The genus Burkholderia is composed of functionally diverse species and it can be divided into several groups. One of these, designated as the plant-beneficialenvironmental (PBE) Burkholderia group, is formed by non-pathogenic species, which in most cases have been found to be associated with plants. It was previously established that members of the PBE group share an N-acyl-homoserine lactone (AHL) quorum-sensing (QS) system designated BraVR that produces and responds to 3-oxoCwHSL (OC14-HSL). In the present study, we further studied the BraIlR system in several members of the PBE group determining the AHL production profile as well its regulons. Major results include that different levels of AHLs are produced by different species and that the regulon is species specific. The biosynthesis of exopolysaccharide was the only common phenotype found to be regulated by BraVR in several species of the PBE cluster. In addition, BraIIR was shown not to be important for plant nodulation by B. phymafum spp. nor for endophytic colonization and growth promotion of maize by B. phyfofirmans PslN. Moreover, the genome of the rice endophyte B. kururiensis M130 was sequenced and analysed in order to detect potential loci involved in its endophytic lifestyle and plant growth promotion. Two experimental approaches (plate screening of a transposon-promoter probe library and RNAseq) were then performed in order to identify loci that were regulated in response to plant macerate. The results indicated that B. kururiensis undergoes major regulatory changes affecting the expression of 27.7% of its protein coding genes. Interestingly, a great number of differentially expressed genes encode membrane transporters and secretion systems, which indicates that the exchange of molecules is an important aspect in planfa. In addition, genes related to mobility, chemotaxis and adhesion were also over-represented suggesting recognition and an intimate interaction between bacteria and plant. This work highlights the close signalling taking place between plants and bacteria and helps us to understand the adaptation of an endophyte in planta.

The objective of this thesis is to use metabolic modelling techniques to investigate primary and secondary metabolism in S. erythraea and from this to identify key factors controlling flux distribution during secondary metabolism. S. erythraea is a member of the actinomycetes a group of bacteria responsible for the production of a number of commercially important small molecules. Actinomycete physiology is considerably more complicated than that seen in "simple" bacteria such as E. coli. The conjecture investigated in this thesis is that metabolic modelling techniques that take into account this extra complexity should be more useful in designing strategies for overproduction of desired metabolites than simpler models. The thesis gives the first detailed description of the dynamic changes in biomass composition seen during the batch cultivation of S. erythraea. It further shows that incorporation of this information into a flux balance model of the organism's metabolism significantly improves the flux distributions generated especially in the stationary phase. Using this improved technique growth phase and stationary phase metabolism are investigated. Some of the unusual stationary phase behaviour is shown to be the result of glucose uptake being independent of demand. Rigid control of branch points in the metabolic network is not found suggesting that the organism's metabolism is flexible. A reverse metabolic engineering strategy is applied, two variants of the wild type organism are compared with an industrial strain. The industrial strain is found to have a considerably lower glucose uptake rate than the parental strain. The relationship between TCA cycle flux, oxidative phosphorylation and organic acid secretion is investigated using an uncoupler. This project demonstrates that applied correctly flux balance analysis is a powerful tool for investigating actinomycete physiology. The insights gained are of direct relevance to the commercial production of secondary metabolites in S. erythraea.

This thesis reports on the use of solid-phase and liquid model food systems to estimate dynamic growth data for Listeria monocytogenes and Yersinia enterocolitica and the survival of Campylobacter jejuni under modified atmospheres and refrigeration temperatures. Modified atmospheres included 5% O2: l0% CO2:85% N2 (MA1), 30% CO2:70% N2 (MA2) and aerobic conditions at 4oC and 8oC. Computer image analysis recorded macro-colony growth on agar surfaces. Correlation of bacterial colony radius to viable cell number allowed estimation of dynamic growth data for L. monocytogenes and Y. enterocolitica. Image analysis techniques were developed allowing estimation of dynamic growth data for micro-colonies under MA1 and aerobic conditions, using area to estimate colony size. Morphological change in Camp. jejuni cells on agar surfaces was determined after storage under atmospheres and temperatures of study and survival after storage was determined by incubating the cells under ideal conditions (microaerophilic; 37 oC). All solid culture models were compared with liquid culture. 30% CO2 significantly (p<0.01) suppressed the growth of L. monocytogenes and Y. enterocolitica macro-colonies especially at 4oC (3 and 2.5 fold decrease in specific growth rate, respectively compared with aerobic conditions). A significant (p<0.05) decrease was observed for Y. enterocolitica micro-colonies (approximate 1.5 fold) at 4oC compared with aerobic conditions and identical rates were obtained for L. monocytogenes (0.02 h-1). At 8oC similar growth rates were obtained under MA1 and aerobic conditions for both organisms. Growth in liquid culture showed similar findings to macro- and micro- colony growth. Survival of Carnp. jejuni was enhanced under 30% CO2 as compared with aerobic conditions. Modifying the atmosphere reduced the morphological shift of the cells to the coccoid form on agar surfaces during resuscitation at37 oC. This thesis extends the knowledge of the behaviour of foodborne pathogens in foods under MAP. The micro-colony technique coupled with image analysis improves the understanding of the change in cell morphology and growth under MAs and refrigeration temperatures.

Epsilon toxin (Etx) and necrotic enteritis toxin B (NetB) are two pore-forming toxins produced by C. perfringens. While Etx has been shown to be the key virulence factor for enterotoxemia in goats and sheep, NetB has been associated with the pathogenesis of avian necrotic enteritis (NE), a gastro-intestinal disease causing economic damage to the poultry industry worldwide. The crystal structure of Etx H149A (an Etx variant with 6x reduced toxicity relative to wild type toxin) was solved to 2.4 Å and showed that the H149A mutation in domain III does not affect organization of the receptor binding region in domain I. The Etx H149A structure also revealed a second putative glycan binding site in domain III. In addition, site-directed mutagenesis in domain I of Etx H149A affirmed the important role of tyrosine residues for toxin binding and demonstrated the capability of Etx H149A to be used as a platform for further receptor binding studies in the future. The crystal structure of the pore-form of NetB was solved to 3.9 Å and revealed high similarities to the Staphylococcus aureus α-hemolysin heptameric structure. However, in particular the region thought to interact with the target cell membrane showed some interesting divergence in amino acid composition. Site-directed mutagenesis within this domain significantly affected binding and toxicity of NetB to target cells. Mutagenesis within the β-sandwich domain of NetB revealed important amino acid residues for toxin oligomerisation and pore-formation. In order to test NetB toxoids as candidate vaccines, a NetB genetic toxoid and a formaldehyde NetB toxoid were used to immunise poultry in an in vivo NE disease model. Vaccination with any of the two antigens resulted in the induction of specific antibody responses against NetB and provided significant protection against disease.

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