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Microbial catabolic diversity in soils contaminated with hydrocarbons and heavy metals.

Understanding indigenous microbial function in contaminated soil is crucial to the successful development and use of bioremediation technologies. We measured the catabolic diversity of indigenous microbial communities in soils with a 30-yr history of Pb, Cr, and hydrocarbon (HC) contamination using a modified substrate-induced respiration method. There were characteristic differences of microbial respirations in the response of highly versus less contaminated soils to the range of organic substrates used. The catabolic response to glucose as compared to succinic acid was approximately 1:5 in less contaminated soils, but 1:25 in highly contaminated soils. In contrast, the response ratio to glucose versus aromatics was about 1:0.4 in less contaminated soils and 1:1 in highly contaminated soils. Principal components analysis (PCA) of the responses confirmed that catabolic diversity differed between highly and less contaminated soils. Univariate analysis also indicated that catabolic diversity was reduced in highly contaminated soils. This catabolic difference was strongly associated with the alteration of microbial community composition. Statistical analyses suggested that the variation in microbial community catabolic diversity was attributed to HCs more than to Pb and Cr.

Bacteria↗

DNA-based, culture-independent strategies for evaluating microbial communities in food-associated ecosystems.

Culture-independent molecular techniques are now available to study microbial ecosystems. They are opening interesting perspectives to problems related to composition and population dynamics of microbial communities in various environmental niches (e.g., soil, water) and foods. In fermented food products, estimates of true microbial diversity is often difficult chiefly on account of the inability to cultivate most of the viable bacteria. The increasing knowledge of gene sequences and the concomitant development of new culture-independent molecular techniques are providing new and effective tools to compare the diversity of microbial communities and to monitor population dynamics in minimally disturbed samples. In this review, recent advances in these techniques are reported. Possible applications to food-associated microbial ecosystems are emphasised.

Bacteria↗

A quantitative assay for linking microbial community function and structure of a naphthalene-degrading microbial consortium.

A comprehensive culture-independent assay, called Q-FAST, was developed for concurrent identification and quantification of active microorganisms involved a specific function in a given microbial community. The development of Q-FAST was achieved by integrating the concept of stable isotope probing technique into a new quantitative fingerprinting assay called real-time-t-RFLP for microbial community structure analysis. The Q-FAST was successfully validated by using a three-member artificial microbial community containing a known naphthalene-utilizing bacterium (Pseudomonas putida G7) and two nonnaphthalene-degrading bacteria (Escherichia coli and Bacillus thuringiensis). The application of Q-FAST to identify and quantify a guild of naphthalene-utilizing microorganisms in soils revealed the involvement of eight members, with six members relating to several phylogenetic groups of eubacteria (three in beta-proteobacteria, two in gamma-proteobacteria, and one in genera Intrasporangium of Gram-positive bacteria) and two members showing no close phylogenetic affiliation to any known bacterial sequences deposited in GenBank. The quantity of three members belonging to beta-proteobacteria accounted for 34% of total 16S rDNA copies measured from the "heavier" fraction of DNA that was contributed from the DNA of microorganisms capable of incorporating 13C-labeled naphthalene into their genetic biomarkers. The other five members composed 66% of total 16S rDNA copies of active naphthalene-utilizing populations measured. Offering a powerful tool for studying microbial ecology, Q-FAST thus opens a new avenue for deeper exploration of microbial-mediated processes, mainly the quantitative relationship between microbial diversity and microbial activity in a given environment.

Bacillus thuringiensis↗

Cultivation of the ubiquitous SAR11 marine bacterioplankton clade.

The alpha-proteobacterial lineage that contains SAR11 and related ribosomal RNA gene clones was among the first groups of organisms to be identified when cultivation-independent approaches based on rRNA gene cloning and sequencing were applied to survey microbial diversity in natural ecosystems. This group accounts for 26% of all ribosomal RNA genes that have been identified in sea water and has been found in nearly every pelagic marine bacterioplankton community studied by these methods. The SAR11 clade represents a pervasive problem in microbiology: despite its ubiquity, it has defied cultivation efforts. Genetic evidence suggests that diverse uncultivated microbial taxa dominate most natural ecosystems, which has prompted widespread efforts to elucidate the geochemical activities of these organisms without the benefit of cultures for study. Here we report the isolation of representatives of the SAR11 clade. Eighteen cultures were initially obtained by means of high-throughput procedures for isolating cell cultures through the dilution of natural microbial communities into very low nutrient media. Eleven of these cultures have been successfully passaged and cryopreserved for future study. The volume of these cells, about 0.01 micro m(3), places them among the smallest free-living cells in culture.

