Established and potential impacts of eukaryotic mycelial decomposers in marine/terrestrial ecotones

Marine mycelial decomposers (eumycotes, members of Kingdom Fungi, and oomycotes, zoosporic members of the Kingdom Protoctista) are highly adapted for capture of solid substrate by pervasion and digestion from within. Thus they exert their influence in areas of large input of litter of vascular plants, especially at some types of terrestrial/marine ecosystemic interfaces (ecotones). Unavailability of methods easily used by general microbial ecologists has hampered progress in the study of marine mycelial decomposers, and there are still pockets of difficulty in this regard (especially for oomycotes). Recently published or refined methods for measuring fungal mass and productivity have begun to allow us to realize the impacts of fungi in marine ecotones. For example, it is now clear that the older paradigm reflecting negligible contribution of microbial mass to litter nitrogen content is false for the standing-decay system of saltmarsh grasses — in these decay systems, fungal mass can account for virtually all of the nitrogen present at some point(s) in the standing-decay period. Another generally held belief about marine fungi has also been reversed — ascomycetes (Fungi) of a saltmarsh grass (smooth cordgrass) clearly do digest lignocellulose under natural-decay circumstances. Much more work is needed to clarify the situation, but at present it appears that major types of marine ecotones (e.g., saltmarshes and mangroves) differ sharply in the balance among major groups of decomposers (eumycotes, oomycotes, and bacteria) with regard to their utilization of vascular-plant litter. In saltmarshes, microbial production in standing grass litter is strongly dominated by fungi, and oomycotes do not show evidence of a substantial role in decomposition. In mangroves, submerged fallen leaves appear to support minor fungal occupancy, but ubiquitous and rapid occupancy by oomycotes (especially Halophytophthora vesicula). Many exciting areas of research are now more open than ever before to marine microbial ecologists interested in working with mycelial decomposers.     

Fluorescence in situ hybridization of uncultured zoosporic fungi: Testing with clone-FISH and application to freshwater samples using CARD-FISH

Recently, molecular environmental surveys of the eukaryotic microbial community in lakes have revealed a high diversity of sequences belonging to uncultured zoosporic fungi commonly known as chytrids. These microorganisms have two different stages in their life cycle and are known as algal parasites (i.e. host-attached infective sporangia) and as food sources for zooplankton (i.e. free-living zooflagellate propagules) in aquatic systems. However, because of their small size and their lack of distinctive morphological features, traditional microscopy does not allow the detection of chytrids, particularly of zoospores which have probably been misidentified as phagotrophic flagellates in previous studies. Hence, quantitative data on chytrids in natural environments is missing. We have adapted a clone-FISH approach known from prokaryotes to optimize the hybridization conditions of a designed oligonucleotidic probe specific to Chytridiales (i.e. the largest group of the true-fungal division of Chytridiomycota), before application to natural samples using the CARD-FISH approach. When these conditions were applied, the CARD-FISH assay demonstrated high specificity and sensitivity, and offers a promising tool for quantitative assessment of natural zoosporic fungi, primarily of zoospores which contributed up to 60% of the total abundance of heterotrophic flagellates. Although the field results from the CARD-FISH approach were considered preliminary and mainly as ‘proof of concept’, findings were consistent with ecological considerations known from pelagic habitats and host versus parasite populations, with recurrent ecological patterns in two contrasting lake ecosystems. We conclude that this approach will contribute to a better understanding of the ecological significance of zoosporic organisms in microbial food webs of pelagic ecosystems.     

MetaMetaDB: A Database and Analytic System for Investigating Microbial Habitability

MetaMetaDB (http://mmdb.aori.u-tokyo.ac.jp/) is a database and analytic system for investigating microbial habitability, i.e., how a prokaryotic group can inhabit different environments. The interaction between prokaryotes and the environment is a key issue in microbiology because distinct prokaryotic communities maintain distinct ecosystems. Because 16S ribosomal RNA (rRNA) sequences play pivotal roles in identifying prokaryotic species, a system that comprehensively links diverse environments to 16S rRNA sequences of the inhabitant prokaryotes is necessary for the systematic understanding of the microbial habitability. However, existing databases are biased to culturable prokaryotes and exhibit limitations in the comprehensiveness of the data because most prokaryotes are unculturable. Recently, metagenomic and 16S rRNA amplicon sequencing approaches have generated abundant 16S rRNA sequence data that encompass unculturable prokaryotes across diverse environments; however, these data are usually buried in large databases and are difficult to access. In this study, we developed MetaMetaDB (Meta-Metagenomic DataBase), which comprehensively and compactly covers 16S rRNA sequences retrieved from public datasets. Using MetaMetaDB, users can quickly generate hypotheses regarding the types of environments a prokaryotic group may be adapted to. We anticipate that MetaMetaDB will improve our understanding of the diversity and evolution of prokaryotes.     

