Showing papers in "The ISME Journal in 2009"

A comprehensive survey of soil acidobacterial diversity using pyrosequencing and clone library analyses

Abstract: Acidobacteria are ubiquitous and abundant members of soil bacterial communities. However, an ecological understanding of this important phylum has remained elusive because its members have been difficult to culture and few molecular investigations have focused exclusively on this group. We generated an unprecedented number of acidobacterial DNA sequence data using pyrosequencing and clone libraries (39 707 and 1787 sequences, respectively) to characterize the relative abundance, diversity and composition of acidobacterial communities across a range of soil types. To gain insight into the ecological characteristics of acidobacterial taxa, we investigated the largescale biogeographic patterns exhibited by acidobacterial communities, and related soil and site characteristics to acidobacterial community assemblage patterns. The 87 soils analyzed by pyrosequencing contained more than 8600 unique acidobacterial phylotypes (at the 97% sequence similarity level). One phylotype belonging to Acidobacteria subgroup 1, but not closely related to any cultured representatives, was particularly abundant, accounting for 7.4% of bacterial sequences and 17.6% of acidobacterial sequences, on average, across the soils. The abundance of Acidobacteria relative to other bacterial taxa was highly variable across the soils examined, but correlated strongly with soil pH (R ¼� 0.80, Po0.001). Soil pH was also the best predictor of acidobacterial community composition, regardless of how the communities were characterized, and the relative abundances of the dominant Acidobacteria subgroups were readily predictable. Acidobacterial communities were more phylogenetically clustered as soil pH departed from neutrality, suggesting that pH is an effective habitat filter, restricting community membership to progressively more narrowly defined lineages as pH deviates from neutrality.

Towards the definition of a core of microorganisms involved in anaerobic digestion of sludge

Abstract: The microbial consortium involved in anaerobic digestion has not yet been precisely characterized and this process remains a ‘black box’ with limited efficiency. In this study, seven anaerobic sludge digesters were selected based on technology, type of sludge, process and water quality. The prokaryotic community of these digesters was examined by constructing and analysing a total of 9890 16S rRNA gene clones. Libraries were constructed using primers specific for the Bacteria and Archaea domains for each digester, respectively. After phylogenetic affiliation, the libraries were compared using statistical tools to determine the similarities or differences among the seven digesters. Results show that the prokaryotic community of an anaerobic digester is composed of phylotypes commonly found in all anaerobic digesters sampled and also of specific phylotypes. The Archaea community is represented by an equilibrium among a restricted number of operational taxonomic units (OTUs). These OTUs are affiliated with Methanosarcinales, Methanomicrobiales and Arc I phylogenetic groups. Statistical analysis revealed that the Bacteria community can be described as a three component model: one-third making up a core group of phylotypes common to most of the digesters, one-third are phylotypes shared among a few digesters and another one-third are specific phylotypes. The core group is composed of only six OTUs affiliated with Chloroflexi, Betaproteobacteria, Bacteroidetes and Synergistetes. Its role in anaerobic degradation appears critical to investigate. This comparison of anaerobic digester populations is a first step towards a future understanding of the relationship among biodiversity, operating conditions and digester efficiency.

16S rRNA gene-based analysis of fecal microbiota from preterm infants with and without necrotizing enterocolitis

Abstract: Neonatal necrotizing enterocolitis (NEC) is an inflammatory intestinal disorder affecting preterm infants. Intestinal bacteria have an important function; however no causative pathogen has been identified. The purpose of this study was to determine if there are differences in microbial patterns that may be critical to the development of this disease. Fecal samples from 20 preterm infants, 10 with NEC and 10 matched controls (including 4 twin pairs) were obtained from patients in a single site level III neonatal intensive care unit. Bacterial DNA from individual fecal samples was PCR-amplified and subjected to terminal restriction fragment length polymorphism analysis and library sequencing of the 16S rRNA gene to characterize diversity and structure of the enteric microbiota. The distribution of samples from NEC patients distinctly clustered separately from controls. Intestinal bacterial colonization in all preterm infants was notable for low diversity. Patients with NEC had even less diversity, an increase in abundance of Gammaproteobacteria, a decrease in other bacteria species, and had received a higher mean number of previous days of antibiotics. Our results suggest that NEC is associated with severe lack of microbiota diversity that may accentuate the impact of single dominant microorganisms favored by empiric and widespread use of antibiotics.

Relationship between N-cycling communities and ecosystem functioning in a 50-year-old fertilization experiment.

Abstract: The relative importance of size and composition of microbial communities in ecosystem functioning is poorly understood. Here, we investigated how community composition and size of selected functional guilds in the nitrogen cycle correlated with agroecosystem functioning, which was defined as microbial process rates, total crop yield and nitrogen content in the crop. Soil was sampled from a 50-year fertilizer trial and the treatments comprised unfertilized bare fallow, unfertilized with crop, and plots with crop fertilized with calcium nitrate, ammonium sulfate, solid cattle manure or sewage sludge. The size of the functional guilds and the total bacterial community were greatly affected by the fertilization regimes, especially by the sewage sludge and ammonium sulfate treatments. The community size results were combined with previously published data on the composition of the corresponding communities, potential ammonia oxidation, denitrification, basal and substrate-induced respiration rates, in addition to crop yield for an integrated analysis. It was found that differences in size, rather than composition, correlated with differences in process rates for the denitrifier and ammonia-oxidizing archaeal and total bacterial communities, whereas neither differences in size nor composition was correlated with differences in process rates for the ammonia-oxidizing bacterial community. In contrast, the composition of nitrate-reducing, denitrifying and total bacterial communities co-varied with primary production and both were strongly linked to soil properties.

A comparison of homologous recombination rates in bacteria and archaea.

Abstract: It is a standard practice to test for the signature of homologous recombination in studies examining the genetic diversity of bacterial populations. Although it has emerged that homologous recombination rates can vary widely between species, comparing the results from different studies is made difficult by the diversity of estimation methods used. Here, Multi Locus Sequence Typing (MLST) datasets from a wide variety of bacteria and archaea are analyzed using the ClonalFrame method. This enables a direct comparison between species and allows for a first exploration of the question whether phylogeny or ecology is the primary determinant of homologous recombination rate.

Functional metagenomics reveals diverse beta-lactamases in a remote Alaskan soil.

Abstract: Despite the threat posed by antibiotic resistance in infectious bacteria, little is known about the diversity, distribution and origins of resistance genes, particularly among the as yet unculturable environmental bacteria. One potentially rich but largely unstudied environmental reservoir is soil. The complexity of its microbial community coupled with its high density of antibiotic-producing bacteria makes the soil a likely origin for diverse antibiotic resistance determinants. To investigate antibiotic resistance genes among uncultured bacteria in an undisturbed soil environment, we undertook a functional metagenomic analysis of a remote Alaskan soil. We report that this soil is a reservoir for b-lactamases that function in Escherichia coli, including divergent b-lactamases and the first bifunctional b-lactamase. Our findings suggest that even in the absence of selective pressure imposed by anthropogenic activity, the soil microbial community in an unpolluted site harbors unique and ancient b-lactam resistance determinants. Moreover, despite their evolutionary distance from previously known genes, the Alaskan b-lactamases confer resistance on E. coli without manipulating its gene expression machinery, demonstrating the potential for soil resistance genes to compromise human health, if transferred to pathogens.

Shotgun metaproteomics of the human distal gut microbiota.

Abstract: The human gut contains a dense, complex and diverse microbial community, comprising the gut microbiome. Metagenomics has recently revealed the composition of genes in the gut microbiome, but provides no direct information about which genes are expressed or functioning. Therefore, our goal was to develop a novel approach to directly identify microbial proteins in fecal samples to gain information about the genes expressed and about key microbial functions in the human gut. We used a non-targeted, shotgun mass spectrometry-based whole community proteomics, or metaproteomics, approach for the first deep proteome measurements of thousands of proteins in human fecal samples, thus demonstrating this approach on the most complex sample type to date. The resulting metaproteomes had a skewed distribution relative to the metagenome, with more proteins for translation, energy production and carbohydrate metabolism when compared to what was earlier predicted from metagenomics. Human proteins, including antimicrobial peptides, were also identified, providing a non-targeted glimpse of the host response to the microbiota. Several unknown proteins represented previously undescribed microbial pathways or host immune responses, revealing a novel complex interplay between the human host and its associated microbes.

Systematic artifacts in metagenomes from complex microbial communities

Abstract: Metagenomics is providing an unprecedented view of the taxonomic diversity, metabolic potential and ecological role of microbial communities in biomes as diverse as the mammalian gastrointestinal tract, the marine water column and soils. However, we have found a systematic error in metagenomes generated by 454-based pyrosequencing that leads to an overestimation of gene and taxon abundance; between 11% and 35% of sequences in a typical metagenome are artificial replicates. Here we document the error in several published and original datasets and offer a web-based solution (http://microbiomes.msu.edu/replicates) for identifying and removing these artifacts.

Skin microbes on frogs prevent morbidity and mortality caused by a lethal skin fungus

Abstract: Emerging infectious diseases threaten human and wildlife populations. Altered ecological interactions between mutualistic microbes and hosts can result in disease, but an understanding of interactions between host, microbes and disease-causing organisms may lead to management strategies to affect disease outcomes. Many amphibian species in relatively pristine habitats are experiencing dramatic population declines and extinctions due to the skin disease chytridiomycosis, which is caused by the chytrid fungus Batrachochytrium dendrobatidis. Using a randomized, replicated experiment, we show that adding an antifungal bacterial species, Janthinobacterium lividum, found on several species of amphibians to the skins of the frog Rana muscosa prevented morbidity and mortality caused by the pathogen. The bacterial species produces the anti-chytrid metabolite violacein, which was found in much higher concentrations on frog skins in the treatments where J. lividum was added. Our results show that cutaneous microbes are a part of amphibians' innate immune system, the microbial community structure on frog skins is a determinant of disease outcome and altering microbial interactions on frog skins can prevent a lethal disease outcome. A bioaugmentation strategy may be an effective management tool to control chytridiomycosis in amphibian survival assurance colonies and in nature.

