A Novel DNA-Binding Protein,PhaR, Plays a Central Role in the Regulation of Polyhydroxyalkanoate Accumulation and Granule Formation in the Haloarchaeon Haloferax mediterranei

Polyhydroxyalkanoates (PHAs) are synthesized and assembled as PHA granules that undergo well-regulated formation in many microorganisms. However, this regulation remains unclear in haloarchaea. In this study, we identified a PHA granule-associated regulator (PhaR) that negatively regulates the expression of both its own gene and the granule structural gene phaP in the same operon (phaRP) in Haloferax mediterranei. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assays demonstrated a significant interaction between PhaR and the phaRP promoter in vivo. Scanning mutagenesis of the phaRP promoter revealed a specific cis-element as the possible binding position of the PhaR. The haloarchaeal homologs of the PhaR contain a novel conserved domain that belongs to a swapped-hairpin barrel fold family found in AbrB-like proteins. Amino acid substitution indicated that this AbrB-like domain is critical for the repression activity of PhaR. In addition, the phaRP promoter had a weaker activity in the PHA-negative strains, implying a function of the PHA granules in titration of the PhaR. Moreover, the H. mediterranei strain lacking phaR was deficient in PHA accumulation and produced granules with irregular shapes. Interestingly, the PhaR itself can promote PHA synthesis and granule formation in a PhaP-independent manner. Collectively, our results demonstrated that the haloarchaeal PhaR is a novel bifunctional protein that plays the central role in the regulation of PHA accumulation and granule formation in H. mediterranei.     

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

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.     

Expression of four <Emphasis Type="Italic">pha</Emphasis> genes involved in poly-β-hydroxybutyrate production and accumulation in <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> FJ1

A cluster of genes encoding polyhydroxybutyrate (PHB) depolymerase (phaZ), PHB synthase (phaC), phasin (phaP), and the regulator protein (phaR) was previously identified in Rhodobacter sphaeroides FJ1 (R. sphaeroides FJ1). In this study, we investigated the role of the PhaR protein on the expression of the pha genes. Immunoblot analysis revealed that the expressions of phaP, phaZ and phaR genes in wild-type cells of R. sphaeroides FJ1 are repressed during the active growth phase, with the exception of phaC. A phaR deletion mutant of R. sphaeroides FJ1 was constructed, and the basal level of phaP and phaZ expression in this mutant was markedly increased. Electrophoretic mobility shift assays demonstrated that PhaR binds to the promoter region of phaP as well as those of phaR and phaZ. These results suggest that the PhaR protein is a repressor of phaP, phaR, and phaZ genes in R. sphaeroides FJ1.     

A repressor protein,PhaR, regulates polyhydroxyalkanoate (PHA) synthesis via its direct interaction with PHA

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-chain-length PHA-producing bacteria suggests a common and important role of PhaR-mediated regulation of PHA synthesis.     

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.     

Binding of the major phasin, PhaP1, from Ralstonia eutropha H16 to poly(3-hydroxybutyrate) granules

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.     

Microbial polyhydroxyalkanote synthesis repression protein PhaR as an affinity tag for recombinant protein purification

Background  

PhaR which is a repressor protein for microbial polyhydroxyalkanoates (PHA) biosynthesis, is able to attach to bacterial PHA granules in vivo, was developed as an affinity tag for in vitro protein purification. Fusion of PhaR-tagged self-cleavable Ssp DnaB intein to the N-terminus of a target protein allowed protein purification with a pH and temperature shift. During the process, the target protein was released to the supernatant while PhaR-tagged intein was still immobilized on the PHA nanoparticles which were then separated by centrifugation.     

