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Characterization and identification of the proteins

bound to two types of polyhydroxyalkanoate
granules in Pseudomonas sp. 61-3
a

Ayaka Hokamura , Kanako Fujino , Yoshiko Isoda , Koji Arizono , Hideki Shiratsuchi &
a

Hiromi Matsusaki
a

Faculty of Environmental and Symbiotic Sciences, Department of Food and Health

Sciences, Prefectural University of Kumamoto, Kumamoto, Japan
Published online: 14 May 2015.

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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 in
Pseudomonas sp. 61-3, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2015.1023250
To link to this article: http://dx.doi.org/10.1080/09168451.2015.1023250

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Bioscience, Biotechnology, and Biochemistry, 2015

Characterization and identication of the proteins bound to two types of

polyhydroxyalkanoate granules in Pseudomonas sp. 61-3
Ayaka Hokamura, Kanako Fujino, Yoshiko Isoda, Koji Arizono, Hideki Shiratsuchi and
Hiromi Matsusaki*
Faculty of Environmental and Symbiotic Sciences, Department of Food and Health Sciences, Prefectural University
of Kumamoto, Kumamoto, Japan
Received January 15, 2015; accepted February 9, 2015

Downloaded by [University of the Punjab] at 06:53 05 June 2015

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

Pseudomonas sp. 61-3 accumulates two types of

polyhydroxyalkanoates (PHAs), poly(3-hydroxybutyrate) [P(3HB)], and poly(3HB-co-3-hydroxyalkanoates) [P(3HB-co-3HA)], and some proteins
associated with their PHA granules have been identied. To date, PhaFPs (GA36) and PhaIPs (GA18)
were identied from P(3HB-co-3HA) granules. In
this study, the gene encoding GA24 associated with
P(3HB) granule was identied as phbPPs. PhbPPs
was composed of 192 amino acids with a calculated
molecular mass of 20.4 kDa and was assumed to be
a phasin. phbFPs gene and unknown ORF were also
found on phb locus. PhbFPs was anticipated to be
the transcriptional repressor of phbPPs gene. PhbPPs
was bound to the P(3HB-co-3HA) granules with
3HB composition of more than 87 mol%, and
PhaIPs and PhaFPs were bound to the P(3HB-co3HA) granules with 3HA (C6C12) composition of
more than 13 mol% in the producing cells, suggesting that localization of these proteins is attributed to
the monomer compositions of the copolymers.
Key words:

polyhydroxyalkanoate (PHA); phasin;

polyhydroxyalkanoate granule-associated
protein; biodegradable plastics

Polyhydroxyalkanoates (PHAs) are accumulated in

many bacteria as intracellular carbon and energy storage materials under nutrient-limited conditions with
excess carbon.13) Within the cells, PHAs are accumulated as granules which contain proteins and lipid
involved in their synthesis and regulation.4) There are
typically 812 granules/cell and the diameter of granules is 0.2 to 0.5 m in Ralstonia eutropha (formerly
Alcaligenes eutrophus).1,57) Studies on PHA granules
using13C NMR spectroscopy, X-ray diffraction, and
electron microscopy have indicated that PHAs are
in vivo mobile amorphous and elastomeric state.68)
To date, some works have revealed the formation

mechanism of granules.911) Gerngross et al. have

reported the localization of the R. eutropha polyhydroxybutyrate (PHB) synthase at the surface of the
granules by immunocytochemical methods.12) The
granules in the cell are surrounded by a membrane of
about 2 nm thickness containing the intracellular
enzyme, such as PHA synthase, PHA depolymerase,
phasin, and PHA-specic regulator protein.5,1316) The
primary function of phasins, which represent the major
components of PHA granule-associated proteins, is to
control the surface properties of PHA granules. Phasins
strongly bind to the hydrophobic surfaces of growing
PHA granules to block the binding of other proteins.
The phasins are amphiphilic proteins, which promote
PHA biosynthesis and their abundance makes an
impact on the PHA granule size.11,17,18) phaP1Re, one
of the structural genes of phasin, has been identied in
Ralstonia eutropha.17) Overexpression of PhaP1Re
results in the formation of many small P(3HB) granules, while the phaP1Re mutant forms only one large P
(3HB) granule per cell.17)
Other phaP genes have been identied from
Rhodococcus rubber, Acinetobacter sp., Chromatium
vinosum, Bacillus megaterium, and Paracoccus denitrificans.1923) PhaRPd from P. denitricans is also associated with PHB granules, whose role has been assumed
to regulate the expression of phaPPd gene.23) In
Pseudomonas oleovorans, two PHA granule-associated
proteins, PhaFPo and PhaIPo, have been characterized
and the corresponding genes have been cloned and
identied.24) Furthermore, PhaFPo negatively regulates
the transcription of pha genes, that is, PhaFPo, which is
a histone H1-like protein with C-terminal AAKP repeating units, was suggested to repress the expression of
phaC1Po and phaIFPo genes.24) These studies have been
suggested that the PHA granule-associated proteins not
only stabilize PHA granules in the cells, but also regulate the related genes for PHA biosynthesis. On the
other hand, in Aeromonas caviae, the phasin (PhaPAc)
enhances PHA accumulation and alters P(3HB-co-3-hy-

