title: The Ralstonia eutropha PhaR protein couples synthesis of the PhaP phasin to the presence of polyhydroxybutyrate in cells and promotes polyhydroxybutyrate production.
abstract: Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by many bacteria and that accumulate as intracellular granules. Phasins (PhaP) are proteins that accumulate during PHA synthesis, bind PHA granules, and promote further PHA synthesis. Interestingly, PhaP accumulation seems to be strictly dependent on PHA synthesis, which is catalyzed by the PhaC PHA synthase. Here we have tested the effect of the Ralstonia eutropha PhaR protein on the regulation of PhaP accumulation. R. eutropha strains with phaR, phaC, and/or phaP deletions were constructed, and PhaP accumulation was measured by immunoblotting. The wild-type strain accumulated PhaP in a manner dependent on PHA production, and the phaC deletion strain accumulated no PhaP, as expected. In contrast, both the phaR and the phaR phaC deletion strains accumulated PhaP to higher levels than did the wild type. This result implies that PhaR is a negative regulator of PhaP accumulation and that PhaR specifically prevents PhaP from accumulating in cells that are not producing PHA. Transfer of the R. eutropha phaR, phaP, and PHA biosynthesis (phaCAB) genes into a heterologous system, Escherichia coli, was sufficient to reconstitute the PhaR/PhaP regulatory system, implying that PhaR both regulates PhaP accumulation and responds to PHA directly. Deletion of phaR caused a decrease in PHA yields, and a phaR phaP deletion strain exhibited a more severe PHA defect than a phaP deletion strain, implying that PhaR promotes PHA production and does this at least partially through a PhaP-independent pathway. Models for regulatory roles of PhaR in regulating PhaP and promoting PHA production are presented.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Gregory M
authors.initials: GM
authors.lastname: York
authors.forename: JoAnne
authors.initials: J
authors.lastname: Stubbe
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Sinskey
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D020118:Cupriavidus necator / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D006885:Hydroxybutyrates / Q000378:metabolism*; D011091:Polyesters / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins; D000094122:Polyhydroxybutyrates
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D006885:Hydroxybutyrates; D011091:Polyesters; D011994:Recombinant Proteins; D012097:Repressor Proteins; C118828:PHAP protein, Bacteria; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates