L. Anne Glover

Spatial Analysis of Archaeal Community Structure in Grassland Soil

ABSTRACT The complex structure of soil and the heterogeneity of resources available to microorganisms have implications for sampling regimens when the structure and diversity of microbial communities are analyzed. To assess the heterogeneity in community structure, archaeal communities, which typically contain sequences belonging to the nonthermophilic Crenarchaeota, were examined at two contrasting spatial scales by using PCR-denaturing gradient gel electrophoresis (DGGE) analysis followed by unweighted pair group method with arithmetic mean analysis of 16S rRNA- and ribosomal DNA-derived profiles. A macroscale analysis was carried out with soil cores taken at 2-m intervals along triplicate 8-m transects from both managed (improved) and natural (unimproved) grassland rhizosphere soils. A microscale analysis was carried out with a single soil core by assessing the effects of both sample size (10, 1, and 0.1 g) and distance between samples. The much reduced complexity of archaeal profiles compared to the complexity typical of the bacterial community facilitated visual comparison of profiles based on band presence and revealed different levels of heterogeneity between sets of samples. At the macroscale level, heterogeneity over the transect could not be related to grassland type. Substantial heterogeneity was observed across both improved and unimproved transects, except for one improved transect that exhibited substantial homogeneity, so that profiles for a single core were largely representative of the entire transect. At the smaller scale, the heterogeneity of the archaeal community structure varied with sample size within a single 8- by 8-cm core. The archaeal DGGE profiles for replicate 10-g soil samples were similar, while those for 1-g samples and 0.1-g samples showed greater heterogeneity. In addition, there was no relationship between the archaeal profiles and the distance between 1- or 0.1-g samples, although relationships between community structure and distance of separation may occur at a smaller scale. Our findings demonstrate the care required when workers attempt to obtain a representative picture of microbial community structure in the soil environment.

Assessment of bioavailability of heavy metals using lux modified constructs of Pseudomonas fluorescens

The bioluminescence response of a genetically modified (lux‐marked) bacterium to potentially toxic elements (PTEs) was monitored using an in vitro assay. Washed cells of Pseudomonas fiuorescens were added to solutions containing various concentrations of metal salts. Bioluminescence, involving either plasmid or chromosomally encoded lux genes, declined as the metal concentration increased. The plasmid marked construct was significantly more sensitive to all metals except Cr. The order of metal sensitivity was found to be Cu = Zn > Cd > Cr > Ni for the chromosomally marked construct and Cu = Zn > Cd > Ni > Cr for the plasmid marked construct. The very sensitive response of lux‐marked terrestrial bacteria to PTEs identified the potential for a rapid and flexible ecotoxicity assay for assessing the pollution of soil or fresh water environments.

Matric potential and the survival and activity of a Pseudomonas fluorescens inoculum in soil

Abstract The survival and activity of a strain of Pseudomonas fluorescens, chromosomally marked with lux genes, was studied following inoculation into microcosms containing autoclaved or non-autoclaved soil adjusted to matric potentials of −30, −750 and −1500 kPa and incubated for up to 3 months. Viable cell concentrations were determined by dilution plate counting and population activity was measured by luminometry radiorespirometry and dehydrogenase activity. Although increased matric stress appeared to reduce survival, measured by viable cell concentration, effects were not statistically significant (P > 0.05) when variability within replicates was taken into account. Similarly, when all data were taken into account, changes in viable cell concentration were statistically insignificant (P > 0.05) during the incubation. Survival was, however, significantly (P

POTENTIAL LUMINESCENCE AS AN INDICATOR OF ACTIVATION OF GENETICALLY-MODIFIED PSEUDOMONAS-FLUORESCENS IN LIQUID CULTURE AND IN SOIL

Potential luminescence was used to assess the ability of luminescence-marked cells of Pseudomonas fluorescens to regain activity following starvation in soil or in liquid culture. In soil, potential luminescence was measured by determining luminescence periodically during incubation of samples of inoculated soil with double-strength complex medium supplemented with sodium citrate. Luminescence increased to a maximum during incubation. Following starvation of cells in soil, maximum luminescence decreased and the time taken to reach the maximum increased. Activity measured by dehydrogenase assays did not vary during the incubation and was not significantly affected by starvation of the cells. Viable cell concentration correlated well with final potential luminescence values and with luminescence in the absence of nutrient amendment, but not with potential dehydrogenase activity. Growth of P. fluorescens, previously starved in liquid medium, was preceded by a lag which increased in length as the duration of the starvation period increased. Although luminescence of the starved cultures decreased with increased starvation period, there was no detectable lag in luminescence following addition of nutrients. Potential luminescence therefore enables rapid, non-extractive and selective determination of changes in activity of luminescence-marked microorganisms in soil, the size of the active population and the time taken to recover from periods of starvation.

Bridging the phenotypic gap: Real-time assessment of mitochondrial function and metabolism of the nematode Caenorhabditis elegans

BackgroundThe ATP levels of an organism are an important physiological parameter that is affected by genetic make up, ageing, stress and disease.ResultsWe have generated luminescent C. elegans through ubiquitous, constitutive expression of firefly luciferase, widely used for in vitro ATP determination. We hypothesise that whole animal luminescence reflects its intracellular ATP levels in vivo. To test this, we characterised the bioluminescence response of C. elegans during sublethal exposure to, and recovery from azide, a treatment that inhibits mitochondrial respiration reversibly, and causes ATP depletion. Consistent with our expectations, in vivo luminescence decreased with increasing sublethal azide levels, and recovered fully when worms were removed from azide. Firefly luciferase expression levels, stability and activity did not influence the final luminescence. Bioluminescence also reflected the lowered activity of the electron transport chain achieved with RNA interference (RNAi) of genes encoding respiratory chain components.ConclusionResults indicated that C. elegans luminescence reports on ATP levels in real-time. For the first time, we are able to directly assess the metabolism of a whole, living, multicellular organism by determination of the relative ATP levels. This will enable genetic analysis based on a readily quantifiable metabolic phenotype and will provide novel insights into mechanisms of fitness and disease that are likely to be of relevance for other organisms, as well as the worm.

