Collembolan Transcriptomes Highlight Molecular Evolution of Hexapods and Provide Clues on the Adaptation to Terrestrial Life.

BACKGROUND: Collembola (springtails) represent a soil-living lineage of hexapods in between insects and crustaceans. Consequently, their genomes may hold key information on the early processes leading to evolution of Hexapoda from a crustacean ancestor. METHOD: We assembled and annotated transcriptomes of the Collembola Folsomia candida and Orchesella cincta, and performed comparative analysis with protein-coding gene sequences of three crustaceans and three insects to identify adaptive signatures associated with the evolution of hexapods within the pancrustacean clade. RESULTS: Assembly of the springtail transcriptomes resulted in 37,730 transcripts with predicted open reading frames for F. candida and 32,154 for O. cincta, of which 34.2% were functionally annotated for F. candida and 38.4% for O. cincta. Subsequently, we predicted orthologous clusters among eight species and applied the branch-site test to detect episodic positive selection in the Hexapoda and Collembola lineages. A subset of 250 genes showed significant positive selection along the Hexapoda branch and 57 in the Collembola lineage. Gene Ontology categories enriched in these genes include metabolism, stress response (i.e. DNA repair, immune response), ion transport, ATP metabolism, regulation and development-related processes (i.e. eye development, neurological development). CONCLUSIONS: We suggest that the identified gene families represent processes that have played a key role in the divergence of hexapods within the pancrustacean clade that eventually evolved into the most species-rich group of all animals, the hexapods. Furthermore, some adaptive signatures in collembolans may provide valuable clues to understand evolution of hexapods on land.

De novo transcriptome characterization and development of genomic tools for Scabiosa columbaria L. using next-generation sequencing techniques.

Next-generation sequencing (NGS) technologies are increasingly applied in many organisms, including nonmodel organisms that are important for ecological and conservation purposes. Illumina and 454 sequencing are among the most used NGS technologies and have been shown to produce optimal results at reasonable costs when used together. Here, we describe the combined application of these two NGS technologies to characterize the transcriptome of a plant species of ecological and conservation relevance for which no genomic resource is available, Scabiosa columbaria. We obtained 528,557 reads from a 454 GS-FLX run and a total of 28,993,627 reads from two lanes of an Illumina GAII single run. After read trimming, the de novo assembly of both types of reads produced 109,630 contigs. Both the contigs and the >75 bp remaining singletons were blasted against the Uniprot/Swissprot database, resulting in 29,676 and 10,515 significant hits, respectively. Based on sequence similarity with known gene products, these sequences represent at least 12,516 unique genes, most of which are well covered by contig sequences. In addition, we identified 4320 microsatellite loci, of which 856 had flanking sequences suitable for PCR primer design. We also identified 75,054 putative SNPs. This annotated sequence collection and the relative molecular markers represent a main genomic resource for S. columbaria which should contribute to future research in conservation and population biology studies. Our results demonstrate the utility of NGS technologies as starting point for the development of genomic tools in nonmodel but ecologically important species.

Increased susceptibility of serum amyloid A 1.1 to degradation by MMP-1: potential explanation for higher risk of type AA amyloidosis.

OBJECTIVE: Genetic polymorphisms in serum amyloid A (SAA) have been shown to substantially influence the risk of developing type AA amyloidosis. Recently, a role for MMP-1 has been suggested in the pathogenesis of AA amyloidosis. Therefore, we investigated if the SAA1 isotypes are differentially degraded by MMP-1. METHODS: Degradation of different SAA isotypes by MMP-1 was assessed by immunoblotting. MALDI-TOF mass spectrometry was used to identify degradation fragments. RESULTS: We found that SAA1.5 is more resistant to degradation by MMP-1 than SAA1.1. This difference is caused by the capacity of MMP-1 to cleave at the site of the polymorphism at position 57. CONCLUSION: These results may explain the higher risk of amyloidosis in patients with a SAA1.1/1.1 genotype vs SAA1.5/1.5 or SAA1.1/1.5 genotype. In addition, the impaired degradation of SAA1.5 by MMP-1 could also explain the higher serum SAA concentrations in persons with a SAA1.5 genotype.

