Lesley Anne Glover

Numerical Analysis of Grassland Bacterial Community Structure under Different Land Management Regimens by Using 16S Ribosomal DNA Sequence Data and Denaturing Gradient Gel Electrophoresis Banding Patterns

ABSTRACT Bacterial diversity in unimproved and improved grassland soils was assessed by PCR amplification of bacterial 16S ribosomal DNA (rDNA) from directly extracted soil DNA, followed by sequencing of ∼45 16S rDNA clones from each of three unimproved and three improved grassland samples (A. E. McCaig, L. A. Glover, and J. I. Prosser, Appl. Environ. Microbiol. 65:1721–1730, 1999) or by denaturing gradient gel electrophoresis (DGGE) of total amplification products. Semi-improved grassland soils were analyzed only by DGGE. No differences between communities were detected by calculation of diversity indices and similarity coefficients for clone data (possibly due to poor coverage). Differences were not observed between the diversities of individual unimproved and improved grassland DGGE profiles, although considerable spatial variation was observed among triplicate samples. Semi-improved grassland samples, however, were less diverse than the other grassland samples and had much lower within-group variation. DGGE banding profiles obtained from triplicate samples pooled prior to analysis indicated that there was less evenness in improved soils, suggesting that selection for specific bacterial groups occurred. Analysis of DGGE profiles by canonical variate analysis but not by principal-coordinate analysis, using unweighted data (considering only the presence and absence of bands) and weighted data (considering the relative intensity of each band), demonstrated that there were clear differences between grasslands, and the results were not affected by weighting of data. This study demonstrated that quantitative analysis of data obtained by community profiling methods, such as DGGE, can reveal differences between complex microbial communities.

Spatial structure in soil chemical and microbiological properties in an upland grassland

We characterised the spatial structure of soil microbial communities in an unimproved grazed upland grassland in the Scottish Borders. A range of soil chemical parameters, cultivable microbes, protozoa, nematodes, phospholipid fatty acid (PLFA) profiles, community-level physiological profiles (CLPP), intra-radical arbuscular mycorrhizal community structure, and eubacterial, actinomycete, pseudomonad and ammonia-oxidiser 16S rRNA gene profiles, assessed by denaturing gradient gel electrophoresis (DGGE) were quantified. The botanical composition of the vegetation associated with each soil sample was also determined. Geostatistical analysis of the data revealed a gamut of spatial dependency with diverse semivariograms being apparent, ranging from pure nugget, linear and non-linear forms. Spatial autocorrelation generally accounted for 40-60% of the total variance of those properties where such autocorrelation was apparent, but accounted for 97% in the case of nitrate-N. Geostatistical ranges extending from approximately 0.6-6 m were detected, dispersed throughout both chemical and biological properties. CLPP data tended to be associated with ranges greater than 4.5 m. There was no relationship between physical distance in the field and genetic similarity based on DGGE profiles. However, analysis of samples taken as close as 1 cm apart within a subset of cores suggested some spatial dependency in community DNA-DGGE parameters below an 8 cm scale. Spatial correlation between the properties was generally weak, with some exceptions such as between microbial biomass C and total N and C. There was evidence for scale-dependence in the relationships between properties. PLFA and CLPP profiling showed some association with vegetation composition, but DGGE profiling did not. There was considerably stronger association between notional sheep urine patches, denoted by soil nutrient status, and many of the properties. These data demonstrate extreme spatial variation in community-level microbiological properties in upland grasslands, and that despite considerable numeric ranges in the majority of properties, overarching controlling factors were not apparent.

Plasmid and chromosomally encoded luminescence marker systems for detection of Pseudomonas fluorescens in soil

Luminescent strains of Pseudomonas fluorescens 10586 were constructed in which luciferase production was constitutive by introduction of Vibrio fischeri luxABE genes on the chromosome and on a multicopy plasmid. Light production in liquid batch culture was directly proportional to biomass concentration during exponential growth and enabled detection by luminometry of 1.7 × 103 and 8.9 × 104 cells/ml for the plasmid and chromosomally marked strains, respectively. Luminescent colonies of both strains were detectable by eye, enabling viable cell enumeration on solid media against a background of non‐luminescent strains. Following inoculation into sterile and non‐sterile soil lower levels of detection were increased but detection of 8.1–59 × 103and 2.2–30 × 103 cells per g of soil was possible for plasmid and chromosomally marked strains. Maximum specific growth rate in liquid culture was unaffected by introduction of lux marker genes on the chromosome, but was reduced in the plasmid marked strain. The chromosomally encoded marker was stable in both liquid culture and in soil, but the plasmid was unstable during continuous subculturing in liquid medium and during growth in soil. The chromosomally encoded luminescence‐marker system therefore provides a convenient, non‐extractive technique for quantification of genetically modified soil microbial inocula.

Novel cyanobacterial biosensor for detection of herbicides

ABSTRACT The aim of this work was to generate a cyanobacterial biosensor that could be used to detect herbicides and other environmental pollutants. A representative freshwater cyanobacterium, Synechocystis sp. strain PCC6803, was chromosomally marked with the luciferase gene luc (from the firefly Photinus pyralis) to create a novel bioluminescent cyanobacterial strain. Successful expression of the luc gene during growth of Synechocystis sp. strain PCC6803 cultures was characterized by measuring optical density and bioluminescence. Bioluminescence was optimized with regard to uptake of the luciferase substrate, luciferin, and the physiology of the cyanobacterium. Bioassays demonstrated that a novel luminescent cyanobacterial biosensor has been developed which responded to a range of compounds including different herbicide types and other toxins. This biosensor is expected to provide new opportunities for the rapid screening of environmental samples or for the investigation of potential environmental damage.

