Polymer Degradation and Stability 83 (2004) 281–287

www.elsevier.com/locate/polydegstab

Direct observation of polyhydroxyalkanoate

granule-associated-proteins on native granules and on
poly(3-hydroxybutyrate) single crystals by atomic force microscopy
Kumar Sudesha,*, Akira Maeharab, Zhihua Ganb, Tadahisa Iwatab, Yoshiharu Doib,c
a
School of Biological Sciences, Universiti Sains Malaysia 11800, Penang, Malaysia
b
Polymer Chemistry Laboratory, RIKEN Institute, Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan
c
Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta,
Midori-ku, Yokohama 226-8502, Japan

Received 22 April 2003; accepted 5 June 2003

Abstract
Polyhydroxyalkanoate (PHA) granules isolated from Comamonas acidovorans revealed the presence of globular particles on the
granule surface when imaged using atomic force microscopy (AFM). The globular particles appeared to form a monolayer on the
granule surface. Repeated washing with pure water resulted in granules with smooth surface without any globular particles. Purified
granule-associated-proteins and PHA single crystals were used to construct a model representing native granule surface for AFM
imaging. For this purpose, PhaR and PhaP proteins, purified from Paracoccus denitrificans were adsorbed onto poly(3-hydro-
xybutyrate) [P(3HB)] single crystals. PhaR is a 22 kDa protein involved in the regulation of PhaP (phasin) protein (16 kDa). Both
purified proteins were morphologically different but adsorbed uniformly on the single crystal surface forming a monolayer.
Immunogold-labeling was used to further confirm the adsorption and identity of PhaR protein on P(3HB) single crystals. The study
shows for the first time, that proteins associated with PHA granules can be imaged directly and characterised using AFM. In
addition, AFM images obtained in this study provide direct evidence for the binding of PhaR and PhaP proteins to the hydro-
phobic PHA surface.
# 2003 Elsevier Ltd. All rights reserved.
Keywords: Polyhydroxyalkanoate (PHA) granules; Atomic force microscopy (AFM)

1. Introduction In a previous study we have shown that AFM offers

Atomic force microscopy (AFM) [1] offers a novel
method of imaging for biologists to study the surface
architecture of cells and cellular components. The abil-
ity of AFM to provide real-time three-dimensional
images under natural conditions with molecular resolu-
tion has been used to obtain images of various samples
[2–4]. Besides providing high resolution images of sur-
face topography, AFM can also be used to obtain force
measurements to probe the physical properties of sam-
ples, such as molecular interactions and mechanical
properties [5].
* Corresponding author. Tel.: +60-4-6533888; fax: +60-4-
6565125.
E-mail address: ksudesh@usm.my (K. Sudesh).
0141-3910/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0141-3910(03)00273-8
a novel approach to study the surface structure of
polyhydroxyalkanoate (PHA) granules [6]. Earlier
attempts to elucidate the native surface structure of
PHA granules using conventional microscopic techni-
ques have proven to be inconclusive. This is because
preparation of the PHA granules for characterisation
using electron microscopy usually involves staining and
dehydration. These treatments, especially dehydration,
can greatly affect the native structure of PHA granules
(author’s unpublished observation).
PHAs are high molecular weight carbon and energy
reserve materials that exist in the form of water-inso-
luble granules in the cell cytoplasm of many micro-
organisms. Although most PHAs exhibit high
crytallinity when extracted from the cells, in vivo the
granules are intriguingly maintained in an amorphous
state [7,8]. Besides the enzymes involved in the bio-
282 K. Sudesh et al. / Polymer Degradation and Stability 83 (2004) 281–287
synthesis (PHA synthase) and mobilization of PHAs
(intracellular PHA depolymerases), the native granules
are associated with several kinds of proteins termed
phasins (PhaP), that are important in the formation and
stabilisation of the granules [9]. These proteins are
located on the granule surface [10,11]. However, it is not
known whether the proteins form a continuous layer
around the PHA granules, thus creating a boundary
between the hydrophobic granule and the hydrophilic
cytoplasm.
Interest in the surface architecture of native PHA
granules has been gaining attention in recent years.
Although it is known that several phasin species are
bound to the granule surface their function(s) still
remain elusive [12]. Phasins have been shown to influ-
ence the size and number of PHA granules in the cell
[13–15]. For the binding of these phasins to the PHA
granules, two different models have been proposed
based on the phasins amino acid sequence information.
The first model assumes the presence of a phospholipid
monolayer to which the phasins are anchored [16,17]. In
the second model, it was proposed that the phasins are
directly bound to the PHA granules because all of them
contain at least one markedly hydrophobic region [18].
Despite the many uncertainties in their actual function,
phasins clearly affect PHA biosynthesis and accumula-
tion in the cell [19]. Recent studies have shown that
some phasins are regulated by specific DNA-binding
proteins that can also bind to the PHA granules [20–22].
PhaR has been identified as the protein involved in the
regulation of phasin expression. It has been proposed
that PhaR derepresses phaP expression by binding to
the PHA granule [20]. PhaR is therefore the first class
IV granule-associated-protein [9] to be identified and
characterised at the molecular level.
The primary interest of this study is to show that
AFM can be used to observe directly and characterise
proteins associated with native PHA granules. AFM is
probably the best imaging tool for this purpose because
of the minimum sample pretreatment that is required
and also because AFM in the tapping mode is gentle
enough to avoid sample damage. Evidence has been
obtained to show that the proteins associated with PHA
granules are capable of forming a continuous mono-
layer on PHA granules and single crystals. The finding in
this study strongly supports the idea that PhaR protein
binds directly to the hydrophobic PHA granule surface.
2. Materials and methods
2.1. Biosynthesis, purification and adsorption of PHA
granules
Detailed information on the biosynthesis, purifica-
tion and adsorption of the PHA granules have been
described in our recent publication [6]. Briefly, the
PHA granules used in this study was isolated from
Comamonas acidovorans (JCM 10181) that was culti-
vated in the presence of 1% (wt./vol.) sodium pentano-
ate. Gas chromatography analysis revealed that the
PHA is a copolymer of poly(3-hydroxybutyrate-co-75
mol% 3-hydroxyvalerate) [P(3HB-co-75 mol% 3HV)].
Crude cell lysate of C. acidovorans was prepared by
passage of the harvested cells through a French pressure
cell twice at 96 MPa. PHA granules were purified by
centrifuging the lysate on a discontinuous sucrose den-
sity gradient (210,000 g, 90 min, 4  C). The purified
granules were washed once with 0.1 M Tris–HCl (pH 7.4)
before adsorption onto a glass cover slip. The glass cover
slips used in this study were layered with ultrathin poly-
cationic film for firm adsorption of the PHA granules.
2.2. Preparation of P(3HB) single crystals
Detailed description of the P(3HB) single crystal pre-
paration is available elsewhere [23]. The method was
derived from that of Marchessault and coworkers [24].
2.3. Purification of PhaR, PhaP proteins and preparation
of antibodies
PhaR (22 kDa) protein was purified from recombi-
nant Escherichia coli BL21 (DE3) cells overexpressing
the phaR gene while PhaP (16 kDa) protein was pur-
ified from E. coli XL1 Blue (pPDPK1.7) cells over-
expressing the phaP gene [15,25]. Antibodies raised
