Essential Role of the <i>hprK</i> Gene in <i>Ralstonia eutropha</i> H16

Abstract

<i>Ralstonia eutropha</i> H16 possesses an incomplete phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) composed of EI, HPr, EIIA<sup>Ntr</sup> (PtsN) and EIIA<sup>Man</sup> (PtsM). We could show that in vitro the incomplete PTS phosphorylation cascade is partially functional. HPr becomes phosphorylated by PEP and EI, and transfers the phosphoryl group to EIIA<sup>Ntr</sup>, but only extremely slowly to EIIA<sup>Man</sup>. Components of this system have previously been shown to regulate the metabolism of polyhydroxybutyrate. Downstream from <i>ptsN</i> this organism contains an <i>hprK</i> gene, which codes for a homologue of HPr kinase/phosphorylase. We show that this enzyme phosphorylates HPr using ATP as phosphoryl donor. Interestingly, <i>hprK</i> appeared to be essential in <i>R. eutropha</i> because this gene could not be deleted in the wild-type strain, but could be deleted in mutants lacking <i>ptsH</i> or <i>ptsI</i>. This suggests that an increase in the HPr and/or P∼His-HPr concentrations might be responsible for the growth defect. To test this hypothesis, various <i>ptsH</i> alleles were introduced into the <i>ptsH hprK</i> double mutant. Complementation of this mutant was possible only with the <i>ptsH</i>(His15Ala) allele, but not with the wild-type or <i>ptsH</i>(Ser46Ala) alleles. We conclude that elevated amounts of His-15-phosphorylated HPr, formed in the <i>hprK</i> mutant, are responsible for its growth defect.