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Characterization and identification of the proteinsbound to two types of polyhydroxyalkanoategranules in Pseudomonas sp. 61-3Ayaka Hokamuraa, Kanako Fujinoa, Yoshiko Isodaa, Koji Arizonoa, Hideki Shiratsuchia &Hiromi Matsusakiaa Faculty of Environmental and Symbiotic Sciences, Department of Food and HealthSciences, Prefectural University of Kumamoto, Kumamoto, JapanPublished online: 14 May 2015.

To cite this article: Ayaka Hokamura, Kanako Fujino, Yoshiko Isoda, Koji Arizono, Hideki Shiratsuchi & Hiromi Matsusaki(2015): Characterization and identification of the proteins bound to two types of polyhydroxyalkanoate granules inPseudomonas sp. 61-3, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2015.1023250

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Characterization and identication of the proteins bound to two types ofpolyhydroxyalkanoate granules in Pseudomonas sp. 61-3

Ayaka Hokamura, Kanako Fujino, Yoshiko Isoda, Koji Arizono, Hideki Shiratsuchi andHiromi Matsusaki*

Faculty of Environmental and Symbiotic Sciences, Department of Food and Health Sciences, Prefectural Universityof Kumamoto, Kumamoto, Japan

Received January 15, 2015; accepted February 9, 2015

http://dx.doi.org/10.1080/09168451.2015.1023250

Pseudomonas sp. 61-3 accumulates two types ofpolyhydroxyalkanoates (PHAs), poly(3-hydroxybuty-rate) [P(3HB)], and poly(3HB-co-3-hydroxyalkano-ates) [P(3HB-co-3HA)], and some proteinsassociated with their PHA granules have been iden-tied. To date, PhaFPs (GA36) and PhaIPs (GA18)were identied from P(3HB-co-3HA) granules. Inthis study, the gene encoding GA24 associated withP(3HB) granule was identied as phbPPs. PhbPPswas composed of 192 amino acids with a calculatedmolecular mass of 20.4 kDa and was assumed to bea phasin. phbFPs gene and unknown ORF were alsofound on phb locus. PhbFPs was anticipated to bethe transcriptional repressor of phbPPs gene. PhbPPswas bound to the P(3HB-co-3HA) granules with3HB composition of more than 87 mol%, andPhaIPs and PhaFPs were bound to the P(3HB-co-3HA) granules with 3HA (C6C12) composition ofmore than 13 mol% in the producing cells, suggest-ing that localization of these proteins is attributed tothe monomer compositions of the copolymers.

Key words: polyhydroxyalkanoate (PHA); phasin;polyhydroxyalkanoate granule-associatedprotein; biodegradable plastics

Polyhydroxyalkanoates (PHAs) are accumulated inmany bacteria as intracellular carbon and energy stor-age materials under nutrient-limited conditions withexcess carbon.13) Within the cells, PHAs are accumu-lated as granules which contain proteins and lipidinvolved in their synthesis and regulation.4) There aretypically 812 granules/cell and the diameter of gran-ules is 0.2 to 0.5 m in Ralstonia eutropha (formerlyAlcaligenes eutrophus).1,57) Studies on PHA granulesusing13C NMR spectroscopy, X-ray diffraction, andelectron microscopy have indicated that PHAs arein vivo mobile amorphous and elastomeric state.68)

To date, some works have revealed the formation

mechanism of granules.911) Gerngross et al. havereported the localization of the R. eutropha poly-hydroxybutyrate (PHB) synthase at the surface of thegranules by immunocytochemical methods.12) Thegranules in the cell are surrounded by a membrane ofabout 2 nm thickness containing the intracellularenzyme, such as PHA synthase, PHA depolymerase,phasin, and PHA-specic regulator protein.5,1316) Theprimary function of phasins, which represent the majorcomponents of PHA granule-associated proteins, is tocontrol the surface properties of PHA granules. Phasinsstrongly bind to the hydrophobic surfaces of growingPHA granules to block the binding of other proteins.The phasins are amphiphilic proteins, which promotePHA biosynthesis and their abundance makes animpact on the PHA granule size.11,17,18) phaP1Re, oneof the structural genes of phasin, has been identied inRalstonia eutropha.17) Overexpression of PhaP1Reresults in the formation of many small P(3HB) gran-ules, while the phaP1Re mutant forms only one large P(3HB) granule per cell.17)

Other phaP genes have been identied fromRhodococcus rubber, Acinetobacter sp., Chromatiumvinosum, Bacillus megaterium, and Paracoccus denitrif-icans.1923) PhaRPd from P. denitricans is also associ-ated with PHB granules, whose role has been assumedto regulate the expression of phaPPd gene.

23) InPseudomonas oleovorans, two PHA granule-associatedproteins, PhaFPo and PhaIPo, have been characterizedand the corresponding genes have been cloned andidentied.24) Furthermore, PhaFPo negatively regulatesthe transcription of pha genes, that is, PhaFPo, which isa histone H1-like protein with C-terminal AAKP repeat-ing units, was suggested to repress the expression ofphaC1Po and phaIFPo genes.