Autoregulator protein PhaR for biosynthesis of polyhydroxybutyrate [P(3HB)] possibly has two separate domains that bind to the target DNA and P(3HB): Functional mapping of amino acid residues responsible for DNA binding

PhaR from Paracoccus denitrificans functions as a repressor or autoregulator of the expression of genes encoding phasin protein (PhaP) and PhaR itself, both of which are components of polyhydroxyalkanoate (PHA) granules (A. Maehara, S. Taguchi, T. Nishiyama, T. Yamane, and Y. Doi, J. Bacteriol. 184:3992-4002, 2002). PhaR is a unique regulatory protein in that it also has the ability to bind tightly to an effector molecule, PHA polyester. In this study, by using a quartz crystal microbalance, we obtained direct evidence that PhaR binds to the target DNA and poly[(R)-3-hydroxybutyrate] [P(3HB)], one of the PHAs, at the same time. To identify the PhaR amino acid residues responsible for DNA binding, deletion and PCR-mediated random point mutation experiments were carried out with the gene encoding the PhaR protein. PhaR point mutants with decreased DNA-binding abilities were efficiently screened by an in vivo monitoring assay system coupled with gene expression of green fluorescent protein in Escherichia coli. DNA-binding abilities of the wild-type and mutants of recombinant PhaR expressed in E. coli were evaluated using a gel shift assay and a surface plasmon resonance analysis. These experiments revealed that basic amino acids and a tyrosine in the N-terminal region, which is highly conserved among PhaR homologs, are responsible for DNA binding. However, most of the mutants with decreased DNA-binding abilities were unaffected in their ability to bind P(3HB), strongly suggesting that PhaR has two separate domains capable of binding to the target DNA and P(3HB).     

Morphological Change in Cellular Granule Formation of Poly[(R)-3-hydroxybutyrate] Caused by DNA-Binding-Related Mutations of an Autoregulated Repressor PhaR

PhaP is a major poly[(R)-3-hydroxybutyrate] [P(3HB)]-granule-associated protein. Its gene expression is controlled by an autoregulated repressor, PhaR, in Paracoccus denitrificans. The packing force of the P(3HB) granule by PhaP is greatly influenced by the number of PhaP molecules. In this study, the effects of DNA-binding-ability-reduced mutations of PhaR on morphological change in the cellular granule formation of P(3HB) were examined under a transmission electron microscope using an Escherichia coli recombinant system. Microscopic observation indicated that stronger packing of P(3HB) granules took place when the number of PhaP molecules was increased by reduction in the DNA-binding ability of PhaR.     

Effects of Granule-Associated Protein PhaP on Glycerol-Dependent Growth and Polymer Production in Poly(3-Hydroxybutyrate)-Producing Escherichia coli

Polyhydroxyalkanoates (PHAs) are accumulated as intracellular granules by many bacteria under unfavorable conditions, enhancing their fitness and stress resistance. Poly(3-hydroxybutyrate) (PHB) is the most widespread and best-known PHA. Apart from the genes that catalyze polymer biosynthesis, natural PHA producers have several genes for proteins involved in granule formation and/or with regulatory functions, such as phasins, that have been shown to affect polymer synthesis. This study evaluates the effect of PhaP, a phasin, on bacterial growth and PHB accumulation from glycerol in bioreactor cultures of recombinant Escherichia coli carrying phaBAC from Azotobacter sp. strain FA8. Cells expressing phaP grew more, and accumulated more PHB, both using glucose and using glycerol as carbon sources. When cultures were grown in a bioreactor using glycerol, PhaP-bearing cells produced more polymer (2.6 times) and more biomass (1.9 times) than did those without the phasin. The effect of this protein on growth promotion and polymer accumulation is expected to be even greater in high-density cultures, such as those used in the industrial production of the polymer. The recombinant strain presented in this work has been successfully used for the production of PHB from glycerol in bioreactor studies, allowing the production of 7.9 g/liter of the polymer in a semisynthetic medium in 48-h batch cultures. The development of bacterial strains that can efficiently use this substrate can help to make the industrial production of PHAs economically feasible.     

