Binding of the Major Phasin, PhaP1, fromRalstonia eutrophaH16 to Poly(3-Hydroxybutyrate) Granules

Abstract

The surface of polyhydroxybutyrate (PHB) storage granules in bacteria is covered mainly by proteins referred to as phasins. The layer of phasins stabilizes the granules and prevents coalescence of separated granules in the cytoplasm and nonspecific binding of other proteins to the hydrophobic surfaces of the granules. Phasin PhaP1 Reu is the major surface protein of PHB granules in Ralstonia eutropha H16 and occurs along with three homologues (PhaP2, PhaP3, and PhaP4) that have the capacity to bind to PHB granules but are present at minor levels. All four phasins lack a highly conserved domain but share homologous hydrophobic regions. To identify the region of PhaP1 Reu which is responsible for the binding of the protein to the granules, N-terminal and C-terminal fusions of enhanced green fluorescent protein with PhaP1 Reu or various regions of PhaP1 Reu were generated by recombinant techniques. The fusions were localized in the cells of various recombinant strains by fluorescence microscopy, and their presence in different subcellular protein fractions was determined by immunodetection of blotted proteins. The fusions were also analyzed to determine their capacities to bind to isolated PHB granules in vitro. The results of these studies indicated that unlike the phasin of Rhodococcus ruber, there is no discrete binding motif; instead, several regions of PhaP1 Reu contribute to the binding of this protein to the surface of the granules. The conclusions are supported by the results of a small-angle X-ray scattering analysis of purified PhaP1 Reu , which revealed that PhaP1 Reu is a planar, triangular protein that occurs as trimer. This study provides new insights into the structure of the PHB granule surface, and the results should also have an impact on potential biotechnological applications of phasin fusion proteins and PHB granules in nanobiotechnology.

Phasin Proteins Activate Aeromonas caviae Polyhydroxyalkanoate (PHA) Synthase but Not Ralstonia eutropha PHA Synthase

“…On the other hand, PhaCs were observed in oligomeric form at the initial stage of PHA polymerization by atomic force microscope imaging (19,30), but the PhaC dimer is thought to be a minimal functional unit (1,15). Based on the observations in this study and previous works (19,(27)(28)(29)(30), we propose a role for PhaPs in the initial stage of PHA polymerization by PhaC Ac , as shown in Fig. 8.…”

“…PhaPs have been reported to form trimers or tetramers as shown by X-ray analysis (28,29). On the other hand, PhaCs were observed in oligomeric form at the initial stage of PHA polymerization by atomic force microscope imaging (19,30), but the PhaC dimer is thought to be a minimal functional unit (1,15).…”

Localization of Poly(3-Hydroxybutyrate) (PHB) Granule-Associated Proteins during PHB Granule Formation and Identification of Two New Phasins, PhaP6 and PhaP7, in Ralstonia eutropha H16

“…In R. eutropha, PhaP1 is the most abundant phasin and can contribute to approximately 5% of the protein fraction during PHB accumulation (51). Colocalization of PhaP1 with PHB has been previously shown (7,26,51). Three other phasins (PhaP2, PhaP3, and PhaP4) have been postulated from the sequenced R. eutropha genome (32) on the basis of sequence similarity to PhaP1 (22,35), and recently, we identified a fifth phasin (PhaP5) via its interaction with PhaP2 in a two-hybrid approach (30).…”

Physiological and Proteomic Adaptation of “Aromatoleum aromaticum” EbN1 to Low Growth Rates in Benzoate-Limited, Anoxic Chemostats

“…5). Among the various phasins of strain EbN1, EbA1323 displays the highest sequence identity (51%) to PhaP1 of Ralstonia eutropha H16, synthesizing only such amounts of this major phasin that can be bound to the PHB granules (43,78). Opposite abundance profiles were observed for two putative acetoacetyl-CoA reductases (PhbB and Adh) of strain EbN1 at low .…”