Small Players, Large Role: Microbial Influence on Biogeochemical Processes in Pelagic Aquatic Ecosystems

Although prokaryotes are small in size, they are a significant biomass component in aquatic planktonic ecosystems and play a major role in biogeochemical processes. A review of the recent literature shows that the relative importance of prokaryotes to material and energy fluxes is maximized in low-productivity (oligotrophic) ecosystems and decreases in high-productivity (eutrophic) ecosystems. We conclude that competition with eukaryotic autotrophs for dissolved nutrients and competition with phagotrophic heterotrophs and physical processes (sinking, photooxidation) for organic carbon (C) play important roles in determining the relative abundance and impact of prokaryotes in aquatic systems. Oligotrophic systems have low nutrient concentrations, with high proportions of dissolved nutrients in organic form, which favors prokaryotic heterotrophs over phytoplankton. Furthermore, a high proportion of the available organic C is dissolved rather than particulate, which favors prokaryotic heterotrophs over phagotrophic heterotrophs. In eutrophic systems, increased relative concentrations and loading of inorganic nutrients and increased relative concentrations of particulate organic C select for phytoplankton and phagotrophic heterotrophs over prokaryotic heterotrophs. Increased particle sinking fluxes and/or decreased excretion of organic carbon (EOC) may also decrease the relative importance of prokaryotic heterotrophs in eutrophic systems. In oligotrophic systems, interactions between autotrophs and heterotrophs are tightly coupled because the dominant heterotrophs are similar in size and growth rates, as well as having similar nutrient composition to the dominant autotrophs, small phytoplankton. In eutrophic systems, increased productivity passes through zooplankton that are larger and have slower growth rates than the autotrophs, leading to a greater potential for decoupled auto- and heterotrophic production and increased export production. Received 18 July 2000; Accepted 13 September 2001.     

Specific adhesion of primary hepatocytes to a novel glucose-carrying polymer

A new amphiphilic glycopolymer, poly-[N-p-vinylbenzyl-d-glucuronamide] (PV6Gna), was synthesized and characterized. Glucose moieties in the polymer were confirmed to be exposed into outer surface of polystyrene (PS) by direct lectin-enzyme assay. Hepatocytes were specifically interacted with PV6Gna substituted at C-6 of glucose but not poly-[N-p-vinylbenzyl-O--d-glucopyranosyl-[14]-d-gluconamide] (PVMA) and poly-[3-O-p-vinylbenzyl-d-glucose (PVG) substituted at C-1 and C-3 of glucose, respectively, although the glycopolymers were interacted with Con A as lectin for -d-glucose and -d-mannose. The adhesion of hepatocytes was dependent on Ca²⁺ and independent on temperature for the PV6Gna surface unlike integrin-dependent adhesion. Morphologies of hepatocytes on the PV6Gna surface were significantly different from ones on collagen type-I and affected by the coating concentration of PV6Gna onto PS dish and epidermal growth factor (EGF).     

Protein phylogenies and signature sequences: evolutionary relationships within prokaryotes and between prokaryotes and eukaryotes

