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The ecological relationships between amoebae and their bacterial prey has been sparsely investigated. Although these relationships are highly pertinent to the enfierging role played by protozoa in harbouring pathogenic bacteria, research has focussed largely on post-infection genetic and nfiolecular events within the protozoan cell. The v\/ork in this thesis aimed to throw light on the processes taking place prior to such events and the influencing factors. Amoebae are largely surface-dwellers, thus natural or man-made mixed bacterial biofilms where amoebae may be the principle predators, provide an ideal locus for these interactions, and the first paper established that amoebae do feed exclusively in attached mode.

Protozoan grazing is commonly recognised as a major factor in shaping the structure and composition of bacterial communities. However, the complexity in the relationship between these predators and their prey means that ingestion of bacteria is not always coupled with complete digestion. A large number of factors are thought to be involved in determining the outcome of an ingestion event, and is highlighted by the fact that there is a large amount of inconsistency in the literature regarding the digestibility of prey types. Protozoan phagosome processing is poorly understood - which in turn makes it difficult to explain why and how differential digestion of prey occurs. Summation of the research into phagocytosis highlights two main implications for protozoan research. Firstly, in comparison to other phagocytic cell types, particularly macrophage and Dictyostelium discoideum, research in protozoan cells is out-dated, and therefore limited. Secondly, this lag in research means that gaps in the knowledge of protozoan phagosome processing are often filled with information from other model phagocytes; but are they comparable? This study firstly aimed to increase our understanding of phagosome processing by profiling changes in phagosome pH and size over time. Initially attempts were made to update research focusing on the pH profile in protozoa. However, work with both direct and indirect methods, previously utilised in other phagocytic cell types, failed to produce an effective assay for ,pH profiling in ciliates. Therefore, the main conclusions of this work explored the methodological considerations required when working with ciliated protozoa and the transferability of techniques between evolutionary distant phagocytic organisms. This research then progressed to assaying phagosome size over time as a measure of phagosomal processing. Results challenged the current beliefs regarding phagosome processing and produced a new model of phagosome sizing over time. Furthermore, utilising the sizing profile, as a relatively simple measure of phagosome processing events, factors that could potentially lead to an altered outcome were investigated. In this way, the pre-culture conditions of both predator and prey were shown to be significant in affecting phagosome processing. The next study examined the experimental variability that may be affecting the perceived outcome of digestion; rather than the events at the processing level. Specifically, by focusing on the digestibility of prey types as a function of the number of prey cells per phagosome it was shown that this latter parameter could alter the probability of prey digestion, with complete digestion of phagosomal contents being more likely if <6 prey cell contained. Finally, an in silico investigation aimed to take a holistic view to get a measure of how relevant and valid comparisons between phagocytic cell types are. Specifically, by analysing the proteins of a known phagosomal component, the V-ATPase enzyme. This work suggested that care must be taken when drawing general comparisons regarding phagocytosis and future work should strive to incorporate a larger number of protozoan and prey types. Overall the results of this study add further support to the idea that the relationship between prey and protozoan predators is one which has many layers of complexity which in turn has implications for nutrient transfer in environmental models.

Aggregates provide a distinct niche within the water column with elevated levels of nutrients compared to the surrounding water. Differences have been observed between aggregate and planktonic communities of ammonia oxidising bacteria within the environment, but such differences have not been explained. Ammonia oxidising bacteria are important due to their role in the first rate-limiting step of nitrification in which nitrogen is converted from its most reduced to its most oxidised form. The aim of this study was to determine mechanisms responsible for aggregate colonisation using a model system. To achieve this aim, a model aggregate system was developed in which colonisation of aggregates by ammonia oxidising bacteria under different conditions could be determined. Candidate ammonia oxidiser strains, <i>Nitrosomonas europaea </i>and <i>Nitrosospira tenuis, </i>were used to demonstrate the effect of the sampling method on observed distribution of AOB within the model system. The degree of bias of the b-AMO and CTO PCR primer sets was assessed and indicated that the predominance of Nitrosospira species in many environmental surveys was not due to primer bias. The composition of particulate communities of AOB determined the extent of colonisation by other species of AOB. The immediate colonisation of aggregates by <i>N. tenuis </i>suggested that differences in particulate and planktonic AOB communities were not solely due to differences in substrate availability and activity of differing AOB under such conditions, although the ability of attached communities of AOB to respond more quickly when substrate becomes available was again illustrated. This study provides evidence of the differences between particle-attached and free-living AOB and indicates how such differences can be related to the ecology of these organisms in natural environments.

