The complex structure of polyhydroxybutyrate (PHB) granules: four orthologous and paralogous phasins occur in Ralstonia eutropha

Abstract

Analysis of the genome sequence of the polyhydroxyalkanoate-(PHA) accumulating bacterium Ralstonia eutropha strain H16 revealed three homologues (PhaP2, PhaP3 and PhaP4) of the phasin protein PhaP1. PhaP1 is known to constitute the major component of the layer at the surface of poly(3-hydroxybutyrate), poly(3HB), granules. PhaP2, PhaP3 and PhaP4 exhibited 42, 49 and 45 % identity or 61, 62 and 63 % similarity to PhaP1, respectively. The calculated molecular masses of PhaP1, PhaP2, PhaP3 and PhaP4 were 20?0, 20?2, 19?6 and 20?2 kDa, respectively. RT-PCR analysis showed that phaP2, phaP3 and phaP4 were transcribed under conditions permissive for accumulation of poly(3HB). 2D PAGE of the poly(3HB) granule proteome and analysis of the detected proteins by MALDI-TOF clearly demonstrated that PhaP1, PhaP3 and PhaP4 are bound to the poly(3HB) granules in the cells. PhaP3 was expressed at a significantly higher level in PhaP1-negative mutants. Occurrence of an unknown protein with an N-terminal amino-acid sequence identical to that of PhaP2 in crude cellular extracts of R. eutropha had previously been shown by others. Although PhaP2 could not be localized in vivo on poly(3HB) granules, in vitro experiments clearly demonstrated binding of PhaP2 to these granules. Further analysis of complete or partial genomes of other poly(3HB)-accumulating bacteria revealed the existence of multiple phasin homologues in Ralstonia solanacearum, Burkholderia fungorum and Azotobacter vinelandii. These new and unexpected findings should affect our current models of PHA-granule structure and may also have a considerable impact on the establishment of heterologous production systems for PHAs.

Genome sequence of the bioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16

“…In addition, two PHB-oligomer hydrolases, which degrade the trimeric products of the PHB depolymerases, are encoded on chromosome 2 (phaY1 and phaY2) 36 . The foregoing data and the results of systematic protein studies sketch a fascinating picture of the PHB granule as a complex and dynamic organelle 34 .…”

“…The contribution of the respective gene products to PHB synthesis remains to be investigated. Four phasin genes have been identified: phaP1, phaP2, phaP3 and phaP4, all of which are expressed 34 . The phasin proteins build up a layer at the surface of the PHB granules, inhibiting their coalescence and thus regulating their number and size.…”

Solvent Stress Response of the Denitrifying Bacterium “ Aromatoleum aromaticum ” Strain EbN1

“…In addition, three phasin-like proteins (EbA1323, EbA6852, and EbA5033) were up-regulated in the same cultures (up to 27.4-fold) and also during growth with 0.32 mM ethylbenzene (up to 5.7-fold). The sequence similarities of these predicted phasins to their orthologs PhaP1 to PhaP4 in R. eutropha (33) are 42 to 66%. In succinate-utilizing, solvent-shocked cultures of strain EbN1, where the aromatic compounds were not catabolized, either PHB-related proteins were not detected or their abundances were not increased.…”

Properties of a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase with High Specific Activity (PhaZd) in Wautersia eutropha H16

“…PhaZa1 Weu (formerly PhaZ1 Reu ) exists in PHB inclusion bodies and degrades artificial amorphous PHB (20,30). The existence of PhaZa1 Weu homologs has also been reported in W. eutropha H16 (29,44). Additionally, enzymes with properties similar to PhaZa1 Weu have been studied in other bacteria (6,18).…”