The evolutionary relationships within prokaryotes and between prokaryotes and eukaryotes is examined based on protein sequence data. Phylogenies and common signature sequences in some of the most conserved proteins point to a close evolutionary relationship between Archaebacteria and Gram-positive bacteria. The monophyletic nature and distinctness of the Archaebacterial domain is not supported by many of the phylogenies. Within Gram-negative bacteria, cyanobacteria are indicated as the deepest branching lineage, and a clade consisting of Archaebacteria, Gram-positive bacteria and cyanobacteria is supported by signature sequences in many proteins. However, the division within the prokaryotic species viz. Archaebacteria Gram-positive bacteria Cyanobacteria other groups of Gram-negative bacteria, is indicated to be not very rigid but, instead is an evolutionary continuum. It is expected that certain species will be found which represent intermediates in the above transitions. By contrast to the evolutionary relationships within prokaryotes, the eukaryotic species, which are structurally very different, appear to have originated by a very different mechanism. Protein phylogenies and signature sequences provide evidence that the eukaryotic nuclear genome is a chimera which has received major contributions from both an Archaebacterium and a Gram-negative bacterium. To explain these observations, it is suggested that the ancestral eukaryotic cell arose by a symbiotic fusion event between the above parents and that this fusion event led to the origin of both nucleus and endoplasmic reticulum. The monophyletic nature of all extant eukaryotic species further suggests that a 'successful primary fusion' between the prokaryotic species that gave rise to the ancestral eukaryotic cell took place only once in the history of this planet.     

Ontogenetic allometry of the bluemouth, <Emphasis Type="Italic">Helicolenus dactylopterus dactylopterus</Emphasis> (Teleostei: Scorpaenidae), in the Northeast Atlantic and Mediterranean based on geometric morphometrics

Since the emergence of the ‘microbial loop’ concept, heterotrophic flagellates have received particular attention as grazers in aquatic ecosystems. These microbes have historically been regarded incorrectly as a homogeneous group of bacterivorous protists in aquatic systems. More recently, environmental rDNA surveys of small heterotrophic flagellates in the pelagic zone of freshwater ecosystems have provided new insights. (i) The dominant phyla found by molecular studies differed significantly from those known from morphological studies with the light microscope, (ii) the retrieved phylotypes generally belong to well-established eukaryotic clades, but there is a very large diversity within these clades and (iii) a substantial part of the retrieved sequences cannot be assigned to bacterivorous but can be assigned instead to parasitic and saprophytic organisms, such as zoosporic true fungi (chytrids), fungus-like organisms (stramenopiles), or virulent alveolate parasites (Perkinsozoa and Amoebophrya sp.). All these microorganisms are able to produce small zoospores to assure dispersal in water during their life-cycles. Based on the existing literature on true fungi and fungus-like organisms, and on the more recently published eukaryotic rDNA environmental studies and morphological observations, we conclude that previously overlooked microbial diversity and related ecological potentials require intensive investigation (i) for an improved understanding of the roles of heterotrophic flagellates in pelagic ecosystems and (ii) to properly integrate the concept of ‘the microbial loop’ into modern pelagic microbial ecology.     

Microbial community diversity and composition varies with habitat characteristics and biofilm function in macrophyte‐rich streams

Biofilms in streams play an integral role in ecosystem processes and function yet few studies have investigated the broad diversity of these complex prokaryotic and eukaryotic microbial communities. Physical habitat characteristics can affect the composition and abundance of microorganisms in these biofilms by creating microhabitats. Here we describe the prokaryotic and eukaryotic microbial diversity of biofilms in sand and macrophyte habitats (i.e. epipsammon and epiphyton, respectively) in five macrophyte‐rich streams in Jutland, Denmark. The macrophyte species varied in growth morphology, C:N stoichiometry, and preferred stream habitat, providing a range in environmental conditions for the epiphyton. Among all habitats and streams, the prokaryotic communities were dominated by common phyla, including Alphaproteobacteria, Bacteriodetes, and Gammaproteobacteria, while the eukaryotic communities were dominated by Stramenopiles (i.e. diatoms). For both the prokaryotes and eukaryotes, the epipsammon were consistently the most diverse communities and the epiphytic communities were generally similar among the four macrophyte species. However, the communities on the least complex macrophyte, Sparganium emersum, had the lowest richness and evenness and fewest unique OTUs, whereas the macrophyte with the most morphological complexity, Callitriche spp., had the highest number of unique OTUs. In general, the microbial taxa were ubiquitously distributed across the relatively homogeneous Danish landscape as determined by measuring the similarity among communities (i.e. Sørensen similarity index). Furthermore, we found significant correlations between microbial diversity (i.e. Chao1 rarefied richness and Pielou's evenness) and biofilm structure and function (i.e. C:N ratio and ammonium uptake efficiency, respectively); communities with higher richness and evenness had higher C:N ratios and lower uptake efficiency. In addition to describing the prokaryotic and eukaryotic community composition in stream biofilms, our study indicates that 1) physical habitat characteristics influence microbial diversity and 2) the variation in microbial diversity may dictate the structural and functional characteristics of stream biofilm communities.     