Global impact and application of the anaerobic ammonium-oxidizing (anammox) bacteria.

In the anaerobic ammonium oxidation (anammox) process, ammonia is oxidized with nitrite as primary electron acceptor under strictly anoxic conditions. The reaction is catalysed by a specialized group of planctomycete-like bacteria. These anammox bacteria use a complex reaction mechanism involving hydrazine as an intermediate. The reactions are assumed to be carried out in a unique prokaryotic organelle, the anammoxosome. This organelle is surrounded by ladderane lipids, which make the organelle nearly impermeable to hydrazine and protons. The localization of the major anammox protein, hydrazine oxidoreductase, was determined via immunogold labelling to be inside the anammoxosome. The anammox bacteria have been detected in many marine and freshwater ecosystems and were estimated to contribute up to 50% of oceanic nitrogen loss. Furthermore, the anammox process is currently implemented in water treatment for the low-cost removal of ammonia from high-strength waste streams. Recent findings suggested that the anammox bacteria may also use organic acids to convert nitrate and nitrite into dinitrogen gas when ammonia is in short supply.

1994-2004: 10 years of research on the anaerobic oxidation of ammonium.

The obligately anaerobic ammonium oxidation (anammox) reaction with nitrite as primary electron acceptor is catalysed by the planctomycete-like bacteria Brocadia anammoxidans, Kuenenia stuttgartiensis and Scalindua sorokinii. The anammox bacteria use a complex reaction mechanism involving hydrazine as an intermediate. They have a unique prokaryotic organelle, the anammoxosome, surrounded by ladderane lipids, which exclusively contains the hydrazine oxidoreductase as the major protein to combine nitrite and ammonia in a one-to-one fashion. In addition to the peculiar microbiology, anammox was shown to be very important in the oceanic nitrogen cycle, and proved to be a very good alternative for treatment of high-strength nitrogenous waste streams. With the assembly of the K. stuttgartiensis genome at Genoscope, Evry, France, the anammox reaction has entered the genomic and proteomic era, enabling the elucidation of many intriguing aspects of this fascinating microbial process.

Complete conversion of nitrate into dinitrogen gas in co-cultures of denitrifying bacteria.

In the past 10 years many molecular aspects of microbial nitrate reduction have been elucidated, but the ecophysiology of this process is hardly understood. In this contribution, our efforts to study the complex microbial communities and interactions involved in the reduction of nitrate to dinitrogen gas are summarized. The initial work concentrated on emission of the greenhouse gas nitrous oxide during incomplete denitrification by Alcaligenes faecalis. As more research methods became available, the fitness of A. faecalis could be tested in mixed cultures with other denitrifying bacteria, most notably with the nitrate-reducing bacterium Pseudomonas G9. Finally, the advancement of molecular diagnostic tools made it possible to survey complex microbial communities using specific primer sets for/and antibodies raised against the various NO(x) reductases. Given the enormous complexity of substrates and environmental conditions, it is evident that mixed cultures rather than single species are responsible for denitrification in man-made and natural ecosystems. However, it is surprising that even for the breakdown of a single compound, such as acetate, mixed cultures are responsible, and that the consecutive denitrification steps are commonly performed by mutualistic co-operating species. Our observations also indicate that we seldom know the identity of the major key players in the nitrogen cycle of these ecosystems.

Anaerobic ammonium oxidation by marine and freshwater planctomycete-like bacteria.