Alphaproteobacteria↗

Importance of extra- and intracellular domains of TLR1 and TLR2 in NFkappa B signaling.

Recognition of ligands by toll-like receptor (TLR) 2 requires interactions with other TLRs. TLRs form a combinatorial repertoire to discriminate between the diverse microbial ligands. Diversity results from extracellular and intracellular interactions of different TLRs. This paper demonstrates that TLR1 and TLR2 are required for ara-lipoarabinomannan- and tripalmitoyl cysteinyl lipopeptide-stimulated cytokine secretion from mononuclear cells. Confocal microscopy revealed that TLR1 and TLR2 cotranslationally form heterodimeric complexes on the cell surface and in the cytosol. Simultaneous cross-linking of both receptors resulted in ligand-independent signal transduction. Using chimeric TLRs, we found that expression of the extracellular domains along with simultaneous expression of the intracellular domains of both TLRs was necessary to achieve functional signaling. The domains from each receptor did not need to be contained within a single contiguous protein. Chimeric TLR analysis further defined the toll/IL-1R domains as the area of crucial intracellular TLR1-TLR2 interaction.

Antibodies, Monoclonal↗

Diversity of the human intestinal microbial flora.

The human endogenous intestinal microflora is an essential "organ" in providing nourishment, regulating epithelial development, and instructing innate immunity; yet, surprisingly, basic features remain poorly described. We examined 13,355 prokaryotic ribosomal RNA gene sequences from multiple colonic mucosal sites and feces of healthy subjects to improve our understanding of gut microbial diversity. A majority of the bacterial sequences corresponded to uncultivated species and novel microorganisms. We discovered significant intersubject variability and differences between stool and mucosa community composition. Characterization of this immensely diverse ecosystem is the first step in elucidating its role in health and disease.

Adult↗

Microbial communities in the larval midgut of laboratory and field populations of cotton bollworm (Helicoverpa armigera).

We compared the bacterial communities in the larval midgut of field and laboratory populations of a polyphagous pest, the cotton bollworm (Helicoverpa armigera), using denaturing gradient gel electrophoresis (DGGE) of amplified 16S rDNA sequences and 16S library sequence analysis. DGGE profiles and 16S rDNA library sequence analysis indicated similar patterns of midgut microbial community structure and diversity: specific bacterial types existed in both populations, and a more diverse microbial community was observed in caterpillars obtained from the field. The laboratory population harbored a rather simple gut microflora consisting mostly of phylotypes belonging to Enterococcus (84%). For the field population, phylotypes belonging to Enterococcus (28%) and Lactococcus (11%), as well as Flavobacterium (10%), Acinetobacter (19%), and Stenotrophomonas (10%) were dominant members. These results provided the first comprehensive description of the microbial diversity of the midgut of the important pest cotton bollworm and suggested that the environment and food supply might influence the diversity of the gut bacterial community.

Animals↗

Effects of above-ground plant species composition and diversity on the diversity of soil-borne microorganisms.

A coupling of above-ground plant diversity and below-ground microbial diversity has been implied in studies dedicated to assessing the role of macrophyte diversity on the stability, resilience, and functioning of ecosystems. Indeed, above-ground plant communities have long been assumed to drive below-ground microbial diversity, but to date very little is known as to how plant species composition and diversity influence the community composition of micro-organisms in the soil. We examined this relationship in fields subjected to different above-ground biodiversity treatments and in field experiments designed to examine the influence of plant species on soil-borne microbial communities. Culture-independent strategies were applied to examine the role of wild or native plant species composition on bacterial diversity and community structure in bulk soil and in the rhizosphere. In comparing the influence of Cynoglossum officinale (hound's tongue) and Cirsium vulgare (spear thistle) on soil-borne bacterial communities, detectable differences in microbial community structure were confined to the rhizosphere. The colonisation of the rhizosphere of both plants was highly reproducible, and maintained throughout the growing season. In a separate experiment, effects of plant diversity on bacterial community profiles were also only observed for the rhizosphere. Rhizosphere soil from experimental plots with lower macrophyte diversity showed lower diversity, and bacterial diversity was generally lower in the rhizosphere than in bulk soil. These results demonstrate that the level of coupling between above-ground macrophyte communities and below-ground microbial communities is related to the tightness of the interactions involved. Although plant species composition and community structure appear to have little discernible effect on microbial communities inhabiting bulk soil, clear and reproducible changes in microbial community structure and diversity are observed in the rhizosphere.

Bacteria↗

Measuring soil microbial community diversity using polar lipid fatty acid and denaturing gradient gel electrophoresis data.