24) These studies have beensuggested that the PHA granule-associated proteins notonly stabilize PHA granules in the cells, but also regu-late the related genes for PHA biosynthesis. On theother hand, in Aeromonas caviae, the phasin (PhaPAc)enhances PHA accumulation and alters P(3HB-co-3-hy-

*Corresponding author. Email: matusaki@pu-kumamoto.ac.jpAbbreviations: PHA, polyhydroxyalkanote; P(3HB), poly(3-hydroxybutyrate); 3HB, 3-hydroxybutyrate; 3HA, 3-hydroxyalkanoate; PHB, poly-hydroxybutyrate; NB, nutrient-broth; LB, lysogeny broth; MS, mineral salt; SDS, sodium dodecyl sulfate; SDS-PAGE, SDS-polyacrylamide gelelectrophoresis; PVDF, poly(vinylidene diuoride); ORF, open reading frame; PCR, polymerase chain reaction; LDPE, low-density polyethylene.

Bioscience, Biotechnology, and Biochemistry, 2015

2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry

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droxyhexanoate) copolymer composition,25,26) and ithas revealed to function as an activator for A. caviaePHA synthase (PhaCAc).

27) Thus, phasins are consid-ered to play a role in PHA biosynthesis, however, thefunctions have not been fully understood. Therefore, itis important to investigate the roles of phasins and otherPHA granule-associated proteins for PHA biosynthesisand the effective production.

Pseudomonas sp. 61-3 synthesizes two types ofPHAs, P(3HB) homopolymer and a random copolymer,P(3HB-co-3HA), consisting of 3-hydroxyalkanoate(3HA) units of 412 carbon atoms.2830) In Pseudomo-nas sp. 61-3, the study using freeze-fracture electronmicroscopy revealed that P(3HB) and P(3HB-co-3HA)were accumulated as different granules in the samecell.31) Some proteins associated with the two types ofPHA granules in Pseudomonas sp. 61-3 were identi-ed.32) The proteins of 18 kDa (GA18) and 36 kDa(GA36) were specically bound to P(3HB-co-3HA)granules, whereas the proteins of 24 kDa (GA24) and48 kDa (GA48, porin) were mainly bound to P(3HB).32)

The proteins of 18 and 36 kDa were identied as PhaIPsand PhaFPs, respectively. The amino acid sequences ofthe proteins showed high homology to those of P. oleo-vorans, and the two genes were located downstream ofthe pha locus as described previously (see Fig. 1).32) Inaddition, N-terminal amino acid sequence analyses ofthe associated proteins with PHA granules and immuno-blotting methods revealed that the PHB synthase(PhbCPs) and PHA synthases (PhaC1Ps possibly withPhaC2Ps) from Pseudomonas sp. 61-3 were bound to P(3HB) and P(3HB-co-3HA) granules, respectively.32)

However, the reason why their proteins, except PHBand PHA synthases, can bind to each PHA granule hasbeen yet unknown. There are two hypotheses for thereason. One is that the granule-associated proteins

(phasins) directly recognize the monomer compositionof PHA, and the other is due to the localization causedby the interaction with PHB and/or PHA synthases. Inthis study, we cloned and identied the gene encodingGA24 (PhbPPs) strongly bound to P(3HB) granule inPseudomonas sp. 61-3. In addition, we discussed thelocalization of the proteins associated with P(3HB-co-3HA) granules with various monomer compositionssynthesized by the recombinant strains of Pseudomonassp. 61-3.

Material and methodsBacterial strains, plasmids, and culture conditions.

The bacterial strains and plasmids used in this studyare listed in Table 1. Pseudomonas sp. 61-3 and therecombinant strains were grown at 28 C in NB med-ium consisting of 1% meat extract (Kyokuto Pharma-ceutical Industrial Co., Ltd, Japan), 1% Bactopeptone(Difco, USA) and 0.5% NaCl (pH 7.0). Escherichiacoli strains were grown at 37 C in LB medium.38)

When needed, ampicillin (100 mg/L), kanamycin(50 mg/L), tetracycline (12.5 mg/L), and/or gentamicin(10 mg/L) were added to the medium.

Production and analysis of PHA. Pseudomonassp. 61-3 and the recombinant strains were cultivated ona reciprocal shaker (130 strokes/min) at 28 C for 48 hor 72 h in 500-mL shaking asks containing 100 mLof a nitrogen-limited MS medium.28,30) Filter-sterilizedglucose (2 wt.%) was added to the medium as a solecarbon source. PHA compositions of the isolated gran-ules (see below) were determined by gas chromatogra-phy as described previously.28,32)

EcoR

I

PstI

BglII

SphI

Bam

HI

XbaI

XhoI

BanI

II

EcoR

I

SphI

SphI

SmaI

EcoR

V

SacI

I

SphI

EcoR

I

PstI

PstI

BanI

II

EcoR

IBa

mH

I

PstI

PstI

SacI

I

phaIPs(423 bp)

phaC1Ps(1680 bp)

phaZPs(858 bp)

phaC2Ps(1683 bp)

phaDPs(621 bp)

phaFPs(762 bp)

