day 15 onwards were those of Penicillium roqueforti and Geotrichum candidum.

Cheese↗

PubMed2002

Spatial and resource factors influencing high microbial diversity in soil.

To begin defining the key determinants that drive microbial community structure in soil, we examined 29 soil samples from four geographically distinct locations taken from the surface, vadose zone, and saturated subsurface using a small-subunit rRNA-based cloning approach. While microbial communities in low-carbon, saturated, subsurface soils showed dominance, microbial communities in low-carbon surface soils showed remarkably uniform distributions, and all species were equally abundant. Two diversity indices, the reciprocal of Simpson's index (1/D) and the log series index, effectively distinguished between the dominant and uniform diversity patterns. For example, the uniform profiles characteristic of the surface communities had diversity index values that were 2 to 3 orders of magnitude greater than those for the high-dominance, saturated, subsurface communities. In a site richer in organic carbon, microbial communities consistently exhibited the uniform distribution pattern regardless of soil water content and depth. The uniform distribution implies that competition does not shape the structure of these microbial communities. Theoretical studies based on mathematical modeling suggested that spatial isolation could limit competition in surface soils, thereby supporting the high diversity and a uniform community structure. Carbon resource heterogeneity may explain the uniform diversity patterns observed in the high-carbon samples even in the saturated zone. Very high levels of chromium contamination (e.g., >20%) in the high-organic-matter soils did not greatly reduce the diversity. Understanding mechanisms that may control community structure, such as spatial isolation, has important implications for preservation of biodiversity, management of microbial communities for bioremediation, biocontrol of root diseases, and improved soil fertility.

Bacteria↗

PubMed2006

Accessing the black box of microbial diversity and ecophysiology: recent advances through polyphasic experiments.

The microbial ecology of a range of anaerobic biological assemblages (granular sludge) from full- and laboratory-scale wastewater treatment bioreactors, and of crop-growing and peat soils, was determined using a variety of 16S rRNA gene-based techniques, including clone library, terminal restriction fragment length polymorphism (TRFLP) and denaturing gradient gel electrophoresis (DGGE) analyses. Fluorescent in situ hybridization (FISH) using 16S rRNA gene-targeted probes was employed to complete a "full-cycle rRNA approach" with selected biomass. Genetic fingerprinting (TRFLP and DGGE) was effectively used to elucidate community structure-crop relationships, and to detect and monitor trends in bioreactor sludge and specific enrichment cultures of peat soil. Greater diversity was resolved within bacterial than within archaeal communities, and unexpected reservoirs of uncultured Crenarchaeota were detected in sludge granules. Advanced radiotracer incubations and micro-beta imaging were employed in conjunction with FISH to elucidate the eco-functionalism of these organisms. Crenarchaeota clusters were identified in close associated with methanogenic Archaea and both were localised with acetate uptake in biofilm structure.

Archaea↗

PubMed2004

Characterisation of the microbial diversity in a pig manure storage pit using small subunit rDNA sequence analysis.

The microbial community structure of pig manure slurry (PMS) was determined with comparative analysis of 202 bacterial, 44 archaeal and 33 eukaryotic small subunit (SSU) rDNA partial sequences. Based on a criterion of 97% of sequence similarity, the phylogenetic analyses revealed a total of 108, eight and five phylotypes for the Bacteria, Archaea and Eukarya lineages, respectively. Only 36% of the bacterial phylotypes were closely related (>or=97% similarity) to any previously known sequence in databases. The bacterial groups most often represented in terms of phylotype and clone abundance were the Eubacterium (22% of total sequences), the Clostridium (15% of sequences), the Bacillus-Lactobacillus-Streptococcus subdivision (20% of sequences), theMycoplasma and relatives (10% of sequences) and the Flexibacter-Cytophaga-Bacteroides (20% of sequences). The global microbial community structure and phylotype diversity show a close relationship to the pig gastrointestinal tract ecosystem whereas phylotypes from the Acholeplasma-Anaeroplasma and the Clostridium purinolyticum groups appear to be better represented in manure. Archaeal diversity was dominated by three phylotypes clustering with a group of uncultured microorganisms of unknown activity and only distantly related to the Thermoplasmales and relatives. Other Archaea were methanogenic H2/CO2 utilisers. No known acetoclastic Archaea methanogen was found. Eukaryotic diversity was represented by a pluricellular nematode, two Alveolata, a Blastocystis and an Entamoebidae. Manure slurry physico-chemical characteristics were analysed. Possible inhibitory effects of acetate, sulphide and ammonia concentrations on the microbial anaerobic ecosystem are discussed.

Animals↗

PubMed2026

Plant genetic and root-associated microbial diversity modulate Lactuca sativa responsiveness to a soil inoculum under phosphate deficiency.

Microbial-based approaches offer a promising strategy to decrease the use of chemical fertilizers in agriculture. Among them, arbuscular mycorrhizal fungi (AMF), which extend root surface area and enhance phosphate uptake, and phosphate-solubilizing bacteria (PSB) are particularly relevant. However, their effectiveness depends strongly on plant genetic diversity. To identify genetic markers underlying plant responses to beneficial soil microbes, we studied a panel of 128 fully sequenced Lactuca sativa varieties under controlled phosphate-starvation conditions and treated with AMF and PSB. Lettuce genetic variation showed a strong effect on physiological and morphological responses to microbial inoculation. Genome-wide association studies identified specific genomic regions associated with changes in leaf phosphate content and shoot biomass following treatment. Beyond genetic factors, we observed shifts in fungal β-diversity and increased bacterial α-diversity associated with phenotypic variation. We also identified 44 amplicon sequence variants associated with agriculturally relevant traits. Among these, six bacterial strains were experimentally validated through in vitro and pot experiments for their effects on leaf phosphate concentration and shoot biomass. Overall, we highlighted key genetic, microbial, and physiological mechanisms that may enhance microbial treatments for improved plant phosphate management in lettuce.