The possibility of calculating useful microbial community diversity indices from environmental polar lipid fatty acid and 16S rDNA PCR-DGGE data was investigated. First, the behavior of the species richness, Shannon's, and Simpson's diversity indices were determined on polar lipid fatty acid profiles of 115 pure cultures, communities constructed from those profiles with different numbers of species, and constructed communities with different distributions of species. Differences in the species richness of these artificial communities was detected by all three diversity indices, but they were insensitive to the evenness of the distribution of species. Second, data from a field experiment with substrate addition to soil was used to compare the methods developed for lipid- and DNA-based diversity indices. Very good agreement was found between indices calculated from environmental polar lipid fatty acid profiles and denaturing gradient gel electrophoresis profiles from matched samples (Pearson's correlation coefficient r=0.95-0.96). A method for data pre-treatment for diversity calculations is described.

Bacteria↗

The diversity of microorganisms associated with Acromyrmex leafcutter ants.

BACKGROUND: Molecular biological techniques are dramatically changing our view of microbial diversity in almost any environment that has so far been investigated. This study presents a systematic survey of the microbial diversity associated with a population of Acromyrmex leafcutter ants. In contrast to previous studies on social insects, which targeted specific groups of symbionts occurring in the gut (termites, Tetraponera ants) or in specialised cells (Camponotus ants) the objective of our present study was to do a total screening of all possible micro-organisms that can be found inside the bodies of these leafcutter ants. RESULTS: We amplified, cloned and sequenced SSU rRNA encoding gene fragments from 9 microbial groups known to have insect-associated representatives, and show that: (1) representatives of 5 out of 9 tested groups are present, (2) mostly several strains per group are present, adding up to a total of 33 different taxa. We present the microbial taxa associated with Acromymex ants in a phylogenetic context (using sequences from GenBank) to assess and illustrate to which known microorganisms they are closely related. The observed microbial diversity is discussed in the light of present knowledge on the evolutionary history of Acromyrmex leafcutter ants and their known mutualistic and parasitic symbionts. CONCLUSIONS: The major merits of the screening approach documented here is its high sensitivity and specificity, which allowed us to identify several microorganisms that are promising candidates for further study of their interactions with Acromyrmex leafcutter ants or their gardens.

Animals↗

Influence of soil zinc concentrations on zinc sensitivity and functional diversity of microbial communities.

Pollution induced community tolerance (PICT) is based on the phenomenon that toxic effects reduce survival of the most sensitive organisms, thus increasing community tolerance. Community tolerance for a contaminant is thus a strong indicator for the presence of that contaminant at the level of adverse concentrations. Here we assessed PICT in 11 soils contaminated with zinc runoff from galvanised electricity pylons and 11 reference soils sampled at 10 m distance from these pylons. Using PICT, the influence of background concentration and bioavailability of zinc on zinc sensitivity and functional diversity of microbial communities was assessed. Zinc sensitivity of microbial communities decreased significantly with increasing zinc concentrations in pore water and calcium chloride extracted fraction while no significant relationship was found with total zinc concentration in the soil. It was also found that functional diversity of microbial communities decreased with increasing zinc concentrations, indicating that increased tolerance is indeed an undesirable phenomenon when environmental quality is considered. The hypothesis that zinc sensitivity of microbial communities is related to background zinc concentration in pore water could not be confirmed.

Bacteria↗

Comparative metagenomic analysis of microbial communities: unravelling microbial communities from the great Rann of Kachchh and coastal saltpans, Gujarat, India.

Hypersaline environments exhibit extreme physiochemical conditions yet support diverse microbial communities. These communities are not only ecologically important but also possess substantial potential for biotechnological exploitation. In this study, we employed a comparative metagenomic approach to assess microbial diversity using two distinct methodologies: (1) direct DNA extraction from raw sediment, and (2) DNA extraction following halophilic enrichment in selective media. Sediment samples were collected from multiple sites and pooled together within the Rann of Kachchh and close-by saltpans and were analysed using 16S rRNA sequencing coupled with bioinformatics pipelines. The results revealed pronounced differences in microbial community composition between the two approaches. Raw sediment samples exhibited significantly higher alpha diversity, with dominant taxa including Halobacterota, Cyanobacteria, and Desulfobacterota, with a substantial proportion of unclassified genera. In contrast, enriched samples were dominated by fast-growing, culturable genera such as Halobacterium, Alkalibacillus, and Candidatus haloredivivus. Principal Coordinate Analysis (PCoA) of beta diversity demonstrated distinct clustering between raw and enriched communities, even within samples from the same sites, underscoring the selective bias introduced by enrichment procedures. These findings emphasise that the methodological choice strongly influences the observed microbial diversity. The aim of this study was to compare microbial community composition in raw hypersaline sediments and enrichment cultures using metagenomic sequencing, to evaluate how enrichment selectively favours specific halophilic taxa. This comparative approach allows identification of the microbial groups that rapidly proliferate under controlled hypersaline conditions, thereby complementing direct environmental sequencing. By integrating both direct and enrichment-based metagenomic approaches, a more comprehensive understanding of microbial community structure in hypersaline environments can be achieved.

