Abstract

The putative physiological functions of two related intracellular PHB depolymerases, PhaZd1 and PhaZd2, of R. eutropha H16 were investigated. Purified PhaZd1 and PhaZd2 were active with native PHB granules in vitro. Partial removal of the proteinaceous surface layer of native PHB granules by trypsin treatment or the use of PHB granules isolated from ΔphaP1 or ΔphaP1-phaP5 strains resulted in increased specific PHB depolymerase activity especially for PhaZd2. Constitutive expression of PhaZd1 or PhaZd2 reduced or even prevented the accumulation of PHB under PHB permissive conditions in vivo. Expression of translational fusions of eYfp with PhaZd1/PhaZd2, in which the active site serines (S190/Ser193) were replaced by alanine, resulted only for PhaZd1 fusions in co-localization with PHB granules. C-terminal fusions of inactive PhaZd2 (S193A) with eYfp revealed the presence of spindle-like structures and no co-localization with PHB granules was observed. Chromosomal deletion of phaZd1, phaZd2 or deletion of both depolymerase genes had no significant effect on PHB accumulation and mobilization during growth in NB or NB-gluconate medium. Moreover, neither proteome analysis of purified nPHB granules nor lacZ-fusion studies gave any indication that PhaZd1 or PhaZd2 were detectably present in the PHB granule fraction or were expressed at all during growth on NB-gluconate medium. In conclusion, PhaZd1 and PhaZd2 represent two PHB depolymerases with principally high capacity to degrade PHB when artificially expressed but they are apparently not involved in PHB mobilization in the wild type. The true in vivo functions of PhaZd1 and PhaZd2 remain obscure.