Regulation and assembly of photosynthetic membranes in Rhodobacter sphaeroides

The purple phototrophic bacteria represent a cornerstone in the research of cyclic photosynthetic electron transport chains, being the subject of extensive modelling and structural biology work to determine the mechanisms by which light may be harvested and used to reduce the quinone pool. The work described in this thesis represents an integrated approach to study the regulation and assembly of photosynthetic membrane domains in this organism, taking advantage of the oxygen repression of the photosynthetic gene cluster to perform a time series following oxygen limitation. Using a fluorescent strain of Rhodobacter sphaeroides, samples were taken following the induction in order to track photosynthetic expression and assembly through the use of fluorescence decay measurements; cryo-electron tomography; UV-visible spectroscopy; and multi-omics approaches. Secondarily, a crucial branch point in tetrapyrrole biosynthesis suspected to be a key target for regulation under photosynthetic growth was investigated – the heme/magnesium protoporphyrin IX branch – through structural studies of the Rba. sphaeroides ferrochelatase by single particle cryo-electron microscopy.
The results of this thesis relate the onset of spectral complex assembly to ultrastructural changes in membrane development, through comparisons of the UV-Vis spectra with tomographic reconstructions of focused ion beam (FIB)-milled transitioning cells. An hour after the induction, large lipid rich vesicles can be seen which appear to constrict to a size characteristic of chromatophores after 3 hours – coincident with the assembly of the peripheral light harvesting 2 complex. RNA sequencing data of these adapting cultures reveal strong upregulation of the photosynthetic gene cluster, which succeeds a general anaerobic response mediated by the regulator FnrL. Expression data was validated by label-free quantification (LFQ) proteomics and clustered in order to identify important genes in the assembly process. Finally, chapter 5 describes the structural characterization of the Rba. sphaeroides ferrochelatase in a substrate free and product bound state. It is shown that the enzyme forms a deactive, tetrameric form in the absence of substrate, which increases its solubility and likely sequesters the enzyme away from the membrane to increase flux down the bacteriochlorophyll branch. The intra-dimer interface of the enzyme is shown to bind two allosteric hemes, which appear to have a stabilising role in the structure.