India↗

Environmental Stresses Constrain Soil Microbial Community Functions by Regulating Deterministic Assembly and Niche Width.

Increasing evidence indicates that the loss of soil microbial α-diversity triggered by environmental stress negatively impacts microbial functions; however, the effects of microbial α-diversity on community functions under environmental stress are poorly understood. Here, we investigated the changes in bacterial and fungal α- diversity along gradients of five natural stressors (temperature, precipitation, plant diversity, soil organic C and pH) across 45 grasslands in China and evaluated their connection with microbial functional traits. By quantifying the five environmental stresses into an integrated stress index, we found that the bacterial and fungal α-diversity declined under high environmental stress across three soil layers (0-20 cm, 20-40 cm and 40-60 cm). Metagenomic-based analyses showed that the diversity of functional genes decreased along the stress gradients. High stress enhanced the abundance of genes associated with broad functional categories (e.g., glycolysis/gluconeogenesis, TCA cycle, DNA replication/repair and cell growth/death) but reduced the abundance of genes linked to specialised functional categories (e.g., C, N, S and methane metabolism). Phylogenetic null models and niche analyses indicated that stochastic assembly processes predominated in high-diversity communities, in which bacterial and fungal taxa had a narrow ecological niche. However, in low-diversity communities, deterministic assembly processes were dominant, and taxa had wide niches, correlating with the reduction in gene abundance observed for broad and specialised functional categories. Given the essential role of the microbiome in regulating ecosystem functions, our findings suggest that low-diversity-induced deterministic community assembly processes and a wide niche under high environmental stress may regulate microbial functions. These findings emphasise the ecological mechanisms through which microbial biodiversity regulates terrestrial ecosystem functioning.

Soil Microbiology↗

Influence of an oyster reef on development of the microbial heterotrophic community of an estuarine biofilm.

We characterized microbial biofilm communities developed over two very closely located but distinct benthic habitats in the Pensacola Bay estuary using two complementary cultivation-independent molecular techniques. Biofilms were grown for 7 days on glass slides held in racks 10 to 15 cm over an oyster reef and an adjacent muddy sand bottom. Total biomass and optical densities of dried biofilms showed dramatic differences for oyster reef versus non-oyster reef biofilms. This study assessed whether the observed spatial variation was reflected in the heterotrophic prokaryotic species composition. Genomic biofilm DNA from both locations was isolated and served as a template to amplify 16S rRNA genes with universal eubacterial primers. Fluorescently labeled PCR products were analyzed by terminal restriction fragment length polymorphism, creating a genetic fingerprint of the composition of the microbial communities. Unlabeled PCR products were cloned in order to construct a clone library of 16S rRNA genes. Amplified ribosomal DNA restriction analysis was used to screen and define ribotypes. Partial sequences from unique ribotypes were compared with existing database entries to identify species and to construct phylogenetic trees representative of community structures. A pronounced difference in species richness and evenness was observed at the two sites. The biofilm community structure from the oyster reef setting had greater evenness and species richness than the one from the muddy sand bottom. The vast majority of the bacteria in the oyster reef biofilm were related to members of the gamma- and delta-subdivisions of Proteobacteria, the Cytophaga-Flavobacterium -Bacteroides cluster, and the phyla Planctomyces and Holophaga-Acidobacterium. The same groups were also present in the biofilm harvested at the muddy sand bottom, with the difference that nearly half of the community consisted of representatives of the Planctomyces phylum. Total species richness was estimated to be 417 for the oyster reef and 60 for the muddy sand bottom, with 10.5% of the total unique species identified being shared between habitats. The results suggest dramatic differences in habitat-specific microbial diversity that have implications for overall microbial diversity within estuaries.

Animals↗

Dilution/extinction of community phenotypic characters to estimate relative structural diversity in mixed communities.

