A systems-level insight into PHB-driven metabolic adaptation orchestrated by the PHB-binding transcriptional...

Poly(3-hydroxybutyrate) (PHB) is a carbon and energy storage polymer, whose accumulation under nutrient imbalances with excess carbon is common in bacteria. PhaR is a conserved transcriptional regulator that associates with PHB granules in several species. Although its role in modulating PHB storage and metabolism has been extensively studied across the bacterial phylogeny, a systems-level view of PhaR's dual function as a metabolic sensor and regulator is lacking. Here, we integrated co-expression network analysis with proteome profiling across multiple mutant backgrounds (lack of PhaR [AniA] and/or PHB synthesis) in the free-living state of the PHB-accumulating α-proteobacterial root nodule symbiont Sinorhizobium meliloti. This analysis was enriched by identifying direct regulatory targets of PhaR through a regulon-centric computational multistep search for DNA-binding site motifs combined with PhaR-DNA-binding and promoter-reporter assays. We confirmed that the model of accumulated PHB sequestering PhaR, and thereby relieving phasin and PHB depolymerase gene repression to control cellular PHB levels, also applies to S. meliloti and showed that PhaR-mediated regulation also occurs in the symbiotic state. Our integrated analyses of the impact of PHB-mediated PhaR titration on cellular functions revealed exopolysaccharide production as well as central carbon metabolism (pdh and bkd), gluconeogenesis (ppdK and pyc), entry into the TCA cycle (gltA), and the initial steps of the Entner-Doudoroff (ED) pathway (zwf, pgl, and edd) as major regulatory targets, along with target genes of yet unknown function. Our findings highlight a pivotal role for PhaR in orchestrating carbon metabolism.IMPORTANCEPoly(3-hydroxybutyrate) (PHB) is a carbon and energy storage polymer typically associated with bacterial survival under nutrient-limited conditions. Its accumulation reflects the cellular metabolic balance, and the transcriptional regulator PhaR has been shown to bind PHB and control the expression of genes involved in its metabolism. At the same time, PhaR has been implicated in broader regulatory roles affecting global gene expression, although the connection between this function and its ability to sense PHB has remained unresolved. In this study, we used the model legume symbiont Sinorhizobium meliloti to bridge this gap. We demonstrated that PhaR modulates global gene expression in response to the metabolic state signaled by PHB accumulation. Our findings highlight PHB not only as a storage compound, but also as a key integrator of metabolic status that links nutrient availability to coordinated transcriptional responses.