Biomacromolecules 2002, 3, 787-792
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Isolation and Characterization of Polyhydroxyalkanoates Inclusions and Their Associated Proteins in Pseudomonas sp. 61-3 Ken’ichiro Matsumoto,†,‡ Hiromi Matsusaki,§ Kazunori Taguchi,† Minoru Seki,‡ and Yoshiharu Doi*,† Polymer Chemistry Laboratory, RIKEN Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan, Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan, and Division of Food and Health Environment, Faculty of Environmental and Symbiotic Sciences, Prefectural University of Kumamoto, 3-1-100, Tsukide, Kumamoto 862-8502, Japan Received February 4, 2002; Revised Manuscript Received March 28, 2002
Two types of polyester inclusions of poly(3-hydroxybutyrate) [P(3HB)] and poly(3HB-co-3-hydroxyalkanoates) [P(3HB-co-3HA)] were isolated from crude extract of Pseudomonas sp. 61-3. Proteins associated with each inclusion were separated by SDS-PAGE. PHA synthase 1 (PhaC1Ps), PhaFPs, and PhaIPs were identified from P(3HB-co-3HA) inclusions by N-terminal amino acid sequences analyses, as well as PHB synthase (PhbCPs) and 24-kDa unknown protein were identified from P(3HB) inclusions. The structural genes of PhaFPs and PhaIPs were located downstream of the pha locus. The relative PHA/PHB synthase activities of each inclusion were measured for various 3-hydroxyacyl-coenzyme As of 4-12 carbon atoms. Direct atomic force microscopy observation of P(3HB) and P(3HB-co-3HA) inclusions demonstrated that the two types of inclusions had different morphologies. Introduction Polyhydroxyalkanoates (PHAs) are storage polyesters synthesized by various bacteria as intracellular carbon and energy reserve material.1-6 PHAs are accumulated as waterinsoluble inclusions within the cells. Much work has been performed to reveal the structure and the function of PHA inclusions.7-9 In the Ralstonia eutropha cell, there are typically 8-12 inclusions and the diameter of inclusions is 0.2-0.5 µm. The characterization by means of 13C NMR spectroscopy, X-ray diffraction, and electron microscopy revealed that native PHA inclusions remain in an amorphous and elastomeric state in vivo.10,11 Gerngross et al. have confirmed the presence of proteins in the boundary region of PHA inclusions.12 Four classes of proteins associated with PHA inclusions have been identified in bacteria: PHA synthase (I), intracellular PHA depolymerase (II), phasins (III) which probably have a function analogous to that of oleosins in oilseed plants, and other proteins (IV) which have been found to be associated with the inclusions but do not belong to classes I-III.7 In particular, the phasins are assumed to form a close protein layer at the surface of the inclusions, providing the interface between the hydrophilic cytoplasm and the much more hydrophobic core of the PHA inclusion. The structural genes of phasins, phaP, have been identified from Rhodococcus ruber,13 Ralstonia eutropha,14 Acineto* Corresponding author. E-mail:
[email protected]. Tel.: +8148-4769402. Fax: +81-48-4624667. † RIKEN Insutitute. ‡ The University of Tokyo. § Prefectural University of Kumamoto.
