Intracellular mechanisms of fungal space searching in microenvironments.

Filamentous fungi that colonize microenvironments, such as animal or plant tissue or soil, must find optimal paths through their habitat, but the biological basis for negotiating growth in constrained environments is unknown. We used time-lapse live-cell imaging of Neurospora crassa in microfluidic environments to show how constraining geometries determine the intracellular processes responsible for fungal growth. We found that, if a hypha made contact with obstacles at acute angles, the Spitzenkörper (an assembly of vesicles) moved from the center of the apical dome closer to the obstacle, thus functioning as an internal gyroscope, which preserved the information regarding the initial growth direction. Additionally, the off-axis trajectory of the Spitzenkörper was tracked by microtubules exhibiting "cutting corner" patterns. By contrast, if a hypha made contact with an obstacle at near-orthogonal incidence, the directional memory was lost, due to the temporary collapse of the Spitzenkörper-microtubule system, followed by the formation of two "daughter" hyphae growing in opposite directions along the contour of the obstacle. Finally, a hypha passing a lateral opening in constraining channels continued to grow unperturbed, but a daughter hypha gradually branched into the opening and formed its own Spitzenkörper-microtubule system. These observations suggest that the Spitzenkörper-microtubule system is responsible for efficient space partitioning in microenvironments, but, in its absence during constraint-induced apical splitting and lateral branching, the directional memory is lost, and growth is driven solely by the isotropic turgor pressure. These results further our understanding of fungal growth in microenvironments relevant to environmental, industrial, and medical applications.

Comparative genomics and transduction potential of Enterococcus faecalis temperate bacteriophages.

To determine the relative importance of temperate bacteriophage in the horizontal gene transfer of fitness and virulence determinants of Enterococcus faecalis, a panel of 47 bacteremia isolates were treated with the inducing agents mitomycin C, norfloxacin, and UV radiation. Thirty-four phages were purified from culture supernatants and discriminated using pulsed-field gel electrophoresis (PFGE) and restriction mapping. From these analyses the genomes of eight representative phages were pyrosequenced, revealing four distinct groups of phages. Three groups of phages, PhiFL1 to 3, were found to be sequence related, with PhiFL1A to C and PhiFL2A and B sharing the greatest identity (87 to 88%), while PhiFL3A and B share 37 to 41% identity with PhiFL1 and 2. PhiFL4A shares 3 to 12% identity with the phages PhiFL1 to 3. The PhiFL3A and B phages possess a high DNA sequence identity with the morphogenesis and lysis modules of Lactococcus lactis subsp. cremoris prophages. Homologs of the Streptococcus mitis platelet binding phage tail proteins, PblA and PblB, are encoded on each sequenced E. faecalis phage. Few other phage genes encoding potential virulence functions were identified, and there was little evidence of carriage of lysogenic conversion genes distal to endolysin, as has been observed with genomes of many temperate phages from the opportunist pathogens Staphylococcus aureus and Streptococcus pyogenes. E. faecalis JH2-2 lysogens were generated using the eight phages, and these were examined for their relative fitness in Galleria mellonella. Several lysogens exhibited different effects upon survival of G. mellonella compared to their isogenic parent. The eight phages were tested for their ability to package host DNA, and three were shown to be very effective for generalized transduction of naive host cells of the laboratory strains OG1RF and JH2-2.

Real-time monitoring of the development and stability of biofilms of Streptococcus mutans using the quartz crystal microbalance with dissipation monitoring.

Quartz crystal microbalance with dissipation monitoring (QCM-D) was used for continuous in-situ monitoring of cell attachment and growth of Streptococcus mutans as biofilms. Cell attachment and proliferation were monitored within an overnight period of 20 h. Biofilms generated using a 'continuous flow' method had a greater mass and were more dissipative (more viscoelastic) than those established using an 'attach and flow' strategy. Cell numbers (as colony forming units, c.f.u.) in biofilms formed inside the QCM-D device after a 2-h attachment phase and during a 20-h growth period could be related to frequency (f) changes. The percentage surface coverage on the QCM-D crystals by bacteria was estimated using the surface analysis features of the atomic force microscope and image analysis software. Both mean percentage coverage and c.f.u increased after growth of S. mutans. The energy losses displayed by the increases in the dissipative factor (D) indicated an increase in 'softness' of the attached cells. The ratio of D/f was used to provide information of the way in which viscoelasticity changed per unit mass. Flow conditions over the cells on the surface appeared to be important in creating biofilms of a greater complexity and stability and the QCM-D enabled properties of cells during attachment and binding, proliferation and removal to be monitored continuously.

Suppression of insect pest populations and damage to plants by vermicomposts.

