Interaction of liposomes with bile salts investigated by asymmetric flow field-flow fractionation (AF4): A novel approach for stability assessment of oral drug carriers.

For oral drug delivery the stability of liposomes against intestinal bile salts is of key importance. Here, asymmetric flow field-flow fractionation (AF4) coupled to multi-angle laser light scattering (MALLS) and a differential refractive index (dRI) detector was employed to monitor structural re-arrangement of liposomes upon exposure to the model bile salt taurocholate. For comparison, a conventional stability assay was employed using a hydrophilic marker and size exclusion chromatography (SEC) to separate released from liposome-entrapped dye. Calcein-containing liposomes with and without cholesterol were compared in terms of their in vitro stability upon exposure to bile salts by separating liposomes from co-existing colloidal species emerging after stress test using AF4/MALLS/dRI. Dynamic light scattering (DLS) was utilized in parallel. Our AF4/MALLS/dRI results suggested that exposure of egg-phospholipid liposomes to bile salts at physiological concentrations led to the formation of two new species of colloidal associates, likely (mixed) micelles. Subjecting cholesterol-containing liposomes to the same bile media did not lead to any new colloidal structures, indicating increased stability of these liposomes. Our SEC-based release assay largely confirmed these findings, indicating that AF4/MALLS/dRI is a suitable technique for prediction of in vitro oral stability of liposomal formulations. Moreover, the powerful AF4/MALLS/dRI technique appears promising to improve the understanding of the underlying mechanisms during bile salt-induced liposomal breakdown.

Freshwater copepod carcasses as pelagic microsites of dissimilatory nitrate reduction to ammonium.

A considerable fraction of freshwater zooplankton was recently found to consist of dead specimens that sink to the lake bottom. Such carcasses host intense microbial activities that may promote oxygen depletion at the microscale. Therefore, we tested the hypothesis that sinking zooplankton carcasses are microsites of anaerobic nitrogen cycling that contribute to pelagic fixed-nitrogen loss even in the presence of ambient oxygen. Incubation experiments were performed with the ubiquitous copepods Eudiaptomus sp. and Megacyclops gigas at different ambient oxygen levels that sinking carcasses encounter during their descent in stratified lakes. 15N-stable-isotope incubations revealed intense carcass-associated anaerobic nitrogen cycling only at low ambient oxygen levels (<25% air saturation). Dissimilatory nitrate reduction to ammonium (DNRA) dominated over denitrification and thus the potential for fixed-nitrogen loss was low. Consistent with this partitioning of anaerobic nitrogen cycling, the relative abundance of the carcass-associated marker gene for DNRA (nrfA) was ∼20-400 times higher than that for denitrification (nirS). Additionally, the relative nrfA and nirS abundances were ∼90-180 times higher on copepod carcasses than in lake water. This functional distinctiveness of carcass-associated bacterial communities was further substantiated by 16S rDNA-based fingerprinting. We conclude that the unique bacterial communities and microenvironments provided by zooplankton carcasses influence pelagic nitrogen cycling in lakes, but mainly at seasonally low ambient O2 levels in the bottom water.

Responses of an Agricultural Soil Microbiome to Flooding with Seawater after Managed Coastal Realignment.

Coastal areas have become more prone to flooding with seawater due to climate-change-induced sea-level rise and intensified storm surges. One way to cope with this issue is by "managed coastal realignment", where low-lying coastal areas are no longer protected and instead flooded with seawater. How flooding with seawater impacts soil microbiomes and the biogeochemical cycling of elements is poorly understood. To address this, we conducted a microcosm experiment using soil cores collected at the nature restoration project site Gyldensteen Strand (Denmark), which were flooded with seawater and monitored over six months. Throughout the experiment, biogeochemical analyses, microbial community fingerprinting and the quantification of marker genes documented clear shifts in microbiome composition and activity. The flooding with seawater initially resulted in accelerated heterotrophic activity that entailed high ammonium production and net removal of nitrogen from the system, also demonstrated by a concurrent increase in the abundances of marker genes for ammonium oxidation and denitrification. Due to the depletion of labile soil organic matter, microbial activity decreased after approximately four months. The event of flooding caused the largest shifts in microbiome composition with the availability of labile organic matter subsequently being the most important driver for the succession in microbiome composition in soils flooded with seawater.

