Anthony G. O'Donnell

An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids

Abstract A simple small-scale procedure for the sequential extraction of isoprenoid quinones and polar lipids from bacterial cells was developed. Extraction with a biphasic mixture of petroleum ether (b.p. 60–80°C) and methanolic saline gave an upper phase containing isoprenoid quinones. The lower phase, containing the partially extracted organisms, was processed according to the Bligh and Dyer extraction method to give a polar lipid extract. As examples of the procedure, the isoprenoid quinones and polar lipids of Bacillus subtilis, Mycobacterium avium, Pseudomonas diminuta and Streptomyces griseus were extracted and analyzed.

Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA- and rRNA-based microbial community composition

ABSTRACT A rapid protocol for the extraction of total nucleic acids from environmental samples is described. The method facilitates concomitant assessment of microbial 16S rRNA diversity by PCR and reverse transcription-PCR amplification from a single extraction. Denaturing gradient gel electrophoresis microbial community analysis differentiated the active component (rRNA derived) from the total bacterial diversity (ribosomal DNA derived) down the horizons of an established grassland soil.

Plants and fertilisers as drivers of change in microbial community structure and function in soils

Here we report on a range of studies designed to understand the link between diversity and function in soils and in particular how plants and fertilisers might interact with microbial community dynamics in soils. The data presented indicate that although plants and fertilisers do impact on microbial community structure, the relationship between diversity, community structure and function remains complex and difficult to interpret using currently available chemical and molecular fingerprinting techniques. The paper assesses plants and management practices as drivers of change in soil and argues that whilst understanding diversity per se is unlikely to contribute to our understanding of function, an appreciation of what causes communities to change and also the relative importance of such drivers, could lead to new insights into the sustainable management and conservation of soils and natural resources.

A dispersion and differential centrifugation technique for representatively sampling microorganisms from soil

Abstract A multi-stage dispersion and differential centrifugation technique for repesentatively sampling non-filamentous microorganisms from soil was developed. The individual steps of the method aimed to disperse soil aggregates and dissociate microorganisms from soil particles. The released microorganisms were then separated by low-speed centrifugation. The dispersion and dissociation efficiency of the individual steps and the method as a whole were tested turbidometrically using a clay loam, a sandy loam and a peat soil. The evaluation indicated that individual steps rarely achieved more than 50% dispersion and. that the sandy loam was more readily dispersed than the other two soils and that the peat soil was particularly difficult to disperse. The multi-stage method was very efficient at dispersing all three soils. The efficiency of the method at sampling microorganisms was assessed by determining the biomass recovered in extracts and residues compared to the soil at the outset. Biomass was determined using direct microscopic cell counts, ATP, phospholipid, lipopolysaccharide, ergoslerol contents and viable counts. The method yielded extracts which were enriched with microorganisms as determined by direct microscopic counts, ATP, phospholipid and lipopolysaccharidc contents and residues enriched with the fungal biomarker, ergosterol. The sandy loam soil provided samples of microorganisms that were the least contaminated with soil material. Difficulties in separating either the smaller mineral particles or the less dense organic particles resulted in lower recoveries from the clay and peat soils. Density gradient ccntrifugution of the soil extracts indicated that a large number of the cells counted by direct microscopy in the extracts were associated with relatively dense soil material.

Assessing the survival of transgenic plant DNA in the human gastrointestinal tract

The inclusion of genetically modified (GM) plants in the human diet has raised concerns about the possible transfer of transgenes from GM plants to intestinal microflora and enterocytes. The persistence in the human gut of DNA from dietary GM plants is unknown. Here we study the survival of the transgene epsps from GM soya in the small intestine of human ileostomists (i.e., individuals in which the terminal ileum is resected and digesta are diverted from the body via a stoma to a colostomy bag). The amount of transgene that survived passage through the small bowel varied among individuals, with a maximum of 3.7% recovered at the stoma of one individual. The transgene did not survive passage through the intact gastrointestinal tract of human subjects fed GM soya. Three of seven ileostomists showed evidence of low-frequency gene transfer from GM soya to the microflora of the small bowel before their involvement in these experiments. As this low level of epsps in the intestinal microflora did not increase after consumption of the meal containing GM soya, we conclude that gene transfer did not occur during the feeding experiment.

Physiological and Community Responses of Established Grassland Bacterial Populations to Water Stress

ABSTRACT The effects of water stress upon the diversity and culturable activity of bacterial communities in the rhizosphere of an established upland grassland soil have been investigated. Intact monoliths were subjected to different watering regimens over a 2-month period to study community adaptation to moisture limitation and subsequent response to stress alleviation following rewetting. Genetic diversity was analyzed with 16S-based denaturing gradient gel electrophoresis (DGGE) of total soil-extracted DNA (rRNA genes) and RNA (rRNA transcripts) in an attempt to discriminate between total and active communities. Physiological response was monitored by plate counts, total counts, and BIOLOG-GN2 substrate utilization analyses. Controlled soil drying decreased the total number of CFU on all the media tested and also decreased the substrate utilization response. Following rewetting of dried soil, culture-based analyses indicated physiological recovery of the microbial population by the end of the experiment. In contrast, DGGE analyses of community 16S rRNA genes, rRNA transcripts and cultured communities did not reveal any changes relating to the moisture regimens, despite the observed physiological effects. We conclude that the imposed moisture regimen modulated the physiological status of the bacterial community and that bacterial communities in this soil are resistant to water stress. Further, we highlight the need for a reexamination of rRNA transcript-based molecular profiling techniques as a means of describing the active component of soil bacterial communities.