The effects of commercial vermicomposts, produced from food waste, on infestations and damage by aphids, mealy bugs and cabbage white caterpillars were studied in the greenhouse. Vermicomposts were used at substitution rates into a soil-less plant growth medium, MetroMix 360 (MM360), at rates of 100% MM360 and 0% vermicompost, 80% MM360 and 20% vermicompost, and 60% MM360 and 40% vermicompost to grow peppers (Capsicum annuum L.), tomatoes (Lycopersicon esculentum Mill.) and cabbages (Brassica oleracea L.), in pots. Groups of 10 pots containing young plants were distributed randomly in nylon mesh cages (40 cm x 40 cm x 40 cm). Groups of 10 pepper seedlings in a single cage were infested with either 100 aphids (Myzus persicae Sulz.) or 50 mealy bugs (Pseudococcus spp.) per cage. Similar groups of tomato seedlings were infested with 50 mealy bugs per cage. Groups of four cabbage seedlings in pots in cages were infested with 16 cabbage white caterpillars (Pieris brassicae L.). Populations of aphids and mealy bugs were counted after 20 days and the shoot dry weights of peppers, tomatoes and cabbages were measured at harvest. Numbers of cabbage white caterpillars and loss in shoot weights were measured after 15 days. The substitution rates of 20% and 40% vermicomposts suppressed populations of both aphids and mealy bugs on peppers, and mealy bugs on tomatoes, significantly. Substitutions with vermicomposts into MM360 decreased losses of dry weights of peppers, in response to both aphid and mealy bug infestations, decreased losses in shoot dry weights of tomatoes after mealy bug infestations significantly. There were significantly decreased losses in leaf areas of cabbage seedlings in response to the cabbage white caterpillar infestations.

Effects of Metals on a Range of Streptomyces Species.

A plate diffusion method was used to assess the tolerance of 28 mesophilic and 4 thermophilic species of streptomycetes to eight metals. This method allowed a qualitative screen of the effects of the metals on growth, on sporulation, and in some cases, on secondary metabolite production. Tolerant and sensitive species were identified, some of which exhibited the same response (i.e., tolerance or sensitivity) to a number of metals. Experiments in liquid culture were used to quantify the precise concentration ranges of the metals at which the most tolerant or sensitive species could grow. Distribution profiles of the sensitivities of all species examined toward different metals revealed that the order of toxicity was Hg > Cd > Co > Zn > Ni > Cu > Cr > Mn.

Salmonella typhimurium displays cyclical patterns of sensitivity to UV-C killing during prolonged incubation in the stationary phase of growth.

Stationary phase cells of Salmonella typhimurium were more resistant to killing by UV-C irradiation than those from the exponential phase. Analysis of the tolerance of cells taken at different stages of prolonged incubation as batch cultures to 60 or 100 J m(2) doses of UV-C revealed cycles of resistance and tolerance. The possible involvement of rpoS-controlled functions in mediating these cycles could be discounted because they were also detected in an rpoS minus mutant of S. typhimurium. The results are discussed in the context of heterogeneity in cells of stationary phase cultures of S. typhimurium.

The potential of site-specific recombinases as novel reporters in whole-cell biosensors of pollution.

DNA recombinases show some promise as reporters of pollutants providing that appropriate promoters are used and that the apparent dependence of expression on cell density can be solved. Further work is in progress using different recombinases and other promoters to optimize recombinase expression as well as to test these genetic constructs in contaminated environmental samples such as soil and water. It may be that a graded response reflecting pollutant concentration may not be possible. However, they show great promise for providing definitive detection systems for the presence of a pollutant and may be applicable to address the problem of bioavailability of pollutants in complex environments such as soil.

Effects of vermicomposts produced from food waste on the growth and yields of greenhouse peppers.

Vermicomposts, produced commercially from food wastes, were substituted at a range of different concentrations into a soil-less commercial bedding plant container medium, Metro-Mix 360 (MM360), to evaluate their effects on the growth and yields of peppers in the greenhouse. Six-week-old peppers (Capsicum annum L. var. California) were transplanted into 100%, 80%, 60%, 40%, 20% or 10% MM360 substituted with 0%, 10%, 20%, 40%, 60%, 80% and 100% vermicompost. All plants were watered three times weekly with 200 ppm Peter's Nutrient Solution from the time of transplanting up to 107 days. Peppers grown in potting mixtures containing 40% food waste vermicomposts and 60% MM360 yielded 45% more fruit weights and had 17% greater mean number of fruits than those grown in MM360 only. The mean heights, numbers of buds and numbers of flowers of peppers grown in potting mixtures containing 10-80% vermicompost although greater did not differ significantly from those of peppers grown in MM360. There were no positive correlations between the increases in pepper yields, and the amounts of mineral-N and microbial biomass-N in the potting mixtures, or the concentrations of nitrogen in the shoot tissues of peppers. Factors such as: an improvement of the physical structure of the potting medium, increases in populations of beneficial microorganisms and the potential availability of plant growth-influencing-substances produced by microorganisms in vermicomposts, could have contributed to the increased pepper yields obtained.