Novel anammox bacteria and nitrogen loss from Lake Superior.

Anaerobic ammonium oxidizing (anammox) bacteria own a central position in the global N-cycle, as they have the ability to oxidize NH4+ to N2 under anoxic conditions using NO2-. They are responsible for up to 50% of all N2 released from marine ecosystems into the atmosphere and are thus indispensible for balancing the activity of N-fixing bacteria and completing the marine N-cycle. The contribution, diversity, and impact of anammox bacteria in freshwater ecosystems, however, is largely unknown, confounding assessments of their role in the global N-cycle. Here we report the activity and diversity of anammox bacteria in the world's largest freshwater lake-Lake Superior. We found that anammox performed by previously undiscovered bacteria is an important contributor to sediment N2 production. We observed striking differences in the anammox bacterial populations found at different locations within Lake Superior and those described from other locations. Our data thus reveal that novel anammox bacteria underpin N-loss from Lake Superior, and if more broadly distributed across inland waters would play an important role in continental N-cycling and mitigation of fixed nitrogen transfer from land to the sea.

Archaeal lipids in oral delivery of therapeutic peptides.

Archaea contain membrane lipids that differ from those found in the other domains of life (Eukarya and Bacteria). These lipids consist of isoprenoid chains attached via ether bonds to the glycerol carbons at the sn-2,3 positions. Two types of ether lipids are known, polar diether lipids and bipolar tetraether lipids. The inherent chemical stability and unique membrane-spanning characteristics of tetraether lipids render them interesting for oral drug delivery purposes. Archaeal lipids form liposomes spontaneously (archaeosomes) and may be incorporated in conventional liposomes (mixed vesicles). Both types of liposomes are promising to protect their drug cargo, such as therapeutic peptides, against the acidic environment of the stomach and proteolytic degradation in the intestine. They appear to withstand lipolytic enzymes and bile salts and may thus deliver orally administered therapeutic peptides to distant sections of the intestine or to the colon, where they may be absorbed, eventually by the help of absorption enhancers. Archaeal lipids and their semisynthetic derivatives may thus serve as biological source for the next generation oral drug delivery systems. The aim of this review is to present a systematic overview over existing literature on archaea carrying diether and tetraether lipids, lipid diversity, means of lipid extraction and purification, preparation and in vitro stability studies of archaeal lipid-based liposomal drug carriers and in vivo proof-of concepts studies.

The Effects of Temperature and Growth Phase on the Lipidomes of Sulfolobus islandicus and Sulfolobus tokodaii.

The functionality of the plasma membrane is essential for all organisms. Adaption to high growth temperatures imposes challenges and Bacteria, Eukarya, and Archaea have developed several mechanisms to cope with these. Hyperthermophilic archaea have earlier been shown to synthesize tetraether membrane lipids with an increased number of cyclopentane moieties at higher growth temperatures. Here we used shotgun lipidomics to study this effect as well as the influence of growth phase on the lipidomes of Sulfolobus islandicus and Sulfolobus tokodaii for the first time. Both species were cultivated at three different temperatures, with samples withdrawn during lag, exponential, and stationary phases. Three abundant tetraether lipid classes and one diether lipid class were monitored. Beside the expected increase in the number of cyclopentane moieties with higher temperature in both archaea, we observed previously unreported changes in the average cyclization of the membrane lipids throughout growth. The average number of cyclopentane moieties showed a significant dip in exponential phase, an observation that might help to resolve the currently debated biosynthesis pathway of tetraether lipids.

Liposomes containing lipids from Sulfolobus islandicus withstand intestinal bile salts: An approach for oral drug delivery?