*Corresponding author. Email: matusaki@pu-kumamoto.ac.jp

Abbreviations: PHA, polyhydroxyalkanote; P(3HB), poly(3-hydroxybutyrate); 3HB, 3-hydroxybutyrate; 3HA, 3-hydroxyalkanoate; PHB, polyhydroxybutyrate; NB, nutrient-broth; LB, lysogeny broth; MS, mineral salt; SDS, sodium dodecyl sulfate; SDS-PAGE, SDS-polyacrylamide gel
electrophoresis; PVDF, poly(vinylidene diuoride); ORF, open reading frame; PCR, polymerase chain reaction; LDPE, low-density polyethylene.
2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry

A. Hokamura et al.

ORF
(2439 bp)

Material and methods

Bacterial strains, plasmids, and culture conditions.
The bacterial strains and plasmids used in this study
are listed in Table 1. Pseudomonas sp. 61-3 and the
recombinant strains were grown at 28 C in NB medium consisting of 1% meat extract (Kyokuto Pharmaceutical Industrial Co., Ltd, Japan), 1% Bactopeptone
(Difco, USA) and 0.5% NaCl (pH 7.0). Escherichia
coli 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.

phbBPs
(744 bp)

SacII

PstI
PstI
SacII
phbAPs
(1176 bp)

ApaI

XbaI

ApaI
PstI

BanIII
SacI
BanIII

BamHI

BglII

BanIII

SphI
SmaI

EcoRV
PstI
BanIII
PstI
phbRPs
(1137 bp)

phaIPs
(423 bp)

phaFPs
(762 bp)

phaDPs
(621 bp)

SphI

PstI

HindIII
EcoRI

EcoRV

Production and analysis of PHA.

Pseudomonas
sp. 61-3 and the recombinant strains were cultivated on
a reciprocal shaker (130 strokes/min) at 28 C for 48 h
or 72 h in 500-mL shaking asks containing 100 mL
of a nitrogen-limited MS medium.28,30) Filter-sterilized
glucose (2 wt.%) was added to the medium as a sole
carbon source. PHA compositions of the isolated granules (see below) were determined by gas chromatography as described previously.28,32)

BanIII

SmaI
PstI

PstI

BamHI

EcoRV
EcoRI

KpnI
SalI

EcoRV

PstI

HindIII

SalI

SalI
PstI

SacI
PstI
phbPPs
(579 bp)

(phasins) directly recognize the monomer composition

of PHA, and the other is due to the localization caused
by the interaction with PHB and/or PHA synthases. In
this study, we cloned and identied the gene encoding
GA24 (PhbPPs) strongly bound to P(3HB) granule in
Pseudomonas sp. 61-3. In addition, we discussed the
localization of the proteins associated with P(3HB-co3HA) granules with various monomer compositions
synthesized by the recombinant strains of Pseudomonas
sp. 61-3.

phaC2Ps
(1683 bp)

phaZPs
(858 bp)

phaC1Ps
(1680 bp)

phbFPs
(534 bp)

SphI
EcoRI

SphI
SphI

XbaI
XhoI

EcoRI

BanIII

SphI

PstI

BglII

EcoRI

droxyhexanoate) copolymer composition,

and it
has revealed to function as an activator for A. caviae
PHA synthase (PhaCAc).27) Thus, phasins are considered to play a role in PHA biosynthesis, however, the
functions have not been fully understood. Therefore, it
is important to investigate the roles of phasins and other
PHA granule-associated proteins for PHA biosynthesis
and the effective production.
Pseudomonas sp. 61-3 synthesizes two types of
PHAs, P(3HB) homopolymer and a random copolymer,
P(3HB-co-3HA), consisting of 3-hydroxyalkanoate
(3HA) units of 412 carbon atoms.2830) In Pseudomonas sp. 61-3, the study using freeze-fracture electron
microscopy revealed that P(3HB) and P(3HB-co-3HA)
were accumulated as different granules in the same
cell.31) Some proteins associated with the two types of
PHA granules in Pseudomonas sp. 61-3 were identied.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) and
48 kDa (GA48, porin) were mainly bound to P(3HB).32)
The proteins of 18 and 36 kDa were identied as PhaIPs
and PhaFPs, respectively. The amino acid sequences of
the proteins showed high homology to those of P. oleovorans, and the two genes were located downstream of
the pha locus as described previously (see Fig. 1).32) In
addition, N-terminal amino acid sequence analyses of
the associated proteins with PHA granules and immunoblotting methods revealed that the PHB synthase
(PhbCPs) and PHA synthases (PhaC1Ps possibly with
PhaC2Ps) 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 PHB
and PHA synthases, can bind to each PHA granule has
been yet unknown. There are two hypotheses for the
reason. One is that the granule-associated proteins