ApaI

EcoR

V

SalI

PstI

SalI

SacI

PstI

Hin

dIII

PstI

EcoR

V

KpnI

SalI

EcoR

VEc

oRI

PstI

Hin

dIII

EcoR

I

EcoR

VPs

tIBa

nIII

PstI

SmaI

BanI

II

BglII

Bam

HI

BanI

IISa

cIBa

nIII

PstI

XbaI

ApaI

SphI

SphI

phbFPs(534 bp)

phbPPs(579 bp)

ORF(2439 bp)

phbRPs(1137 bp)

phbBPs(744 bp)

phbAPs(1176 bp)

phbCPs(1701 bp)

4.2 kb EcoRV-SphI region was sequenced in thisstudy

Fig. 1. Organization of pha and phb loci in Pseudomonas sp. 61-3.Notes: The genes located on pha and phb loci in Pseudomonas sp. 61-3 are involved in the biosynthesis of P(3HB-co-3HA) and P(3HB),respectively. In pha locus, the genes encoding PHA synthase 1 (PhaC1), PHA depolymerase (PhaZ), PHA synthase 2 (PhaC2), an unknownfunction protein (PhaD), and PHA granule-associated proteins (PhaI and PhaF) are located. In phb locus, the genes encoding a putative negativeregulator protein (PhbF) related to the transcription of phbP gene, phasin (PhbP), an unknown function protein (ORF), a putative regulator protein(PhbR) related to the transcription of phbBAC, NADPH-dependent acetoacetyl coenzyme A reductase (PhbB), -ketothiolase (PhbA), and PHBsynthase (PhbC) are located.

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Isolation of PHA granules and SDS-PAGE analy-sis. Cells cultivated in MS medium were harvestedby centrifugation (7,700 g, 10 min, 4 C), and thenwashed twice and resuspended in 2.0 mL of 0.1 MTrisHCl buffer (pH 7.5). Finally, the cells were dis-rupted by the treatment of ultrasonication (30 W, 10s, 20 times). Approximately 1 mL of the broken cellsuspension was layered on a discontinuous sucrosegradient from 1 mL each of 2.0, 1.67, 1.33, and1.0 M sucrose in 0.1 M TrisHCl buffer (pH 7.5) asdescribed previously.32) After ultracentrifugation(210,000 g, 160 min, 4 C), the white layers contain-ing PHA granules [P(3HB) and/or P(3HB-co-3HA)]were isolated. The isolated PHA granules werewashed twice with 0.1 M Tris-HCl (pH 7.5) by cen-trifugation (24,000 g, 30 min, 4 C). Samples of thepuried granules were mixed with 2-fold gel buffer(12% of -mercaptoethanol, 4% of SDS, 20% ofglycerol, 0.001% of bromophenol blue, and 0.125 Mof Tris-HCl [pH 6.8]), and the proteins were dena-tured and released from the granules by heating thesuspension at 98 C for 10 min. The proteins wereseparated by SDS-PAGE with 14% polyacrylamidegels as described by Laemmli39) and stained with aBio-safe Coomassie (Bio-Rad Laboratories, USA).After SDS-PAGE, the proteins were blotted from thepolyacrylamide gels onto PVDF membranes and wereidentied by N-terminal amino acid sequencing,except PhbCPs was detected by immunoblotting, asdescribed previously.32)

DNA manipulations. Isolation of the total genomicDNA and plasmids, digestion of DNA with restrictionendonucleases, agarose gel electrophoresis, and trans-formation of E. coli were performed by standard proce-dures.38) The genomic DNA library of Pseudomonassp. 61-3 was prepared as described previously.30) DNArestriction fragments were extracted from agarose gelsusing a GENECLEAN Kit (BIO 101, Inc., USA). Con-jugation of Pseudomonas sp. 61-3 or the mutant strains

with E. coli S17-1 harboring broad-host-range plasmidswas performed as described by Friedrich et al.40)

Cloning of the gene encoding the protein associatedwith P(3HB) granule. For cloning of the gene(phbPPs) encoding the protein (GA24) associated withP(3HB) granule, PCR was performed with primer pairsphbRDS-f1 (5-TTCCTTGTGAAGGCTCATTGAGGC-GTTCAT-3) and GA24-r1 (5-TG(T/C)TCIACIG(A/T)IGC(A/G)AA(A/G/T)AT(T/C)TT-3) using genomicDNA of Pseudomonas sp. 61-3 as a template. Theprimer phbRDS-f1 was synthesized based on thesequence of the downstream region of phbRPs gene.The degenerate primer GA24-r1 was designed based onthe N-terminal amino acid sequence, (M)TFFNLEKLQ-DAQKANLDLLQ, of GA24 described previously.32)