In an attempt to design an oral drug delivery system, suited to protect labile drug compounds like peptides and proteins against the harsh environment in the stomach and upper intestine, we have prepared liposomes from phospholipids, cholesterol and archaeal lipids. As source for the archaeal lipids we used Sulfolobus islandicus, a hyperthermophilic archaeon, whose lipids have not been used in liposomes before. Culturing conditions and extraction protocols for its membrane lipids were established and the lipid composition of the crude lipid extract was characterized. The extracted membrane lipid fraction of S. islandicus consisted primarily of diether lipids with only a small fraction of tetraether lipids. Small unilamellar liposomes with 18% (mol/mol) of crude archaeal lipid extract were from S. islandicus were produced, for the first time and proven to be stabilized against aggressive bile salts as determined by loss of entrapped marker (calcein). At 4.4mM taurocholate (physiological taurocholate level) liposomes containing archaeal lipids retained entrapped marker better than liposomes made of egg phosphatidylcholine (PC) alone and to an extent similar to liposomes made of egg PC and cholesterol. Our findings showed that crude archaeal lipid extracts have, to a certain extent, stabilizing effects on liposomes similar to purified tetraether lipid fractions tested previously.

Structural characterization of ether lipids from the archaeon Sulfolobus islandicus by high-resolution shotgun lipidomics.

The molecular structures, biosynthetic pathways and physiological functions of membrane lipids produced by organisms in the domain Archaea are poorly characterized as compared with that of counterparts in Bacteria and Eukaryota. Here we report on the use of high-resolution shotgun lipidomics to characterize, for the first time, the lipid complement of the archaeon Sulfolobus islandicus. To support the identification of lipids in S. islandicus, we first compiled a database of ether lipid species previously ascribed to Archaea. Next, we analyzed the lipid complement of S. islandicus by high-resolution Fourier transform mass spectrometry using an ion trap-orbitrap mass spectrometer. This analysis identified five clusters of molecular ions that matched ether lipids in the database with sub-ppm mass accuracy. To structurally characterize and validate the identities of the potential lipid species, we performed structural analysis using multistage activation on the ion trap-orbitrap instrument as well as tandem mass analysis using a quadrupole time-of-flight machine. Our analysis identified four ether lipid species previously reported in Archaea, and one ether lipid species that had not been described before. This uncharacterized lipid species features two head group structures composed of a trisaccharide residue carrying an uncommon sulfono group (-SO3) and an inositol phosphate group. Both head groups are linked to a glycerol dialkyl glycerol tetraether core structure having isoprenoid chains with a total of 80 carbon atoms and 4 cyclopentane moieties. The shotgun lipidomics approach deployed here defines a novel workflow for exploratory lipid profiling of Archaea.

Effects of ecological engineered oxygenation on the bacterial community structure in an anoxic fjord in western Sweden.

Oxygen-depleted bodies of water are becoming increasingly common in marine ecosystems. Solutions to reverse this trend are needed and under development, for example, by the Baltic deep-water OXygenation (BOX) project. In the framework of this project, the Swedish Byfjord was chosen for a pilot study, investigating the effects of an engineered oxygenation on long-term anoxic bottom waters. The strong stratification of the water column of the Byfjord was broken up by pumping surface water into the deeper layers, triggering several inflows of oxygen-rich water and increasing oxygen levels in the lower water column and the benthic zone up to 110 µmol l(-1).We used molecular ecologic methods to study changes in bacterial community structure in response to the oxygenation in the Byfjord. Water column samples from before, during and after the oxygenation as well as from two nearby control fjords were analyzed. Our results showed a strong shift in bacterial community composition when the bottom water in the Byfjord became oxic. Initially dominant indicator species for oxygen minimum zones such as members of the SUP05 clade declined in abundance during the oxygenation event and nearly vanished after the oxygenation was accomplished. In contrast, aerobic species like SAR11 that initially were restricted to surface waters could later be detected deep into the water column. Overall, the bacterial community in the formerly anoxic bottom waters changed to a community structure similar to those found in oxic waters, showing that an engineered oxygenation of a large body of anoxic marine water is possible and emulates that of a natural oxygenation event.

Marine bacterioplankton community turnover within seasonally hypoxic waters of a subtropical sound: Devil's Hole, Bermuda.