What is microbial community ecology

Abstract: The activities of complex communities of microbes affect biogeochemical transformations in natural, managed and engineered ecosystems. Meaningfully defining what constitutes a community of interacting microbial populations is not trivial, but is important for rigorous progress in the field. Important elements of research in microbial community ecology include the analysis of functional pathways for nutrient resource and energy flows, mechanistic understanding of interactions between microbial populations and their environment, and the emergent properties of the complex community. Some emergent properties mirror those analyzed by community ecologists who study plants and animals: biological diversity, functional redundancy and system stability. However, because microbes possess mechanisms for the horizontal transfer of genetic information, the metagenome may also be considered as a community property.

Abundance, composition, diversity and novelty of soil Proteobacteria

Abstract: Small subunit (16S) rRNA gene surveys generating near full-length 16S rRNA clones offer a unique opportunity for in-depth phylogenetic analysis to highlight the breadth of diversity within various major bacterial phyla encountered in soil. This study offers a detailed phylogenetic analysis of the Proteobacteria-affiliated clones identified from 13,001 nearly full-length 16S rRNA gene clones derived from Oklahoma tall-grass prairie soil. Proteobacteria was the most abundant phylum in the community, and comprised 25% of the total clones. The most abundant and diverse class within the Proteobacteria was Alphaproteobacteria, followed by the Delta-, Beta- and Gammaproteobacteria. Members of the Epsilon- and Zetaproteobacteria were not detected in the dataset. Our analysis identified 15 novel order-level and 48 novel family-level Proteobacteria lineages. In addition, we show that the majority of Proteobacteria clones in the dataset belong to orders and families containing no described cultivated representatives (50% and 65%, respectively). An examination of the ecological distribution of the six most abundant Proteobacteria lineages in this dataset with no characterized pure culture representatives provided important information regarding their global distribution and environmental preferences. This level of novel phylogenetic diversity indicates that our understanding of the functions of soil microorganisms, even those belonging to phyla with numerous and diverse well-characterized cultured representatives such as the Proteobacteria, remains far from adequate.

Bacterial diversity and White Plague Disease-associated community changes in the Caribbean coral Montastraea faveolata.

Abstract: Increasing evidence confirms the crucial role bacteria and archaea play within the coral holobiont, that is, the coral host and its associated microbial community. The bacterial component constitutes a community of high diversity, which appears to change in structure in response to disease events. In this study, we highlight the limitation of 16S rRNA gene (16S rDNA) clone library sequencing as the sole method to comprehensively describe coral-associated communities. This limitation was addressed by combining a high-density 16S rRNA gene microarray with, clone library sequencing as a novel approach to study bacterial communities in healthy versus diseased corals. We determined an increase in diversity as well as a significant shift in community structure in Montastraea faveolata colonies displaying phenotypic signs of White Plague Disease type II (WPD-II). An accumulation of species that belong to families that include known coral pathogens (Alteromonadaceae, Vibrionaceae), bacteria previously isolated from diseased, stressed or injured marine invertebrates (for example, Rhodobacteraceae), and other species (for example, Campylobacteraceae) was observed. Some of these species were also present in healthy tissue samples, but the putative primary pathogen, Aurantimonas corallicida, was not detected in any sample by either method. Although an ecological succession of bacteria during disease progression after causation by a primary agent represents a possible explanation for our observations, we also discuss the possibility that a disease of yet to be determined etiology may have affected M. faveolata colonies and resulted in (or be a result of) an increase in opportunistic pathogens.

Selective progressive response of soil microbial community to wild oat roots.

Abstract: Roots moving through soil enact physical and chemical changes that differentiate rhizosphere from bulk soil, and the effects of these changes on soil microorganisms have long been a topic of interest. Use of a high-density 16S rRNA microarray (PhyloChip) for bacterial and archaeal community analysis has allowed definition of the populations that respond to the root within the complex grassland soil community; this research accompanies previously reported compositional changes, including increases in chitinase and protease specific activity, cell numbers and quorum sensing signal. PhyloChip results showed a significant change in 7% of the total rhizosphere microbial community (147 of 1917 taxa); the 7% response value was confirmed by16S rRNA T-RFLP analysis. This PhyloChip-defined dynamic subset was comprised of taxa in 17 of the 44 phyla detected in all soil samples. Expected rhizosphere-competent phyla, such as Proteobacteria and Firmicutes, were well represented, as were less-well-documented rhizosphere colonizers including Actinobacteria, Verrucomicrobia and Nitrospira. Richness of Bacteroidetes and Actinobacteria decreased in soil near the root tip compared to bulk soil, but then increased in older root zones. Quantitative PCR revealed {beta}-Proteobacteria and Actinobacteria present at about 10{sup 8} copies of 16S rRNA genes g{sup -1} soil, with Nitrospira having about 10{sup 5} copies g{sup -1} soil. This report demonstrates that changes in a relatively small subset of the soil microbial community are sufficient to produce substantial changes in function in progressively more mature rhizosphere zones.