Identification of β 1 → 4 glucan chains as part of a fraction of slime synthesized within the dictyosomes of maize root caps

Summary The protective polysaccharides synthesized by the outer root-cap cells of maize have been prepared in radioactive and non-radioactive form and studied using the techniques oftrans-elimination, gel filtration and partial hydrolysis under acid and alkaline conditions. The results indicate that the slime consists of a central 1 4 linked glucan rendered soluble by a coating of hydrophilic polysaccharides linked both covalently and non-covalently. The covalently-linked polysaccharide is relatively rich in galacturonic acid and fucose in regions near the central glucan. It is likely that the synthesis of the slime, including the glucan component, takes place within the dictyosome sacs and vesicles and this has important consequences for ideas on the sites of 1 4 glucan synthesis within plant cells.     

Characterization and improvement of oxygen transfer in pilot plant external air-lift bioreactor for mycelial biomass production

The oxygen transfer dynamics in a pilot plant external air-lift bioreactor (EALB) during the cultivation of mycelial biomass were characterized with respect to hydrodynamic parameters of gas holdup (), oxygen transfer coefficient (K_La) and superficial gas velocity (U _g), and dissolved oxygen (DO). An increased flow rate of air supply was required to meet the increased oxygen demand with mycelial biomass growth. Consequently, an increase in air flow rate led to an increase in , K_La and the DO level. The enhancement of oxygen transfer rate in the cultivated broth system, however, was limited with highly increased viscosity of the mycelial broth. An increase in air flow rate from 1.25 to 2.00 v/v/m resulted in a low increment of oxygen transfer. The newly designed pilot plant EALB with two air spargers significantly improved processing reliability, aeration rate and K_La. The pilot plant EALB process, operated under a top pressure from 0 to 1.0 bars, also demonstrated a significant improvement of oxygenation efficiency by more than 20% in DO and K_La. The performance of the two sparger EALB process under top pressure demonstrated an efficient and economical aerobic system with fast mycelial growth and high biomass productivity in mycelial biomass production and wastewater treatment.     

New advances in coenzyme Q biosynthesis

Summary Coenzyme Q (or ubiquinone) is the product of two distinct biosynthetic pathways: the lipid tail of coenzyme Q is formed via the isoprene biosynthetic pathway, and the quinone ring derives from the metabolism of either shikimic acid or tyrosine. In general, eukaryotic organisms use the classical mevalonate pathway to form isopentenyl- and dimethylallyl-diphosphate, the five carbon building blocks of the polyisoprenoid tail, and prokaryotes use 1-deoxy-D-xylulose-5-phosphate, formed via the Rohmer pathway. The quinone ring precursor is 4-hydroxybenzoic acid, which is formed directly from chorismate inSaccharomyces cerevisiae andEscherichia coli, or from tyrosine in animal cells. Ring modification steps including prenylation, decarboxylation, and successive hydroxylation and methylation steps form the fully substituted benzoquinone ring of coenzyme Q. Many of the genes and polypeptides involved in coenzyme Q biosynthesis have been isolated and characterized by utilizing strains ofE. coli andS. cerevisiae with mutations in theubi andCOQ genes, respectively. This article reviews recent progress in characterizing the biosynthesis of coenzyme Q inE. coli, S. cerevisiae, and other eukaryotic organisms.     

Microbial production,enzyme activity,and carbon turnover in surface sediments of the Hudson River estuary