bacter sp.,15 Chromatium Vinosum,16 Paracoccus denitrificans,17 and Bacillus megaterium.18 In Pseudomonas putida and P. oleoVorans, two phasins, PhaF and PhaI, have been found and the corresponding genes have been cloned.19,20 Furthermore, it has been proposed that phasins act as a transcriptional regulator of PHA biosynthesis genes. In P. oleoVorans, PhaF has been proposed to repress expression of the phaC1 gene and the phaIF operon.19 PhaR of Paracoccus denitrificans is associated with PHA inclusion (unpublished data) and represses phaP expression.21 Thus, it has been suggested that some phasins not only stabilize PHA inclusions but also regulate the related genes for PHA biosynthesis. Pseudomonas sp. 61-3 is capable of producing simultaneously two types of PHAs, that is, poly(3-hydroxybutyrate) [P(3HB)] homopolymer and poly(3HB-co-3-hydroxyalkanoates) [P(3HB-co-3HA)] copolymer consisting of monomeric units with 4-12 carbon atoms.22,23 Freeze-fracture electron microscopy studies revealed that two types of PHAs are stored in separate inclusions in the same cell.24,25 However, it has not been reported which type of proteins associate with each inclusion. The regulative role of phasins in Pseudomonas sp. 61-3 is unknown. In this strain, the genes in phb and pha loci are respectively involved in P(3HB) and P(3HB-co-3HA) biosyntheses.23 The genes encoding β-ketothiolase (PhbAPs), acetoacetyl-coenzyme A (CoA) reductase (PhbBPs), PHB synthase (PhbCPs), and putative transcriptional activator (PhbRPs) of the phbBACPs genes are located in the phb locus. As for the pha locus, the genes encoding two PHA synthases (PhaC1Ps and PhaC2Ps) and intracellular PHA depolymerase (PhaZPs) have been identified.23
10.1021/bm025516k CCC: $22.00 © 2002 American Chemical Society Published on Web 04/30/2002
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In this report, we isolated two types of P(3HB) and P(3HBco-3HA) inclusions from the cells of Pseudomonas sp. 61-3 and identified six proteins associated with the inclusions. In addition, the relative synthase activities of both inclusions were measured for various 3-hydroxyacyl-CoAs of 4-12 carbon atoms, and the composition and morphologies of the inclusions have been discussed. Materials and Methods Production of PHA and Isolation of Native PHA Inclusions. Pseudomonas sp. 61-3 (JCM 10015) was grown at 28 °C in nutrient-rich (NR) medium. Cells grown in NR medium for 12 h were transferred to 500 mL flasks with 100 mL of mineral salt (MS) medium at 0.05 of initial absorbance at 600 nm and were cultivated on a reciprocal shaker (130 strokes/min) at 28 °C for 48 h.23 Glucose (2 wt %) was added to the medium as a sole carbon source. Cells were harvested by centrifugation (4000g, 10 min, 4 °C) and then washed and resuspended in 1.5 mL of 0.1 M Tris-HCl buffer (pH 7.5). Finally, the cells were disrupted by two passages through a French Press (96 MPa). 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 Tris-HCl buffer (pH 7.5). After centrifugation (210000g, 1.5 h, 4 °C), the two layers containing PHA inclusions [P(3HB) and P(3HB-co-3HA)] were isolated. PHA compositions of the inclusions were determined by gas chromatography (GC) after methanolysis of the lyophilized inclusions in the presence of 15% sulfuric acid as described previously.26 SDS-PAGE Analysis, N-terminal Amino Acid Determination and Immunoblotting. Isolated inclusions were washed twice with 0.1 M Tris-HCl buffer (pH 7.5). To 10 µL of the inclusion suspension, 10 µL of gel sample buffer was added, and the proteins were denatured and released from the inclusions by heating the suspension at 98 °C for 10 min or 60 °C for 1 h. The proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with 7.5-15% polyacrylamide gels as described by Laemmli27 and stained with a Rapid CBB KANTO kit (Kanto Chemical, Tokyo, Japan). To determine the N-terminal amino acid sequence, the proteins were blotted from the polyacrylamide gels onto poly(vinylidene difluoride) (PVDF) membranes (Immoblion-PSQ, Millipore, Bedford, MA) according to the method described by Towbin et al.28 The bands representing proteins were cut out, and the N-terminal amino acid sequences were determined by automated Edman degradation. PhbCPs (PHB synthase of Pseudomonas sp. 61-3) was detected using an anti-PhbCRe (PHB synthase of R. eutropha) antiserum and an alkaline-phosphate-antibody conjugate. The specific antibody was produced by immunizing rabbits with synthetic oligopeptides (PhbCRe 568-587 amino acids). The specificity of the rabbit anti-oligopeptide serum was examined by Western blot analysis using an E. coli-expressed PhbCRe as an antigen (data not shown). Bound antibodies were detected using nitro blue tetrazolium chloride (Kanto Chemical) and toluidine salt of 5-bromo-4-chloro-3-indolyl phosphate (Wako Chemicals, Osaka, Japan).
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Cloning and Sequence Analysis of the ORF2 and ORF3 in Pseudomonas sp. 61-3. Escherichia coli strains for DNA manipulations were grown at 37 °C in Luria-Bertani (LB) medium.29 The genes encoding 18 kDa and 36 kDa proteins associated with P(3HB-co-3HA) inclusions were found downstream of phaDPs gene on pha locus. A clone containing the genes was isolated from the genomic library of Pseudomonas sp. 61-3 using the downstream region of phaDPs gene, which was generated by PCR, as a probe. Southern blot and colony hybridization were performed as described by Southern.30 The 2.8-kb EcoRV-SacII region was completely sequenced with a 310 Genetic Analyzer (Perkin-Elmer). Preparation of 3-Hydroxyacyl-CoAs. The chemical synthesis of (R)-3-hydroxybutyryl-CoA was performed as described by Schubert et al.31 For the chemical synthesis of trans-hexenoyl-CoA, the coupling reaction of CoA (Wako Chemicals) with trans-2-hexenoic acid (Tokyo Kasei, Tokyo, Japan) was performed using Schubert’s method.31 transHexenoyl-CoA was purified using a reverse phase column (Sephasil Peptide C18 12µ ST 4.6/250, Pharmacia Biotech) by HPLC.32 (R)-3-Hydroxyhexanoyl-CoA was obtained by enzymatic conversion of trans-hexenoyl-CoA using (R)specific enoyl-CoA hydratase from Aeromonas caViae.33 A 16.7 µmol portion of trans-hexenoyl-CoA was incubated at 28 °C for 12 h in 1 mL of 0.1 M Tris-HCl buffer (pH 8.0) containing approximately 0.5 U of enoyl-CoA hydratase. (RS)-3-Hydroxyoctanoyl-CoA, (RS)-3-hydroxydecanoylCoA, and (RS)-3-hydroxydodecanoyl-CoA were obtained by enzymatic coupling of CoA with corresponding (RS)-3hydroxyalkanoic acids (Wako Chemicals) using a modified method of Kraak et al.32 These 3-hydroxyacyl-CoAs were purified using the same column by HPLC.32 Assay of PHA Synthase Activity. PHA synthase activities were determined by a modified spectroscopic assay based on the method described by Valentin and Steibu¨chel.34 The suspensions of inclusions were incubated at 30 °C in 10 mM Tris-HCl buffer (pH 7.0) containing 1 mM 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) plus 0.08 mM (R)-3-hydroxybutyryl-CoA, 0.08 mM (R)-3-hydroxyhexanoyl-CoA, 0.16 mM (RS)-3-hydroxyoctanoyl-CoA, 0.16 mM (RS)-3-hydroxydecanoyl-CoA, or 0.16 mM (RS)-3-hydroxydodecanoyl-CoA, respectively, and the time-dependent changes in the absorbance at 412 nm were recorded. The suspension of inclusion in the same buffer containing only DTNB was used as a reference. Atomic Force Microscope (AFM) Measurement. For AFM observation, the isolated PHA inclusions were washed twice with pure water and diluted 100-fold in pure water, and a 50 µL aliquot of the suspension was directly deposited onto a glass cover slip that had been cleaned by soaking in piranha solution (H2SO4:H2O2 ) 3:1). Upon standing at room temperature for 5 min, the glass cover slip adsorbed with the inclusions was rinsed gently with pure water and allowed to dry until no traces of water were visible (about 15 min). AFM analysis was performed with a SPI3800/SPA400 (Seiko Instruments Inc., Chiba, Japan) using the dynamic force mode. Pyramid-like silicon tips, made of Si3N4 mounted on 200 µm long microcantilevers with spring constants of 13 N m-1 were applied for the dynamic force mode
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Polyester Inclusions in Proteins Table 1. Compositions of Two Types of Inclusions Isolated from Pseudomonas sp. 61-3a PHA composition (mol %) PHA inclusions
3HB (C4)
3HHx (C6)
3HO (C8)
3HD (C10)
3HDD (C12)
3H5DD (C12′)
lower band upper band
100 40
1
13
25
9
12
a Cells were cultivated at 28 °C for 48 h in MS medium containing glucose (2 wt %/vol) as the sole carbon source. Key: -, not detectable; 3HB, 3-hydroxybutyrate; 3HHx, 3-hydroxyhexanoate; 3HO, 3-hydroxyoctanoate; 3HD, 3-hydroxydecanoate; 3HDD, 3-hydroxydodecanoate; 3H5DD, 3-hydroxy-cis-5-dodecenoate.