From the information in the nucleotide sequence of the3-kb amplied fragment, the primers GA24-f2 (5-AACTTGGAGAAATTGCAAGACGCT-3) and GA24-f3 (5-CAACCTAGACCTCCTGCAGCAAAT-3) weresynthesized, and the nested PCR was performed usingLA PCRTM in vitro Cloning Kit (TAKARA BIO,Japan) for cloning of the whole phbPPs gene and thedownstream region. The nested PCR was rstly per-formed with primers GA24-f2 and Cassette Primer C1using Pseudomonas sp. 613 genomic DNA digestedwith SalI as a template. Subsequently, second PCR wasperformed with primers GA24-f3 and Cassette PrimerC2 using the rst PCR-amplied product as a template.Finally, the amplied 0.7-kb fragment including phbPPsgene was ligated to pT7Blue T-vector to givepT7-GA24(LA)-F. Furthermore, the 0.5-kb product,including the part of phbPPs gene was amplied byPCR with primer pairs GA24-f3 and GA24-r3 (5-TT-ACTTGTTACCGCTTGTTGCCTTGCCAGT-3) usingpT7-GA24(LA)-F as a template and was used for thesubsequent hybridization experiments as a probe.Southern hybridization was performed as described

by Southern.41) Preparation of an alkaline phosphatase-labeled probe and detection of hybridization signals on

Table 1. Bacterial strains and plasmids used in this study.

Strain or plasmid Relevant characteristicsSource orreference

StrainsPseudomonas sp. 61-3 Wild type JCM 1001530)

Pseudomonas sp. 61-3(phbC::tet)

Inactivation of chromosomal phbCPs by integration of Tcr; phbCPs-negative mutant

30)

Pseudomonas sp. AC1-TnK phaC1Ps-negative mutant, phaC1Ps::kan (Tn10), Kmr This study

Pseudomonas sp. BCG-TcGm Inactivation of chromosomal phbCPs and phaGPs by integration of Tcr and

Gmr, respectively; phbCPs- and phaGPs-negative mutant

33)

E. coli DH5 deoR endA1 gyrA96 hsdR17 (rK mK

+) relA1 supE thi-1 (lacZYA-argFV169) 80lacZM15F ClontechE. coli S17-1 recA and tra genes of plasmid RP4 integrated into the chromosome; auxotrophic

for proline and thiamine

34)

E. coli S17-1 (pir) protein encoded by R6K integrated into chromosome 35)

plasmidspBluescript II KS+ Apr lacPOZ T7 and T3 promoter StratagenepT7Blue T-vector Apr, lacPOZ NovagenpJASc22 pJRD215 derivative; phaC1Ps

30)

pJKSc54-phab pJRD215 derivative; phaPs promoter, phaC1Ps, phbRe promoter, phbARe, phbBRe36,37)

pJKSc46-pha pJRD215 derivative; phaPs promoter, phaC1Ps, phbARe, phbBRe36,37)

pBSEX22 pBluescript II KS+ derivative; phaPs promoter, phaC1Ps36,37)

pBSL180 Apr, Kmr, R6K replicon, suicide, lacIq, tnp (Tn10), mob+, IS10 35)

pSLBE13dC1 pBSL180 derivative containing the 1.3-kb BglII-EcoRI fragment of pBSEX22 This study

The proteins bound to two types of polyhydroxyalkanoates 3

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membranes were carried out with Gene Images Alk-phos Direct Labelling and Detection System (GEHealthcare, USA). Colony hybridization of genomicDNA libraries of Pseudomonas sp. 61-3 was performedwith the probe as described previously.30)

DNA sequencing analysis. DNA fragments to besequenced were subcloned into pBluescript II KS+.DNA was sequenced by the modied dideoxy-chaintermination method basically as described by Sangeret al.42) with a NEN Global Edition IR2 System,LIC4200L (LI-COR, USA). The sequencing reactionwas carried out according to the manual supplied withthe Thermo Sequenase Cycle Sequencing Kit (USB,USA). The resulting nucleotide sequence was analyzedwith SDC-GENETYX information processing software(GENETYX CORPORATION, Japan).

Disruption of phaC1Ps gene. pBSL180 vector wasused as an integration to disrupt the chromosomalphaC1Ps gene of Pseudomonas sp. 61-3. pBSEX22 wasdigested with BglII and EcoRI, and the 1.3-kb BglII-EcoRI fragment (5- and 3-truncated phaC1Ps) wasthen ligated with pBSL180 at the same restriction sitesto construct pSLBE13dC1. Conjugation of Pseudomo-nas sp. 61-3 with E. coli S17-1 (pir) harboringpSLBE13dC1 was carried out as described previ-ously.33) Southern hybridization analysis was performedusing the phaC1Ps gene as a probe to conrm the genedisruption.

Nucleotide sequence accession number. The nucle-otide sequence data reported in this study will appearin EMBL, GenBank, and DDBJ database with acces-sion No. LC019127.