The detrital food web is a major nexus of energy flow in nearly all aquatic ecosystems. Energy enters this nexus by microbial assimilation of detrital carbon. To link microbiological variables with ecosystem process, it is necessary to understand the regulatory hierarchy that controls the distribution of microbial biomass and activity. Toward that goal, we investigated variability in microbial abundance and activities within the tidal freshwater estuary of the Hudson River. Surface sediments were collected from four contrasting sites: a mid-channel shoal, two types of wetlands, and a tributary confluence. These samples, collected in June to August 1992, were sorted into two to four size fractions, depending on the particle size distribution at each site. Each fraction was analyzed for bacterial biomass (by acridine orange direct counting), bacterial production (by ³H-thymidine incorporation into DNA), fungal biomass (by ergosterol extraction), fungal production (by biomass accrual), and the potential activities of seven extracellular enzymes involved in the degradation of detrital structural molecules. Decomposition rates for particulate organic carbon (POC) were estimated from a statistical model relating mass loss rates to endocellulase activity. Within samples, bacterial biomass and productivity were negatively correlated with particle size: Standing stocks and rates in the <63-m class were roughly twofold greater than in the >4-mm class. Conversely, fungal biomass was positively correlated with particle size, with standing stocks in the largest size class more than 1OX greater than in the smallest. Extracellular enzyme activities also differed significantly among size classes, with high carbohydrase activities associated with the largest particles, while oxidative activities predominated in the smallest size classes. Among sites, the mid-channel sediments had the lowest POC standing stock (2% of sediment dry mass) and longest turnover time (approximately 1.7 years), with bacterial productivity approximately equal to fungal (56 vs. 46 g C per gram POC per day, respectively). In the Typha wetland, POC standing stock was high (10%); turnover time was about 0.3 years; and 90% of the microbial productivity was fungal (670 vs. 84 g C per gram POC per day). The other two sites, a Trapa wetland and a tributary confluence, showed intermediate values for microbial productivity and POC turnover. Differences among sites were described by regression models that related the distribution of microbial biomass (r ² = 0.98) and productivity (r ² = 0.81) to particle size and carbon quality. These factors also determined POC decomposition rates. Net microbial production efficiency (production rate/decomposition rate) averaged 10.6%, suggesting that the sediments were exporting large quantities of unassimilated dissolved organic carbon into the water column. Our results suggest that studies of carbon processing in large systems, like the Hudson River estuary, can be facilitated by regression models that relate microbial dynamics to more readily measured parameters. Correspondence to: R.L. Sinsabaugh     

Evolutionary Constraints of Phosphorylation in Eukaryotes,Prokaryotes, and Mitochondria

