New Insight into the Role of the PhaP Phasin of
            <i>Ralstonia eutropha</i>
            in Promoting Synthesis of Polyhydroxybutyrate

York, Gregory M.; Stubbe, JoAnne; Sinskey, Anthony J.

doi:10.1128/jb.183.7.2394-2397.2001

Cited by 121 publications

(150 citation statements)

References 18 publications

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“…The deletion strains in R. eutropha H16 were created by homologous recombination by a standard procedure (13,21). The phaZ replacement plasmids pGY96, pJOE39, and pPK51 were introduced into the appropriate R. eutropha strains to generate ⌬phaZ1, ⌬phaZ2, ⌬phaZ3, ⌬phaZ1⌬phaZ2, ⌬phaZ1⌬phaZ3, ⌬phaZ2⌬phaZ3, and ⌬phaZ1⌬phaZ2⌬phaZ3 by conjugation from donor strain E. coli S17-1.…”

Section: Methodsmentioning

confidence: 99%

Ralstonia eutrophaH16 Encodes Two and Possibly Three Intracellular Poly[d-(−)-3-Hydroxybutyrate] Depolymerase Genes

York¹,

Lupberger²,

et al. 2003

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Intracellular poly[D-(؊)-3-hydroxybutyrate] (PHB) depolymerases degrade PHB granules to oligomers and monomers of 3-hydroxybutyric acid.Recently an intracellular PHB depolymerase gene (phaZ1) from Ralstonia eutropha was identified. We now report identification of candidate PHB depolymerase genes from R. eutropha, namely, phaZ2 and phaZ3, and their characterization in vivo. phaZ1 was used to identify two candidate depolymerase genes in the genome of Ralstonia metallidurans. phaZ1 and these genes were then used to design degenerate primers. These primers and PCR methods on the R. eutropha genome were used to identify two new candidate depolymerase genes in R. eutropha: phaZ2 and phaZ3. Inverse PCR methods were used to obtain the complete sequence of phaZ3, and library screening was used to obtain the complete sequence of phaZ2. PhaZ1, PhaZ2, and PhaZ3 share ϳ30% sequence identity. The function of PhaZ2 and PhaZ3 was examined by generating R. eutropha H16 deletion strains (⌬phaZ1, ⌬phaZ2, ⌬phaZ3, ⌬phaZ1⌬phaZ2, ⌬phaZ1⌬phaZ3, ⌬phaZ2⌬phaZ3, and ⌬phaZ1⌬phaZ2⌬phaZ3). These strains were analyzed for PHB production and utilization under two sets of conditions. When cells were grown in rich medium, PhaZ1 was sufficient to account for intracellular PHB degradation. When cells that had accumulated ϳ80% (cell dry weight) PHB were subjected to PHB utilization conditions, PhaZ1 and PhaZ2 were sufficient to account for PHB degradation. PhaZ2 is thus suggested to be an intracellular depolymerase. The role of PhaZ3 remains to be established.Polyhydroxyalkanoates (PHAs) are polyoxoesters produced by a wide range of bacteria when they find themselves in an environment with an available carbon source but limited in additional nutrient(s) required for growth (9). The shortchain-length PHAs, where R is a methyl or ethyl, have properties of thermoplastics and are biodegradable (Fig. 1). Much effort has focused on understanding the biology of PHA homeostasis for several reasons. First, this understanding could lead to expression of the appropriate gene set in heterologous systems to make PHA production economically competitive with oil-based polymers. Second, understanding PHA homeostasis serves as a paradigm for understanding the mechanism of homopolymerization reactions in which the product undergoes a phase transition during its formation, generating insoluble inclusions (granules). The intracellular PHAs can be degraded when the bacteria require carbon but are in otherwise nutrientreplete conditions, and the monomers and energy released can be reused to allow the bacteria to grow (17). The insoluble PHA granules must, therefore, be biosynthesized in a controlled fashion to facilitate enzymatic degradation. A variety of proteins associated with PHA homeostasis have been identified and are being characterized (3,8,10,12,20,22). As part of our research to understand the mechanisms that control polymer size and reuse, we have been interested in identifying the intracellular depolymerases that degrade poly[D-(Ϫ)-3-hydroxybutyrate] (PHB) w...

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Section: Methodsmentioning

confidence: 99%

Ralstonia eutrophaH16 Encodes Two and Possibly Three Intracellular Poly[d-(−)-3-Hydroxybutyrate] Depolymerase Genes

York¹,

Lupberger²,

et al. 2003

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“…PHA depolymerases hydrolyze PHAs to yield oligomers and/or monomers of hydroxyalkanoates (5,7,22). Phasins accumulate during PHA synthesis, bind PHA granules, and promote further PHA synthesis in a manner that is poorly understood (6,13,15,17,25,27). Despite similarities in regulation and function, phasins from different bacterial species are not homologs.…”

mentioning

confidence: 99%

“…Ralstonia eutropha is an excellent system to study phasin regulation because it is amenable to standard gene replacement techniques (20,24), it is the only strain for which phasin mutants have been generated (25,27), and it produces polyhydroxybutyrate (PHB) under a range of cultivation conditions. In R. eutropha the phaR gene (originally designated ORF1 [24]) is located downstream of the PHA biosynthetic operon phaCAB and is not linked to phaP.…”

mentioning

confidence: 99%

The Ralstonia eutropha PhaR Protein Couples Synthesis of the PhaP Phasin to the Presence of Polyhydroxybutyrate in Cells and Promotes Polyhydroxybutyrate Production

York¹,

Stubbe

Sinskey³

2002

J Bacteriol

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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.

