Marine metagenomics: strategies for the discovery of novel enzymes with biotechnological applications from marine environments

Metagenomic based strategies have previously been successfully employed as powerful tools to isolate and identify enzymes with novel biocatalytic activities from the unculturable component of microbial communities from various terrestrial environmental niches. Both sequence based and function based screening approaches have been employed to identify genes encoding novel biocatalytic activities and metabolic pathways from metagenomic libraries. While much of the focus to date has centred on terrestrial based microbial ecosystems, it is clear that the marine environment has enormous microbial biodiversity that remains largely unstudied. Marine microbes are both extremely abundant and diverse; the environments they occupy likewise consist of very diverse niches. As culture-dependent methods have thus far resulted in the isolation of only a tiny percentage of the marine microbiota the application of metagenomic strategies holds great potential to study and exploit the enormous microbial biodiversity which is present within these marine environments.     

Strain persistence of invasive Candida albicans in chronic hyperplastic candidosis that underwent malignant change

Objectives: The aim of this study was to assess persistence and tissue invasion of Candida albicans strains isolated from a 65 year‐old patient with chronic hyperplastic candidosis (CHC), that subsequently developed into squamous cell carcinoma (SCC). Materials and Methods: C. albicans (n=7) were recovered from the oral cavity of the patient over seven years. Confirmation of CHC and SCC in this patient was achieved by histopathological examination of incisional biopsy tissue. DNA fingerprinting was performed on the seven isolates from the CHC patient together with a further eight isolates from patients with normal oral mucosa (n=2), chronic atrophic candidosis (n=1), SCC (n=1) and CHC (n=4). Genotyping involved the use of inter‐repeat PCR using the eukaryotic repeat primer 1251. Characterisation of the tissue invasive abilities of the isolates was achieved by infecting a commercially available reconstituted human oral epithelium (RHE; SkinEthic, Nice, France). After 24 h. C. albicans tissue invasion was assessed by histopathological examination. Results: DNA fingerprinting demonstrated strain persistence of C. albicans in the CHC patient over a seven year period despite provision of systemic antifungal therapy. The strain of C. albicans isolated from this patient was categorised as a high invader within the RHE compared to other isolates. Conclusions: Candidal strain persistence was evident in a patient with CHC over seven years. This persistence may be due to incomplete eradication from the oral cavity following antifungal therapy or subsequent recolonisation from other body sites or separate exogenous sources. The demonstration of enhanced in vitro tissue invasion by this particular strain may, in part, explain the progression to carcinoma.     

A measure of conformational entropy change during thermal protein unfolding using neutron spectroscopy

Thermal unfolding of proteins at high temperatures is caused by a strong increase of the entropy change which lowers Gibbs free energy change of the unfolding transition (DeltaG(unf) = DeltaH - TDeltaS). The main contributions to entropy are the conformational entropy of the polypeptide chain itself and ordering of water molecules around hydrophobic side chains of the protein. To elucidate the role of conformational entropy upon thermal unfolding in more detail, conformational dynamics in the time regime of picoseconds was investigated with neutron spectroscopy. Confined internal structural fluctuations were analyzed for alpha-amylase in the folded and the unfolded state as a function of temperature. A strong difference in structural fluctuations between the folded and the unfolded state was observed at 30 degrees C, which increased even more with rising temperatures. A simple analytical model was used to quantify the differences of the conformational space explored by the observed protein dynamics for the folded and unfolded state. Conformational entropy changes, calculated on the basis of the applied model, show a significant increase upon heating. In contrast to indirect estimates, which proposed a temperature independent conformational entropy change, the measurements presented here, demonstrated that the conformational entropy change increases with rising temperature and therefore contributes to thermal unfolding.     

Arbuscular mycorrhizal community composition associated with two plant species in a grassland ecosystem

Arbuscular mycorrhizal (AM) fungi are biotrophic symbionts colonizing about two-thirds of land plant species and found in all ecosystems. They are of major importance in plant nutrient supply and their diversity is suggested to be an important determinant of plant community composition. The diversity of the AM fungal community composition in the roots of two plant species (Agrostis capillaris and Trifolium repens) that co-occurred in the same grassland ecosystem was characterized using molecular techniques. We analysed the small subunit (SSU) ribosomal RNA gene amplified from a total root DNA extract using AM fungal-specific primers. A total of 2001 cloned fragments from 47 root samples obtained on four dates were analysed by restriction fragment length polymorphism, and 121 of them were sequenced. The diversity found was high: a total of 24 different phylotypes (groups of phylogenetically related sequences) colonized the roots of the two host species. Phylogenetic analyses demonstrate that 19 of these phylotypes belonged to the Glomaceae, three to the Acaulosporaceae and two to the Gigasporaceae. Our study reveals clearly that the AM fungal community colonizing T. repens differed from that colonizing A. capillaris, providing evidence for AM fungal host preference. In addition, our results reveal dynamic changes in the AM fungal community through time.     

Are microorganisms more effective than plants at competing for nitrogen?

Plant scientists have long debated whether plants or microorganisms are the superior competitor for nitrogen in terrestrial ecosystems. Microorganisms have traditionally been viewed as the victors but recent evidence that plants can take up organic nitrogen compounds intact and can successfully acquire N from organic patches in soil raises the question anew. We argue that the key determinants of 'success' in nitrogen competition are spatial differences in nitrogen availability and in root and microbial distributions, together with temporal differences in microbial and root turnover. Consequently, it is not possible to discuss plant-microorganism competition without taking into account this spatiotemporal context.     

Bacteriorhodopsin: the functional details of a molecular machine are being resolved

The photon-driven proton translocator bacteriorhodopsin is considered to be the best understood membrane protein so far. It is nowadays regarded as a model system for photosynthesis, ion pumps and seven transmembrane receptors. The profound knowledge came from the applicability of a variety of modern biophysical techniques which have often been further developed with research on bacteriorhodopsin and have delivered major contributions also to other areas. Most prominent examples are electron crystallography, solid-state NMR spectroscopy and time-resolved vibrational spectroscopy. The recently introduced method of crystallising a membrane protein in the lipidic cubic phase led to high-resolution structures of ground state bacteriorhodopsin and some of the photocycle intermediates. This achievement in combination with spectroscopic results will strongly advance our understanding of the functional mechanism of bacteriorhodopsin on the atomic level. We present here the current knowledge on specific aspects of the structural and functional dynamics of the photoreaction of bacteriorhodopsin with a focus on techniques established in our institute.     

Impact of temperature on the relationship between respiration and nitrogen concentration in roots: an analysis of scaling relationships, Q10 values and thermal acclimation ratios

* The impact of nitrogen (N) supply on the temperature response of root respiratory O(2) uptake (R) was assessed in several herbaceous species grown in solution culture. Warm-grown (25 : 20 degrees C, day:night) plants differing in root N concentration were shifted to 13 : 8 degrees C for 7 d to cold-acclimate. * Log-log plots of root R vs root N concentration both showed that R increased with increasing tissue N concentration, irrespective of the growth temperature. Although the regression slopes of the log-log plots did not differ between the warm-grown and cold-acclimated plants, cold-acclimated plants did exhibit a higher y-axis intercept than their warm-grown counterparts. This suggests that cold acclimation of root R is not entirely dependent on cold-induced increases in tissue N concentration and that scaling relationships (i.e. regression equations fitted to the log-log plots) between root R and N concentration are not fixed. * No systematic differences were found in the short-term Q(10) (proportional change in R per 10 degrees C change in temperature), or degree of cold acclimation (as measured by the proportional difference between warm- and cold-acclimated roots) among roots differing in root N concentration. The temperature response of root R is therefore insensitive to tissue N concentration. * The insensitivity of Q(10) values and acclimation to tissue N concentration raises the possibility that root R and its temperature sensitivity can be predicted for a range of N supply scenarios.     

Non-motile mini-transposon mutants of Bordetella bronchiseptica exhibit altered abilities to invade and survive in eukaryotic cells

Non-motile mutants of Bordetella bronchiseptica were generated after mini-transposon mutagenesis. One non-motile mutant (designated VMM1) was derived from the bvg-positive strain BB7865 and four mutants (designated AMM1–4) were derived from the isogenic bvg-negative strain BB7866. Southern hybridisation analysis indicated that loss of motility was not due to the disruption of the flagellin subunit gene. Western blot and transmission electron microscopic analysis indicated that three of the five mutants expressed neither the flagellin subunit (40 kDa) nor flagella whereas one mutant expressed intact flagella under all conditions tested. One unique bvg-negative mutant, AMM4, exhibited temperature-dependent repression of flagella biosynthesis and motility at 37°C. The ability of AMM4 to invade and survive in HeLa cells was significantly decreased.     

Why plants bother: root proliferation results in increased nitrogen capture from an organic patch when two grasses compete

We investigated whether the capacities of Lolium perenne L. and Poa pratensis L. roots to proliferate locally and to alter local nitrogen (N) inflows in a decomposing organic matter patch were important in their capture of N when grown together. In the presence of a patch, plants of both species were significantly heavier and contained more N. Root length and weight densities increased in the patch, but specific root length was unaltered. Although both species proliferated roots in the patch, L. perenne produced greater root length densities than P. pratensis , and also captured more N from the patch. Indeed, total N uptake from the patch was related to root length density within the patch. N inflows (rate of N uptake per unit root length) in the patch were no faster than in the whole root system for both species. Under the conditions of this study, root proliferation in an organic patch was more important for N capture from the patch than alterations in N inflows. Local proliferation of roots may be a key factor in interspecific competition for non-uniformly distributed supplies of N in natural habitats, so resolving the previous uncertainty as to the 'adaptive' nature of root proliferation.