Transport functions dominate the SAR11 metaproteome at low-nutrient extremes in the Sargasso Sea.

Abstract: The northwestern Sargasso Sea undergoes annual cycles of productivity with increased production in spring corresponding to periods of upwelling, and oligotrophy in summer and autumn, when the water column becomes highly stratified. The biological productivity of this region is reduced during stratified periods as a result of low concentrations of phosphorus and nitrogen in the euphotic zone. To better understand the mechanisms of microbial survival in this oligotrophic environment, we used capillary liquid chromatography (LC)-tandem mass spectrometry to detect microbial proteins in surface samples collected in September 2005. A total of 2215 peptides that mapped to 236 SAR11 proteins, 1911 peptides that mapped to 402 Prochlorococcus proteins and 2407 peptides that mapped to 404 Synechococcus proteins were detected. Mass spectra from SAR11 periplasmic substrate-binding proteins accounted for a disproportionately large fraction of the peptides detected, consistent with observations that these extremely small cells devote a large proportion of their volume to periplasm. Abundances were highest for periplasmic substrate-binding proteins for phosphate, amino acids, phosphonate, sugars and spermidine. Proteins implicated in the prevention of oxidative damage and protein refolding were also abundant. Our findings support the view that competition for multiple nutrients in oligotrophic systems is extreme, but nutrient flux is sufficient to sustain microbial community activity.

Changes in land use alter the structure of bacterial communities in Western Amazon soils.

Abstract: Here we show how agricultural practices by indigenous peoples as well as forest recovery relate to the structure and composition of Amazon soil bacterial communities. Soil samples were collected in different land use systems and bacterial community composition and diversity were explored by T-RFLP, cloning and sequencing, and data were analyzed with multivariate techniques. The main differences in bacterial community structure were related to changes in the soil attributes that, in turn, were correlated to land use. Community structure changed significantly along gradients of base saturation, [Al3+] and pH. The relationship with soil attributes accounted for about 31% of the variation of the studied communities. Clear differences were observed in community composition as shown by the differential distribution of Proteobacteria, Bacteroidetes, Firmicutes, Acidobacteria and Actinobacteria. Similarity between primary and secondary forest communities indicates the recovery of bacterial community structure during succession. Pasture and crop soil communities were among the most diverse, showing that these land use types did not deplete bacterial diversity under the conditions found in our sites.

The biofilm life cycle and virulence of Pseudomonas aeruginosa are dependent on a filamentous prophage

Abstract: Mature Pseudomonas aeruginosa biofilms undergo specific developmental events. Using a bacteriophage mutant, generated by deletion of the entire filamentous Pf4 prophage, we show that the phage is essential for several stages of the biofilm life cycle and that it significantly contributes to the virulence of P. aeruginosa in vivo. Here, we show for the first time that biofilms of the Pf4 phage-deficient mutant did not develop hollow centres or undergo cell death, typical of the differentiation process of wild-type (WT) P. aeruginosa PAO1 biofilms. Furthermore, microcolonies of the Pf4 mutant were significantly smaller in size and less stable compared with the WT biofilm. Small colony variants (SCVs) were detectable in the dispersal population of the WT biofilm at the time of dispersal and cell death, whereas no SCVs were detected in the effluent of the Pf4 mutant biofilm. This study shows that at the time when cell death occurs in biofilms of the WT, the Pf4 phage converts into a superinfective form, which correlates with the appearance of variants in the dispersal population. Unexpectedly, mice infected with the Pf4 mutant survived significantly longer than those infected with its isogenic WT strain, showing that Pf4 contributes to the virulence of P. aeruginosa. Hence, a filamentous prophage is a major contributor to the life cycle and adaptive behaviour of P. aeruginosa and offers an explanation for the prevalence of phage in this organism.