Understanding bacterioplankton community dynamics in coastal hypoxic environments is relevant to global biogeochemistry because coastal hypoxia is increasing worldwide. The temporal dynamics of bacterioplankton communities were analysed throughout the illuminated water column of Devil's Hole, Bermuda during the 6-week annual transition from a strongly stratified water column with suboxic and high-pCO2 bottom waters to a fully mixed and ventilated state during 2008. A suite of culture-independent methods provided a quantitative spatiotemporal characterization of bacterioplankton community changes, including both direct counts and rRNA gene sequencing. During stratification, the surface waters were dominated by the SAR11 clade of Alphaproteobacteria and the cyanobacterium Synechococcus. In the suboxic bottom waters, cells from the order Chlorobiales prevailed, with gene sequences indicating members of the genera Chlorobium and Prosthecochloris--anoxygenic photoautotrophs that utilize sulfide as a source of electrons for photosynthesis. Transitional zones of hypoxia also exhibited elevated levels of methane- and sulfur-oxidizing bacteria relative to the overlying waters. The abundance of both Thaumarcheota and Euryarcheota were elevated in the suboxic bottom waters (> 10(9) cells l(-1)). Following convective mixing, the entire water column returned to a community typical of oxygenated waters, with Euryarcheota only averaging 5% of cells, and Chlorobiales and Thaumarcheota absent.

An experiment with forced oxygenation of the deepwater of the anoxic By Fjord, western Sweden.

In a 2.5-year-long environmental engineering experiment in the By Fjord, surface water was pumped into the deepwater where the frequency of deepwater renewals increased by a factor of 10. During the experiment, the deepwater became long-term oxic, and nitrate became the dominating dissolved inorganic nitrogen component. The amount of phosphate in the water column decreased by a factor of 5 due to the increase in flushing and reduction in the leakage of phosphate from the sediments when the sediment surface became oxidized. Oxygenation of the sediments did not increase the leakage of toxic metals and organic pollutants. The bacterial community was the first to show changes after the oxygenation, with aerobic bacteria also thriving in the deepwater. The earlier azoic deepwater bottom sediments were colonized by animals. No structural difference between the phytoplankton communities in the By Fjord and the adjacent Havsten Fjord, with oxygenated deepwater, could be detected during the experiment.

Oxygen requirements of the earliest animals.

A rise in the oxygen content of the atmosphere and oceans is one of the most popular explanations for the relatively late and abrupt appearance of animal life on Earth. In this scenario, Earth's surface environment failed to meet the high oxygen requirements of animals up until the middle to late Neoproterozoic Era (850-542 million years ago), when oxygen concentrations sufficiently rose to permit the existence of animal life for the first time. Although multiple lines of geochemical evidence support an oxygenation of the Ediacaran oceans (635-542 million years ago), roughly corresponding with the first appearance of metazoans in the fossil record, the oxygen requirements of basal animals remain unclear. Here we show that modern demosponges, serving as analogs for early animals, can survive under low-oxygen conditions of 0.5-4.0% present atmospheric levels. Because the last common ancestor of metazoans likely exhibited a physiology and morphology similar to that of a modern sponge, its oxygen demands may have been met well before the enhanced oxygenation of the Ediacaran Period. Therefore, the origin of animals may not have been triggered by a contemporaneous rise in the oxygen content of the atmosphere and oceans. Instead, other ecological and developmental processes are needed to adequately explain the origin and earliest evolution of animal life on Earth.

High-resolution SAR11 ecotype dynamics at the Bermuda Atlantic Time-series Study site by phylogenetic placement of pyrosequences.

Advances in next-generation sequencing technologies are providing longer nucleotide sequence reads that contain more information about phylogenetic relationships. We sought to use this information to understand the evolution and ecology of bacterioplankton at our long-term study site in the Western Sargasso Sea. A bioinformatics pipeline called PhyloAssigner was developed to align pyrosequencing reads to a reference multiple sequence alignment of 16S ribosomal RNA (rRNA) genes and assign them phylogenetic positions in a reference tree using a maximum likelihood algorithm. Here, we used this pipeline to investigate the ecologically important SAR11 clade of Alphaproteobacteria. A combined set of 2.7 million pyrosequencing reads from the 16S rRNA V1-V2 regions, representing 9 years at the Bermuda Atlantic Time-series Study (BATS) site, was quality checked and parsed into a comprehensive bacterial tree, yielding 929 036 Alphaproteobacteria reads. Phylogenetic structure within the SAR11 clade was linked to seasonally recurring spatiotemporal patterns. This analysis resolved four new SAR11 ecotypes in addition to five others that had been described previously at BATS. The data support a conclusion reached previously that the SAR11 clade diversified by subdivision of niche space in the ocean water column, but the new data reveal a more complex pattern in which deep branches of the clade diversified repeatedly across depth strata and seasonal regimes. The new data also revealed the presence of an unrecognized clade of Alphaproteobacteria, here named SMA-1 (Sargasso Mesopelagic Alphaproteobacteria, group 1), in the upper mesopelagic zone. The high-resolution phylogenetic analyses performed herein highlight significant, previously unknown, patterns of evolutionary diversification, within perhaps the most widely distributed heterotrophic marine bacterial clade, and strongly links to ecosystem regimes.