against the PhaR protein were prepared in rabbit as
described previously [15]. Anti-rabbit immunoglobulin
G (IgG) (whole molecule) gold conjugate (10 nm nom-
inal particle) was purchased from Sigma Chemical
Company (St. Louis, Mo.). Four microlitres of the
purified PhaR (and PhaP) protein (0.2 mg/ml) were
mixed with a 1 ml suspension of P(3HB) single crystals
(0.4 mg/ml) in 50 mM Tris–HCl buffer (pH 7.0). Fol-
lowing a 30 min incubation at 37  C to allow the pro-
teins to adsorb on to the P(3HB) single crystals, the
mixture was washed three times by centrifugation with
Buffer 1 (0.1 M maleic acid, 0.15 M NaCl, pH 7.5)
supplemented with 1% skim milk and 0.5 M NaCl.
The washed P(3HB) single crystals were then resus-
pended in 1.0 ml of the same buffer mixture before
adding 10 ml antiserum against PhaR and incubating at
room temperature for 3 h. To remove unbound anti-
serum, the P(3HB) single crystals were washed once
with the buffer mixture, followed by twice with Buffer
1 containing only 0.5 M NaCl and finally once with
only Buffer 1. The single crystals were then resus-
pended in 200 ml Buffer 1 added with 0.1% poly-
ethylene glycol (PEG) (Mw=20,000). Ten microlitres of
anti-rabbit IgG gold conjugate were then added to the
mixture and incubated at room temperature for 30 min.
 K. Sudesh et al. / Polymer Degradation and Stability 83 (2004) 281–287
The gold labelled preparation was washed once with
Buffer 1 containing 0.05% PEG, once with Buffer 1
containing 0.02% PEG, and finally resuspended in
Buffer 1 containing 0.02% PEG for deposition on the
silicon substrate.
2.4. Atomic force microscopy observation
AFM analysis was performed with an SPI3800/
SPA400 (Seiko Instruments Inc.) using the tapping
mode. Pyramid-like silicon tips, made of Si3N4 mounted
on 200 mm long microcantilever with spring constants of
13 Nm 1 were used for the dynamic force mode mea-
surements. Simultaneous registration was performed for
height (topographic) and deflection images. Although
the deflection image could not give any quantitative
height information, it is a useful mode to reveal height
differences in the sample surface, especially when the
sample surface is rough.
3. Results and discussion
3.1. Globular particles on the PHA granule surface
In a previous study, we have tested various substrates
(glass cover slip, glass cover slip coated with poly-
anionic film and/or polycationic film, silicon, freshly
cleaved mica) to adsorb the freshly isolated native PHA
granules [26]. The granules have to be firmly adsorbed
to a flat substrate in a gentle manner, for scanning by
the AFM tip. PHA granules were successfully imaged
on all the tested substrates. However, for repeated, long
term observation, PHA granules adsorbed on glass
cover slips coated with polycationic ultrathin film
proved superior [6]. Fig. 1 shows AFM images of
P(3HB-co-3HV) granule adsorbed on glass cover slip
coated with polycationic film. The granule is char-
acterised by the presence of globular particles on its
surface (Fig. 1A). Such globular particles were not
observed on the polyelectrolyte film surface, suggesting
that the particles bind preferentially to the P(3HB-co-
3HV) granule. Fig. 1B is a higher magnification AFM
deflection image of the globular particles on the granule.
The magnified image shows that the globular particles
are adsorbed homogeneously, almost forming a con-
tinuous layer on the granule surface.
To this end, the adsorbed granules have not been
subjected to repeated rinsing with pure water (MiliQ).
The AFM deflection image in Fig. 1C shows the surface
structure of a P(3HB-co-3HV) granule that was rinsed
several times using a gentle stream of pure water. This
treatment resulted in the disappearance of the globular
particles, revealing a rather smooth surface structure.
We have obtained evidence to show that this smooth
surface structure is the characteristic of all amorphous
283
Fig. 1. (A) AFM deflection images of P(3HB-co-3HV) granule iso-
lated from C. acidovorans showing the presence of globular particles
on the granule surface. (B) Close up observation of the globular par-
ticles in (A). (C) P(3HB-co-3HV) granule that has been subjected to
repeated washing with water, showing the absence of globular par-
ticles. (D) AFM height image of the globular particles in (B). Inset
showing the apparent size of a globular particle in (D).
PHA granules prior to the onset of crystallisation
(manuscript in preparation). The smooth surfaced,
washed PHA granule in Fig. 1C eventually developed
crystal-like lamellar structures as observed in a previous
study [6]. It should be noted that the PHA granules are
adsorbed very firmly on the polycationic film and can
withstand repeated washing. However, the washing
process can sometime result in granule deformation
which is characterized by an apparent orientation of the
adsorbed granules in the direction of the water stream
used for washing [6].
Proteins associated with PHA granules are bound
quite firmly to the granule surface. This is based on the
observation that SDS-PAGE analysis of washed gran-
ules reveals the firmly adsorbed proteins. In this study,
we found that the globular particles on the P(3HB-co-
3HV) granule surface could be removed by repeated
washing with pure water. The relative ease at which the
globular particles could be removed by gentle washing
maybe because the granules exist singly on the substrate
and not in bulk as is in the preparation for SDS-PAGE
analysis. In a separate study we have treated freshly
isolated PHA granules with aqueous acetone, which
also resulted in the removal of surface particles. In
addition, acetone treatment also resulted in the swelling
284 K. Sudesh et al. / Polymer Degradation and Stability 83 (2004) 281–287