Species-specific structural and functional diversity of bacterial communities in lichen symbioses

Abstract: Lichens are generally considered as mutualisms between fungi and green algae or cyanobacteria. These partnerships allow light-exposed and long-living joint structures. The unique organization of lichens provides still unexplored environments for microbial communities. To study lichen-associated bacterial communities, we analyze samples, by a polyphasic approach, from three lichen species (Cladonia arbuscula, Lecanora polytropa and Umbilicaria cylindrica) from alpine environments. Our results indicate that bacteria can form highly structured, biofilm-like assemblages on fungal surfaces and reach considerable abundances of up to 108 cells per gram fresh weight. Fluorescence in situ hybridization reveals the predominance of Alphaproteobacteria. Microbial fingerprints performed by PCR-single-strand conformation polymorphism analysis using universal and group-specific primers show distinct patterns for each lichen species. Characterization of cultivable strains and presence of functional genes in the total fraction suggest the involvement of associated bacteria in nutrient cycling. Ubiquitous nifH genes, which encode the nitrogenase reductase, show a high diversity and are assigned to Alphaproteobacteria and Firmicutes, for example, Paenibacillus. Cultivable strains mainly belonging to the genera Acinetobacter, Bacillus, Burkholderia, Methylobacterium and Paenibacillus show lytic (chitinolytic, glucanolytic, and proteolytic) activities, hormone production (indole-3-acetic acid) as well as phosphate mobilization and antagonistic activity toward other microorganisms. The traditional concept of lichens has to be expanded to consider multiple bacterial partners.

Polymerase chain reaction primers miss half of rRNA microbial diversity.

Abstract: The rRNA approach is the principal tool to study microbial diversity, but it has important biases. These include polymerase chain reaction (PCR) primers bias, and relative inefficiency of DNA extraction techniques. Such sources of potential undersampling of microbial diversity are well known, but the scale of the undersampling has not been quantified. Using a marine tidal flat bacterial community as a model, we show that even with unlimited sampling and sequencing effort, a single combination of PCR primers/DNA extraction technique enables theoretical recovery of only half of the richness recoverable with three such combinations. This shows that different combinations of PCR primers/DNA extraction techniques recover in principle different species, as well as higher taxa. The majority of earlier estimates of microbial richness seem to be underestimates. The combined use of multiple PCR primer sets, multiple DNA extraction techniques, and deep community sequencing will minimize the biases and recover substantially more species than prior studies, but we caution that even this—yet to be used—approach may still leave an unknown number of species and higher taxa undetected.

High diversity of cultivable heterotrophic bacteria in association with cyanobacterial water blooms.

Abstract: Cyanobacterial mass occurrences (water blooms) cause ecological, economic and health problems worldwide. Still, little is known about heterotrophic bacteria associated with cyanobacteria and the interactions between those organisms. We isolated 460 bacterial strains from more than 40 lakes and rivers (151 samples), Baltic Sea (32 samples) and treated drinking water of seven treatment plants (29 samples). The water bodies and the raw water of the treatment plants were frequently dominated by high numbers of cyanobacteria. Various growth media were used to isolate the strains. Analysis of partial 16S rRNA gene fragments (701–905 bp for 358 strains and 413–497 bp for 102 strains) classified the isolated bacteria as Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes and Deinococcus-Thermus. Some of these isolates represented possible new bacterial orders, families, genera or species. We isolated various potentially pathogenic bacteria, such as Aeromonas, Vibrio, Acinetobacter and Pseudomonas, that may cause adverse health effects in humans and animals and should be taken into consideration when assessing the risks caused by cyanobacterial blooms. Several strains also inhibited or enhanced the growth of cyanobacteria. Most of such strains had an enhancing effect on the cyanobacterial growth. Other isolates were affiliated with genera such as Sphingomonas or Flavobacterium, which include strains that are capable of degrading cyanobacterial toxins or other recalcitrant and problematic organic compounds. The isolated strains provide a large group of bacteria that could be used in assessing and controlling the harmful effects of cyanobacteria.

Seasonal dynamics of SAR11 populations in the euphotic and mesopelagic zones of the northwestern Sargasso Sea.

Abstract: Bacterioplankton belonging to the SAR11 clade of a-proteobacteria were counted by fluorescence in situ hybridization (FISH) over eight depths in the surface 300 m at the Bermuda Atlantic Time-series Study (BATS) site from 2003 to 2005. SAR11 are dominant heterotrophs in oligotrophic systems; thus, resolving their temporal dynamics can provide important insights to the cycling of organic and inorganic nutrients. This quantitative time-series data revealed distinct annual distribution patterns of SAR11 abundance in the euphotic (0-120) and upper mesopelagic (160-300 m) zones that were reproducibly correlated with seasonal mixing and stratification of the water column. Terminal restriction fragment length polymorphism (T-RFLP) data generated from a decade of samples collected at BATS were combined with the FISH data to model the annual dynamics of SAR11 subclade populations. 16S rRNA gene clone libraries were constructed to verify the correlation of the T-RFLP data with SAR11 clade structure. Clear vertical and temporal transitions were observed in the dominance of three SAR11 ecotypes. The mechanisms that lead to shifts between the different SAR11 populations are not well understood, but are probably a consequence of finely tuned physiological adaptations that partition the populations along physical and chemical gradients in the ecosystem. The correlation between evolutionary descent and temporal/spatial patterns we describe, confirmed that a minimum of three SAR11 ecotypes occupy the Sargasso Sea surface layer, and revealed new details of their population dynamics.

