Phosphotransferase protein EIIANtr interacts with SpoT, a key enzyme of the stringent response, in Ralstonia eutropha H16

Abstract

EIIANtr is a member of a truncated phosphotransferase (PTS) system that serves regulatory functions and exists in many Proteobacteria in addition to the sugar transport PTS. In Escherichia coli, EIIANtr regulates K+ homeostasis through interaction with the K+ transporter TrkA and sensor kinase KdpD. In the β-Proteobacterium Ralstonia eutropha H16, EIIANtr influences formation of the industrially important bioplastic poly(3-hydroxybutyrate) (PHB). PHB accumulation is controlled by the stringent response and induced under conditions of nitrogen deprivation. Knockout of EIIANtr increases the PHB content. In contrast, absence of enzyme I or HPr, which deliver phosphoryl groups to EIIANtr, has the opposite effect. To clarify the role of EIIANtr in PHB formation, we screened for interacting proteins that co-purify with Strep-tagged EIIANtr from R. eutropha cells. This approach identified the bifunctional ppGpp synthase/hydrolase SpoT1, a key enzyme of the stringent response. Two-hybrid and far-Western analyses confirmed the interaction and indicated that only non-phosphorylated EIIANtr interacts with SpoT1. Interestingly, this interaction does not occur between the corresponding proteins of E. coli. Vice versa, interaction of EIIANtr with KdpD appears to be absent in R. eutropha, although R. eutropha EIIANtr can perfectly substitute its homologue in E. coli in regulation of KdpD activity. Thus, interaction with KdpD might be an evolutionary ‘ancient’ task of EIIANtr that was subsequently replaced by interaction with SpoT1 in R. eutropha. In conclusion, EIIANtr might integrate information about nutritional status, as reflected by its phosphorylation state, into the stringent response, thereby controlling cellular PHB content in R. eutropha.