Phytoplankton distribution patterns in the northwestern Sargasso Sea revealed by small subunit rRNA genes from plastids.

Phytoplankton species vary in their physiological properties, and are expected to respond differently to seasonal changes in water column conditions. To assess these varying distribution patterns, we used 412 samples collected monthly over 12 years (1991-2004) at the Bermuda Atlantic Time-Series Study site, located in the northwestern Sargasso Sea. We measured plastid 16S ribosomal RNA gene abundances with a terminal restriction fragment length polymorphism approach and identified distribution patterns for members of the Prymnesiophyceae, Pelagophyceae, Chrysophyceae, Cryptophyceae, Bacillariophyceae and Prasinophyceae. The analysis revealed dynamic bloom patterns by these phytoplankton taxa that begin early in the year, when the mixed layer is deep. Previously, unreported open-ocean prasinophyte blooms dominated the plastid gene signal during convective mixing events. Quantitative PCR confirmed the blooms and transitions of Bathycoccus, Micromonas and Ostreococcus populations. In contrast, taxa belonging to the pelagophytes and chrysophytes, as well as cryptophytes, reached annual peaks during mixed layer shoaling, while Bacillariophyceae (diatoms) were observed only episodically in the 12-year record. Prymnesiophytes dominated the integrated plastid gene signal. They were abundant throughout the water column before mixing events, but persisted in the deep chlorophyll maximum during stratified conditions. Various models have been used to describe mechanisms that drive vernal phytoplankton blooms in temperate seas. The range of taxon-specific bloom patterns observed here indicates that different 'spring bloom' models can aptly describe the behavior of different phytoplankton taxa at a single geographical location. These findings provide insight into the subdivision of niche space by phytoplankton and may lead to improved predictions of phytoplankton responses to changes in ocean conditions.

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

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.

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

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.

Novel genes for nitrite reductase and Amo-related proteins indicate a role of uncultivated mesophilic crenarchaeota in nitrogen cycling.

Mesophilic crenarchaeota are frequently found in terrestrial and marine habitats worldwide, but despite their considerable abundance the physiology of these as yet uncultivated archaea has remained unknown. From a 1.2 Gb large-insert environmental fosmid library of a calcareous grassland soil, a 43 kb genomic fragment was isolated with a ribosomal RNA that shows its affiliation to group 1.1b of crenarchaeota repeatedly found in soils. The insert encoded a homologue of a copper-containing nitrite reductase with an unusual C-terminus that encoded a potential amicyanin-like electron transfer domain as well as two proteins related to subunits of ammonia monooxygenases or particulate methane monooxygenases (AmoAB/PmoAB) respectively. Expression of nirK and the amoA-like gene was shown by reverse transcription polymerase chain reaction (PCR) analyses in soil samples, the latter being found at higher levels when the soil was incubated with ammonia (measured by quantitative PCR). Further variants of both genes were amplified from soil samples and were found in the environmental database from the Sargasso Sea plankton. Taken together, our findings suggest that mesophilic terrestrial and marine crenarchaeota might be capable of ammonia oxidation under aerobic and potentially also under anaerobic conditions.

Characterization of large-insert DNA libraries from soil for environmental genomic studies of Archaea.