Despite strong seasonal responses, soil microbial consortia are more resilient to long-term changes in rainfall than overlying grassland.

Abstract: Climate change impacts on soil microbial communities could alter the structure of terrestrial ecosystems and biogeochemical cycles of the Earth. We used 16S rRNA gene microarrays to evaluate changes in the composition of grassland soil microbial communities under rainfall amendments simulating alternative climate change scenarios, and to compare these to responses of overlying plants and invertebrates. Following 5 years of rainfall manipulation, soil bacteria and archaea in plots where natural rain was supplemented differed little from ambient controls, despite profound treatment-related changes in the overlying grassland. During the sixth and seventh year, seasonal differences in bacterial and archaeal assemblages emerged among treatments, but only when watering exacerbated or alleviated periods of particularly aberrant conditions in the ambient climate. In contrast to effects on plants and invertebrates, effects on bacteria and archaea did not compound across seasons or years, indicating that soil microbial communities may be more robust than associated aboveground macroorganisms to certain alterations in climate.

Culture-independent identification of gut bacteria correlated with the onset of diabetes in a rat model.

Abstract: Bacteria associated with the onset of type 1 diabetes in a rat model system were identified. In two experiments, stool samples were collected at three time points after birth from bio-breeding diabetes-prone (BB-DP) and bio-breeding diabetes-resistant (BB-DR) rats. DNA was isolated from these samples and the 16S rRNA gene was amplified using universal primer sets. In the first experiment, bands specific to BB-DP and BB-DR genotypes were identified by automated ribosomal intergenic spacer analysis at the time of diabetes onset in BB-DP. Lactobacillus and Bacteroides strains were identified in the BB-DR- and BB-DP-specific bands, respectively. Sanger sequencing showed that the BB-DP and BB-DR bacterial communities differed significantly but too few reads were available to identify significant differences at the genus or species levels. A second experiment confirmed these results using higher throughput pyrosequencing and quantitative PCR of 16S rRNA with more rats per genotype. An average of 4541 and 3381 16S rRNA bacterial reads were obtained from each of the 10 BB-DR and 10 BB-DP samples collected at time of diabetes onset. Nine genera were more abundant in BB-DP whereas another nine genera were more abundant in BB-DR. Thirteen and eleven species were more abundant in BB-DP and BB-DR, respectively. An average of 23% and 10% of all reads could be classified at the genus and species levels, respectively. Quantitative PCR verified the higher abundance of Lactobacillus and Bifidobacterium in the BB-DR samples. Whether these changes are caused by diabetes or are involved in the development of the disease is unknown.

Seasonality and vertical structure of microbial communities in an ocean gyre.

Abstract: Vertical, seasonal and geographical patterns in ocean microbial communities have been observed in many studies, but the resolution of community dynamics has been limited by the scope of data sets, which are seldom up to the task of illuminating the highly structured and rhythmic patterns of change found in ocean ecosystems. We studied vertical and temporal patterns in the microbial community composition in a set of 412 samples collected from the upper 300 m of the water column in the northwestern Sargasso Sea, on cruises between 1991 and 2004. The region sampled spans the extent of deep winter mixing and the transition between the euphotic and the upper mesopelagic zones, where most carbon fixation and reoxidation occurs. A bioinformatic pipeline was developed to de-noise, normalize and align terminal restriction fragment length polymorphism (T-RFLP) data from three restriction enzymes and link T-RFLP peaks to microbial clades. Non-metric multidimensional scaling statistics resolved three microbial communities with distinctive composition during seasonal stratification: a surface community in the region of lowest nutrients, a deep chlorophyll maximum community and an upper mesopelagic community. A fourth microbial community was associated with annual spring blooms of eukaryotic phytoplankton that occur in the northwestern Sargasso Sea as a consequence of winter convective mixing that entrains nutrients to the surface. Many bacterial clades bloomed in seasonal patterns that shifted with the progression of stratification. These richly detailed patterns of community change suggest that highly specialized adaptations and interactions govern the success of microbial populations in the oligotrophic ocean.

Evidence for the functional significance of diazotroph community structure in soil.