Probing the growth dynamics of Neurospora crassa with microfluidic structures.

Despite occupying physically and chemically heterogeneous natural environments, the growth dynamics of filamentous fungi is typically studied on the surface of homogeneous laboratory media. Fungal exploration and exploitation of complex natural environments requires optimal survival and growth strategies at the colony, hyphal, and intra hyphal level, with hyphal space-searching strategies playing a central role. We describe a new methodology for the characterisation and analysis of hyphal space-searching strategies, which uses purposefully designed three-dimensional microfluidics structures mimicking some of the characteristics of natural environments of the fungi. We also demonstrate this new methodology by running a comparative examination of two Neurospora crassa strains, i.e., the wild type of N. crassa -- a commonly used model organism for the study of filamentous fungi -- and the N. crassa ro-1 mutant strain -- which is deficient in hyphal and mycelial growth. Continuous live imaging showed that both strains responded actively to the geometrically confined microstructured environments without any detectable temporal delay or spatial adjustment. While both strains navigated the test structures exhibiting similar geometry-induced space-searching mechanisms, they presented fundamentally different growth patterns that could not be observed on geometrically unconfined, flat agar surfaces.

Identification of carbohydrate metabolism genes in the metagenome of a marine biofilm community shown to be dominated by gammaproteobacteria and bacteroidetes.

Polysaccharides are an important source of organic carbon in the marine environment and degradation of the insoluble and globally abundant cellulose is a major component of the marine carbon cycle. Although a number of species of cultured bacteria are known to degrade crystalline cellulose, little is known of the polysaccharide hydrolases expressed by cellulose-degrading microbial communities, particularly in the marine environment. Next generation 454 Pyrosequencing was applied to analyze the microbial community that colonizes and degrades insoluble polysaccharides in situ in the Irish Sea. The bioinformatics tool MG-RAST was used to examine the randomly sampled data for taxonomic markers and functional genes, and showed that the community was dominated by members of the Gammaproteobacteria and Bacteroidetes. Furthermore, the identification of 211 gene sequences matched to a custom-made database comprising the members of nine glycoside hydrolase families revealed an extensive repertoire of functional genes predicted to be involved in cellulose utilization. This demonstrates that the use of an in situ cellulose baiting method yielded a marine microbial metagenome considerably enriched in functional genes involved in polysaccharide degradation. The research reported here is the first designed to specifically address the bacterial communities that colonize and degrade cellulose in the marine environment and to evaluate the glycoside hydrolase (cellulase and chitinase) gene repertoire of that community, in the absence of the biases associated with PCR-based molecular techniques.

Adsorption of calf thymus DNA on Au(110) studied by reflection anisotropy spectroscopy.

Reflection anisotropy spectroscopy (RAS) was used to investigate the adsorption of single-stranded (ss-) and double-stranded (ds-) calf thymus DNA on Au(110) in an electrochemical cell. Both types of DNA form ordered structures for electrode potentials in the range from +0.6 to -0.4 V. Both types of DNA desorb at -0.6 V and may start desorbing at lower negative potentials. When adsorbed at +0.6 V, both forms give rise to a similar RAS signal and adsorb through the phosphate groups. As the potential is reduced, the RAS intensity observed from ss-DNA increases to roughly twice that observed from ds-DNA, a result that is interpreted as due to a change in the adsorption of the ss-DNA from sites involving the phosphate groups to sites involving the bases.

Reflection anisotropy spectroscopy study of the adsorption of sulfur-containing amino acids at the Au(110)/electrolyte interface.

Protein interactions with surfaces are key to understanding the behavior of implantable medical devices. The optical technique of reflection anisotropy spectroscopy (RAS) has considerable potential for the study of interactions between important biological molecules and surfaces. This study used RAS to investigate the adsorption of S amino acids onto Au(110) in a liquid environment under different conditions of potential and pH. Certain spectral features can be associated with the Au(110), as reported previously, while other features are assigned to bonds between the amino acids and the Au surface. The RA spectra are shown to be influenced by the structure of the amino acid, the solution pH, and the applied electrode potential. This work has assigned the negative feature at 2.5 eV to the Au-thiolate, bond while the positive feature at 2.5 eV is assigned to the disulfide bond. The broad spectral feature at 3.5 eV is attributed to the Au-amino interaction, while the sharper feature at slightly higher energy is associated with the Au-carboxylate interaction. Sulfur-containing amino acids are frequently found on the outside of protein molecules and could be used to anchor the protein to the surface.