Polyhydroxyalkanoate Inclusion Body-Associated Proteins and Coding Region in Bacillus megaterium

Polyhydroxyalkanoic acids (PHA) are carbon and energy storage polymers that accumulate in inclusion bodies in many bacteria and archaea in response to environmental conditions. This work presents the results of a study of PHA inclusion body-associated proteins and an analysis of their coding region in Bacillus megaterium 11561. A 7,917-bp fragment of DNA was cloned and shown to carry a 4,104-bp cluster of 5 pha genes, phaP, -Q, -R, -B, and -C. The phaP and -Q genes were shown to be transcribed in one orientation, each from a separate promoter, while immediately upstream, phaR, -B, and -C were divergently transcribed as a tricistronic operon. Transfer of this gene cluster to Escherichia coli and to a PhaC⁻ mutant of Pseudomonas putida gave a Pha⁺ phenotype in both strains. Translational fusions to the green fluorescent protein localized PhaP and PhaC to the PHA inclusion bodies in living cells. The data presented are consistent with the hypothesis that the extremely hydrophilic protein PhaP is a storage protein and suggests that PHA inclusion bodies are not only a source of carbon, energy, and reducing equivalents but are also a source of amino acids.     

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

Poly(3-hydroxybutyrate) (PHB) granules are covered by a surface layer consisting of mainly phasins and other PHB granule-associated proteins (PGAPs). Phasins are small amphiphilic proteins that determine the number and size of accumulated PHB granules. Five phasin proteins (PhaP1 to PhaP5) are known for Ralstonia eutropha. In this study, we identified three additional potential phasin genes (H16_B1988, H16_B2296, and H16_B2326) by inspection of the R. eutropha genome for sequences with “phasin 2 motifs.” To determine whether the corresponding proteins represent true PGAPs, fusions with eYFP (enhanced yellow fluorescent protein) were constructed. Similar fusions of eYFP with PhaP1 to PhaP5 as well as fusions with PHB synthase (PhaC1), an inactive PhaC1 variant (PhaC1-C319A), and PhaC2 were also made. All fusions were investigated in wild-type and PHB-negative backgrounds. Colocalization with PHB granules was found for all PhaC variants and for PhaP1 to PhaP5. Additionally, eYFP fusions with H16_B1988 and H16_B2326 colocalized with PHB. Fusions of H16_B2296 with eYFP, however, did not colocalize with PHB granules but did colocalize with the nucleoid region. Notably, all fusions (except H16_B2296) were soluble in a ΔphaC1 strain. These data confirm that H16_B1988 and H16_B2326 but not H16_B2296 encode true PGAPs, for which we propose the designation PhaP6 (H16_B1988) and PhaP7 (H16_B2326). When localization of phasins was investigated at different stages of PHB accumulation, fusions of PhaP6 and PhaP7 were soluble in the first 3 h under PHB-permissive conditions, although PHB granules appeared after 10 min. At later time points, the fusions colocalized with PHB. Remarkably, PHB granules of strains expressing eYFP fusions with PhaP5, PhaP6, or PhaP7 localized predominantly near the cell poles or in the area of future septum formation. This phenomenon was not observed for the other PGAPs (PhaP1 to PhaP4, PhaC1, PhaC1-C319A, and PhaC2) and indicated that some phasins can have additional functions. A chromosomal deletion of phaP6 or phaP7 had no visible effect on formation of PHB granules.     

Accumulation of the PhaP Phasin of Ralstonia eutropha Is Dependent on Production of Polyhydroxybutyrate in Cells

Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by diverse bacteria and that accumulate as intracellular granules. Phasins are granule-associated proteins that accumulate to high levels in strains that are producing PHAs. The accumulation of phasins has been proposed to be dependent on PHA production, a model which is now rigorously tested for the phasin PhaP of Ralstonia eutropha. R. eutropha phaC PHA synthase and phaP phasin gene replacement strains were constructed. The strains were engineered to express heterologous and/or mutant PHA synthase alleles and a phaP-gfp translational fusion in place of the wild-type alleles of phaC and phaP. The strains were analyzed with respect to production of polyhydroxybutyrate (PHB), accumulation of PhaP, and expression of the phaP-gfp fusion. The results suggest that accumulation of PhaP is strictly dependent on the genetic capacity of strains to produce PHB, that PhaP accumulation is regulated at the level of both PhaP synthesis and PhaP degradation, and that, within mixed populations of cells, PhaP accumulation within cells of a given strain is not influenced by PHB production in cells of other strains. Interestingly, either the synthesis of PHB or the presence of relatively large amounts of PHB in cells (>50% of cell dry weight) is sufficient to enable PhaP synthesis. The results suggest that R. eutropha has evolved a regulatory mechanism that can detect the synthesis and presence of PHB in cells and that PhaP expression can be used as a marker for the production of PHB in individual cells.     

Functional role of granule-associated genes, phaP and phaR, in poly-β-hydroxybutyrate biosynthesis in recombinant E. coli harboring phbCAB operon

The expression characteristic of two granule-associated genes, phaP and phaR, in a recombinant E. coli harboring the phbCAB operon was investigated. Polybetamydroxybutyrate (PHB) accumulation increased up from 16% to 57% (w/w) after transformation of the granule-associated genes due to the stabilization of PHB granules rather than by a direct effect on PHB biosynthetic enzymes. The morphology of PHB granules also varied depending on the transformed phaP and phaR genes.     

PhaP is involved in the formation of a network on the surface of polyhydroxyalkanoate inclusions in Cupriavidus necator H16

Polyhydroxyalkanoate (PHA) inclusions are polymeric storage inclusions formed in some bacterial species when carbon levels are high but levels of another essential nutrient, such as nitrogen, are low. Though much is known about PHA synthesis, little is known about inclusion structure. In this study, atomic force microscopy (AFM) was employed to elucidate the structure of PHA inclusions at the nanoscale level, including the characterization of different layers of structure. AFM data suggest that underneath the inclusion envelope, there is a 2- to 4-nm-thick network layer that resides on top of a harder layer that is likely to be a crystalline lamellar polymer. The network is comprised of ~20-nm-wide linear segments and junctions that are typically formed by the joining of three to four of the linear segments. In some cases, ~50-nm globular structures that are raised ~1 to 2 nm above the network are present at the junctions. These globular structures always have a central pore that is ~15 nm in diameter. To determine if the major surface protein of PHA inclusions, PhaP, is involved in the structure of this network, inclusions from Cupriavidus necator H16 ΔphaP were examined. No network structure was detected. Instead, apparently random globular structures were found on the surfaces of the inclusions. When PhaP levels were reconstituted in this strain by the addition of phaP on a plasmid, the network was also reconstituted, albeit in a slightly different arrangement from that of the wild-type network. We conclude that PhaP participates in the formation of the inclusion network.     

Morphological change in cellular granule formation of poly[(R)-3-hydroxybutyrate] caused by DNA-binding-related mutations of an autoregulated repressor PhaR

PhaP is a major poly[(R)-3-hydroxybutyrate] [P(3HB)]-granule-associated protein. Its gene expression is controlled by an autoregulated repressor, PhaR, in Paracoccus denitrificans. The packing force of the P(3HB) granule by PhaP is greatly influenced by the number of PhaP molecules. In this study, the effects of DNA-binding-ability-reduced mutations of PhaR on morphological change in the cellular granule formation of P(3HB) were examined under a transmission electron microscope using an Escherichia coli recombinant system. Microscopic observation indicated that stronger packing of P(3HB) granules took place when the number of PhaP molecules was increased by reduction in the DNA-binding ability of PhaR.     