Abstract: Microbial ecologists continue to seek a greater understanding of the factors that govern the ecological significance of microbial community structure. Changes in community structure have been shown to have functional significance for processes that are mediated by a narrow spectrum of organisms, such as nitrification and denitrification, but in some cases, functional redundancy in the community seems to buffer microbial ecosystem processes. The functional significance of microbial community structure is frequently obscured by environmental variation and is hard to detect in short-term experiments. We examine the functional significance of free-living diazotrophs in a replicated long-term tillage experiment in which extraneous variation is minimized and N-fixation rates can be related to soil characteristics and diazotroph community structure. Soil characteristics were found to be primarily impacted by tillage management, whereas N-fixation rates and diazotroph community structure were impacted by both biomass management practices and interactions between tillage and biomass management. The data suggest that the variation in diazotroph community structure has a greater impact on N-fixation rates than do soil characteristics at the site. N-fixation rates displayed a saturating response to increases in diazotroph community diversity. These results show that the changes in the community structure of free-living diazotrophs in soils can have ecological significance and suggest that this response is related to a change in community diversity.

Exogenous glucosinolate produced by Arabidopsis thaliana has an impact on microbes in the rhizosphere and plant roots

Abstract: A specificity of Brassicaceous plants is the production of sulphur secondary metabolites called glucosinolates that can be hydrolysed into glucose and biocidal products. Among them, isothiocyanates are toxic to a wide range of microorganisms and particularly soil-borne pathogens. The aim of this study was to investigate the role of glucosinolates and their breakdown products as a factor of selection on rhizosphere microbial community associated with living Brassicaceae. We used a DNA-stable isotope probing approach to focus on the active microbial populations involved in root exudates degradation in rhizosphere. A transgenic Arabidopsis thaliana line producing an exogenous glucosinolate and the associated wild-type plant associated were grown under an enriched 13CO2 atmosphere in natural soil. DNA from the rhizospheric soil was separated by density gradient centrifugation. Bacterial (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Acidobacteria), Archaea and fungal community structures were analysed by DGGE fingerprints of amplified 16S and 18S rRNA gene sequences. Specific populations were characterized by sequencing DGGE fragments. Roots of the transgenic plant line presented an altered profile of glucosinolates and other minor additional modifications. These modifications significantly influenced microbial community on roots and active populations in the rhizosphere. Alphaproteobacteria, particularly Rhizobiaceae, and fungal communities were mainly impacted by these Brassicaceous metabolites, in both structure and composition. Our results showed that even a minor modification in plant root could have important repercussions for soil microbial communities.

Microbial community structure in the North Pacific ocean.

Abstract: We report a ribosomal tag pyrosequencing study of the phylogenetic diversity of Archaea, Bacteria and Eucarya over a depth profile at the Hawaii Ocean Time-Series Station, ALOHA. The V9 region of the SSU rRNA gene was amplified from samples representing the epi- (10 m), meso- (800 m) and bathy- (4400 m) pelagia. The primers used are expected to amplify representatives of approximately 80% of known phylogenetic diversity across all three domains. Comparisons of unique sequences revealed a remarkably low degree of overlap between communities at each depth. The 444 147 sequence tags analyzed represented 62 975 unique sequences. Of these, 3707 (5.9%) occurred at two depths, and only 298 (0.5%) were observed at all three depths. At this level of phylogenetic resolution, Bacteria diversity decreased with depth but was still equivalent to that reported elsewhere for different soil types. Archaea diversity was highest in the two deeper samples. Eucarya observations and richness estimates are almost one order of magnitude higher than any previous marine microbial Eucarya richness estimates. The associations of many Eucarya sequences with putative parasitic organisms may have significant impacts on our understanding of the mechanisms controlling host population density and diversity, and point to a more significant role for microbial Eucarya in carbon flux through the microbial loop. We posit that the majority of sequences detected from the deep sea that have closest matches to sequences from non-pelagic sources are indeed native to the marine environment, and are possibly responsible for key metabolic processes in global biogeochemical cycles.

Phenazine antibiotics produced by fluorescent pseudomonads contribute to natural soil suppressiveness to Fusarium wilt.

Abstract: Natural disease-suppressive soils provide an untapped resource for the discovery of novel beneficial microorganisms and traits. For most suppressive soils, however, the consortia of microorganisms and mechanisms involved in pathogen control are unknown. To date, soil suppressiveness to Fusarium wilt disease has been ascribed to carbon and iron competition between pathogenic Fusarium oxysporum and resident non-pathogenic F. oxysporum and fluorescent pseudomonads. In this study, the role of bacterial antibiosis in Fusarium wilt suppressiveness was assessed by comparing the densities, diversity and activity of fluorescent Pseudomonas species producing 2,4-diacetylphloroglucinol (DAPG) (phlD+) or phenazine (phzC+) antibiotics. The frequencies of phlD+ populations were similar in the suppressive and conducive soils but their genotypic diversity differed significantly. However, phlD genotypes from the two soils were equally effective in suppressing Fusarium wilt, either alone or in combination with non-pathogenic F. oxysporum strain Fo47. A mutant deficient in DAPG production provided a similar level of control as its parental strain, suggesting that this antibiotic does not play a major role. In contrast, phzC+ pseudomonads were only detected in the suppressive soil. Representative phzC+ isolates of five distinct genotypes did not suppress Fusarium wilt on their own, but acted synergistically in combination with strain Fo47. This increased level of disease suppression was ascribed to phenazine production as the phenazine-deficient mutant was not effective. These results suggest, for the first time, that redox-active phenazines produced by fluorescent pseudomonads contribute to the natural soil suppressiveness to Fusarium wilt disease and may act in synergy with carbon competition by resident non-pathogenic F. oxysporum.