ResultsCloning and identication of phbPPs and phbFPs

genesWe previously reported that pha and phb loci in

Pseudomonas sp. 61-3 involved in the biosyntheses ofP(3HB-co-3HA) and P(3HB), respectively.30,32) Thegenes encoding the P(3HB-co-3HA) granule-associatedproteins, GA18 and GA36 were identied as PhaIPsand PhaFPs, and the two genes were located in thedownstream region of phaC1ZC2D gene cluster in phalocus.32) Whereas, the gene encoding the P(3HB) gran-ule-associated protein (GA24) remained unidentiedalthough the N-terminal amino acid sequence wasdetermined.It was reported that Azotobacter vinelandii UW136

had a P(3HB) biosynthetic gene cluster (phbRBAC) assame as phb locus in Pseudomonas sp. 61-3,43) andphbPAv gene encoding a putative P(3HB) granule-associated protein phasin was present downstream ofphbRAv in the same orientation. Similarly, the regiondownstream of phbRPs gene of Pseudomonas sp. 61-3was explored to nd GA24 gene (phbPPs). Firstly, weattempted PCR with primer pairs phbRDS-f1, whichcorresponded to the downstream sequence of phbRPs

gene, and GA24-r1, which was a degenerate primerbased on the N-terminal amino acid sequence of GA24,using genomic DNA of Pseudomonas sp. 61-3 as atemplate. As a result, approximately 3-kb DNA frag-ment was amplied by PCR. From the information inthe nucleotide sequence of the PCR product, further-more, nested PCR and colony hybridization with geno-mic DNA library of Pseudomonas sp. 61-3 wereperformed with the probe, including the gene encodingGA24 (PhbPPs) for cloning of the downstream regionof phbPPs gene as described in materials and methodssection. A positive clone isolated by colony hybridiza-tion was used for southern hybridization analysis. Thepositive 7.6-kb HindIII and 6.9-kb SacI fragments werecloned into pBluescript II KS+ and partially sequenced.From the information in the nucleotide sequencesobtained from the positive clones and the nucleotidesequence of the 3-kb PCR product amplied with prim-ers phbRDS-f1 and GA24-r1 as described above, the4.2 kb EcoRV-SphI region downstream of phbRPs genewas completely sequenced. The restriction maps ofPHA biosynthesis genes (pha and phb loci), whichhave been so far elucidated, are shown in Fig. 1. In thedownstream region of phbRPs gene, three potentialORFs were identied by computer analysis. The nucle-otide sequence revealed homologies to genes encodingphasin GA24 (PhbPPs) and the transcriptional negativeregulator (PhbFPs) in A. vinelandii UW136 and Azoto-bacter sp. FA8.43,44) phbPPs and phbFPs encoded puta-tive proteins composed of 192 amino acids with acalculated molecular mass of 20.4 kDa and 177 aminoacids with a calculated molecular mass of 19.6 kDa,respectively. The deduced amino acid sequence ofPhbPPs (GA24) showed high homologies to PhaP ofAzotobacter sp. FA8 (57% identity)44) and PhbP of A.vinelandii AvOP (54% identity).43) Furthermore, thededuced amino acid sequence of PhbFPs revealed highhomologies to PhaF of Azotobacter sp. FA8 (69% iden-tity)44) and PhbF of A. vinelandii AvOP (68% iden-tity).43) PhbPPs (GA24) of Pseudomonas sp. 61-3 wasexpected to have a function of phasin protein, such asreported elsewhere.11,17,18) PhbFPs also showed 37.5%identity of amino acid homology to PhaR of P. deni-tricans.23) The role of PhaR protein in P. denitricansis assumed to negatively regulate the transcriptionalexpression of phaP gene. In addition, Pfam programshowed that PhbFPs comprised three domains, PHB/PHA accumulation regulator DNA-binding domain(amino acid positions 10 to 73), PHB accumulationregulatory domain (amino acid positions 75 to 114),and PHB accumulation regulatory domain (amino acidpositions 116 to 154). Therefore, PhbFPs of Pseudomo-nas sp. 61-3 is probably a negative transcriptional regu-lator to repress the expression of phbPPs gene.Interestingly, another ORF was found between

phbPPs and phbRPs genes in phb locus of Pseudomonassp. 61-3. Such ORF has not been found in the P(3HB)biosynthesis gene clusters of A. vinerandii UW136 andAzotobacter sp. FA8 (Fig. 1).43,44) The ORF encoded aputative protein composed of 812 amino acids with acalculated molecular mass of 90.2 kDa. The deducedamino acid sequence of the ORF showed high homolo-gies to putative poly(3-hydroxyalkanoate) synthetasesof Pseudomonas sp. GM48 (92% identity, accession

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No. WP_007988013) and P. putida (87% identity,accession No. WP_033040191), and putative poly(3-hydroxybutyrate) depolymerase of A. vinelandii DJ(59% identity) according to the genomic informations.Pfam program showed that the function (DUF3141) ofthese putative proteins having / hydrolase domainwas unknown. Function of the ORF found in this studyremains unknown although the possibility of PHA syn-thase or PHA depolymerase have been investigatedin vivo (data not shown).

