This thesis concentrates on the Bacillus cereus group of organisms and interactions that they may encounter in their natural environment. Inorganic polyphosphate has been identified as an important factor of stress and survival in B. cereus. One of the aims of this project was to create knock out mutants of certain enzymes involved in polyphosphate metabolism in B. anthracis, the etiological agent of anthrax. Unfortunately, even though B. anthracis is veryclosely related to B. cereus and despite the application of published methods it was not possible to create these B. anthracis knockout mutants. In order to address the importance of inorganic polyphosphate in B. anthracis, a real time RT‐PCR assay was developed to monitor the mRNA levels of these enzymes when the bacterium is faced with harsh nutrient environments Real time RT‐PCR analysis showed that mRNA levels of the metabolizing enzymes were upregulated in low nutrient conditions but that the profiles of gene expressionwere varied when grown in a chemically defined media.In addition to abiotic stresses such as low nutrients, B. anthracis is also likely to face biotic stress such as predation by amoeba in the soil. Investigations were performed into the outcome of the interaction of B. cereus group bacteria with a model amoeba, Acanthamoeba polyphaga. Amoebae are bacterial predators but can also be utilised as hosts by bacterial symbionts and pathogens, such as Legionella pneumophila. It was theorised that amoebae may provide a host environment similar to that of the professional macrophages, which B. anthracis encounters in mammalian infection. These investigations confirmed that the B. cereus group bacteria demonstrate a range of interactions with amoeba cells, from surface attachment through to intracellular persistence. These studies went on to show that B. cereus, B. thuringiensis and B. anthracis can all be engulfed by amoebae when challenged in their vegetative form and that spores were able to survive, and apparently germinate. Finally these studies have identified a new developmental stage of the B. cereus group bacteria. When grown in static conditions, especially in the presence of amoeba, the bacterial cells cease to septate and large (often motile) continuous hyphae like filaments form. These filaments can be seen to “weave” together to form large “rope” like macrofibre structures which can even become visible by eye. Previously this macrofibre growth has also been seen in B. subtilis, suggesting it may be common to the whole genus. In the light of these findings we speculate that this group of pathogens have evolved complex behaviours to interact with soil amoeba in order to facilitate survival in harsh environmental conditions.
|Date of Award||29 Oct 2010|
|Supervisor||M Browne (Supervisor), David Clarke (Supervisor) & Nicholas Waterfield (Supervisor)|