Recently, two fresh water species, " Candidatus Brocadia anammoxidans" and " Candidatus Kuenenia stuttgartiensis", and one marine species, " Candidatus Scalindua sorokinii", of planctomycete anammox bacteria have been identified. " Candidatus Scalindua sorokinii" was discovered in the Black Sea, and contributed substantially to the loss of fixed nitrogen. All three species contain a unique organelle--the anammoxosome--in their cytoplasm. The anammoxosome contains the hydrazine/hydroxylamine oxidoreductase enzyme, and is thus the site of anammox catabolism. The anammoxosome is surrounded by a very dense membrane composed almost exclusively of linearly concatenated cyclobutane-containing lipids. These so-called 'ladderanes' are connected to the glycerol moiety via both ester and ether bonds. In natural and man-made ecosystems, anammox bacteria can cooperate with aerobic ammonium-oxidising bacteria, which protect them from harmful oxygen, and provide the necessary nitrite. The cooperation of these two groups of ammonium-oxidising bacteria is the microbial basis for a sustainable one reactor system, CANON (completely autotrophic nitrogen-removal over nitrite) to remove ammonia from high strength wastewater.

Microbial cycling of volatile organic sulfur compounds in anoxic environments.

Microbial cycling of volatile organic sulfur compounds (VOSC) is investigated due to the impact these compounds are thought to have on environmental processes like global temperature control, acid precipitation and the global sulfur cycle. Moreover, in several kinds of industries like composting plants and the paper industry VOSC are released causing odor problems. Waste streams containing these compounds must be treated in order to avoid the release of these compounds to the atmosphere. This paper describes the general mechanisms for the production and degradation of methanethiol (MT) and dimethyl sulfide (DMS), two ubiquitous VOSC in anaerobic environments. Slurry incubations indicated that methylation of sulfide and MT resulting in MT and DMS, respectively, is one of the major mechanisms for VOSC in sulfide-rich anaerobic environments. An anaerobic bacterium that is responsible for the formation of MT and DMS through the anaerobic methylation of H2S and MT was isolated from a freshwater pond after enrichment with syringate as a methyl group donating compound and sole carbon source. In spite of the continuous formation of MT and DMS, steady state concentrations are generally very low. This is due to the microbial degradation of these compounds. Experiments with sulfate-rich and sulfate-amended sediment slurries demonstrated that besides methanogens, sulfate-reducing bacteria can also degrade MT and DMS, provided that sulfate is available. A methanogen was isolated that is able to grow on DMS as the sole carbon source. A large survey of sediments slurries of various origin demonstrated that both isolates are commonly occurring inhabitants of anaerobic environments.

Microbial cycling of volatile organic sulfur compounds.

Microbial cycling of volatile organic sulfur compounds (VOSCs), especially dimethyl sulfide (DMS) and methanethiol (MT), is intensively studied because these compounds play an important role in the processes of global warming, acid precipitation, and the global sulfur cycle. VOSC concentrations in freshwater sediments are low due to the balance between the formation and degradation of these compounds. These reactions occur for the greater part at the oxic/anoxic interphase of sediment and water column. In contrast to marine ecosystems, where dimethylsulfoniopropionate is the main precursor of MT and DMS, in freshwater ecosystems, VOSCs are formed mainly by methylation of sulfide and to a lesser extent from the degradation of S-containing amino acids. One of the major routes for DMS and MT formation through sulfide methylation is anaerobic O-demethylation of methoxylated aromatic compounds. Inhibition studies have revealed that the major part of the endogenously produced MT and DMS is degraded anaerobically by methanogens. The major bacterial groups involved in formation and consumption of VOSCs are described.

Purine degradation in the edible mushroom Agaricus bisporus.

Agaricus bisporus is able to use urate, allantoin, allantoate, urea and alloxanate as nitrogen sources for growth. The presence of urate oxidase, allantoinase, ureidoglycolase and urease activities, both in fruit bodies and mycelia, points to a degradative pathway for urate similar to that found in various microorganisms. So far all efforts to demonstrate the enzyme responsible for allantoate degradation failed. A urease inhibitor appeared to be present in cell-free extracts from fruit bodies.

The major component of the cellulosomes of anaerobic fungi from the genus Piromyces is a family 48 glycoside hydrolase.