Analysis of PHA granules and localization of PHAgranule-associated proteins

phbCPs- or phaC1Ps-disrupted strains of Pseudomo-nas sp. 61-3 was used as a host in order to synthesizethe only one type of PHA, since the wild-type strainaccumulated two types of PHAs, P(3HB) homopolymerand P(3HB-co-3HA) copolymer in the same cells(Fig. 1).30,31,37) The P(3HB-co-3HA) granules with var-ious monomer compositions were synthesized by therecombinant strains of Pseudomonas sp. 61-3, andPHA granules accumulated in the cells were isolatedby sonication and a subsequent sucrose density gradientmethod as described in materials and methods section.Only one white band was observed at the interfaces of01.0 M or 1.31.67 sucrose from each of the cellextracts of all recombinant strains of Pseudomonas sp.61-3, indicating that only one type of PHA is synthe-sized in the cells. P(3HB-co-3HA) granules with

relatively low 3HB compositions (less than 66 mol%)were collected at the interface of 01.0 M sucrose, andthe copolymer granules with high 3HB compositions(more than 87 mol%) were collected at the interface of1.331.67 M sucrose. The monomer compositions ofthe copolymer granules accumulated by the recombi-nant strains of Pseudomonas sp. 61-3 were determinedby gas chromatography (Table 2) and the proteins asso-ciated with the granules were separated by SDS-PAGE(Fig. 2). The relationship between the monomer com-positions of the isolated PHA granules and the proteinsbound to the respective PHA granules is shown inTable 2.In Fig. 2, the 6070 kDa proteins were identied as

PHB synthase (PhbCPs) or PHA synthase 1 (PhaC1Ps)from Pseudomonas sp. 61-3 by analyses of the N-ter-minal amino acid sequences and immunoblotting asdescribed previously.32) Whereas, PHA synthase 2(PhaC2Ps) of Pseudomonas sp. 61-3 was not able to bedetected from P(3HB-co-3HA) granules, suggestingthat PhaC1Ps was the major PHA providing enzyme inPseudomonas sp. 61-3 as described previously.32)

GA18, GA36, and GA48 proteins were also identiedas PhaIPs, PhaFPs, and porin D, respectively.

32) Asshown in Fig. 2 and Table 2, PhaC1Ps was detectedwith the PHA granules isolated from ve strains exceptPseudomonas sp. AC1-TnK, and PhbCPs was weaklydetected with the PHA granule in Pseudomonas sp.AC1-TnK. PhaIPs and PhaFPs were detected with thePHA granules isolated from Pseudomonas sp. 61-3

PHB th PhbC97.0

(kDa)1 2 3 4 5 6 7

syn ase; PhbCPsPHA synthase 1; PhaC1PsGA48; Porin

66.0

45.0

GA36; PhaFPs

GA24; PhbPPs

30.0

; Ps

GA18; PhaIPs20.1

14.4

Fig. 2. SDS-PAGE analysis of native PHA granules isolated from the recombinant strains of Pseudomonas sp. 613. Lane 1, molecular weightmarkers; lane 2, Pseudomonas sp. 613 (phbC::tet); lane 3, Pseudomonas sp. 613 (phbC::tet)/pJASc22; lane 4, Pseudomonas sp. 613 (phbC::tet)/pJKSc46-pha; lane 5, Pseudomonas sp. 613 (phbC::tet)/pJKSc54-phab; lane 6, Pseudomonas sp. AC1-TnK; and lane 7, Pseudomonas sp.BCG-TcGm/pJKSc54-phab.

Table 2. Relationship of the monomer composition of PHA accumulated by recombinant strains of Pseudomonas sp. 61-3 and the granule-associated proteins.

Strain plasmid

PHA composition (mol%)Molecular weight of granule-associated

protein (kDa)3HB (C4) 3HA (C6C12)

Pseudomonas sp. 61-3 (phbC::tet) none 30 70 62 48 36 18Pseudomonas sp. 61-3 (phbC::tet) pJASc22 49 51 62 48 36 18Pseudomonas sp. 61-3 (phbC::tet) pJKSc46-pha 66 34 62 48 36 18Pseudomonas sp. 61-3 (phbC::tet) pJKSc54-phab 87 13 62 48 36 24 18Pseudomonas sp. AC1-TnK none 95 5 (69) (48) 24Pseudomonas sp. BCG-TcGm pJKSc54-phab 99 1 62 (48) 24

Notes: Cells were cultivated at 28 C for 48 h or 72 h (Pseudomonas sp. AC1-TnK) in MS medium containing 2% (wt./vol) glucose as a sole carbon source. Minorbands are indicated in parentheses. 3HB, 3-hydroxybutyrate; 3HA, medium-chain-length 3-hydroxyalkanoate units (C6C12).

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(phbC::tet) and the recombinant strains harboringpJASc22, pJKSc46-pha, and pJKSc54-phab. GA24identied as PhbPPs in this study was conrmed withthe PHA granules isolated from Pseudomonas sp. 61-3(phbC::tet)/pJKSc54-phab, Pseudomonas sp. AC1-TnK,and Pseudomonas sp. BCG-TcGm/pJKSc54-phab.However, PhaIPs and PhaFPs were not able to be asso-ciated with the granules in Pseudomonas sp. AC1-TnKand Pseudomonas sp. BCG-TcGm/pJKSc54-phab. Inother words, PhbPPs (GA24) could bind to P(3HB-co-3HA) granules with 3HB composition of more than87 mol%, but could not be almost bind to the copoly-mer granule with 3HB composition of less than at least66 mol%. Whereas, PhaIPs (GA18) and PhaFPs (GA36)could bind to P(3HB-co-3HA) granules with 3HA (C6-C12) composition of more than 13 mol%. GA48 (porin)was considered to be nonspecically bound to allPHAs regardless of the monomer compositions. Inter-estingly, the all phasin or phasin-like proteins (PhbPPs,PhaIPs, and PhaFPs) were associated with P(87% 3HB-co-13% 3HA) granule.PhbPPs (GA24) was rstly found as the protein asso-