Recent interest in microbial diversity has led to increased emphasis on the development of appropriate techniques. Structural diversity encompasses the number and distribution of separate or interacting biological entities responsible for a given function within the overall set of functions of a community. This study evaluated an approach for estimating the relative degree of structural diversity in heterotrophic microbial communities by dilution to extinction of community phenotypic traits. Serial dilutions of environmental samples (rhizosphere, stream) were tested for community phenotypic traits (i.e. carbon source respiration). The non-linear relationship between the number of positive responses (i.e. functional richness or R) and inoculum density in each sample dilution (I) fit the simple rectangular hyperbola model, allowing estimation of the maximal richness (R(max)) and the inoculum density at half-maximal richness (K(I)). The later term appears to be useful in assessing relative structural diversity as evidenced by significantly higher values for communities with higher predicted species diversity. The examination of community functional characteristics across a series of dilutions, particularly in conjunction with other techniques, may be a useful approach for the study of microbial diversity and related ecological parameters such as niche width and metabolic redundancy.

Journal Article↗

Genetic Diversity of Microbial Eukaryotes in Anoxic Sediment of the Saline Meromictic Lake Namako-ike (Japan): On the Detection of Anaerobic or Anoxic-tolerant Lineages of Eukaryotes.

Available sequence data on eukaryotic small-subunit ribosomal DNA (SSU rDNA) directly retrieved from various environments have increased recently, and the diversity of microbial eukaryotes (protists) has been shown to be much greater than previously expected. However, the molecular information accumulated to date does still not thoroughly reveal ecological distribution patterns of microbial eukaryotes. In the ongoing challenge to detect anaerobic or anoxic-tolerant lineages of eukaryotes, we directly extracted DNA from the anoxic sediment of a saline meromictic lake, constructed genetic libraries of PCR-amplified SSU rDNA, and performed phylogenetic analyses with the cloned SSU rDNA sequences. Although a few sequences could not be confidently assigned to any major eukaryotic groups in the analyses and are debatable regarding their taxonomic positions, most sequences obtained have affiliations with known major lineages of eukaryotes (Cercozoa, Alveolata, Stramenopiles, and Opisthokonta). Among these sequences, some branched with lineages predominantly composed of uncultured environmental clones retrieved from other anoxic environments, while others were closely related to those of eukaryotic parasites (e.g. Phytomyxea of Cercozoa, Gregarinea of Alveolata, and Ichthyosporea of Opisthokonta).

Anaerobiosis↗

Theoretical and practical aspects of the quantification of biodiversity among microorganisms.

The quantification of biodiversity among microorganisms has to address both theoretical and practical aspects. Species concepts are often at variance with those applied in macroorganisms, and satisfactory concepts suitable for general use in bacteria and fungi have yet to be formulated. Molecular approaches have not yet provided a universal solution to this key issue. Quantification in habitats such as soil is difficult as isolation procedures yield only a small and skewed selection of the microorganisms present. Indices of taxonomic or phylogenetic diversity have potential in the quantification of microbial diversity at a range of ranks, but the non-equivalence of ranks and representatives of the taxa detected have to be addressed. Chemical and molecular methods have immense potential in the quantification of microbial diversity in environmental samples; 16S rRNA has shown particular promise with bacteria, but as yet the fungi lack a universal probe. A greater awareness of the limitations of existing approaches and methodologies used by microbiologists is needed, but significant progress can be anticipated as new technologies are developed and become more widely adopted.

Bacteria↗

Microbial colonization of beech and spruce litter--influence of decomposition site and plant litter species on the diversity of microbial community.

The present study was conducted to investigate the effect of decomposition site and plant litter species on the colonizing microbial communities. For this, litter bag technique using beech and spruce litter was combined with RNA-based fingerprinting and cloning. Litter bags were incubated for 2 and 8 weeks in the Ah horizon of beech and beech-spruce mixed forest sites. Although sugars and starch were rapidly lost, lignin content increased by more than 40% for beech and more than doubled for spruce litter at both soil sites at the end of the experiment. Denaturing gradient gel electrophoresis analysis of 16S and 18S rRNA RT-PCR products was used for screening of differences between bacterial and fungal communities colonizing the two litter types. Development of the microbial community over time was observed to be specific for each litter type and decomposition site. RT-PCR products from both litter types incubated in beech-spruce mixed forest site were also cloned to identify the bacterial and fungal colonizers. The 16S rRNA clone libraries of beech litter were dominated by gamma-proteobacterial members, whereas spruce libraries were mainly composed of alpha-, beta-, and gamma-proteobacterial members. Ascomycota members dominated the 18S rRNA clone libraries. Clones similar to Zygomycota were absent from spruce, whereas those similar to Basidiomycota and Glomeromycota were absent from beech libraries. Selective effects of litter quality were observed after 8 weeks. The study provides an insight into the bacterial and fungal communities colonizing beech and spruce litter, and the importance of litter quality and decomposition site as key factors in their development and succession.

Bacteria↗