EcoRV

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25,26)

EcoRI
BamHI

phbCPs
(1701 bp)

4.2 kb EcoRV-SphI region was sequenced in this

study

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 unknown
function protein (PhaD), and PHA granule-associated proteins (PhaI and PhaF) are located. In phb locus, the genes encoding a putative negative
regulator 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 PHB
synthase (PhbC) are located.

The proteins bound to two types of polyhydroxyalkanoates

Table 1.

Bacterial strains and plasmids used in this study.

Strain or plasmid
Strains
Pseudomonas
Pseudomonas
(phbC::tet)
Pseudomonas
Pseudomonas

Source or
reference

Relevant characteristics

JCM 1001530)

sp. 61-3
sp. 61-3

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

sp. AC1-TnK
sp. BCG-TcGm

phaC1Ps-negative mutant, phaC1Ps::kan (Tn10), Kmr

Inactivation of chromosomal phbCPs and phaGPs by integration of Tcr and
Gmr, respectively; phbCPs- and phaGPs-negative mutant
deoR endA1 gyrA96 hsdR17 (rK mK+) relA1 supE thi-1 (lacZYA-argFV169) 80lacZM15F
recA and tra genes of plasmid RP4 integrated into the chromosome; auxotrophic
for proline and thiamine
protein encoded by R6K integrated into chromosome

This study

Apr lacPOZ T7 and T3 promoter

Apr, lacPOZ
pJRD215 derivative; phaC1Ps
pJRD215 derivative; phaPs promoter, phaC1Ps, phbRe promoter, phbARe, phbBRe
pJRD215 derivative; phaPs promoter, phaC1Ps, phbARe, phbBRe
pBluescript II KS+ derivative; phaPs promoter, phaC1Ps
Apr, Kmr, R6K replicon, suicide, lacIq, tnp (Tn10), mob+, IS10
pBSL180 derivative containing the 1.3-kb BglII-EcoRI fragment of pBSEX22

Stratagene
Novagen

E. coli DH5
E. coli S17-1

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E. coli S17-1 (pir)

plasmids
pBluescript II KS+
pT7Blue T-vector
pJASc22
pJKSc54-phab
pJKSc46-pha
pBSEX22
pBSL180
pSLBE13dC1

Isolation of PHA granules and SDS-PAGE analysis. Cells cultivated in MS medium were harvested
by centrifugation (7,700 g, 10 min, 4 C), and then
washed twice and resuspended in 2.0 mL of 0.1 M
TrisHCl buffer (pH 7.5). Finally, the cells were disrupted by the treatment of ultrasonication (30 W, 10
s, 20 times). Approximately 1 mL of the broken cell
suspension was layered on a discontinuous sucrose
gradient from 1 mL each of 2.0, 1.67, 1.33, and
1.0 M sucrose in 0.1 M TrisHCl buffer (pH 7.5) as
described previously.32) After ultracentrifugation
(210,000 g, 160 min, 4 C), the white layers containing PHA granules [P(3HB) and/or P(3HB-co-3HA)]
were isolated. The isolated PHA granules were
washed twice with 0.1 M Tris-HCl (pH 7.5) by centrifugation (24,000 g, 30 min, 4 C). Samples of the
puried granules were mixed with 2-fold gel buffer
(12% of -mercaptoethanol, 4% of SDS, 20% of
glycerol, 0.001% of bromophenol blue, and 0.125 M
of Tris-HCl [pH 6.8]), and the proteins were denatured and released from the granules by heating the
suspension at 98 C for 10 min. The proteins were
separated by SDS-PAGE with 14% polyacrylamide
gels as described by Laemmli39) and stained with a
Bio-safe Coomassie (Bio-Rad Laboratories, USA).
After SDS-PAGE, the proteins were blotted from the
polyacrylamide gels onto PVDF membranes and were
identied by N-terminal amino acid sequencing,
except PhbCPs was detected by immunoblotting, as
described previously.32)
DNA manipulations. Isolation of the total genomic
DNA and plasmids, digestion of DNA with restriction
endonucleases, agarose gel electrophoresis, and transformation of E. coli were performed by standard procedures.38) The genomic DNA library of Pseudomonas
sp. 61-3 was prepared as described previously.30) DNA
restriction fragments were extracted from agarose gels
using a GENECLEAN Kit (BIO 101, Inc., USA). Conjugation of Pseudomonas sp. 61-3 or the mutant strains