Heap bioleaching technology has been successfully applied at an industrial scale for over 30 years, and is generally considered to be the most effective engineering option to bio-process low-grade copper sulfide ores. Heap bioleaching was established at the Escondida mine in Chile in 2006, and culture-independent analysis of liquid samples had previously provided some information of the composition of the indigenous microbial communities. In the current project, six prokaryotes belonging to three bacterial genera and one archaeal genus were isolated from the heap bioleaching plant at Escondida and studies carried out comparing their responses to those of known species and strains of mineral- degrading bacteria to some environmental stress factors, in order to understand how the indigenous microflora are able to successfully colonize and exploit the bioheaps. Stress factors examined including elevated concentrations of heavy metals and osmotic potentials. One of the isolates, identified as a strain of Acidithiobacillus ferrooxidans, was able to grow at more extreme conditions of pH and temperature than reported for most other strains of this species. Copper tolerance observed in a Leptospirillum ferriphilum isolate was found to be comparable with other strains of this important mineral-oxidizing acidophile, and far greater than in strains of other Leptospirillum spp. (L. ferrooxidans and UL. ferrodiazotrophum'), which helps to explain why only L. ferriphilum rather than other species is found in this (and many other) copper bioleach operation, even though the relatively low temperatures might favour other species. Studies carried out on osmotic stress response by several mineral-degrading acidophiles identified the disaccharide trehalose as a major organic osmo-protectant synthesized by several unrelated species of bacteria, including L. ferriphilum, At. ferrooxidans, Sulfobacillus thermosulfidooxidans and Acidiphilium sp. SJH. In contrast, the major organic solute that accumulated within the sulfur-oxidizer At. thiooxidans in response to salt stress was the monosaccharide glucose, together with smaller amounts of proline. Micro-representational difference analysis (MRDA) was used to elucidate the genetic response of a L. ferriphilum isolate to copper-induced and osmotic stress. Data obtained suggested that certain metabolisms related to lipopolysacharide synthesis, transport systems and nucleic acid processing were activated in response to these stress factors.

Les bactéries rhizosphériques qualifiées de PGPR (Plant Growth-Promoting Rhizobacteria) forment des symbioses associatives avec les plantes, stimulant la croissance de ces dernières. Les PGPR présentent différents mécanismes phytobénéfiques (production de phytohormones, fixation non symbiotique de l’azote, etc.). Plusieurs PGPR sont susceptibles d’interagir avec la même plante hôte, et il est possible que leurs effets phytobénéfiques soient influencés par les interactions qu’elles auront les unes avec les autres. L’objectif de cette thèse était de caractériser la compatibilité des PGPR dans la rhizosphère d’une même plante hôte, dans le cas de modèles bactériens appartenant aux genres Azospirillum et Pseudomonas. Certains Pseudomonas phytobénéfiques produisant des métabolites antimicrobiens, comme le 2,4-diacétylphloroglucinol (DAPG), nous avons tout d’abord examiné si la capacité à produire du DAPG pouvait inhiber Azospirillum. Les expériences de confrontation réalisées in vivo avec P. fluorescens F113 et un mutant DAPG-négatif, en système gnotobiotique, ont montré que la colonisation racinaire et l’activité phytostimulatrice de certaines PGPR Azospirillum pouvaient effectivement être diminuées en présence de Pseudomonas producteurs de DAPG. Pour évaluer la colonisation racinaire par Azospirillum en sol non stérile, des outils de PCR quantitative en temps réel ont été développés et validés pour trois souches de premier plan (A. lipoferum CRT1, A. brasilense UAP-154 et CFN-535). L’utilisation de ces outils a permis la comparaison de ces trois souches d’Azospirillum, chacune co-inoculée avec la souche P. fluorescens F113 productrice de DAPG, sur du maïs cultivé en sol non stérile. Les niveaux de colonisation racinaire différaient selon la souche d’Azospirillum, et la combinaison de microorganismes phytobénéfiques conduisait à une meilleure croissance du maïs par comparaison avec des plantes non inoculées. Les résultats suggèrent que des PGPR des genres Pseudomonas et Azospirillum peuvent être compatibles dans la rhizosphère d’une même plante, même si les premiers ont le potentiel d’inhiber certains des seconds par la production de métabolites secondaires antimicrobiens / Plant Growth-Promoting Rhizobacteria (PGPR) can form an associative symbiosis with plants, which results in stimulation of plant growth. PGPR harbour different phytobeneficial mechanisms (non-symbiotic nitrogen fixation, phytohormone synthesis, etc.). Various PGPR can interact with the same host plant, and it is possible that their phytobeneficial effects will be influenced by the interactions between these PGPR. The objective of this doctoral work was to characterize PGPR compatibility in the rhizosphere of the same host plant, in the case of model bacteria belonging to the genera Azospirillum and Pseudomonas. Because certain phytobeneficial Pseudomonas produce antimicrobial metabolites, such as 2,4-diacetylphloroglucinol (DAPG), we have first examined if DAPG production capacity could be involved in Azospirillum inhibition. In vivo experiments, performed with P. fluorescens F113 and a DAPG-negative mutant in gnotobiotic systems, showed that root colonization and phytostimulation activity of certain Azospirillum PGPR was indeed affected in the presence of DAPG-producing Pseudomonas. In order to evaluate Azospirillum root colonization in non-sterile soil, real-time quantitative PCR tools were developed and validated for three prominent Azospirillum strains (A. lipoferum CRT1, A. brasilense UAP-154 and CFN-535). The use of these real-time PCR tools enabled the comparison of the three Azospirillum strains, each co-inoculated with the DAPG-producing strain P. fluorescens F113, in the rhizosphere of maize grown in non-sterile soil. Root colonization levels differed according to the Azospirillum strain, and the combination of phytobeneficial microorganisms led to enhanced maize growth in comparison with non-inoculated plants. These results suggest that PGPR belonging to the genera Pseudomonas and Azospirillum may be compatible in the rhizosphere of a same plant, even if the former have the potential to inhibit some of the latter by producing antimicrobial secondary metabolites