Structural and Functional Diversity of alpha-Proteobacterial Acidocalcisomes/polyphosphate

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In the proposed project we will determine the structural and functional differences between bacterial acidocalcisomes (polyphosphate inclusions in a vacuolar proton pyro-phosphatase [V-H+-PPase, HppA]-driven acidic membrane-enclosed compartment) and non-membrane-enclosed polyphosphate granules of non-acidocalcisome-forming bacteria. We postulate that the presence of an hppA gene is indicative for the ability to form acidocalcisomes (all genome-sequenced alpha-proteobacteria have a hppA gene and the formation of acidocalcisomes in Agrobacterium tumefaciens and Rhodospirillum rubrum has been shown). We will isolate acidocalcisomes from A. tumefaciens (with membranes) and from Caulobacter crescentus (postulated to have membrane-enclosed acidocalcisomes). We will determine the elemental composition of acidocalcisomes, identify the acidocalcisome-associated proteins and determine the lipid-composition of the acidocalcisome membrane by in vitro and in vivo methods. This knowledge will enable us to compare bacterial acidocalcisomes with acidocalcisomes from eukaryotic unicellular counterparts (e. g. Trypanosomas cruzi) and with polyP granules from bacteria lacking a lipid membrane around polyphosphate granules. We will investigate the function of bacterial acidocalcisomes by a combination of growth and life imaging experiments and phenotype analysis of the wild type and generated mutant strains in which the key gene necessary for the formation of the acidic acidocalcisome compartment (V-H+-PPase, hppA) and/or the key gene of polyP synthesis (polyP kinase gene, ppk) are deleted in A. tumefaciens or C. crescentus. This project will considerably increase our knowledge on bacterial organelles.