Stringent response and phosphorylations of PHB synthase and PHB depolymerase influence the PHB content of Ralstonia eutropha H16

A physiological connection between polyhydroxybutyrate (PHB) metabolism and the formation of polyphosphate (polyP) granules is known in form of the "enhanced biological phosphate removal process" (EBPR) and is used to expurgate phosphate from waste-water. Unfortunately, EBPR bacteria such as candidatus Accumulibacter phosphatis cannot be cultivated in pure cultures. In this work the question of a potential link between PHB and polyP metabolism in Ralstonia eutropha H16, an easy to cultivate β-proteobacterium that is well-known for its ability to synthesise PHB and polyP and that is taxonomically related to candidatus Accumulibacter phosphatis, was addressed. R. eutropha was used to simulate the EBPR process as a model organism and the adaptation to EBPR conditions was monitored. It could be shown that despite its similarities to c. A. phosphatis, R. eutropha is not an appropriate model organism for the EBPR process due to its inability to adapt to anaerobic conditions and to metabolize carbon sources during oxygen depletion. Nevertheless R. eutropha remains a model organism for the accumulation of the intracellular carbon and energy storage compound PHB. PHB helps to survive under starvation and stress conditions. Over the past three decades different laboratories demonstrated the PHB synthesising enzyme PhaC1 and the PHB degrading enzyme PhaZa1 are constitutively expressed. Consequently a different regulation for the activity of PhaC1 and PhaZa1 is necessary to avoid a futile cycle of simultaneous PHB synthesis and PHB degradation. Previous reports suggested a connection between players of stringent response and the regulation of the PHB metabolism but alarmones were never quantified. In this work a method to extract and quantify (p)ppGpp from R. eutropha was established. This enabled to study the relationship between (p)ppGpp concentrations and PHB accumulation in the wild type and in different mutants of genes involved in stringent response. Single-, double-, and triple-gene deletion strains of R. eutropha in (p)ppGpp synthase/hydrolase (spoT1), (p)ppGpp synthase (spoT2) and polyhydroxybutyrate (PHB) depolymerase (phaZa1 or phaZa3) genes were constructed. Absence of ppGpp in a ΔspoT1 ΔspoT2 double deletion strain correlated with elongation of the cells and enhanced degradation of PHB due to PhaZa1-mediated depolymerisation of PHB. In line, the overproduction of ppGpp by treatment with amino acid analogues or over-expression of SpoT2 in absence of SpoT1 correlated with reduced growth and significant overproduction of PHB. Data on a previously constructed ΔspoT2 strain (Brigham et al., 2012) was identified as an experimental error. Post-translational modifications were identified as a further adjusting screw of the PHB metabolism. PhaC1 and PhaZa1 were screened for the presence of phosphorylated residues. Ten different phosphorylations of PhaC1 were identified under PHB degrading conditions (at residues S10, T11, S16, T30, T94, T109, S149, S178, T191, S196, T198 and T373). T30, T94 and T109 were identified reproducibly in two different experiments while T373 was identified in all experiments. Site directed mutagenesis revealed that T373 influences the activity of PhaC1 in vitro, but that a combined mutagenesis of different residues is necessary to have a detectable impact in vivo. The residue S35 of PhaZa1 was phosphorylated at all stages of cell growth and detected in three independent experiments. Phosphorylations at the residues T26 and T28 were identified in one experiment. Modification of the residues T26 and S35 of PhaZa1 strongly reduced the PHB degrading activity