30)

33)

Clontech
34)

35)

30)
36,37)
36,37)
36,37)
35)

This study

with E. coli S17-1 harboring broad-host-range plasmids

was performed as described by Friedrich et al.40)
Cloning of the gene encoding the protein associated
with P(3HB) granule.
For cloning of the gene
(phbPPs) encoding the protein (GA24) associated with
P(3HB) granule, PCR was performed with primer pairs
phbRDS-f1 (5-TTCCTTGTGAAGGCTCATTGAGGCGTTCAT-3) and GA24-r1 (5-TG(T/C)TCIACIG(A/T)
IGC(A/G)AA(A/G/T)AT(T/C)TT-3) using genomic
DNA of Pseudomonas sp. 61-3 as a template. The
primer phbRDS-f1 was synthesized based on the
sequence of the downstream region of phbRPs gene.
The degenerate primer GA24-r1 was designed based on
the N-terminal amino acid sequence, (M)TFFNLEKLQDAQKANLDLLQ, of GA24 described previously.32)
From the information in the nucleotide sequence of the
3-kb amplied fragment, the primers GA24-f2 (5AACTTGGAGAAATTGCAAGACGCT-3) and GA24f3 (5-CAACCTAGACCTCCTGCAGCAAAT-3) were
synthesized, and the nested PCR was performed using
LA PCRTM in vitro Cloning Kit (TAKARA BIO,
Japan) for cloning of the whole phbPPs gene and the
downstream region. The nested PCR was rstly performed with primers GA24-f2 and Cassette Primer C1
using Pseudomonas sp. 613 genomic DNA digested
with SalI as a template. Subsequently, second PCR was
performed with primers GA24-f3 and Cassette Primer
C2 using the rst PCR-amplied product as a template.
Finally, the amplied 0.7-kb fragment including phbPPs
gene was ligated to pT7Blue T-vector to give
pT7-GA24(LA)-F. Furthermore, the 0.5-kb product,
including the part of phbPPs gene was amplied by
PCR with primer pairs GA24-f3 and GA24-r3 (5-TTACTTGTTACCGCTTGTTGCCTTGCCAGT-3) using
pT7-GA24(LA)-F as a template and was used for the
subsequent hybridization experiments as a probe.
Southern hybridization was performed as described
by Southern.41) Preparation of an alkaline phosphataselabeled probe and detection of hybridization signals on

A. Hokamura et al.

membranes were carried out with Gene Images Alkphos Direct Labelling and Detection System (GE
Healthcare, USA). Colony hybridization of genomic
DNA libraries of Pseudomonas sp. 61-3 was performed
with the probe as described previously.30)

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DNA sequencing analysis. DNA fragments to be

sequenced were subcloned into pBluescript II KS+.
DNA was sequenced by the modied dideoxy-chain
termination method basically as described by Sanger
et al.42) with a NEN Global Edition IR2 System,
LIC4200L (LI-COR, USA). The sequencing reaction
was carried out according to the manual supplied with
the Thermo Sequenase Cycle Sequencing Kit (USB,
USA). The resulting nucleotide sequence was analyzed
with SDC-GENETYX information processing software
(GENETYX CORPORATION, Japan).
Disruption of phaC1Ps gene. pBSL180 vector was
used as an integration to disrupt the chromosomal
phaC1Ps gene of Pseudomonas sp. 61-3. pBSEX22 was
digested with BglII and EcoRI, and the 1.3-kb BglIIEcoRI fragment (5- and 3-truncated phaC1Ps) was
then ligated with pBSL180 at the same restriction sites
to construct pSLBE13dC1. Conjugation of Pseudomonas sp. 61-3 with E. coli S17-1 (pir) harboring
pSLBE13dC1 was carried out as described previously.33) Southern hybridization analysis was performed
using the phaC1Ps gene as a probe to conrm the gene
disruption.
Nucleotide sequence accession number. The nucleotide sequence data reported in this study will appear
in EMBL, GenBank, and DDBJ database with accession No. LC019127.