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“…These lipid-bodyassociated proteins exist in several organisms (for a recent review, see reference 30). Phasins also affect the size and the number of PHA granules (15,16,49) and positively affect PHA synthesis (8,15,26,32,33,36,40,42,49,53). The production of phasin in Ralstonia eutropha (formerly designated Alcaligenes eutrophus) is suggested to be dependent on the presence of an intact PHA synthesis apparatus, although the mechanism of their regulation is unknown (49).…”

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confidence: 99%

A Repressor Protein, PhaR, Regulates Polyhydroxyalkanoate (PHA) Synthesis via Its Direct Interaction with PHA

et al. 2002

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Phasins (PhaP) are predominantly polyhydroxyalkanoate (PHA) granule-associated proteins that positively affect PHA synthesis. Recently, we reported that the phaR gene, which is located downstream of phaP in Paracoccus denitrificans, codes for a negative regulator involved in PhaP expression. In this study, DNase I footprinting revealed that PhaR specifically binds to two regions located upstream of phaP and phaR, suggesting that PhaR plays a role in the regulation of phaP expression as well as autoregulation. Many TGC-rich sequences were found in upstream elements recognized by PhaR. PhaR in the crude lysate of recombinant Escherichia coli was able to rebind specifically to poly[(R)-3-hydroxybutyrate] [P(3HB)] granules. Furthermore, artificial P(3HB) granules and 3HB oligomers caused the dissociation of PhaR from PhaR-DNA complexes, but native PHA granules, which were covered with PhaP or other nonspecific proteins, did not cause the dissociation. These results suggest that PhaR is able to sense both the onset of PHA synthesis and the enlargement of the granules through direct binding to PHA. However, free PhaR is probably unable to sense the mature PHA granules which are already covered sufficiently with PhaP and/or other proteins. An in vitro expression experiment revealed that phaP expression was repressed by the addition of PhaR and was derepressed by the addition of P(3HB). Based on these findings, we present here a possible model accounting for the PhaR-mediated mechanism of PHA synthesis. Widespread distribution of PhaR homologs in short-chainlength PHA-producing bacteria suggests a common and important role of PhaR-mediated regulation of PHA synthesis.Polyhydroxyalkanoates (PHAs) are intracellular carbon and energy storage materials produced in various microorganisms under nutrient-limited conditions (2,7,20,23). PHAs can be classified into two groups, short chain length (SCL) (3 to 5 carbons per monomer) and medium chain length (MCL) (6 to 14 carbons per monomer) (43). Paracoccus denitrificans, a facultative methylotrophic bacterium, accumulates SCL-PHAs from several alcohols (54). Two gene clusters involved in PHA synthesis and degradation in P. denitrificans have been cloned and characterized. The first is the phaA-phaB gene cluster, which encodes ␤-ketothiolase and acetoacetyl-coenzyme A (CoA) reductase (50), respectively, and the second is the gene cluster phaZ-phaC-phaPphaR, which encodes PHA depolymerase (9), PHA synthase (47), PHA granule-associated phasin (26), and a DNA-binding regulatory protein (24), respectively.Phasins are the most dominant proteins of relatively small molecular size that are associated with PHA granules. It has been proposed that phasins form an amphiphilic layer between the PHA granule and cytoplasm in a manner similar to that of oleosins, which form a layer at the surface of triacylglycerol inclusion in oilseed plants (32,33,42,49). These lipid-bodyassociated proteins exist in several organisms (for a recent review, see reference 30). Phasins also affect the size and the...

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New Insight into the Role of the PhaP Phasin of Ralstonia eutropha in Promoting Synthesis of Polyhydroxybutyrate

Cited by 121 publications

References 18 publications

Ralstonia eutrophaH16 Encodes Two and Possibly Three Intracellular Poly[d-(−)-3-Hydroxybutyrate] Depolymerase Genes

Ralstonia eutrophaH16 Encodes Two and Possibly Three Intracellular Poly[d-(−)-3-Hydroxybutyrate] Depolymerase Genes

The Ralstonia eutropha PhaR Protein Couples Synthesis of the PhaP Phasin to the Presence of Polyhydroxybutyrate in Cells and Promotes Polyhydroxybutyrate Production

A Repressor Protein, PhaR, Regulates Polyhydroxyalkanoate (PHA) Synthesis via Its Direct Interaction with PHA

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