Iron is an essential element for virtually all forms of life. Complicating matters, it is present in the insoluble ferric form in aerobic environments, while the more soluble ferrous form is found in anaerobic or reducing environments. Vibrio cholerae, the causative agent of the disease cholera, requires iron to survive. In order to meet the need for iron, V. cholerae expresses a variety of iron acquisition systems. One of these systems, Feo, is highly conserved among bacterial species as well as archaea and transports ferrous iron. The Feo system consists of three proteins: FeoA, FeoB, and FeoC. Previous work using the bacterial adenylate cyclase two hybrid system has shown that FeoC interacts with the cytoplasmic N-terminal domain of FeoB. However, the significance of this interaction is not known. In this study, V. cholerae Feo system proteins were analyzed for residues important for the interaction between FeoB and FeoC. In addition, FeoA and FeoC were purified for antibody production. It was found that a residue in the G protein domain of FeoB was not necessary for interaction with FeoC. However, a conserved residue in FeoC did abolish the interaction with FeoB. These results indicate that there is at least one residue important in the interaction of FeoB and FeoC, although further characterization will most likely reveal more. Antibodies to FeoA and FeoC were generated to use them for further characterization of the Feo system. / text

Perceiving and responding to the environment is key to survival. Using the prokaryotic equivalent of a nervous system – the chemotaxis system – bacteria sense chemical stimuli and respond by adjusting their movement accordingly. In chemotactic bacteria, such as the well-studied E. coli, environmental nutrient sensing is achieved through a membrane embedded protein array that specifically clusters at the cell poles. Signalling to the motor is performed by activation of the CheA kinase, which phosphorylates CheY and CheB. CheY-P tunes the activity of the flagellar motor while CheB-P, together with CheR is involved in adaptation to the stimulus. In E. coli, a dedicated phosphatase terminates the signal. Most bacterial species however, have a much more complex chemotaxis network. Rhodobacter sphaeroides, a model organism for complex chemotaxis systems, has one membrane-embedded chemosensory array and one cytoplasmic chemosensory array, plus several homologs of the E. coli chemotaxis proteins. Signals from both arrays are integrated to control the rotation of a single start-stop flagellar motor. The phosphorelay network has been studied extensively through in vitro phosphotransfer while in vivo studies have established the components of each array and the requirements for formation. Mathematical modelling has also contributed towards inferring connectivities within the signalling network. Starting by constructing a two-hybrid-based interaction network focused on the components of the cytoplasmic chemosensory array, this thesis further addresses its associated adaptation network through a series of in vivo techniques. The swimming behaviour of series of deletion mutants involving the adaptation network of R. sphaeroides is characterised under steady state conditions as well as upon chemotactic stimulation. New connectivities within the R. sphaeroides chemotaxis network are inferred from analysing these data together with results from in vivo photoactivation localisation microscopy of CheB₂. The experimental results are used to propose a new model for chemotaxis in R. sphaeroides.