High accuracy mass spectrometry has proven to be a powerful technology for the large scale identification of serine/threonine/tyrosine phosphorylation in the living cell. However, despite many described phosphoproteomes, there has been no comparative study of the extent of phosphorylation and its evolutionary conservation in all domains of life. Here we analyze the results of phosphoproteomics studies performed with the same technology in a diverse set of organisms. For the most ancient organisms, the prokaryotes, only a few hundred proteins have been found to be phosphorylated. Applying the same technology to eukaryotic species resulted in the detection of thousands of phosphorylation events. Evolutionary analysis shows that prokaryotic phosphoproteins are preferentially conserved in all living organisms, whereas-site specific phosphorylation is not. Eukaryotic phosphosites are generally more conserved than their non-phosphorylated counterparts (with similar structural constraints) throughout the eukaryotic domain. Yeast and Caenorhabditis elegans are two exceptions, indicating that the majority of phosphorylation events evolved after the divergence of higher eukaryotes from yeast and reflecting the unusually large number of nematode-specific kinases. Mitochondria present an interesting intermediate link between the prokaryotic and eukaryotic domains. Applying the same technology to this organelle yielded 174 phosphorylation sites mapped to 74 proteins. Thus, the mitochondrial phosphoproteome is similarly sparse as the prokaryotic phosphoproteomes. As expected from the endosymbiotic theory, phosphorylated as well as non-phosphorylated mitochondrial proteins are significantly conserved in prokaryotes. However, mitochondrial phosphorylation sites are not conserved throughout prokaryotes, consistent with the notion that serine/threonine phosphorylation in prokaryotes occurred relatively recently in evolution. Thus, the phosphoproteome reflects major events in the evolution of life.Reversible protein phosphorylation on serines, threonines, and tyrosines plays a crucial role in regulating processes in all living organisms ranging from prokaryotes to eukaryotes (1). Traditionally, phosphorylation has been detected in single, purified proteins using in vitro assays. Recent advances in mass spectrometry (MS)-based proteomics now allow the identification of in vivo phosphorylation sites with high accuracy (2–7). On-line databases such as PhosphoSite (8), Phospho.ELM (9), and PHOSIDA1 (10) have collected and organized thousands of identified phosphosites. These databases as well as dedicated analysis environments such as NetworKIN (11, 12) offer and use contextual information including structural features, potential kinases, and conservation. They constitute resources that should allow the derivation of general patterns for phosphorylation events. Specifically, the recent availability of data for archaeal, prokaryotic, and diverse eukaryotic phosphoproteomes in these databases should enable investigation of the evolutionary history of this post-translational modification.Prokaryotes have two separate classes of phosphorylation events. Apart from the canonical histidine/aspartate phosphorylation, which has been studied for decades, serine/threonine/tyrosine phosphorylation is also present and has recently become amenable to analysis by MS (13). Bacterial phosphoproteins are involved in protein synthesis, carbohydrate metabolism, and the phosphoenolpyruvate-dependent phosphotransferase system. Recent phosphoproteomics studies of Bacillus subtilis, Escherichia coli, and Lactococcus lactis described around 100 phosphorylation sites on serine, threonine, and tyrosine in each of these species (13–15). Bacterial phosphorylation sites can change in response to environmental conditions (16).Interestingly, even archaea have serine/threonine and tyrosine phosphorylation. A recent study of Halobacterium salinarum described 75 serine/threonine/tyrosine phosphorylation sites on 62 proteins involved in a wide range of cellular processes including a variety of metabolic pathways (17).Although only a few hundred phosphorylation events have been found in prokaryotic species, similar experimental conditions and effort have yielded the detection of thousands of phosphorylation events in eukaryotes ranging from yeast to human (7, 18–21). Before the advent of large scale phosphoproteomics, serine/threonine/tyrosine phosphorylation has been estimated to affect one-third of all proteins (22). Recent large scale phosphoproteomics studies now suggest that more than half of all eukaryotic proteins are phosphorylated (23).A key event in evolution was the endosymbiosis of prokaryotes that enabled the development of a much more complex type of life, the eukaryotic cell. Analyses of mitochondrial genes suggest that the α-proteobacterium Rickettsia prowazekii is the endosymbiotic precursor leading to modern mitochondria (24). Almost all of the mitochondrial genes have migrated to the nuclear genome during subsequent evolution, and it is predicted that 10–15% of eukaryotic nuclear genes of organisms encode mitochondrial proteins (25).Thus, mitochondria with their unique evolutionary position between prokaryotes and eukaryotes form an interesting link for the evolutionary analysis of phosphorylation. Several studies investigated the mitochondrial phosphoproteome in different organisms using gel electrophoresis or specific enrichment methods coupled with mass spectrometry (26–28). Those studies established potential mitochondrial phosphoproteins. Three large scale studies based on affinity enrichment of phosphopeptides and mass spectrometry obtained direct experimental evidence of phosphorylation sites in mitochondria. Lee et al. (29) used a combination of different peptide enrichment strategies and found 80 phosphorylation sites of 48 different proteins from mouse liver. Very recently, a study by Deng et al. (30) characterized the murine cardiac mitochondrial mouse phosphoproteome, covering 236 phosphosites on 181 proteins. Investigating yeast, Reinders et al. (31) assigned 84 phosphorylation sites in 62 proteins.To enable comparative analysis of phosphoproteomes between all domains of life and mitochondria, here we experimentally determined a high accuracy mitochondrial mouse phosphoproteome based on technology conditions similar to those applied to the identification of prokaryotic and eukaryotic phosphoproteomes. We then performed a detailed evolutionary study of the conservation of the identified phosphoproteins and phosphorylation sites in prokaryotes and in eukaryotes. This allowed an initial comparison of the phosphoproteomes of prokaryotes, mitochondria, and eukaryotes.     

Immunochemical assay applied to mycotoxin biosynthesis: ELISA comparison of sterigmatocystin production byAspergillus versicolor andAspergillus nidulans