of PHA granules and release of polymer microfibrils

[27]. Similar acetone treatment of some PHA granules
[poly(4-hydroxybutyrate)] resulted in granules with very
smooth surface structure initially, but these granules
soon showed high tendency to crystallize (unpublished
observation).
Many of the globular particles on the P(3HB-co-3HV)
granule showed an apparent lateral dimension of about
31 nm across as is shown in the inset of Fig. 1D. Smaller
globular particles could also be observed. Such globular
particles were also observed in a previous study,
although at a much reduced concentration [6]. In the
previous study the adsorbed granules were washed sev-
eral times with pure water, which explains the reduction
in globular particle concentration on the granule sur-
face. At this stage, it is not known if the globular parti-
cles represent individual protein molecules or complexes
consisting of several protein molecules. However, it is
known that the lateral dimension determined by AFM Fig. 2. (A) AFM deflection image of a P(3HB) single crystal on
is subjected to magnification effect caused by tip geo- silicon substrate. (B) Close up deflection image showing the surface
metry [28]. Further AFM observations were therefore structure of P(3HB) single crystal. (C) AFM height image of a
P(3HB) single crystal. Inset showing the height profile of the P(3HB)
conducted using nanostructures with known dimensions
single crystal in (C).
to determine the extent of magnification in this study.

