Regulation of central carbon metabolism and storage polymers by PTS ^Ntr in Rhizobium leguminosarum

The coordination of intracellular carbon and nitrogen levels is essential for optimal bacterial growth and, in rhizobia, for survival in the soil, root colonization, and symbiotic interactions with their host plants. Here, we show that the phosphotransferase system PTS ^Ntr , a global regulator of carbon and nitrogen metabolism, modulates the tricarboxylic acid (TCA) cycle activity and the accumulation of three major carbon storage polymers: glycogen, polyhydroxybutyrate, and exopolysaccharide in Rhizobium leguminosarum . The unphosphorylated form of the effector protein ManX is sufficient for the full activation of the TCA cycle dehydrogenase enzymes. Accordingly, loss of manX reduced dehydrogenase activity and redirected overflow carbon into storage polymers, phenocopying wild-type cells grown under nitrogen starvation. We further demonstrate that carbon metabolism in R. leguminosarum is also tightly regulated by the HPr kinase (HprK) protein. In an hprK mutant, loss of NPr Ser48 phosphorylation favors phosphotransfer through the histidyl arm and is predicted to increase phosphorylation of downstream PTS ^Ntr components. When grown on glucose, the hprK mutant accumulates elevated intracellular pyrimidine levels and exhibits increased malate dehydrogenase and TCA cycle activity, consistent with a misregulated shift toward gluconeogenesis. This mutant also overproduces exopolysaccharide, an effect potentially mediated by crosstalk between phosphorylated PtsN and the ChvI/ChvG two-component regulatory system. Together, these data suggest that HprK exerts pleiotropic control over processes regulated by PTS ^Ntr , influencing central metabolism while repressing exopolysaccharide production, likely by promoting the unphosphorylated state of PtsN.