Conventional thin layer and instrumental methods for analyzing mycotoxins and their precursors are time-consuming and make the investigation of mycotoxin biosynthesis particularly difficult. As an alternative, sensitive enzyme-liked immunosorbent assays (ELISAs) can be utilized to analyze for these compounds. In this report, sterigmatocystin production in test tube cultures ofAspergillus versicolor ATCC 18643 andAspergillus nidulans ATCC 32610 were compared using competitive ELISA. Polyclonal antiserum that was prepared against a sterigmatocystin hemiacetal-bovine serum albumin conjugate exhibited greatest specificity for sterigmatocystin hemiacetal and sterigmatocystin with less reactivity for O-methylsterigmatocystin. The antiserum could be used to detect as little as 50 ng/ml sterigmatocystin in ELISA. Direct ELISA could be performed on diluted culture broth and on mycelial extracts solubilized with N,N-dimethylformamide.Apergillus versicolor ATCC 18643 produced more sterigmatocystin in SLS medium than in YES medium, and showed maximal levels at between 9 to 12 days incubation. Approximately 75% of sterigmatocystin was detectable in mycelium (254g/ml culture) compared to the extracellular fraction (87g/ml culture).Aspergillus nidulans exhibited qualitatively similar patterns of growth and toxigenesis in SLS medium but accumulated maximal levels of only 15g mycelial sterigmatocystin/ml culture and 5g extracellular sterigmatocystin/ml broth, respectively.     

The effect of polyamine biosynthesis inhibition on growth and differentiation of the phytopathogenic fungus Sclerotinia sclerotiorum

We studied the effects of several polyamine biosynthesis inhibitors on growth, differentiation, free polyamine levels and in vivo and in vitro activity of polyamine biosynthesis enzymes in Sclerotinia sclerotiorum. -Difluoromethylornithine (DFMO) and -difluoromethylarginine (DFMA) were potent inhibitors of mycelial growth. The effect of DFMO was due to inhibition of ornithine decarboxylase (ODC). No evidence for the existence of an arginine decarboxylase (ADC) pathway was found. The effect of DFMA was partly due to inhibition of ODC, presumably after its conversion into DFMO by mycelial arginase, as suggested by the high activity of this enzyme detected both in intact mycelium and mycelial extracts. In addition, toxic effects of DFMA on cellular processes other than polyamine metabolism might have occurred. Cyclohexylamine (CHA) slightly inhibited mycelial growth and caused an important decrease of free spermidine associated with a drastic increase of free putrescine concentration. Methylglyoxal bis-[guanyl hydrazone] (MGBG) had no effect on mycelial growth. Excepting MGBG, all the inhibitors strongly decreased sclerotial formation. Results demonstrate that sclerotial development is much more sensitive to polyamine biosynthesis inhibition than mycelial growth. Our results suggest that mycelial growth can be supported either by spermidine or putrescine, while spermidine (or the putrescine/spermidine ratio) is important for sclerotial formation to occur. Ascospore germination was completely insensitive to the inhibitors.     

Patterns and governing forces in aquatic microbial communities

In this review we survey recent publications employing molecular techniques to investigate the distribution of microbial species in aquatic environments. We analyzed the occurrence of microbial phyla in freshwater and marine habitats and observed patterns of distribution that could be explained by the adaptation of microorganisms to physical and biological parameters that vary in aquatic habitats. The gram-positive bacteria, the Verrucomicrobiales and the - and -subdivisions of the Proteobacteria are distributed throughout a range of aquatic habitats, while other phylogenetic groups appear to be adapted to more narrowly defined environmental niches such as anoxic water and sediments (-Proteobacteria) or floating aggregates (Cytophaga-Flexibacter-Bacteroides phylum). -proteobacterial sequence types have been detected throughout freshwater habitats, but these organisms are largely absent from open ocean environments. Within several of these divisions, clusters of closely related small sub unit ribosomal RNA sequence types have been detected in geographically disparate environments, suggesting that some microbial species are globally distributed. In addition to physical variables such as salinity and pH, biological variables also influence microbial community composition. This was illustrated by changes that occurred in the eukaryotic and bacterial species composition in laboratory mesocosms after a viral outburst. We conclude that physical and biological forces govern the composition of aquatic microbial communities and result in divergent evolutionary histories of the indigenous microbial species.     

Linking Host Prokaryotic Physiology to Viral Lifestyle Dynamics in a Temperate Freshwater Lake (Lake Pavin,France)