Results
Cloning and identication of phbPPs and phbFPs
genes
We previously reported that pha and phb loci in
Pseudomonas sp. 61-3 involved in the biosyntheses of
P(3HB-co-3HA) and P(3HB), respectively.30,32) The
genes encoding the P(3HB-co-3HA) granule-associated
proteins, GA18 and GA36 were identied as PhaIPs
and PhaFPs, and the two genes were located in the
downstream region of phaC1ZC2D gene cluster in pha
locus.32) Whereas, the gene encoding the P(3HB) granule-associated protein (GA24) remained unidentied
although the N-terminal amino acid sequence was
determined.
It was reported that Azotobacter vinelandii UW136
had a P(3HB) biosynthetic gene cluster (phbRBAC) as
same as phb locus in Pseudomonas sp. 61-3,43) and
phbPAv gene encoding a putative P(3HB) granuleassociated protein phasin was present downstream of
phbRAv in the same orientation. Similarly, the region
downstream of phbRPs gene of Pseudomonas sp. 61-3
was explored to nd GA24 gene (phbPPs). Firstly, we
attempted PCR with primer pairs phbRDS-f1, which
corresponded to the downstream sequence of phbRPs

gene, and GA24-r1, which was a degenerate primer

based on the N-terminal amino acid sequence of GA24,
using genomic DNA of Pseudomonas sp. 61-3 as a
template. As a result, approximately 3-kb DNA fragment was amplied by PCR. From the information in
the nucleotide sequence of the PCR product, furthermore, nested PCR and colony hybridization with genomic DNA library of Pseudomonas sp. 61-3 were
performed with the probe, including the gene encoding
GA24 (PhbPPs) for cloning of the downstream region
of phbPPs gene as described in materials and methods
section. A positive clone isolated by colony hybridization was used for southern hybridization analysis. The
positive 7.6-kb HindIII and 6.9-kb SacI fragments were
cloned into pBluescript II KS+ and partially sequenced.
From the information in the nucleotide sequences
obtained from the positive clones and the nucleotide
sequence of the 3-kb PCR product amplied with primers phbRDS-f1 and GA24-r1 as described above, the
4.2 kb EcoRV-SphI region downstream of phbRPs gene
was completely sequenced. The restriction maps of
PHA biosynthesis genes (pha and phb loci), which
have been so far elucidated, are shown in Fig. 1. In the
downstream region of phbRPs gene, three potential
ORFs were identied by computer analysis. The nucleotide sequence revealed homologies to genes encoding
phasin GA24 (PhbPPs) and the transcriptional negative
regulator (PhbFPs) in A. vinelandii UW136 and Azotobacter sp. FA8.43,44) phbPPs and phbFPs encoded putative proteins composed of 192 amino acids with a
calculated molecular mass of 20.4 kDa and 177 amino
acids with a calculated molecular mass of 19.6 kDa,
respectively. The deduced amino acid sequence of
PhbPPs (GA24) showed high homologies to PhaP of
Azotobacter sp. FA8 (57% identity)44) and PhbP of A.
vinelandii AvOP (54% identity).43) Furthermore, the
deduced amino acid sequence of PhbFPs revealed high
homologies to PhaF of Azotobacter sp. FA8 (69% identity)44) and PhbF of A. vinelandii AvOP (68% identity).43) PhbPPs (GA24) of Pseudomonas sp. 61-3 was
expected to have a function of phasin protein, such as
reported elsewhere.11,17,18) PhbFPs also showed 37.5%
identity of amino acid homology to PhaR of P. denitricans.23) The role of PhaR protein in P. denitricans
is assumed to negatively regulate the transcriptional
expression of phaP gene. In addition, Pfam program
showed that PhbFPs comprised three domains, PHB/
PHA accumulation regulator DNA-binding domain
(amino acid positions 10 to 73), PHB accumulation
regulatory domain (amino acid positions 75 to 114),
and PHB accumulation regulatory domain (amino acid
positions 116 to 154). Therefore, PhbFPs of Pseudomonas sp. 61-3 is probably a negative transcriptional regulator to repress the expression of phbPPs gene.
Interestingly, another ORF was found between
phbPPs and phbRPs genes in phb locus of Pseudomonas
sp. 61-3. Such ORF has not been found in the P(3HB)
biosynthesis gene clusters of A. vinerandii UW136 and
Azotobacter sp. FA8 (Fig. 1).43,44) The ORF encoded a
putative protein composed of 812 amino acids with a
calculated molecular mass of 90.2 kDa. The deduced
amino acid sequence of the ORF showed high homologies to putative poly(3-hydroxyalkanoate) synthetases
of Pseudomonas sp. GM48 (92% identity, accession

The proteins bound to two types of polyhydroxyalkanoates

(kDa)
97.0
66.0

PHB synthase; PhbCPs

45.0

GA48; Porin

PHA synthase 1; PhaC1Ps

GA36; PhaFPs
30.0
GA24;; PhbPPs
20.1
GA18; PhaIPs
14.4

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Fig. 2. SDS-PAGE analysis of native PHA granules isolated from the recombinant strains of Pseudomonas sp. 613. Lane 1, molecular weight
markers; 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.

