Identification of a multifunctional protein, PhaM, that determines number, surface to volume ratio, subcellular localization and distribution to daughter cells of poly(3-hydroxybutyrate), PHB, granules in Ralstonia eutropha H16.
A two-hybrid approach was applied to screen for proteins with the ability to interact with PHB synthase (PhaC1) of Ralstonia eutropha. The H16_A0141 gene (phaM) was identified in the majority of positive clones. PhaM (26.6 kDa) strongly interacted with PhaC1 and with phasin PhaP5 but not with PhaP1 or other PHB granule-associated proteins. A ΔphaM mutant accumulated only one or two large PHB granules instead of three to six medium-sized PHB granules of the wild type, and distribution of granules to daughter cells was disordered. All three phenotypes (number, size and distribution of PHB granules) were reversed by reintroduction of phaM. Purified PhaM revealed DNA-binding properties in gel mobility shift experiments. Expression of a fusion of the yellow fluorescent protein (eYfp) with PhaM resulted in formation of many small fluorescent granules that were bound to the nucleoid region. Remarkably, an eYfp-PhaP5 fusion localized at the cell poles in a PHB-negative background and overexpression of eYfp-PhaP5 in the wild type conferred binding of PHB granules to the cell poles. In conclusion, subcellular localization of PHB granules in R. eutropha depends on a concerted expression of at least three PHB granule-associated proteins, namely PhaM, PhaP5 and PHB synthase PhaC1.
Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, 70550 Stuttgart, Germany.
Daniel
D
Pfeiffer
Andreas
A
Wahl
Dieter
D
Jendrossek
D000217:Acyltransferases / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020118:Cupriavidus necator / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D017353:Gene Deletion; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D006885:Hydroxybutyrates / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D011091:Polyesters / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D000094122:Polyhydroxybutyrates
D000217:Acyltransferases; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D006885:Hydroxybutyrates; D008164:Luminescent Proteins; D011091:Polyesters; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C060970:poly-beta-hydroxybutyrate polymerase