ciated with P(3HB) granule, which was one of the twotypes of PHAs, P(3HB), and P(3HB-co-3HA), accumu-lated in Pseudomonas sp. 61-3. In addition, PhbPPscould bind to P(3HB-co-3HA) granules obtained fromthe cells of Pseudomonas sp. 61-3 (phbC::tet)/pJKSc54-phab and Pseudomonas sp. BCG-TcGm/pJKSc54-phab, although the phbCPs gene of thesestrains was disrupted. Therefore, PhbPPs is likely torecognize the monomer units, probably 3HB unit, ofcopolymers without interaction of PHB synthase.

Discussion

Pseudomonas sp. 61-3 produces two types of PHAs,P(3HB) homopolymer and P(3HB-co-3HA) randomcopolymer and accumulates them as different granulesin the cell.28,29,31) The genes involved in P(3HB) and P(3HB-co-3HA) biosyntheses from Pseudomonas sp.61-3 were cloned and identied previously.30,32,33) Inthe previous report, two PHA granules, P(3HB) andP(3HB-co-3HA), were isolated from Pseudomonas sp.61-3, and polyester synthases (PhaC1Ps and PhbCPs)and the proteins (PhaIPs and PhaFPs) associated withPHA granules were identied.32) In this report, anotherprotein (GA24) associated with P(3HB) granule wasidentied. The deduced amino acid sequence of GA24gene revealed high homologies to those of PhaPAs ofAzotobacter sp. FA844) and PhbPAv of A. vinelandiiAvOP.43) Therefore, GA24 was referred to as PhbPPs,and it was probably anticipated to stabilize the granulesof P(3HB) and P(3HB-co-3HA) with high 3HB fraction(more than 87 mol%) in the producing cells as a pha-sin. In this experiment, we also found a potential ORFdownstream of phbPPs gene in the opposite direction(Fig. 1). The putative translational product of the ORFrevealed high homologies to PhaFAs of Azotobacter sp.FA844) and to PhbFAv of A. vinelandii AvOP.

43) There-fore, the ORF was referred to as phbFPs, and PhbFPswas assumed to repress the transcriptional expressionof phbPPs gene in Pseudomonas sp. 61-3, since it alsoshowed 37.5 and 56% identities to PhaRPd of P. deni-

tricans and PhaRRe of R. eutropha, respectively,which were known as transcriptional repressors toregulate the expression of phasins.23,45,46) Moreover,another large ORF was found between phbPPs andphbRPs genes in phb locus of Pseudomonas sp. 61-3(Fig. 1). The deduced amino acid sequence of the ORFshowed high homologies to putative poly(3-hydrox-yalkanoate) synthetases of Pseudomonas sp. GM48(92% identity) and P. putida (87% identity), and aputative poly(3-hydroxybutyrate) depolymerase of A.vinelandii DJ (59% identity) according to the genomicinformations. Additionally, the transcription of the ORFwas conrmed by semi-quantitative RT-PCR (data notshown). Therefore, it was expected that the ORF mightencode PHA synthase or PHA depolymerase, especiallyfor biosynthesis or degradation of P(3HB), since theORF was also found in phb locus of Pseudomonas sp.61-3. However, the function of the ORF remainsunknown, and we will investigate and report it in thenext research.In this study, localization of the proteins (PhbPPs,

PhaIPs, and PhaFPs) associated with the granules of P(3HB) and P(3HB-co-3HA) in the cells of Pseudomonassp. 61-3 was supposed to be attributed to the monomercompositions of polymers. Our ndings are summa-rized and depicted in Fig. 3. PhbPPs (GA24) wasdetected with the granules of P(3HB-co-3HA) copoly-mers with 3HB composition of more than 87 mol%,and both PhaIPs (GA18) and PhaFPs (GA36) weredetected with the granules of the copolymers with3HA (C6-C12) composition of more than 13 mol%(Table 2 and Fig. 2). PhbPPs was detected from thepolyester granules in the cells of Pseudomonas sp. 61-3 (phbC::tet)/pJKSc54-phab and Pseudomonas sp.BCG-TcGm/pJKSc54-phab whose strains were phbCPs-disruptants. PhaIPs and PhaFPs were detected from thepolyester granules in the cells of Pseudomonas sp. 61-3 (phbC::tet), Pseudomonas sp. 61-3 (phbC::tet)/pJASc22, Pseudomonas sp. 61-3 (phbC::tet)/pJKSc46-pha, and Pseudomonas sp. 61-3 (phbC::tet)/pJKSc54-phab, whereas these proteins could not be conrmedon the surface of the polyester granules in the cells ofPseudomonas sp. BCG-TcGm/pJKSc54-phab wherephaC1Ps gene was introduced. Thus, the three granule-associated proteins, PhbPPs (GA24), PhaIPs (GA18),and PhaFPs (GA36), appear to recognize the polyesterchain directly without interaction and support of poly-ester synthases. Additional copies of phbPPs, phaIPs,and/or phaFPs genes did not affect the production andthe monomer compositions of P(3HB-co-3HA) copoly-mers synthesized by the recombinant strains of Pseu-domonas sp. 61-3 (phbC::tet) as a host (data notshown), unlike phaPAc of A. caviae.

