Linking phylogeny to abundant ribotypes of community fingerprints: an exercise on the phylotypic responses to plant species, fertilisation and Lolium perenne ingression.

The present study explores the potential of directly linking phylogenetic identities obtained by cloning and sequencing of ITS sequences to dominant ribotypes of molecular community fingerprints to give further insight into dominant members of the communities in three Irish grassland soils. The ten most abundant bacterial ribotypes of untreated bare soils of three grassland microcosms were chosen to represent the "baseline community" of the respective soil. Identities on phylum and order level were assigned to these ribotypes on a weighted basis, by matching sequence homologies of cloned ITS sequences with ribotypes of the same fragment lengths ±5 bp. Results showed that ribotypes were represented by the phyla Acidobacteria, Actinobacteria, Proteobacteria, and Firmicutes and the distribution of the ribotype and phylotype communities was shown to be highly site-specific. Furthermore the response of dominant bacterial phylotypes to plant species composition, fertilisation and Lolium perenne ingression was investigated within a larger microcosm study (Microb Ecol 63:509-521).

Heterologous overexpression, purification and characterisation of an alcohol dehydrogenase (ADH2) from Halobacterium sp. NRC-1.

Replacement of chemical steps with biocatalytic ones is becoming increasingly more interesting due to the remarkable catalytic properties of enzymes, such as their wide range of substrate specificities and variety of chemo-, stereo- and regioselective reactions. This study presents characterisation of an alcohol dehydrogenase (ADH) from the halophilic archaeum Halobacterium sp. NRC-1 (HsADH2). A hexahistidine-tagged recombinant version of HsADH2 (His-HsADH2) was heterologously overexpressed in Haloferax volcanii. The enzyme was purified in one step by immobilised Ni-affinity chromatography. His-HsADH2 was halophilic and mildly thermophilic with optimal activity for ethanol oxidation at 4 M KCl around 60 °C and pH 10.0. The enzyme was extremely stable, retaining 80 % activity after 30 days. His-HsADH2 showed preference for NADP(H) but interestingly retained 60 % activity towards NADH. The enzyme displayed broad substrate specificity, with maximum activity obtained for 1-propanol. The enzyme also accepted secondary alcohols such as 2-butanol and even 1-phenylethanol. In the reductive reaction, working conditions for His-HsADH2 were optimised for acetaldehyde and found to be 4 M KCl and pH 6.0. His-HsADH2 displayed intrinsic organic solvent tolerance, which is highly relevant for biotechnological applications.

A comparison of two novel alcohol dehydrogenase enzymes (ADH1 and ADH2) from the extreme halophile Haloferax volcanii.

Haloarchaeal alcohol dehydrogenases are exciting biocatalysts with potential industrial applications. In this study, two alcohol dehydrogenase enzymes from the extremely halophilic archaeon Haloferax volcanii (HvADH1 and HvADH2) were homologously expressed and subsequently purified by immobilized metal-affinity chromatography. The proteins appeared to copurify with endogenous alcohol dehydrogenases, and a double Δadh2 Δadh1 gene deletion strain was constructed to prevent this occurrence. Purified HvADH1 and HvADH2 were compared in terms of stability and enzymatic activity over a range of pH values, salt concentrations, and temperatures. Both enzymes were haloalkaliphilic and thermoactive for the oxidative reaction and catalyzed the reductive reaction at a slightly acidic pH. While the NAD(+)-dependent HvADH1 showed a preference for short-chain alcohols and was inherently unstable, HvADH2 exhibited dual cofactor specificity, accepted a broad range of substrates, and, with respect to HvADH1, was remarkably stable. Furthermore, HvADH2 exhibited tolerance to organic solvents. HvADH2 therefore displays much greater potential as an industrially useful biocatalyst than HvADH1.

Influences of plant species composition, fertilisation and Lolium perenne ingression on soil microbial community structure in three Irish grasslands.

Semi-natural grassland soils are frequently fertilised for agricultural improvement. This practice often comes at a loss of the indigenous flora while fast-growing nitrogen-responsive species, such as Lolium perenne, take over. Since soil microbial communities depend on plant root exudates for carbon and nitrogen sources, this shift in vegetation is thought to influence soil microbial community structure. In this study, we investigated the influence of different plant species, fertilisation and L. perenne ingression on microbial communities in soils from three semi-natural Irish grasslands. Bacterial and fungal community compositions were determined by automated ribosomal intergenic spacer analysis, and community changes were linked to environmental factors by multivariate statistical analysis. Soil type had a strong effect on bacterial and fungal communities, mainly correlated to soil pH, as well as soil carbon and nitrogen status. Within each soil type, plant species composition was the main influencing factor followed by nitrogen fertilisation and finally Lolium ingression in the acidic upland and mesotrophic grassland. In the alkaline grassland, however, Lolium ingression had a stronger effect than fertilisation. Our results suggest that a change in plant species diversity strongly influences the microbial community structure, which may subsequently lead to significant changes in ecosystem functioning.

Field preservation and DNA extraction methods for intestinal microbial diversity analysis in earthworms.

We assessed the effect of DNA extraction and sample preservation methods on the DNA yield and quality obtained from earthworm (Aporrectodea caliginosa Savigny) gut samples and on the results obtained by bacterial and fungal automated ribosomal intergenic spacer analysis (ARISA) of DNA extracts. Methods based on a hexadecyltrimethylammonium bromide dithiotreitol (CTAB-DTT) extraction buffer yielded more favourable results than those based on a sodium dodecyl sulphate (SDS) buffer. For both of these buffers, incorporation of a bead-beating during the lysis step increased the ARISA-derived bacterial ribotype numbers and diversity estimates, as determined for gut wall samples (P<0.01). Although spectrophotometric analysis indicated that DNA extracted by the CTAB-DTT and SDS-based methods were of comparable quality (P> or =0.05), the former method yielded >1.5 times more DNA from both gut contents and gut walls of earthworms than the latter method (both incorporating the bead beating step) (P<0.01). ARISA analysis detected more reproducible ribotypes and more microbial diversity in DNA extracted by the CTAB-DTT- as compared to the SDS-based method (P<0.01). Significant difference between bacterial communities of gut contents and gut walls were detected within DNA extracted by the CTAB-DTT (but not by the SDS-based) method (Global R=0.76, P<0.001, analysis of similarity). Using the CTAB-DTT-based method, we showed that earthworm preservation in ethanol yielded higher quality DNA from gut contents than preservation in either chloroform or liquid N, as determined by spectrophotometry, PCR inhibition analysis and bacterial and fungal ARISA (P<0.05). Bacterial or fungal communities in the gut contents of fresh and ethanol-preserved earthworms were more similar and were significantly different from those of earthworms preserved in chloroform or liquid N (Global R=0.79 and 0.83 for bacteria and fungi, respectively; P<0.001, analysis of similarity). We propose that ethanol preservation and the CTAB-DTT-based DNA extraction method described herein are also suitable for the analysis of gut-associated microbiota in other soil and sediment feeding invertebrates.