A model for bacterial conjugal gene transfer on solid surfaces.

Abstract Quantitative models of bacterial conjugation are useful tools in environmental risk assessment and in studies of the ecology and evolution of bacterial communities. We constructed a mathematical model for gene transfer between bacteria growing on a solid surface. The model considers that donor and recipient cells will form separate colonies, which grow exponentially until nutrient exhaustion. Conjugation occurs when donor and recipient colonies meet, all recipient cells becoming transconjugants instantly, after which they act as donors. The model was tested theoretically by computer simulations that followed the histories of individual bacterial colonies and was validated for initial surface coverage of 60% or less, where confluent growth does not occur. Model predictions of final number of donors, recipients and transconjugants were tested experimentally using a filter mating system with two isogenic strains of Pseudomonas fluorescens MON787 acting as donor and recipient of plasmid RP4. Experimental trends resulting from varying donor and recipient inoculum numbers and donor:recipient ratios were well described by the model, although it often overestimated conjugation by 0.5-2 orders of magnitude. Predictions were greatly improved, generally to within half a log unit of experimental values, by consideration of the time for conjugative transfer. The model demonstrates the relationship between spatial separation of cells and nutrient availability on numbers of transconjugants. By providing a mechanistic approach to the study conjugation on surfaces, the model may contribute to the study of gene transfer in natural environments.

Rapid Sublethal Toxicity Assessment Using Bioluminescent Caenorhabditis elegans, a Novel Whole-Animal Metabolic Biosensor

Sublethal metabolic effects are informative toxicological end points. We used a rapid quantitative metabolic end point, bioluminescence of firefly luciferase expressing Caenorhabditis elegans, to assess effects of sublethal chronic exposure (19 h) to the oxidative stress agent and environmental pollutant cadmium (provided as chloride salt). Bioluminescence declined in a concentration-dependent manner in the concentration range tested (0-30 microM Cd), with comparable sensitivity to reproduction and developmental assay end points (after 67 and 72 h, respectively). Cd concentrations that resulted in 20% reduction in bioluminescence (EC(20)) were 11.8-13.0 microM, whereas the reproduction EC(20) (67 h exposure) was 10.2 microM. At low concentrations of Cd (< or = 15 microM), the decline in bioluminescence reflected a drop in ATP levels. At Cd concentrations of 15-30 microM, decreased bioluminescence was attributable both to effects of Cd on ATP levels and decreased production of luciferase proteins, concomitant with a decline in protein levels. We show that whole-animal bioluminescence is a valid toxicological end point and a rapid and sensitive predictor of effects of Cd exposure on development and reproduction. This provides a platform for high-throughput sublethal screening and will potentially contribute to reduction of testing in higher animals.

The role of the earthworm Lumbricus terrestris in the transport of bacterial inocula through soil

Two laboratory experiments were used to investigate the effect of the earthworm Lumbricus terrestris on transport of genetically marked Pseudomonas fluorescens inocula through soil microcosms. The microcosms comprised cylindrical cores of repacked soil with or without earthworms. Late log-phase cells of P. fluorescens, chromosomally marked with lux genes encoding bioluminescence, were applied to the surface of soil cores as inoculated filter paper discs. In one experiment, 5 and 10 days after inoculation, cores were destructively harvested to determine concentrations of marked pseudomonads with depth relative to the initial inoculum applied. Transport of the bacteria occurred only in the presence of earthworms. In a second experiment cores were subjected to simulated rainfall events 18 h after inoculation with lux-marked bacteria at 3-day intervals over a 24-day period. Resulting leachates were analysed for the appearance of the marked bacteria, and after 28 days cores were destructively harvested. Although some marked cells (less than 0.1% of the inoculum applied) were leached through soil in percolating water, particularly in the presence of earthworms, the most important effect of earthworms on cell transport was through burial of inoculated litter rather than an increase in bypass flow due to earthworm channels.

Evidence for a localization signal in the 3'untranslated region of myosin heavy chain messenger RNA.

Localization signals in the 3′untranslated region (3′UTR) of myosin heavy chain mRNA were investigated using hybrid gene constructs. In myoblasts transfected with constructs containing either both coding sequences and 3′UTR of the rabbit β‐globin gene or the β‐globin coding sequences alonein situhybridization showed that globin transcripts were distributed throughout the cytoplasm with no localization. In contrast, in myoblasts transfected with β‐globin coding sequences linked to the myosin heavy chain 3′UTR there was strong perinuclear localization of the hybrid mRNA; this was maintained in myotubes. We conclude that myosin heavy chain 3′UTR contains a localization signal.

Localisation of metallothionein isoform mRNAs in rat hepatoma (H4) cells

The localisation of metallothionein isoform mRNAs in rat hepatoma (H4) cells was investigated using two approaches, namely Northern hybridisation of total RNA extracted from free, cytoskeletal‐bound and membrane‐bound polysomes isolated by a sequential detergent/salt extraction procedure and in situ hybridisation. The cytoskeletal‐bound polysomes were enriched in metallothionein‐I (MT‐I) and c‐myc mRNAs but showed a significantly lower enrichment in MT‐II mRNA. These findings indicate that the MT‐I mRNA is localised to the cytoskeleton during translation. In situ hybridisation using a biotin‐labelled oligonucleotide probe revealed a predominantly perinuclear localisation for the MT‐I mRNA.