No. WP_007988013) and P. putida (87% identity,

accession No. WP_033040191), and putative poly(3hydroxybutyrate) depolymerase of A. vinelandii DJ
(59% identity) according to the genomic informations.
Pfam program showed that the function (DUF3141) of
these putative proteins having / hydrolase domain
was unknown. Function of the ORF found in this study
remains unknown although the possibility of PHA synthase or PHA depolymerase have been investigated
in vivo (data not shown).
Analysis of PHA granules and localization of PHA
granule-associated proteins
phbCPs- or phaC1Ps-disrupted strains of Pseudomonas sp. 61-3 was used as a host in order to synthesize
the only one type of PHA, since the wild-type strain
accumulated two types of PHAs, P(3HB) homopolymer
and P(3HB-co-3HA) copolymer in the same cells
(Fig. 1).30,31,37) The P(3HB-co-3HA) granules with various monomer compositions were synthesized by the
recombinant strains of Pseudomonas sp. 61-3, and
PHA granules accumulated in the cells were isolated
by sonication and a subsequent sucrose density gradient
method as described in materials and methods section.
Only one white band was observed at the interfaces of
01.0 M or 1.31.67 sucrose from each of the cell
extracts of all recombinant strains of Pseudomonas sp.
61-3, indicating that only one type of PHA is synthesized 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, and
the copolymer granules with high 3HB compositions
(more than 87 mol%) were collected at the interface of
1.331.67 M sucrose. The monomer compositions of
the copolymer granules accumulated by the recombinant strains of Pseudomonas sp. 61-3 were determined
by gas chromatography (Table 2) and the proteins associated with the granules were separated by SDS-PAGE
(Fig. 2). The relationship between the monomer compositions of the isolated PHA granules and the proteins
bound to the respective PHA granules is shown in
Table 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-terminal amino acid sequences and immunoblotting as
described previously.32) Whereas, PHA synthase 2
(PhaC2Ps) of Pseudomonas sp. 61-3 was not able to be
detected from P(3HB-co-3HA) granules, suggesting
that PhaC1Ps was the major PHA providing enzyme in
Pseudomonas sp. 61-3 as described previously.32)
GA18, GA36, and GA48 proteins were also identied
as PhaIPs, PhaFPs, and porin D, respectively.32) As
shown in Fig. 2 and Table 2, PhaC1Ps was detected
with the PHA granules isolated from ve strains except
Pseudomonas sp. AC1-TnK, and PhbCPs was weakly
detected with the PHA granule in Pseudomonas sp.
AC1-TnK. PhaIPs and PhaFPs were detected with the
PHA granules isolated from Pseudomonas sp. 61-3

Table 2. Relationship of the monomer composition of PHA accumulated by recombinant strains of Pseudomonas sp. 61-3 and the granuleassociated proteins.
PHA composition (mol%)
Strain
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas

plasmid
sp.
sp.
sp.
sp.
sp.
sp.

61-3 (phbC::tet)
61-3 (phbC::tet)
61-3 (phbC::tet)
61-3 (phbC::tet)
AC1-TnK
BCG-TcGm

none
pJASc22
pJKSc46-pha
pJKSc54-phab
none
pJKSc54-phab

3HB (C4)

3HA (C6C12)

30
49
66
87
95
99

70
51
34
13
5
1

Molecular weight of granule-associated

protein (kDa)
62
62
62
62
(69)
62

48
48
48
48
(48)
(48)

36
36
36
36

24
24
24

18
18
18
18

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. Minor
bands are indicated in parentheses. 3HB, 3-hydroxybutyrate; 3HA, medium-chain-length 3-hydroxyalkanoate units (C6C12).

Downloaded by [University of the Punjab] at 06:53 05 June 2015

A. Hokamura et al.

(phbC::tet) and the recombinant strains harboring

pJASc22, pJKSc46-pha, and pJKSc54-phab. GA24
identied as PhbPPs in this study was conrmed with
the 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 associated with the granules in Pseudomonas sp. AC1-TnK
and Pseudomonas sp. BCG-TcGm/pJKSc54-phab. In
other words, PhbPPs (GA24) could bind to P(3HB-co3HA) granules with 3HB composition of more than
87 mol%, but could not be almost bind to the copolymer granule with 3HB composition of less than at least
66 mol%. Whereas, PhaIPs (GA18) and PhaFPs (GA36)
could bind to P(3HB-co-3HA) granules with 3HA (C6C12) composition of more than 13 mol%. GA48 (porin)
was considered to be nonspecically bound to all
PHAs regardless of the monomer compositions. Interestingly, the all phasin or phasin-like proteins (PhbPPs,
PhaIPs, and PhaFPs) were associated with P(87% 3HBco-13% 3HA) granule.
PhbPPs (GA24) was rstly found as the protein associated with P(3HB) granule, which was one of the two
types of PHAs, P(3HB), and P(3HB-co-3HA), accumulated in Pseudomonas sp. 61-3. In addition, PhbPPs
could bind to P(3HB-co-3HA) granules obtained from
the cells of Pseudomonas sp. 61-3 (phbC::tet)/
pJKSc54-phab and Pseudomonas sp. BCG-TcGm/
pJKSc54-phab, although the phbCPs gene of these
strains was disrupted. Therefore, PhbPPs is likely to
recognize the monomer units, probably 3HB unit, of
copolymers without interaction of PHB synthase.

Discussion
Pseudomonas sp. 61-3 produces two types of PHAs,
P(3HB) homopolymer and P(3HB-co-3HA) random
copolymer and accumulates them as different granules
in 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) In
the previous report, two PHA granules, P(3HB) and
P(3HB-co-3HA), were isolated from Pseudomonas sp.
61-3, and polyester synthases (PhaC1Ps and PhbCPs)
and the proteins (PhaIPs and PhaFPs) associated with
PHA granules were identied.32) In this report, another
protein (GA24) associated with P(3HB) granule was
identied. The deduced amino acid sequence of GA24
gene revealed high homologies to those of PhaPAs of
Azotobacter sp. FA844) and PhbPAv of A. vinelandii
AvOP.43) Therefore, GA24 was referred to as PhbPPs,
and it was probably anticipated to stabilize the granules
of P(3HB) and P(3HB-co-3HA) with high 3HB fraction
(more than 87 mol%) in the producing cells as a phasin. In this experiment, we also found a potential ORF
downstream of phbPPs gene in the opposite direction
(Fig. 1). The putative translational product of the ORF
revealed high homologies to PhaFAs of Azotobacter sp.
FA844) and to PhbFAv of A. vinelandii AvOP.43) Therefore, the ORF was referred to as phbFPs, and PhbFPs
was assumed to repress the transcriptional expression
of phbPPs gene in Pseudomonas sp. 61-3, since it also
showed 37.5 and 56% identities to PhaRPd of P. deni-

tricans and PhaRRe of R. eutropha, respectively,

which were known as transcriptional repressors to
regulate the expression of phasins.23,45,46) Moreover,
another large ORF was found between phbPPs and
phbRPs genes in phb locus of Pseudomonas sp. 61-3
(Fig. 1). The deduced amino acid sequence of the ORF
showed high homologies to putative poly(3-hydroxyalkanoate) synthetases of Pseudomonas sp. GM48
(92% identity) and P. putida (87% identity), and a
putative poly(3-hydroxybutyrate) depolymerase of A.
vinelandii DJ (59% identity) according to the genomic
informations. Additionally, the transcription of the ORF
was conrmed by semi-quantitative RT-PCR (data not
shown). Therefore, it was expected that the ORF might
encode PHA synthase or PHA depolymerase, especially
for biosynthesis or degradation of P(3HB), since the
ORF was also found in phb locus of Pseudomonas sp.
61-3. However, the function of the ORF remains
unknown, and we will investigate and report it in the
next 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 Pseudomonas
sp. 61-3 was supposed to be attributed to the monomer
compositions of polymers. Our ndings are summarized and depicted in Fig. 3. PhbPPs (GA24) was
detected with the granules of P(3HB-co-3HA) copolymers with 3HB composition of more than 87 mol%,
and both PhaIPs (GA18) and PhaFPs (GA36) were
detected with the granules of the copolymers with
3HA (C6-C12) composition of more than 13 mol%
(Table 2 and Fig. 2). PhbPPs was detected from the
polyester granules in the cells of Pseudomonas sp. 613 (phbC::tet)/pJKSc54-phab and Pseudomonas sp.
BCG-TcGm/pJKSc54-phab whose strains were phbCPsdisruptants. PhaIPs and PhaFPs were detected from the
polyester granules in the cells of Pseudomonas sp. 613 (phbC::tet), Pseudomonas sp. 61-3 (phbC::tet)/
pJASc22, Pseudomonas sp. 61-3 (phbC::tet)/pJKSc46pha, and Pseudomonas sp. 61-3 (phbC::tet)/pJKSc54phab, whereas these proteins could not be conrmed
on the surface of the polyester granules in the cells of
Pseudomonas sp. BCG-TcGm/pJKSc54-phab where
phaC1Ps gene was introduced. Thus, the three granuleassociated proteins, PhbPPs (GA24), PhaIPs (GA18),
and PhaFPs (GA36), appear to recognize the polyester
chain directly without interaction and support of polyester synthases. Additional copies of phbPPs, phaIPs,
and/or phaFPs genes did not affect the production and
the monomer compositions of P(3HB-co-3HA) copolymers synthesized by the recombinant strains of Pseudomonas sp. 61-3 (phbC::tet) as a host (data not
shown), unlike phaPAc of A. caviae.2527) It has been
reported that PhaPAc activate A. caviae PHA synthase
(PhaCAc), but not R. eutropha PHA synthase
(PhaCRe).27) This may be due to the low amino acid
sequence identity (13%) between PhaPAc and PhaP1Re
of R. eutropha. Similarly, the PhbPPs shows a low
identity (23%) to PhaPAc. Also, the PHA granuleassociated proteins of Pseudomonas sp. 61-3 may form
multimer. For example, PhaPs of Aeromonas
hydrophila and R. eutropha have been reported to form
trimers and tetramers by X-ray analysis, respectively.11,47) However, the relationship between the

The proteins bound to two types of polyhydroxyalkanoates

GA62 (PHA synthase)

(A)

GA18
P(66% 3HB-co-3HA)
granule

(C)

GA62 (PHA synthase)

(B)

GA36

GA36
GA48 (porin)

GA69 (PHB synthase)

GA48 ((porin)
i )

GA18
P(87% 3HB-co-3HA)
granule

(D)

GA24

GA62 (PHA synthase)

GA48 (porin)

GA48 (porin)

Downloaded by [University of the Punjab] at 06:53 05 June 2015

P(95%
(
3HB-co-3HA))
granule

GA24

P(99%
(
3HB-co-3HA))
granule

GA24

Fig. 3. The localization model of the proteins associated with polyester granules accumulated in (A) Pseudomonas sp. 613 (phbC::tet)/pJKSc46pha, (B) Pseudomonas sp. 613 (phbC::tet)/pJKSc54-phab, (C) Pseudomonas sp. AC1-TnK, and (D) Pseudomonas sp. BCG-TcGm/pJKSc54phab.

multimeric form of phasins and the binding to PHA

granule has not been elucidated yet.
In vivo, PHAs are mobile amorphous and elastomeric
state, and PHA granules are surrounded by a membrane. Several works have been carried out to reveal
the forming mechanism of membrane, and some models have been proposed.4,68,15,20) According to the rst
model, PHA granules are surrounded by a phospholipid
membrane with embedded proteins consisting of PHA
synthase, intracellular PHA depolymerase, phasin protein, and other proteins.4,20) The second model has
been proposed that PHA granule-associated proteins
present on the phospholipid monolayer.15) The third
model has been proposed that PHA granule-associated
proteins present on a much more membrane structure
with phospholipid bilayer.15,48) In Pseudomonas sp. 613, the membrane structure surrounded PHA granules
has not been elucidated, however, it was found that
PhbPPs (GA24), PhaIPs (GA18), and PhaFPs (GA36)
specically bound to P(3HB) and P(3HB-co-3HA)
granules, respectively, in the previous study.32) In addition, our data suggest that binding of their proteins to
PHA granules would be due to recognizing the monomer units of polymers by the proteins. While, Mayer
et al. have reported that the boundary layer structure of
PHA granules in bacteria might vary by PHA monomer
composition.49) Therefore, the specic binding of
PhbPPs, PhaIPs, and PhaFPs to PHA granules might be
due to the difference of boundary layer structure of surrounded PHAs. Possibly, the layer structure might be
attributed to the monomer compositions of P(3HB-co3HA) copolymers. PHB/PHA synthases, PHB/PHA
depolymerases, phasins, and regulatory proteins are
known as major PHA granule-associated proteins.
These proteins are denitely important for biosynthesis
and/or degradation of PHAs. The ndings and the
observations obtained here will lead to the effective
production and the biosynthesis of PHAs, P(3HB-co3HA) copolymers, with favorable monomer compositions. For example, the P(94% 3HB-co-6% 3HA)
copolymer synthesized by the recombinant strain of
Pseudomonas sp. 61-3 is known to have properties

similar to low-density polyethylene (LDPE), and the

copolymer is a practical material for application of biodegradable plastics.37) Thus, to produce practical P
(3HB-co-3HA) copolymer with high 3HB fraction
effectively, sufcient amounts of amphiphilic PhbPPs
might have to be provided in the producing cells for
stabilization of hydrophobic PHA granules in the cells.
Therefore, further studies on PHA biosynthesis and
PHA granule formation in Pseudomonas sp. 61-3 is in
progress.

Acknowledgment
We are grateful to Dr Kenichiro Matsumoto for the
technical assistance and to NBRP (National BioResource Project, Japan) for the plasmid pBSL180 and
E. coli S17-1 (pir).

Disclosure statement
No potential conict of interest was reported by the
authors.

Funding
This work was supported by JSPS KAKENHI [grant
number 14780448], [grant number 16710054].

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