Biogenesis of Polyhydroxyalkanoate Inclusion Bodies in Bacillus Megaterium

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The storage polymer polyhydroxyalkanoic acid (PHA) accumulates in inclusion bodies in most bacteria and archaea. Given the widespread occurrence and influence on the metabolism and physiology of the cell of these inclusion bodies, it is surprising that so little is known about their structure and biogenesis. This project will identify the functions of small proteins (pha proteins) that co-purify with PHA inclusion bodies. The focus is on localizing these proteins throughout growth, and on determining the precise amino acid sequences required for accurate localization to take place, using Bacillus megaterium. Six pha genes at two loci have been identified in B. megaterium. The functions of three of these genes (phaA, B and C) can be predicted based on homology to known genes, while those of the remaining three genes (phaP, Q and R) are unknown. Intracellular localization of phaQ and phaR will be examined using translational fusions to the green fluorescent protein (GFP). The identity of the amino acid sequences required for localization of phaP and phaC (and phaQ and phaR, if appropriate) to the inclusion bodies will be determined. The phenotypes of phaP, Q and R deletion mutants will be characterized, and the interdependence of these proteins on each other for accurate localization will be tested by analyzing subcellular localization of each of the pha proteins in various deletion mutants. The data from these experiments should indicate the extent to which phaP, Q and R are involved in the assembly of PHA inclusion bodies, and how they interact with each other in that process.