Influence of depth and sampling time on bacterial community structure in an upland grassland soil

Abstract Temporal and spatial variation of soil bacterial communities was evaluated with both molecular and metabolic profiling techniques. Soil cores (20 cm deep) were taken from an upland grassland in the Scottish Borders (UK) over 3 days in July 1999, and on single days in October 1999, April 2000, and August 2000. Cores were separated into four 5-cm depths to examine vertical spatial distribution. The 0-5-, 5-10- and 10-15-cm samples represented organic horizons whilst the 15-20-cm depths were from a mineral horizon. The potential metabolic activities were analysed using BIOLOG-GN plates, whereas genotypic diversity was evaluated using molecular profiling of amplified 16S rRNA and 16S rDNA gene fragments (denaturing gradient gel electrophoresis (DGGE)). BIOLOG-GN analysis revealed decreased substrate utilisation in the lowest depths, which was coupled with changes in the DNA and RNA DGGE profiles. Seasonal variation was pronounced in the 5-10-cm and 10-15-cm organic horizons for the July samplings whilst the 15-20-cm depths appeared more stable. Potential factors influencing the observed changes in bacterial communities resulting from soil depth and sampling time are discussed.

Visualization, modelling and prediction in soil microbiology

The introduction of new approaches for characterizing microbial communities and imaging soil environments has benefited soil microbiology by providing new ways of detecting and locating microorganisms. Consequently, soil microbiology is poised to progress from simply cataloguing microbial complexity to becoming a systems science. A systems approach will enable the structures of microbial communities to be characterized and will inform how microbial communities affect soil function. Systems approaches require accurate analyses of the spatio–temporal properties of the different microenvironments present in soil. In this Review we advocate the need for the convergence of the experimental and theoretical approaches that are used to characterize and model the development of microbial communities in soils.

Lipids in the classification of Nocardioides: Reclassification of Arthrobacter simplex (Jensen) lochhead in the genus Nocardioides (Prauser) emend. O'Donnell et al. as Nocardioides simplex comb. nov.

Representative strains of Nocardioides, Arthrobacter simplex and Arthrobacter tumescens were degraded by acid methanolysis and the fatty acid esters released examined by thin-layer and gas chromatography. Branchedchain 14-methylpentadecanoic acid (iso-16) was the predominant component in all but one of the Nocardioides strains. Arthrobacter simplex also contained major amounts of this acid whereas A. tumescens had only minor amounts. All of the test strains possessed 15 and 17 carbon straight chain acids, tuberculostearic acid (10-methyloctadecanoic acid) and its 17 and 18-carbon homologues. The fatty acid profiles of Nocardioides strains lacked 13-methyltetradecanoic and heptadecanoic acids which were both present in Arthrobacter simplex and Arthrobacter tumescens. The profiles of these latter organisms were quantitatively different from each other. The polar lipids of the test strains all contained diphosphatidylglycerol and phosphatidylglycerol but only Arthrobacter tumescens contained phosphatidylinositol and three unidentified polar lipids. Nocardioides and Arthrobacter simplex strains all contained two very characteristic closely related polar lipids.All of the test strains contained tetrahydrogenated menaquinones with eight isoprene units as the major isoprenologue. The results of the present study support the integrity of the genus Nocardioides and provide a reliable way of distinguishing it from other actinomycetes, such as Streptomyces, which also have LL-diaminopimelic acid and glycine in the peptidoglycan. The lipid data, together with results from chemical, genetic and phage host range studies, provide sufficient grounds for the transfer of Arthrobacter simplex to Nocardioides as Nocardioides simplex comb. nov. An emended description of the genus Nocarioides is given.

Soil Security: Solving the Global Soil Crisis

Soil degradation is a critical and growing global problem. As the world population increases, pressure on soil also increases and the natural capital of soil faces continuing decline. International policy makers have recognized this and a range of initiatives to address it have emerged over recent years. However, a gap remains between what the science tells us about soil and its role in underpinning ecological and human sustainable development, and existing policy instruments for sustainable development. Functioning soil is necessary for ecosystem service delivery, climate change abatement, food and fiber production and fresh water storage. Yet key policy instruments and initiatives for sustainable development have under-recognized the role of soil in addressing major challenges including food and water security, biodiversity loss, climate change and energy sustainability. Soil science has not been sufficiently translated to policy for sustainable development. Two underlying reasons for this are explored and the new concept of soil security is proposed to bridge the science–policy divide. Soil security is explored as a conceptual framework that could be used as the basis for a soil policy framework with soil carbon as an exemplar indicator.