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Tn5 insertion behavioural mutants of A. tumefaciens C58C(^1) were available. Cloning of the kanamycin resistance gene allowed isolation of Tn5 flanking sequences from a number of the mutants. Flanking sequences from five mutants were used to isolate cosmids, overlapping the Tn5 insertion sites of the mutantsfrom a C58C(^1) library. Two cosmids, pDUB1900 and 1905 have been characterised. pDUB1900 contains the insertion sites of eight motility mutants, with another immediately adjacent. The pDUB1905 insert overlaps sequences interrupted in another three mutants. Behavioural genes in Agrobacterium are clustered together on the chromosome, as in other motile bacteria. Restriction maps of the isolated cosmids show that none of motility mutants analysed was the result of mactivation of pscA or chvB which would lead to an altered behavioural phenotype. Flanking sequences from three of the mutants hybridised to R. meliloti chromosomal DNA, but not to DNA from other motile genera. DNA adjacent to the insertion site of fla-11 hybridised to the insert of pRZ-2, a cosmid containing behavioural genes from R. meliloti. Experiments were undertaken to investigate the occurrence of proteins homologous to the MCP's of enteric bacteria in C58C(^1) DNA hybridisation to oligonucleotide probes showed DNA that could potentially code for MCP-like proteins exists m both Agrobacterium and Rhizobium spp. In addition, an antibody raised against the E. coli MCP Tar cross-reacted with a protein of approximately 60kDa. in C58C(^1). In vivo protein methylation experiments using C58C(^1) resulted in the labelling of a 45kDa protein, whose methylation pattern did not change upon addition of chemostimuli. Possible reasons for the difference in size between the labelled protein and that revealed by the antibodv are discussed.

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Doctor of Philosophy / Department of Biology / Michael Herman / Soil nematode communities are important components of the micro fauna in grassland ecosystems and their interaction with soil microbes affects important ecological processes such as decomposition and nutrient recycling. To study genetic mechanisms underlying ecologically important traits involved in the response of nematode communities to soil microbes, we employed genomic tools available for the model nematode, Caenorhabditis elegans. Previous work identified 204 C. elegans genes that were differentially expressed in response to growth on four different bacteria: Bacillus megaterium, Pseudomonas sp., Micrococcus luteus and Escherichia coli. For many of the genes the degree of differential gene expression between two bacterial environments predicted the magnitude of the effect of the loss of gene function on life-history traits in those environments. Mutations can have differential effects on fitness in variable environments, which can influence their maintenance in a population. Our fitness assays revealed that bacterial environments had varying magnitude of stress, defined as an environment in which the wild-type has a relatively low fitness. We performed fitness assays as part of a comprehensive analysis of life history traits on thirty five strains that contained mutations in genes involved in the C. elegans response to E. coli, B. megaterium, Pseudomonas sp. We found that many of the mutations had conditionally beneficial effects and led to increased fitness when nematodes bearing them were exposed to stressful bacteria. We compared the relative fitness of strains bearing these mutations across bacterial environments and found that the deleterious effects of many mutations were alleviated in the presence of stressful bacteria. Although transcriptional profiling studies can identify genes that are differentially regulated in response to environmental stimuli, how the expressed genes provide functional specificity to a particular environment remains largely unknown. We focused on defense and metabolism genes involved in C. elegans-bacterial interactions and measured the survivorship of loss-of-function mutants in these genes exposed to different bacteria. We found that genes had both bacteria-specific and bacteria-shared responses. We then analyzed double mutant strains and found bacteria-specific genetic interaction effects. Plasticity in gene interactions and their environment-specific modulation have important implications for host phenotypic differentiation and adaptation to changing environments.

Soil is a complex heterogeneous system comprising of highly variable and dynamic micro-habitats that have significant impacts on the growth and activity of resident microbiota. A question addressed in this research is how soil structure affects the temporal dynamics and spatial distribution of bacteria. Using repacked microcosms, the effect of bulk-density, aggregate sizes and water content on growth and distribution of introduced Pseudomonas fluorescens and Bacillus subtilis bacteria was determined. Soil bulk-density and aggregate sizes were altered to manipulate the characteristics of the pore volume where bacteria reside and through which distribution of solutes and nutrients is controlled. X-ray CT was used to characterise the pore geometry of repacked soil microcosms. Soil porosity, connectivity and soil-pore interface area declined with increasing bulk-density. In samples that differ in pore geometry, its effect on growth and extent of spread of introduced bacteria was investigated. The growth rate of bacteria reduced with increasing bulk-density, consistent with a significant difference in pore geometry. To measure the ability of bacteria to spread thorough soil, placement experiments were developed. Bacteria were capable of spreading several cm’s through soil. The extent of spread of bacteria was faster and further in soil with larger and better connected pore volumes. To study the spatial distribution in detail, a methodology was developed where a combination of X-ray microtopography, to characterize the soil structure, and fluorescence microscopy, to visualize and quantify bacteria in soil sections was used. The influence of pore characteristics on distribution of bacteria was analysed at macro- and microscales. Soil porosity, connectivity and soil-pore interface influenced bacterial distribution only at the macroscale. The method developed was applied to investigate the effect of soil pore characteristics on the extent of spread of bacteria introduced locally towards a C source in soil. Soil-pore interface influenced spread of bacteria and colonization, therefore higher bacterial densities were found in soil with higher pore volumes. Therefore the results in this showed that pore geometry affects the growth and spread of bacteria in soil. The method developed showed showed how thin sectioning technique can be combined with 3D X-ray CT to visualize bacterial colonization of a 3D pore volume. This novel combination of methods is a significant step towards a full mechanistic understanding of microbial dynamics in structured soils.

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