16S and ITS metabarcoding↗

PubMed2005

Microbial diversity of the freshwater sponge Spongilla lacustris.

To provide insight into the phylogenetic bacterial diversity of the freshwater sponge Spongilla lacustris, a 16S rRNA gene libraries were constructed from sponge tissues and from lake water. Restriction fragment length polymorphism (RFLP) analysis of >190 freshwater sponge-derived clones resulted in six major restriction patterns, from which 45 clones were chosen for sequencing. The resulting sequences were affiliated with the Alpha proteobacteria (n=19), the Actinobacteria (n=15), the Beta proteobacteria (n=2), and the Chloroflexi (n=2) lineages. About half of the sequences belonged to previously described actinobacterial (hgc-I) and beta proteobacterial (beta-II) sequence clusters of freshwater bacteria that were also present in the lake water 16S rRNA gene library. At least two novel, deeply rooting alpha proteobacterial lineages were recovered from S. lacustris that showed <89% sequence similarity to known phylogenetic groups. Electron microscopical observations revealed that digested bacterial remnants were contained within food vacuoles of sponge archaeocytes, whereas the extracellular matrix was virtually free of bacteria. This study is the first molecular diversity study of a freshwater sponge and adds to a growing database on the diversity and community composition of sponge-associated microbial consortia.

Animals↗

PubMed2004

Microbial diversity during biodegradation of crude oil in seawater from the North Sea.

Microbial communities were characterized during biodegradation of immobilized oil in seawater from the Statfjord field and the German Bight in the North Sea. Seawater samples were collected at different distances from pollution sources at the two locations. A Statfjord oil was immobilized on hydrophobic synthetic Fluortex fabrics and submerged in closed flasks (no headspace) with natural or sterile seawater and incubated at 13 degrees C for 56 days. Biodegradation of immobilized n-alkanes was measured by gas chromatography, total microbes were enumerated by epifluorescence microscopy, and culturable heterotrophic and oil-degrading microorganisms were quantified by most probable number (MPN) analysis. Polymerase chain reaction (PCR) amplification of bacterial 16S rDNA in water samples was conducted during biodegradation experiments. The amplified 16S rDNA fragments were characterized by denaturing gradient gel electrophoresis (DGGE), and by sequence analysis of cloned inserts. Biodegradation rates of alkanes in seawater collected at different distances from the pollution sources did not differ significantly (P > 0.05). Concentrations of oil-degrading microorganisms showed a temporary peak after 7 days of degradation, with a subsequent decline later in the period. DGGE analysis of 16S rRNA genes showed that community diversity decreased during the first 2-3 weeks of biodegradation, with the emergence of a few dominant bands. Cloning, restriction analysis, and sequence analysis of the 16S rDNA fragments revealed >30 different phylotypes. Abundant types during biodegradation belonged to the alpha-Proteobacteria, in waters from both Statfjord and the German Bight. Cloning and sequencing studies indicated that the most abundant bacteria during biodegradation belonged to the family Rhodobacteraceae, with the closest relationship to the genera Sulfitobacter and Roseobacter.

Alkanes↗

PubMed2002

Molecular and conventional analyses of microbial diversity in mesophilic and thermophilic upflow anaerobic sludge blanket granular sludges.

The microbial community structure of mesophilic (35 degrees C) and thermophilic (55 degrees C) methanogenic granular sludges was surveyed by using both cultivation-independent molecular approach and conventional cultivation technique in order to address the fundamental questions on the microbial populations, i.e. who are present, where they are located, and what they are doing there. To elucidate the microbial constituents within both sludges, we first constructed 16S ribosomal DNA clone libraries, and partial sequencing of the clones was conducted for phylogenetic analysis. In this experiment, we found a number of unidentifiable clones within the domain Bacteria as well as clones that were closely related with 16S rDNAs of cultured microbes. The unidentifiable clones accounted for approximately 60-70% of the total clones in both mesophilic and thermophilic libraries. 16S rRNA-targeted in situ hybridization combined with confocal laser scanning microscopy was subsequently employed to examine where the uncultured populations were located within sludge granules. Spatial organization of uncultured microbes was visualized in thin-sections of both types of granules using fluorescent oligonucleotide probes, which were designed based on the clone sequences of certain novel clusters. This resulted in the detection of two types of uncultured cells in specific locations inside the granules. Finally, the goal-directed conventional cultivation technique was employed to recover such uncultured anaerobes and uncover their physiology and functions. In this approach, a total of five new species of thermophilic microorganisms were isolated, including several types of syntrophs and a novel sugar-fermenting bacterium. In the previous molecular approaches, all of these isolates were suggested to be significant populations within thermophilic granular sludge, hence obtaining these isolates in pure culture decreased the fraction of unknown clones in the previous thermophilic clone library from 70% to 40%. In conclusion, these approaches successfully revealed biodiversity and spatial organization of microbes of interest in sludge granules, and enlarged the fundamental knowledge of microbial constituents functioning as significant populations in the UASB processes.

Bacteria, Anaerobic↗