Soil amoebae rapidly change bacterial community composition in the rhizosphere of Arabidopsis thaliana

Abstract: We constructed an experimental model system to study the effects of grazing by a common soil amoeba, Acanthamoeba castellanii, on the composition of bacterial communities in the rhizosphere of Arabidopsis thaliana. Amoebae showed distinct grazing preferences for specific bacterial taxa, which were rapidly replaced by grazing tolerant taxa in a highly reproducible way. The relative proportion of active bacteria increased although bacterial abundance was strongly decreased by amoebae. Specific bacterial taxa had disappeared already two days after inoculation of amoebae. The decrease in numbers was most pronounced in Betaproteobacteria and Firmicutes. In contrast, Actinobacteria, Nitrospira, Verrucomicrobia and Planctomycetes increased. Although other groups, such as betaproteobacterial ammonia oxidizers and Gammaproteobacteria did not change in abundance, denaturing gradient gel electrophoresis with specific primers for pseudomonads (Gammaproteobacteria) revealed both specific changes in community composition as well as shifts in functional genes (gacA) involved in bacterial defence responses. The resulting positive feedback on plant growth in the amoeba treatment confirms that bacterial grazers play a dominant role in structuring bacteria-plant interactions. This is the first detailed study documenting how rapidly protozoan grazers induce shifts in rhizosphere bacterial community composition.

The alkaline phosphatase PhoX is more widely distributed in marine bacteria than the classical PhoA.

Abstract: Phosphorus (P) is a vital nutrient for all living organisms and may control the growth of bacteria in the ocean. Bacteria induce alkaline phosphatases when inorganic phosphate (P(i)) is insufficient to meet their P-requirements, and therefore bulk alkaline phosphatase activity measurements have been used to assess the P-status of microbial assemblages. In this study, the molecular basis of marine bacterial phosphatases and their potential role in the environment were investigated. We found that only a limited number of homologs to the classical Escherichia coli alkaline phosphatase (PhoA) were present in marine isolates in the Bacteroidetes and gamma-proteobacteria lineages. In contrast, PhoX, a recently described phosphatase, was widely distributed among diverse bacterial taxa, including Cyanobacteria, and frequently found in the marine metagenomic Global Ocean Survey database. These taxa included ecologically important groups such as Roseobacter and Trichodesmium. PhoX was induced solely upon P-starvation and accounted for approximately 90% of the phosphatase activity in the model marine bacterium Silicibacter pomeroyi. Analysis of the available transcriptomic datasets and their corresponding metagenomes indicated that PhoX is more abundant than PhoA in oligotrophic marine environments such as the North Pacific Subtropical Gyre. Those analyses also revealed that PhoA may be important when Bacteroidetes are abundant, such as in algal bloom episodes. However, PhoX appears to be much more widespread. Its identification as a gene that mediates organic P acquisition in ecologically important groups, and as a marker of P(i)-stress, constitutes an important step toward a better understanding of the marine P cycle.

Genomic islands link secondary metabolism to functional adaptation in marine Actinobacteria

Abstract: Genomic islands have been shown to harbor functional traits that differentiate ecologically distinct populations of environmental bacteria. A comparative analysis of the complete genome sequences of the marine Actinobacteria Salinispora tropica and Salinispora arenicola reveals that 75% of the species-specific genes are located in 21 genomic islands. These islands are enriched in genes associated with secondary metabolite biosynthesis providing evidence that secondary metabolism is linked to functional adaptation. Secondary metabolism accounts for 8.8% and 10.9% of the genes in the S. tropica and S. arenicola genomes, respectively, and represents the major functional category of annotated genes that differentiates the two species. Genomic islands harbor all 25 of the species-specific biosynthetic pathways, the majority of which occur in S. arenicola and may contribute to the cosmopolitan distribution of this species. Genome evolution is dominated by gene duplication and acquisition, which in the case of secondary metabolism provide immediate opportunities for the production of new bioactive products. Evidence that secondary metabolic pathways are exchanged horizontally, coupled with earlier evidence for fixation among globally distributed populations, supports a functional role and suggests that the acquisition of natural product biosynthetic gene clusters represents a previously unrecognized force driving bacterial diversification. Species-specific differences observed in clustered regularly interspaced short palindromic repeat sequences suggest that S. arenicola may possess a higher level of phage immunity, whereas a highly duplicated family of polymorphic membrane proteins provides evidence for a new mechanism of marine adaptation in Gram-positive bacteria.