Luminescence-Based Systems for Detection of Bacteria in the Environment

The development of techniques for detection and tracking of microorganisms in natural environments has been accelerated by the requirement for assessment of the risks associated with environmental release of genetically engineered microbial inocula. Molecular marker systems are particularly appropriate for such studies and luminescence-based markers have the broadest range of applications, involving the introduction of prokaryotic (lux) or eukaryotic (luc) genes for the enzyme luciferase. Lux or luc genes can be detected on the basis of unique DNA sequences by gene probing and PCR amplification, but the major advantage of luminescence-based systems is the ability to detect light emitted by marked organisms or by luciferase activity in cell-free extracts. Luminescent colonies can be detected by eye, providing distinction from colonies of indigenous organisms, and the sensitivity of plate counting can be increased greatly by CCD imaging. Single cells or microcolonies of luminescent organisms can also be detected in environmental samples by CCD image-enhanced microscopy, facilitating study of their spatial distribution. The metabolic activity of luminescence-marked populations can be quantified by luminometry and does not require extraction of cells or laboratory growth. Metabolic activity, and potential activity, of marked organisms therefore can be measured during colonization of soil particles and plant material in real time without disturbing the colonization process. In comparison with traditional activity techniques, luminometry provides significant increases in sensitivity, accuracy, and, most importantly, selectivity, as activity can be measured in the presence of indigenous microbial communities. The sensitivity, speed, and convenience of luminescence measurements make this a powerful technique that is being applied to the study of an increasingly wide range of ecological problems. These include microbial survival and recovery, microbial predation, plant pathogenicity, phylloplane and rhizosphere colonization and reporting of gene expression in environmental samples.

A mathematical model for dispersal of bacterial inoculants colonizing the wheat rhizosphere

Abstract A mathematical model has been constructed to describe bacterial growth and movement in the rhizosphere. In the model, bacteria are introduced into the soil on inoculated seeds and growth occurs, after seed germination, on material produced as root exudates. Movement of substrates away from the rhizosphere into the bulk soil is by diffusion and microbial movement is mediated by carriage on the root surface. The relationship between specific growth rate and substrate concentration is described by Monod kinetics and death occurs at a constant specific rate. An important component of the model is treatment of the effects of matric potential on the distribution and activity of bacteria in different microhabitats. Simulation of the model quantifies the distribution of both bacteria and substrate with depth and time in the rhizosphere and demonstrates significant differences between substrate concentrations at high and low matric potentials. Sensitivity analysis of model predictions indicates the parameters which govern microbial growth to be more important determinants of microbial movement than plant-associated parameters. Predictions of the model compared well with experimental data on microbial movement in the rhizosphere of wheat plants grown in microcosms, and inoculated with luminescence-marked Pseudomonas fluorescens, and provide the basis for quantitative risk assessment following environmental release of genetically-engineered microorganisms.

Matric potential in relation to survival and activity of a genetically modified microbial inoculum in soil

Abstract The effects of matric potential on the survival and activity of an inoculum of Escherichia coli in soil were investigated by inoculating cells into sterile soil, with or without glucose, at three different matric potentials (— 5 kPa, — 64 kPa and—1.5 MPa). The E. coli strain used plasmid-borne lux genes to enable expression of luminescence in active cells. Survival of cells was determined over 10 days by dilution plate counting of viable cellsand activity was estimated by luminometry (actual activity) and radio-respirometry (potential activity). Matric potential had a significant effect on both viable cell concentrations and microbial activity. Light output and carbon dioxide production per cell both decreased with greater matric stress in both glucose-amended and unamended soil. Light output decreased with time, however, while substrateamended respiration remained fairly constant, highlighting important differences between actual and potential microbial activities.

Development of an acute and chronic ecotoxicity assay using lux-marked Rhizobium leguminosarum biovar trifolii

A soil isolate of Rhizobium leguminosarum bv. trifolii was marked with a lux CDABE gene cassette to enable the expression of bioluminescence. The suitability of the bacterium as a soil pollution biosensor was assessed using acute and chronic assays. Bacterial bioluminescence responded sensitively to the metals studied. The order of sensitivity was found to be Cd > Ni = Zn > Cu for the acute test and Cd > Ni = Zn = Cu for the chronic test. The sensitive response of the biosensor highlighted its potential for use as an indicator of soil pollution.

Development and application of bioluminescent Caenorhabditis elegans as multicellular eukaryotic biosensors

We describe a novel approach to assess toxicity to the free‐living nematode Caenorhabditis elegans that relies on the ability of firefly luciferase to report on endogenous ATP levels. We have constructed bioluminescent C. elegans with the luc gene under control of a constitutive promoter. Light reduction was observed in response to increasing temperature, concentrations of copper, lead and 3,5‐dichlorophenol. This was due to increased mortality coupled with decreased metabolic activity in the surviving animals. The light emitted by the transgenic nematodes gave a rapid, real‐time indication of metabolic status. This forms the basis of rapid and biologically relevant toxicity tests.

Use of luminescence-marked bacteria to assess copper bioavailability in malt whisky distillery effluent

Abstract Samples were taken from upstream, influent, effluent, and downstream locations of a whisky distillery in north east Scotland, and the concentration of inorganic pollutants determined using inductively coupled plasma mass spectrometry. The principal contaminant was found to be Cu, and three bioluminescence based microbial bioassays were carved out to assess the bioavailability of Cu. One assay involved standard use of a naturally luminescent marine bacterium and two involved use of genetically modified (luminescence-marked) terrestrial bacteria. Use of the luminescence-marked biosensors was found to be the most sensitive and reproducible, offering assessment of toxicity over a wide pH range.