Accumulation of the PhaP phasin of Ralstonia eutropha is dependent on production of polyhydroxybutyrate in cells

Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by diverse bacteria and that accumulate as intracellular granules. Phasins are granul e-associated proteins that accumulate to high levels in strains that are pr oducing PHAs. The accumulation of phasins has been proposed to be dependent on PHA production, a model which is now rigorously tested for the phasin P haP of Ralstonia eutropha. R. eutropha phaC PHA synthase and phaP phasin ge ne replacement strains were constructed. The strains were engineered to exp ress heterologous and/or mutant PHA synthase alleles and a phaP-gfp transla tional fusion in place of the wild-type alleles of phaC and phaP. The strai ns were analyzed with respect to production of polyhydroxybutyrate (PHB), a ccumulation of PhaP, and expression of the phaP-gfp fusion. The results sug gest that accumulation of PhaP is strictly dependent on the genetic capacit y of strains to produce PHB, that PhaP accumulation is regulated at the lev el of both PhaP synthesis and PhaP degradation, and that, within mixed popu lations of cells, PhaP accumulation within cells of a given strain is not i nfluenced by PHB production in cells of other strains. Interestingly, eithe r the synthesis of PHB or the presence of relatively large amounts of PHB i n cells (> 50% of cell dry weight) is sufficient to enable PhaP synthesis. The results suggest that R. eutropha has evolved a regulatory mechanism tha t can detect the synthesis and presence of PHB in cells and that PhaP expre ssion can be used as a marker for the production of PHB in individual cells .