Sequencing of two cDNAs from the anaerobic fungi Piromyces equi and Piromyces sp. strain E2 revealed that they both encode a glycoside hydrolase (GH) family 48 cellulase, containing two C-terminal fungal dockerin domains. N-terminal sequencing of the major component of the Piromyces multi-enzyme cellulase/hemicellulase complex, termed the cellulosome, showed that these 80 kDa proteins corresponded to the GH family 48 enzyme. These data show for the first time that GH family 48 cellulases are not confined to bacteria, and that bacterial and fungal cellulosomes share the same pivotal component.

Noncatalytic docking domains of cellulosomes of anaerobic fungi.

A method is presented for the specific isolation of genes encoding cellulosome components from anaerobic fungi. The catalytic components of the cellulosome of anaerobic fungi typically contain, besides the catalytic domain, mostly two copies of a 40-amino-acid cysteine-rich, noncatalytic docking domain (NCDD) interspaced by short linkers. Degenerate primers were designed to anneal to the highly conserved region within the NCDDs of the monocentric fungus Piromyces sp. strain E2 and the polycentric fungus Orpinomyces sp. strain PC-2. Through PCR using cDNA from Orpinomyces sp. and genomic DNA from Piromyces sp. as templates, respectively, 9 and 19 PCR products were isolated encoding novel NCDD linker sequences. Screening of an Orpinomyces sp. cDNA library with four of these PCR products resulted in the isolation of new genes encoding cellulosome components. An alignment of the partial NCDD sequence information obtained and an alignment of database-accessible NCDD sequences, focusing on the number and position of cysteine residues, indicated the presence of three structural subfamilies within fungal NCDDs. Furthermore, evidence is presented that the NCDDs in CelC from the polycentric fungus Orpinomyces sp. strain PC-2 specifically recognize four proteins in a cellulosome preparation, indicating the presence of multiple scaffoldins.

Microbial populations involved in cycling of dimethyl sulfide and methanethiol in freshwater sediments.

Although several microorganisms that produce and degrade methanethiol (MT) and dimethyl sulfide (DMS) have been isolated from various habitats, little is known about the numbers of these microorganisms in situ. This study reports on the identification and quantification of microorganisms involved in the cycling of MT and DMS in freshwater sediments. Sediment incubation studies revealed that the formation of MT and DMS is well balanced with their degradation. MT formation depends on the concentrations of both sulfide and methyl group-donating compounds. A most-probable number (MPN) dilution series with syringate as the growth substrate showed that methylation of sulfide with methyl groups derived from syringate is a commonly occurring process in situ. MT appeared to be primarily degraded by obligately methylotrophic methanogens, which were found in the highest positive dilutions on DMS and mixed substrates (methanol, trimethylamine [TMA], and DMS). Amplified ribosomal DNA restriction analysis (ARDRA) and 16S rRNA gene sequence analysis of the total DNA isolated from the sediments and of the DNA isolated from the highest positive dilutions of the MPN series (mixed substrates) revealed that the methanogens that are responsible for the degradation of MT, DMS, methanol, and TMA in situ are all phylogenetically closely related to Methanomethylovorans hollandica. This was confirmed by sequence analysis of the product obtained from a nested PCR developed for the selective amplification of the 16S rRNA gene from M. hollandica. The data from sediment incubation experiments, MPN series, and molecular-genetics detection correlated well and provide convincing evidence for the suggested mechanisms for MT and DMS cycling and the common presence of the DMS-degrading methanogen M. hollandica in freshwater sediments.

In vitro metabolism of fumonisin B1 by ruminal microflora.

Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium moniliforme and F. proliferatum. Little is known of its metabolic fate after oral ingestion in ruminants, but these animals are reported to be tolerant towards FB1. The metabolism of this mycotoxin was evaluated following incubation (1 microg/ml) in ruminal fluid for up to 72 h, in the presence or absence of alfalfa as a substrate for microbial growth, using a model rumen (sealed flask, anaerobic conditions, exclusion of light, gentle agitation, 39 degrees C). The decrease in FB1 concentration and the production of short-chain fatty acids were determined. FB1 had no effect on SCFA production. After 72 h incubation, FB1 depletion was 12% and 18% in samples with and without alfalfa, respectively. No hydrolysed metabolites (aminopolyols or aminopentol) were detected. These results indicate that FB1 is poorly metabolized in the rumen and suggest that such metabolism is not the cause of the tolerance to this toxin displayed by ruminants.