The inherent property of polyhydroxyalkanoate synthase to form spherical PHA granules at the cell poles: the core region is required for polar localization

Only the PHA synthase is required for formation of spherical intracellular PHA granules emerging at cell poles. This study aims to assign the polar targeting signal in the PHA synthase and to provide insight into molecular mechanisms of granule formation. Random in-frame insertion mutagenesis indicated dispensable and essential regions suggesting that only the N terminus (<100 aa) is dispensable and forms a random coil structure. The inactive PHA synthase (C319A) is still localized to cell poles, indicating that the nascent PHA chain does not serve as an anchor or signal for subcellular localization and granule formation. Deletion of the N terminus did neither affect subcellular localization nor PHA granule formation. The deletion of the hydrophobic C terminus (68 aa) did not impact on subcellular localization of the PHA synthase, but abolished PHA synthase activity. The structural protein PhaP1 was found to be not required for subcellular localization and initiation of granule formation. PhaP1 only localizes to the cell poles, when PHA granules are formed. These data suggested that the PHA synthase itself localizes to the cell poles via its core region (93-521 aa), which is structurally constraint and comprises the polar positional information for self-assembly of PHA granules at the cell poles.     

Influence of the poly-3-hydroxybutyrate (PHB) granule-associated proteins (PhaP1 and PhaP2) on PHB accumulation and symbiotic nitrogen fixation in Sinorhizobium meliloti Rm1021

Sinorhizobium meliloti cells store excess carbon as intracellular poly-3-hydroxybutyrate (PHB) granules that assist survival under fluctuating nutritional conditions. PHB granule-associated proteins (phasins) are proposed to regulate PHB synthesis and granule formation. Although the enzymology and genetics of PHB metabolism in S. meliloti have been well characterized, phasins have not yet been described for this organism. Comparison of the protein profiles of the wild type and a PHB synthesis mutant revealed two major proteins absent from the mutant. These were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) as being encoded by the SMc00777 (phaP1) and SMc02111 (phaP2) genes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins associated with PHB granules followed by MALDI-TOF confirmed that PhaP1 and PhaP2 were the two major phasins. Double mutants were defective in PHB production, while single mutants still produced PHB, and unlike PHB synthesis mutants that have reduced exopolysaccharide, the double mutants had higher exopolysaccharide levels. Medicago truncatula plants inoculated with the double mutant exhibited reduced shoot dry weight (SDW), although there was no corresponding reduction in nitrogen fixation activity. Whether the phasins are involved in a metabolic regulatory response or whether the reduced SDW is due to a reduction in assimilation of fixed nitrogen rather than a reduction in nitrogen fixation activity remains to be established.     

Nonspecific hydrophobic interactions of a repressor protein, PhaR, with poly[(R)-3-hydroxybutyrate] film studied with a quartz crystal microbalance

The gene expression for phasins (PhaP), which are predominantly polyhydroxyalkanoates (PHAs) granule-associated proteins, is regulated by a repressor protein of PhaR through the dual binding abilities of PhaR to the target DNAs and the granules. In this study, the binding functions of PhaR to poly[(R)-3-hydroxybutyrate] (P(3HB)) were investigated quantitatively by using a quartz crystal microbalance (QCM) technique. Adsorption of PhaR onto a melt-crystallized film of P(3HB) (cr-P(3HB)) was detected as a negative frequency shift of the QCM. The time course of the frequency changes observed for PhaR adsorption was composed of a quick frequency decrease at an initial stage and a subsequent slower frequency decrease for several hours, indicating multilayered adsorption of PhaR molecules onto cr-P(3HB). The initial rapid adsorption, which corresponds to direct adsorption of PhaR molecules onto a bare surface of cr-P(3HB), was a diffusion-controlled process. Strong interactions between PhaR and cr-P(3HB) were also observed as apparently irreversible adsorption. The comparative QCM measurement of PhaR adsorption onto various types of polymers with different aliphatic chemical structures revealed that PhaR was adsorbed onto the surfaces of polymers, including cr-P(3HB), mainly by nonspecific hydrophobic interactions. These results illustrate the high affinity and low specificity for adsorption of PhaR to P(3HB).