A systems-level insight into PHB-driven metabolic adaptation orchestrated by the PHB-binding transcriptional regulator AniA (PhaR)

Polyhydroxybutyrate (PHB) is a carbon and energy storage polymer, whose accumulation under nutrient imbalances with excess carbon is a widespread phenomenon in bacteria. PhaR is a conserved transcriptional regulator found to be associated to PHB granules in several species. Although its role in modulating PHB storage and metabolism has been extensively studied across the bacterial phylogeny, a comprehensive analysis of the PhaR regulon within the context of its dual role as a metabolic sensor and regulator remains missing. To bridge this gap, 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 alphaproteobacterial root nodule symbiont Sinorhizobium meliloti . This analysis was enriched by identifying direct regulatory targets of PhaR through a regulon-centric computational multi-step 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 the symbiotic state. A comprehensive picture 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 pathway ( zwf, pgl and edd ) as major regulatory targets, and beyond carbon metabolism several target genes of yet unknown function. Our findings highlight a pivotal role for PhaR in orchestrating carbon metabolism.