Methanosarcina semesiae sp. nov., a dimethylsulfide-utilizing methanogen from mangrove sediment.

Methanosarcina semesiae MD1T (T = type strain), a novel obligately methylotrophic methanogenic archaeon is described. Strain MD1T was isolated from an enrichment on dimethylsulfide inoculated with mangrove sediment. The cells were irregularly coccoid, non-motile, 1.4+/-0.2 microm in diameter and stained Gram-positive. The catabolic substrates used included dimethylsulfide, methanethiol, methanol and methylated amines, but not acetate, formate, H2/CO2 or a combination of these substrates. When cells grown on dimethylsulfide were transferred to trimethylamine or methanol and vice versa, a lag phase was observed. The same lag phase occurred when cells grown on trimethylamine were transferred to methanol and vice versa, indicating that for each substrate different enzymes were induced. Fastest growth occurred within a temperature range of 30-35 degrees C and a pH of 6.5-7.5. Both Na+ and Mg2+ were required for growth, with maximum growth rates at 200-600 mM Na+ and 20-100 mM Mg2+. The cells exhibited specific growth rates (h-1) of 0.07+/-0.02, 0.15+/-0.04 and 0.18-/+0.05 on dimethylsulfide, methanol and trimethylamine, respectively. Analysis of the 16S rRNA gene sequence showed that strain MD1T was phylogenetically closely related to members of the genus Methanosarcina, but clearly differed from all described species of this genus (94-97% sequence similarity).

HPLC detection of soluble carbohydrates involved in mannitol and trehalose metabolism in the edible mushroom Agaricus bisporus.

A convenient and sensitive method was developed to separate and detect various types of carbohydrates (polyols, mono- and disaccharides, and phosphorylated sugars) simultaneously using high-performance liquid chromatography (HPLC). The method consists of a chromatographic separation on a CarboPac PA1 anion-exchange analytical column followed by pulsed amperometric detection. In a single run (43 min) 13 carbohydrates were readily resolved. Calibration plots were linear over the ranges of 5-25 microM to 1. 0-1.5 mM. The reliable and fast analysis technique, avoiding derivatization steps and long run times, was used to determine the levels of carbohydrates involved in mannitol and trehalose metabolism in the edible mushroom Agaricus bisporus. Moreover, the method was used to study the trehalose phosphorylase reaction.

Purification and characterization of an acid phosphatase from the commercial mushroom Agaricus bisporus.

Acid phosphatase [AP; EC 3.1.3.2], a key enzyme involved in the synthesis of mannitol in Agaricus bisporus, was purified to homogeneity and characterized. The native enzyme appeared to be a high molecular weight type glycoprotein. It has a molecular weight of 145 kDa and consists of four identical 39-kDa subunits. The isoelectric point of the enzyme was found at 4.7. Maximum activity occurred at 65 degrees C. The optimum pH range was between 3.5 and 5.5, with maximum activity at pH 4.75. The enzyme was unaffected by EDTA, and inhibited by tartrate and inorganic phosphate. The enzyme exhibits a Km for p-nitrophenylphosphate and fructose-6-phosphate of 370 microM and 3.1 mM, respectively. A broad substrate specificity was observed with significant activities for fructose-6-phosphate, glucose-6-phosphate, mannitol-1-phosphate, AMP and beta-glycerol phosphate. Only phosphomonoesters were dephosphorylated. Antibodies raised against the purified enzyme could precipitate AP activity from a cell-free extract in an anticatalytic immunoprecipitation test.

A beta-1,4-endoglucanase-encoding gene from Cellulomonas pachnodae.

