Transcription factor with ambivalent role – Ralstonia eutropha’s PhaR is a repressor of the phasin-gene phaP1 and an activator of phaP3

Polyhydroxyalkanoates (PHAs) serve as intracellular carbon and energy storage compounds in many prokaryotes. They accumulate in granules composed of a hydrophobic polymer core surrounded by a surface layer of proteins that regulate granule formation, stability, and degradation. Among these, the most abundant are low-molecular-weight, amphiphilic proteins known as phasins. In Ralstonia eutropha (Cupriavidus necator), one of the best-studied model organisms for PHA biosynthesis, the phasin PhaP1 controls the surface-to-volume ratio of the granule. Under non-accumulating conditions, the transcriptional repressor PhaR negatively regulates phaP1 expression. A similar PhaR-mediated repression has also been proposed for another phasin gene, phaP3, though direct evidence has so far been lacking. In this study, the phaP3 transcription start site was determined and three putative PhaR binding sites upstream and within the phaP3 gene identified. Electrophoretic mobility shift assays confirmed PhaR binding to all three sites. Strikingly, β-galactosidase reporter-gene assays revealed that, unlike its repressive effect on phaP1, PhaR activates phaP3 expression. Our findings provide the first direct evidence of the dual regulatory role of PhaR in R. eutropha, functioning as a repressor and activator of different phasin encoding genes. This suggests distinct physiological roles for PhaP1 and PhaP3 in PHA granule formation and turnover, further underscoring the complexity of the regulation of PHA metabolism.