In aquatic ecosystems, fluctuations in environmental conditions and prokaryotic host physiological states can strongly affect the dynamics of viral life strategies. The influence of prokaryote physiology and environmental factors on viral replication cycles (lytic and lysogeny) was investigated from April to September 2011 at three different strata (epi, meta, and hypolimnion) in the mixolimnion of deep volcanic temperate freshwater Lake Pavin (France). Overall, the euphotic region (epi and metalimnion) was more dynamic and showed significant variation in microbial standing stocks, prokaryotic physiological state, and viral life strategies compared to the aphotic hypolimnion which was stable within sampled months. The prokaryotic host physiology as inferred from the nucleic acid content of prokaryotic cells (high or low nucleic acid) was strongly regulated by the chlorophyll concentration. The predominance of the high nucleic acid (HNA) prokaryotes (cells) over low nucleic acid (LNA) prokaryotes (cells) in the spring (HNA/LNA?=?1.2) and vice versa in the summer period (HNA/LNA?=?0.4) suggest that the natural prokaryotic communities underwent major shifts in their physiological states during investigated time period. The increase in the percentage of inducible lysogenic prokaryotes in the summer period was associated with the switch in the dominance of LNA over HNA cells, which coincided with the periods of strong resource (nutrient) limitation. This supports the idea that lysogeny represents a maintenance strategy for viruses in unproductive or harsh nutrient/host conditions. A negative correlation of percentage of lysogenic prokaryotes with HNA cell abundance and chlorophyll suggest that lysogenic cycle is closely related to prokaryotic cells which are stressed or starved due to unavailability of resources for its growth and activity. Our results provide support to previous findings that changes in prokaryote physiology are critical for the promotion and establishment of lysogeny in aquatic ecosystems, which are prone to constant environmental fluctuations.     

Diversity,role in decomposition,and succession of zoosporic fungi and straminipiles on submerged decaying leaves in a woodland stream

Leaf litter is a very important primary source of energy in woodland streams. Decomposition of leaf litter is a process mediated by many groups of microorganisms which release extracellular enzymes for the degradation of complex macromolecules. In this process, true fungi and straminipiles are considered to be among the most active groups, more active than the bacteria, at least during the early stages of the process. Colonization increases the quality of the leaves as a food resource for detritivores. In this way, matter and energy enter detritus-based food chains. Previously, aquatic hyphomycetes were considered to be the major fungal group responsible for leaf litter decomposition. Although zoosporic fungi and straminipiles are known to colonize and decompose plant tissues in various environments, there is scant information on their roles in leaf decomposition. This study focuses on the communities of zoosporic fungi and straminipiles in a stream which are involved in the decomposition of leaves of two plant species, Ligustrum lucidum and Pouteria salicifolia, in the presence of other groups of fungi. A characteristic community dominated by Nowakowskiella elegans, Phytophthora sp., and Pythium sp. was found. Changes in the fungal community structure over time (succession) was observed: terrestrial mitosporic fungi appeared during the early stages, zoosporic fungi, straminipiles, and aquatic Hyphomycetes in early-to-intermediate stages, while representatives of the phylum Zygomycota were found at early and latest stages of the decomposition. These observations highlight the importance of zoosporic fungi and straminipiles in aquatic ecosystems.     

The composition of the chitinolytic microbial complex and its effect on chitin decomposition at various humidity levels

The dynamics of assimilation of chitin by soil microorganisms (primarily prokaryotes) as a source of carbon and nitrogen has been determined by gas chromatography and fluorescence microscopy. The highest rates of chitin decomposition in chernozem were detected at humidity levels corresponding to the pressure of soil moisture (P) of ?1.4 atm. The rate of microbial consumption of chitin is three times higher than that of the carbon of soil organic matter. Fluorescence microscopy revealed that an increase in the pressure of soil moisture from P = ?10 atm to P = ?0.7 atm resulted in a considerable increase in the proportion of the specific surface of mycelial bacteria (actinomycetes).     

The border fresidues of the dihydrofolate reductase domain inEscherichia coli β-galactosidase correspond to the positions of introns 1 and 5 of dihydrofolate reductase of chicken

Summary In-galactosidase ofEscherichia coli residues 820–934 are similar to residues in dihydrofolate reductase ofE. coli. Dihydrofolate reductase ofE. coli and chicken are also similar and have identical tertiary structures. I used the similarity of the three-dimensional structure of prokaryotic and eukaryotic dihydrofolage reductases to align the chicken dihydrofolate reductase and the similar residues of-galactosidase. The positions of introns 1 and 5 of the chicken dihydrofolate reductase gene correspond exactly to the start and the end of the dihydrofolate reductase-like domain in the-galactosidase polypeptide chain. This equivalence of intron positions in a eukaryotic gene and domain structure in a prokaryotic protein was interpreted as evidence for a common origin of both genes.