A gene library of Cellulomonas pachnodae was constructed in Escherichia coli and was screened for endoglucanase activity. Five endoglucanase-positive clones were isolated that carried identical DNA fragments. The gene, designated cel6A, encoding an endoglucanase enzyme, belongs to the glycosyl hydrolase family 6 (cellulase family B). The recombinant Cel6A had a molecular mass of 53 kDa, a pH optimum of 5.5, and a temperature optimum of 50-55 degrees C. The recombinant endoglucanase Cel6A bound to crystalline cellulose and beech litter. Based on amino acid sequence similarity, a clear cellulose-binding domain was not distinguished. However, the regions in the Cel6A amino acid sequence at the positions 262-319 and 448-473, which did not show similarity to any of the known family-6 glycosyl hydrolases, may be involved in substrate binding.

Molecular and biochemical characterization of two xylanase-encoding genes from Cellulomonas pachnodae.

Two xylanase-encoding genes, named xyn11A and xyn10B, were isolated from a genomic library of Cellulomonas pachnodae by expression in Escherichia coli. The deduced polypeptide, Xyn11A, consists of 335 amino acids with a calculated molecular mass of 34,383 Da. Different domains could be identified in the Xyn11A protein on the basis of homology searches. Xyn11A contains a catalytic domain belonging to family 11 glycosyl hydrolases and a C-terminal xylan binding domain, which are separated from the catalytic domain by a typical linker sequence. Binding studies with native Xyn11A and a truncated derivative of Xyn11A, lacking the putative binding domain, confirmed the function of the two domains. The second xylanase, designated Xyn10B, consists of 1,183 amino acids with a calculated molecular mass of 124,136 Da. Xyn10B also appears to be a modular protein, but typical linker sequences that separate the different domains were not identified. It comprises a N-terminal signal peptide followed by a stretch of amino acids that shows homology to thermostabilizing domains. Downstream of the latter domain, a catalytic domain specific for family 10 glycosyl hydrolases was identified. A truncated derivative of Xyn10B bound tightly to Avicel, which was in accordance with the identified cellulose binding domain at the C terminus of Xyn10B on the basis of homology. C. pachnodae, a (hemi)cellulolytic bacterium that was isolated from the hindgut of herbivorous Pachnoda marginata larvae, secretes at least two xylanases in the culture fluid. Although both Xyn11A and Xyn10B had the highest homology to xylanases from Cellulomonas fimi, distinct differences in the molecular organizations of the xylanases from the two Cellulomonas species were identified.

Trehalose phosphorylase activity and carbohydrate levels during axenic fruiting in three Agaricus bisporus strains.

Three strains of Agaricus bisporus (B430, 116, and 155.8), which share the ability to form hyphal aggregates on solid media under axenic conditions, were investigated with respect to carbohydrate levels and activities of enzymes involved in their carbon metabolism. The size and macroscopic appearance of the aggregates, when grown on diluted medium, suggest that substrate limitation plays a role in the process of fruiting body development in A. bisporus. The enzymes trehalose phosphorylase (TP), mannitol dehydrogenase (MD), and glucose-6-phosphate dehydrogenase (G6PD) seem to be developmentally regulated, in contrast to hexokinase (HK). Activities of TP (measured in the direction of trehalose degradation), MD, and G6PD were higher in the hyphal aggregates compared with the mycelium, whereas HK activity varied little. In the period preceding the axenic formation of hyphal aggregates, synthesis of trehalose by TP approximately doubled in the mycelium. The carbohydrate levels, which were measured by HPLC, varied in a way similar to their corresponding enzymes. The results indicate synthesis of trehalose in the mycelium of A. bisporus before the hyphal aggregates arise. Subsequently, translocation of the trehalose takes place from the mycelium to the emerging aggregates. In these small aggregates the trehalose is rapidly broken down to yield glucose and glucose-1-phosphate, serving as carbon and energy sources for further growth of the aggregates and for the synthesis of the osmolyte mannitol.