Complex genomic libraries are increasingly being used to retrieve complete genes, operons or large genomic fragments directly from environmental samples, without the need to cultivate the respective microorganisms. We report on the construction of three large-insert fosmid libraries in total covering 3 Gbp of community DNA from two different soil samples, a sandy ecosystem and a mixed forest soil. In a fosmid end sequencing approach including 5376 sequence tags of approximately 700 bp length, we show that mostly bacterial and, to a much lesser extent, archaeal and eukaryotic genome fragments (approximately 1% each) have been captured in our libraries. The diversity of putative protein-encoding genes, as reflected by their distribution into different COG clusters, was comparable to that encoded in complete genomes of cultivated microorganisms. A huge variety of genomic fragments has been captured in our libraries, as seen by comparison with sequences in the public databases and by the large variation in G+C contents. We dissect differences between the libraries, which relate to the different ecosystems analysed and to biases introduced by different DNA preparations. Furthermore, a range of taxonomic marker genes (other than 16S rRNA) has been identified that allows the assignment of genome fragments to specific lineages. The complete sequences of two genome fragments identified as being affiliated with Archaea, based on a gene encoding a CDC48 homologue and a thermosome subunit, respectively, are presented and discussed. We thereby extend the genomic information of uncultivated crenarchaeota from soil and offer hints to specific metabolic traits present in this group.

Acidobacteria form a coherent but highly diverse group within the bacterial domain: evidence from environmental genomics.

Acidobacteria have been established as a novel phylum of Bacteria that is consistently detected in many different habitats around the globe by 16S rDNA-based molecular surveys. The phylogenetic diversity, ubiquity and abundance of this group, particularly in soil habitats, suggest an important ecological role and extensive metabolic versatility. However, the genetic and physiological information about Acidobacteria is scarce. In order to gain insight into genome structure, evolution and diversity of these microorganisms we have initiated an environmental genomic approach by constructing large insert libraries directly from DNA of a calcerous grassland soil. Genomic fragments of Acidobacteria were identified with specific 16S rDNA probes and sequence analyses of six independently identified clones were performed, representing in total more than 210,000 bp. The 16S rRNA genes of the genomic fragments differed between 2.3% and 19.9% and were placed into two different subgroups of Acidobacteria (groups III and V). Although partial co-linearity was found between genomic fragments, the gene content around the rRNA operons was generally not conserved. Phylogenetic reconstructions with orthologues that were encoded on two of the six genomic fragments (PurF, PurL, PurB and formamidopyrimidine-DNA glycosylase) confirmed the coherence of the acidobacterial phylum. One genomic fragment harboured a cluster of eight genes which was syntenic and highly homologous to genomic regions in Rhodopseudomonas palustris and Bradyrhizobium japonicum, including a conserved two-component system. Phylogenetic analysis of the putative response regulator confirmed that this similarity between Rhizobiales and Acidobacteria might be due to a horizontal gene transfer. In total, our data give first insight into the genome content and diversity of the ubiquitously distributed but poorly characterized phylum of Acidobacteria. Furthermore they support the phylogenetic inferences made from 16S rRNA gene libraries, suggesting that Acidobacteria form a broad group in the same sense and with a similar diversity as that of many well-studied bacterial phyla.

First insight into the genome of an uncultivated crenarchaeote from soil.

Molecular phylogenetic surveys based on the characterization of 16S rRNA genes have revealed that soil is an environment particularly rich in microbial diversity. A clade of crenarchaeota (archaea) has frequently been detected among many other novel lineages of uncultivated bacteria. In this study we have initiated a genomic approach for the characterization of uncultivated microorganisms from soil. We have developed a procedure based on a two-phase electrophoresis technique that allows the fast and reliable purification of concentrated and clonable, high molecular weight DNA. From this DNA we have constructed complex large-insert genomic libraries. Using archaea-specific 16S rRNA probes we have isolated a 34 kbp fragment from a 900 Mbp fosmid library of soil DNA. The clone contained a complete 16S/23S rRNA operon and 17 genes encoding putative proteins. Phylogenetic analyses of the rRNA genes and of several protein encoding genes (e.g. DNA polymerase, FixAB, glycosyl transferase) confirmed the specific affiliation of the genomic fragment with the non-thermophilic clade of the crenarchaeota. Content and structure of the genomic fragment indicated that the archaea from soil differ significantly from their previously studied uncultivated marine relatives. The protein encoding genes gave the first insights into the physiological potential of these organisms and can serve as a basis for future genomic and functional genomic studies.