Development of temporal temperature gradient electrophoresis for characterising methanogen diversity.

Temporal temperature gradient electrophoretic (TTGE) analysis of 16S rDNA sequences was optimized to monitor the methanogen population present in water and sediments of a small eutrophic lake, Priest Pot, in the English Lake district. The production of nonrepresentative TTGE profiles due to the generation of polymerase chain reaction (PCR) artifacts initially proved problematical. The use of a proofreading polymerase in the PCR was found to be essential and fully optimized protocols were established and tested to ensure confidence that the TTGE profiles truly reflected sequence diversity. TTGE analysis revealed the methanogen population to be less diverse in water than in sediment. The most genetic diversity was observed in TTGE profiles of sediment DNA isolated in winter and the least was in sediment DNA isolated in summer. DNA sequencing analysis of bands recovered from TTGE gels revealed the presence of two methanogen communities. One clustered with Methanosaeta species and the other with the Methanomicrobiales. Many sequences showed low DNA sequence similarity to known methanogens, suggesting that Priest Pot harbors previously undescribed methanogen species.

The use of integrated soil microcosms to assess the impact of carbendazim on soil ecosystems.

Our investigation used carbendazim as a representative pesticide for testing an integrated soil microcosm (ISM) test protocol. Microcosms, set up in a greenhouse, consisted of cylinders made from high-density polyethylene (HDPE) pipe, 7.5 cm (i.d.) x 15 cm high. A fine nylon mesh was placed across the bottom of each microcosm for leachate collection. Field soil, (silty clay loam), collected from Flörsheim, Germany, was sieved through a 5 mm screen and mixed thoroughly. Earthworms, enchytraeids, and microarthropods were added to each microcosm. Each microcosm contained five wheat seedlings, and was maintained at a 12 h-12 h light-dark cycle. Artificial rainwater was used to water microcosms as required. Soil microcosms were treated with carbendazim at concentrations 1, 3, 9, 27, and 81 times higher than the predicted environmental concentration (PEC) of 0.76 mg a.i./kg soil dry weight. A water-only control treatment was also used. The key soil processes used as endpoints were microbial activity, nitrogen mineralization. soil enzymatic activity, ammonium and nitrate leaching, organic matter decomposition and biological feeding activity. Key structural parameters measured were microbial biomass, nematode communities, microarthropod populations and diversity, enchytraeid and earthworm populations and plant growth. Pesticide degradation, leaching and uptake into plants and earthworms were also assessed. Carbendazim had significant effects on several key soil processes including soil ammonium-N and nitrate-N concentrations and soil dehydrogenase activity. Wheat growth, nematode and earthworm populations, and invertebrate feeding activity were soil structural parameters affected significantly by carbendazim. Earthworm biomass was the most sensitive parameter measured with an EC50 of 1.9 mg a.i./kg soil dry weight 28 days after treatment. A comparison of these results with results from single-species tests, small microcosms, large terrestrial model ecosystems, and field tests indicated that the ISM protocol may adequately predict environmental effects.

Ring-testing and field-validation of a terrestrial model ecosystem (TME)--an instrument for testing potentially harmful substances: conceptual approach and study design.

During spring and summer 1999 a ring-test and field-validation study with an open, intact Terrestrial Model Ecosystem (TME) was conducted at four different European sites (Amsterdam, The Netherlands; Bangor, U.K.; Coimbra, Portugal; Flörsheim, Germany). The objective of the study was to establish a standardised method which allows the impact of chemical stressors on terrestrial compartments at ecosystem level to be investigated and possible uses of such data in existing Environmental Risk Assessments (ERAs) for chemicals to be evaluated. This issue of Ecotoxicology presents in a series of papers the results of the TME ring-test and field-validation study. Additionally, results derived from an open-homogeneous terrestrial microcosm (Integrated Soil Microcosm, ISM) are included in this series as a separate paper. In this first paper of the series background information on the planning and organisation of the study are given. The conceptual approach and the design of the study with TMEs are briefly outlined, based on the scientific discussion on the use of terrestrial microcosms in ecology and applied environmental sciences during the last 25 years. Further, some suggestions are presented on the selection of measurement endpoints to quantify structural and functional aspects of terrestrial ecosystems. Finally, the main results of the TME-study are summarised and conclusions are drawn on the technical feasibility of TMEs, their comparability with field studies and the potential use of TMEs in ERA.