3.2. Globular particles of PhaR proteins on P(3HB)

single crystals

In order to determine further the ability of AFM to

image granule-associated-proteins and to estimate their
sizes, we have used well known nanostructures such as
P(3HB) single crystals as substrates for the adsorption
of purified granule-associated-proteins. In recent years,
AFM has gained popularity as an additional tool to
study the enzymatic degradation of PHAs [23,29].
P(3HB) single crystals have been widely used as a model
substrate for this purpose. Fig. 2A shows the AFM
deflection image of a P(3HB) single crystal on a silicon
substrate. At higher magnification (Fig. 2B), the surface
of the single crystal appeared rather smooth under the
imaging conditions used in this study. Fig. 3C shows the
AFM height image of the P(3HB) single crystal. The
inset gives the height profile at the location indicated by
dotted lines in Fig. 3C. The lamellar thickness of the
Fig. 3. (A) AFM deflection image of PhaR proteins adsorbed on
P(3HB) single crystal used in this study is approximately
P(3HB) single crystals. (B) Close up observation of the globular PhaR
5 nm. The morphology and dimensions of P(3HB) sin- proteins on P(3HB) single crystal. (C) AFM height image of (B). Inset
gle crystals observed in this study are in close agreement showing the height profile of P(3HB) single crystal adsorbed with
with previously reported observation using contact PhaR proteins.
mode AFM [23].
Based on current knowledge of native PHA granule esis and binds to the intracellular PHA granules [20]. In
surface, we attempted to construct a possible model of contrast to the smooth surface of P(3HB) single crystals
the granule surface for AFM observation. For this pur- in Fig. 2, the single crystals are now characterized by a
pose, we adsorbed purified granule-associated-proteins surface covered with globular particles (Fig. 3A). At
onto the P(3HB) single crystals. Fig. 3 shows the AFM higher magnification (Fig. 3B), it can be seen that the
images of P(3HB) single crystals adsorbed with PhaR globular particles are adsorbed homogeneously, almost
protein. PhaR protein has been shown to be a reg- forming a monolayer on the single crystal surface.
ulatory protein that indirectly controls PHA biosynth- Fig. 3C shows the AFM height image of Fig. 3B and its
 K. Sudesh et al. / Polymer Degradation and Stability 83 (2004) 281–287
height profile is shown in the inset. The thickness of the
P(3HB) single crystal together with the monolayer of
PhaR proteins is now 12.5 nm. Considering that the
PhaR proteins would adsorb homogeneously on the top
as well as on the bottom surfaces of the P(3HB) single
crystals, the PhaR proteins had formed a layer of
approximately 4 nm thick on each side. In a previous
study, a 14 kDa PHA granule-associated-protein of
Rhodococcus ruber was shown to be a globular protein
with a diameter of 3 nm using electron microscopy [17].
To further confirm that the globular particles on the
P(3HB) single crystals are indeed PhaR proteins,
immunogold labeling against the PhaR protein was
performed. Fig. 4A and B show the AFM deflection and
height images, respectively, of immunogold labeled
PhaR proteins on P(3HB) single crystal. As expected,
the immunogold labels were detected on the P(3HB)
single crystals. This was confirmed by transmission
electron microscopy, where it was observed that the
gold particles were distributed homogeneously on the
Fig. 4. (A) AFM deflection image of immunogold-labeled PhaR pro-
teins on P(3HB) single crystal. (B) AFM height image of (A). (C)
AFM deflection image of immunogold complex on silicone substrate.
(D) AFM height image of (C). Inset (top) showing the apparent lateral
size of gold particles on the complex surface. Inset (bottom) showing
the height profile and apparent lateral size of immunogold complex.
Gold particle in the complex centre is clearly distinguishable by its
regular height and shape.
285
single crystal surface (results not shown). It is interest-
ing to note that the globular particles now seen on the
P(3HB) single crystals are very regular not only in size
but also in shape. Such particles can also be observed
scattered on the silicon substrate albeit at a much
reduced concentration (Fig. 4A). Higher magnification
of the globular particles on the substrate (Fig. 4C)
shows that each globular particle is identical in size and
shape (inset at the bottom). These identical structures
were interpreted as individual IgG–gold complexes. At
higher magnification it was clear that the globular
structures on the P(3HB) single crystal were the same as
those on the silicon substrate (results not shown).
The average height of the globular particles on the
silicon substrate was about 14 nm (inset at the bottom).
This is the combined height of gold particle (10 nm) and
the IgG protein. The IgG protein in this case appears as
the flattened structure around the gold particle (Fig. 4C).
The highest point in the IgG complex is interpreted as
the 10 nm gold particle. Colloidal gold particles have
been used as incompressible imaging standards for
AFM [28]. They are known to give constantly accurate
height value when measured by AFM. However, the
lateral dimension of the gold particle is affected by the
AFM tip geometry. The magnification effect of AFM is
a well-known phenomenon that needs to be considered
especially when measuring objects smaller than the tip
size. Using the imaging conditions in this study, the
apparent lateral dimension of the 10 nm gold particles
was approximately 29 nm (inset at the top in Fig. 4).
On the other hand, the apparent lateral dimension of a
single IgG–gold complex was approximately 65 nm
(inset at the bottom in Fig. 4). Repeated scanning of
the IgG–gold complex on the silicon substrate resulted
in structural deformation of the IgG protein but the
gold particle constantly gave similar height values
(results not shown).
3.3. Globular particles of PhaP proteins on P(3HB)
single crystals
We were also able to image another granule-asso-
ciated-protein, PhaP (16 kDa), purified from P. deni-
trificans. Fig. 5 shows the AFM deflection images of
PhaP protein adsorbed on P(3HB) single crystals. The
PhaP protein like PhaR, adsorbed homogeneously on
the single crystals (Fig. 5A). Comparison of the image
in Fig. 5A with that in Fig. 3B, shows that both proteins
are morphologically different. The globular particles of
PhaP protein appear relatively smaller than those of
PhaR protein. Both proteins however seem to form a
continuous monolayer on the P(3HB) single crystal.
Recent studies have shown that phaP expression is
repressed by the binding of PhaR to a region upstream
of phaP. When P(3HB) granules start to form in the
cell cytoplasm, PhaR is titrated from phaP, possibly by
286 K. Sudesh et al. / Polymer Degradation and Stability 83 (2004) 281–287
Fig. 5. (A) AFM deflection image of PhaP proteins adsorbed on
P(3HB) single crystals. (B) Close up image of the globular PhaP pro-
teins on P(3HB) single crystal.
binding to the P(3HB) granules [22]. Strong evidence
for the interaction of PhaR to amorphous as well as
crystalline P(3HB) granules was also presented [20]. The
present AFM study has provided conclusive evidence
for the direct binding of both PhaR and PhaP proteins
to the hydrophobic surface of P(3HB). The results also
indicate that both PhaR and PhaP interact directly with
the hydrophobic PHA material without any need for
additional factors in the bacterial cell.
4. Conclusion
To the best of our knowledge, this is the first study to
show that proteins associated with native PHA granules
can be visualised directly using AFM. We were able to
obtain three-dimensional images of protein particles on
native PHA granules as well as on P(3HB) single crystals.
The results have clearly shown that granule-associated-
proteins are capable of binding directly to the hydro-
phobic PHA material to form a continuous monolayer.
With the help of immunogold-labeling, it should be pos-
sible to map the location and distribution of various
granule-associated-proteins more precisely. Direct visua-
lisation would enable us to understand in more detail the
molecular mechanism of PHA granule formation, mobi-
lization and the regulatory machinery involved.
Acknowledgements
This work was supported in part by Ecomolecular
Science Research (RIKEN Institute). KS acknowledges
financial support from the Fundamental Research
Grant Scheme of Universiti Sains Malaysia (304/PBIO-
LOGI/670038).
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