2527) It has beenreported that PhaPAc activate A. caviae PHA synthase(PhaCAc), but not R. eutropha PHA synthase(PhaCRe).

27) This may be due to the low amino acidsequence identity (13%) between PhaPAc and PhaP1Reof R. eutropha. Similarly, the PhbPPs shows a lowidentity (23%) to PhaPAc. Also, the PHA granule-associated proteins of Pseudomonas sp. 61-3 may formmultimer. For example, PhaPs of Aeromonashydrophila and R. eutropha have been reported to formtrimers and tetramers by X-ray analysis, respec-tively.11,47) However, the relationship between the

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multimeric form of phasins and the binding to PHAgranule has not been elucidated yet.

In vivo, PHAs are mobile amorphous and elastomericstate, and PHA granules are surrounded by a mem-brane. Several works have been carried out to revealthe forming mechanism of membrane, and some mod-els have been proposed.4,68,15,20) According to the rstmodel, PHA granules are surrounded by a phospholipidmembrane with embedded proteins consisting of PHAsynthase, intracellular PHA depolymerase, phasin pro-tein, and other proteins.4,20) The second model hasbeen proposed that PHA granule-associated proteinspresent on the phospholipid monolayer.15) The thirdmodel has been proposed that PHA granule-associatedproteins present on a much more membrane structurewith phospholipid bilayer.15,48) In Pseudomonas sp. 61-3, the membrane structure surrounded PHA granuleshas not been elucidated, however, it was found thatPhbPPs (GA24), PhaIPs (GA18), and PhaFPs (GA36)specically bound to P(3HB) and P(3HB-co-3HA)granules, respectively, in the previous study.32) In addi-tion, our data suggest that binding of their proteins toPHA granules would be due to recognizing the mono-mer units of polymers by the proteins. While, Mayeret al. have reported that the boundary layer structure ofPHA granules in bacteria might vary by PHA monomercomposition.49) Therefore, the specic binding ofPhbPPs, PhaIPs, and PhaFPs to PHA granules might bedue to the difference of boundary layer structure of sur-rounded PHAs. Possibly, the layer structure might beattributed to the monomer compositions of P(3HB-co-3HA) copolymers. PHB/PHA synthases, PHB/PHAdepolymerases, phasins, and regulatory proteins areknown as major PHA granule-associated proteins.These proteins are denitely important for biosynthesisand/or degradation of PHAs. The ndings and theobservations obtained here will lead to the effectiveproduction and the biosynthesis of PHAs, P(3HB-co-3HA) copolymers, with favorable monomer composi-tions. For example, the P(94% 3HB-co-6% 3HA)copolymer synthesized by the recombinant strain ofPseudomonas sp. 61-3 is known to have properties

similar to low-density polyethylene (LDPE), and thecopolymer is a practical material for application of bio-degradable plastics.37) Thus, to produce practical P(3HB-co-3HA) copolymer with high 3HB fractioneffectively, sufcient amounts of amphiphilic PhbPPsmight have to be provided in the producing cells forstabilization of hydrophobic PHA granules in the cells.Therefore, further studies on PHA biosynthesis andPHA granule formation in Pseudomonas sp. 61-3 is inprogress.

Acknowledgment

We are grateful to Dr Kenichiro Matsumoto for thetechnical assistance and to NBRP (National BioRe-source Project, Japan) for the plasmid pBSL180 andE. coli S17-1 (pir).

Disclosure statement

No potential conict of interest was reported by theauthors.

FundingThis work was supported by JSPS KAKENHI [grant

number 14780448], [grant number 16710054].

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(A) (B)GA62 (PHA synthase)

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Fig. 3. The localization model of the proteins associated with polyester granules accumulated in (A) Pseudomonas sp. 613 (phbC::tet)/pJKSc46-pha, (B) Pseudomonas sp. 613 (phbC::tet)/pJKSc54-phab, (C) Pseudomonas sp. AC1-TnK, and (D) Pseudomonas sp. BCG-TcGm/pJKSc54-phab.

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Abstract Material and methods Bacterial strains, plasmids, and culture conditions Production and analysis of PHA Isolation of PHA granules and SDS-PAGE analysis DNA manipulations Cloning of the gene encoding the protein associated with P(3HB) granule DNA sequencing analysis Disruption of phaC1Ps gene Nucleotide sequence accession number

Results Cloning and identification of phbPPs and phbFPs genes Analysis of PHA granules and localization of PHA granule-associated proteins

DiscussionAcknowledgment Disclosure statementFundingReferences