Figure 1. SDS-PAGE analysis of native PHA inclusions isolated from Pseudomonas sp. 61-3 grown on 2% glucose for 48 h at 28 °C. M: Lane 1, lower band [P(3HB) inclusions]; lane 2, upper band [P(3HB-co-3HA) inclusions].
measurements. Images were taken either at 512 × 512 or 256 × 256 pixels. Simultaneous registration was performed for height and deflection images. Nucleotide Sequence Accession Number. The nucleotide sequence data reported here will be appear in EMBL, GeneBank, and DDBJ nucleotide sequence database with accession no. AB073926. Results Analyses of PHA Inclusions and Associated Proteins. The constituents of cell extract of Pseudomonas sp. 61-3 (grown on glucose) were separated by sucrose densitygradient ultracentrifugation. Two white bands, at the interface of 0-1.0 M sucrose (upper band) and at the interface of 1.33-1.67 M sucrose (lower band), were collected. The PHA compositions of the isolated PHA inclusions were determined by GC analysis (Table 1). The upper and lower bands were found to be a P(3HB-co-3HA) copolymer and a P(3HB) homopolymer, respectively. Analyses of proteins in PHA inclusions were performed by applying the suspensions of inclusions to SDS-PAGE. As shown in Figure 1 (lane 1), separation of proteins associated with P(3HB) inclusions indicated the presence of two major bands representing proteins of 24 kDa and 48
kDa with some minor additional proteins. On the other hand, two major bands at 18 kDa and 36 kDa as well as two minor bands at 24 kDa and 70 kDa were detected on P(3HB-co3HA) inclusions (see lane 2 in Figure 1). The N-terminal amino acid sequences of these proteins were determined by automated Edman degradation. The sequence of the 24 kDa protein in P(3HB) inclusions was (M)TFFNLEKLQDAQKANLDLLQ, indicating no significant homology from database. Thus, the protein was designated as GA24. The sequence of the 48 kDa protein in P(3HB) inclusions was APFVGNQADAKGFVEDSKLD, which has 72% identity to porin D precursor of P. aeruginosa by database search. An appearance of the 48 kDa protein is possibly due to contamination of the P(3HB) inclusion fraction by membrane fraction. The sequence of minor protein at 70 kDa in P(3HB) inclusion was MDNNAHTFKTYWSGQVPFI, which was identical with the deduced amino acid sequence of PHB synthase (PhbCPs). The sequence of the 24 kDa protein in P(3HB-co-3HA) inclusions was TFFNLEKLQDAQKANLDLLQ, indicating that this protein was the same GA24 in P(3HB) inclusion. The sequences of the 18 kDa and 36 kDa proteins in P(3HB-co-3HA) inclusions were AKVSLKKEIDVQPTTLS and AGKKNTEKEGS, respectively. These two sequences corresponded to the deduced amino acid sequences of ORF3 and ORF2 on the pha locus except for the initial Met residue, respectively (see Figure 2). The sequence of the 70 kDa protein in P(3HB-co-3HA) inclusions was SNKNSDDLNRQASENTLGLN, which was identical with the deduced amino acid sequence of PHA synthase 1 (PhaC1Ps) except for initial Met residue. PHA synthase 2 (PhaC2Ps) was not able to be confirmed from P(3HB-co3HA) inclusions. According to the N-terminal amino acid sequences of the 18 kDa and 36 kDa proteins, their corresponding genes were found to be located in the downstream regions of phaDPs gene on pha locus, which had already been cloned.23 Since the gene of the 36 kDa protein had been incompletely cloned, a 2.8-kb EcoRV-SacII region was cloned in this study by Southern blot analysis using the downstream region of phaDPs gene as a probe. As a result, DNA sequence of the entire ORF2 and ORF3 was identified as shown in Figure 2. ORF2 encoded a protein of 253 amino acids with a molecular mass of 25.6 kDa, and the deduced amino acid sequence was similar to those of GA2 (67% identity, 75% similarity) of P. putida BMO120 and PhaF (70% identity, 77% similarity) of P. oleoVorans GPo1.19 ORF3 encoded a protein of 140 amino acids with a molecular mass of 15.4 kDa, and the deduced amino acid sequence was similar to those of GA1 (64% identity, 79% similarity) of P. putida BMO1 and PhaI (65% identity, 79% similarity) of P. oleoVorans GPo1. Hence, ORF2 and ORF3 were referred to phaFPs and phaIPs, respectively. The calculated molecular masses of PhaFPs and PhaIPs were lower than the estimated ones with SDS-PAGE. This phenomenon was also observed in GA1 and GA2 of P. putida BMO1.20 The deduced amino acid sequence of truncated ORF4 was very similar to ubiquinone biosynthesis methyltransferase (UbiE) of P.
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Figure 2. Organization of PHA biosynthesis genes (pha locus) of Pseudomonas sp. 61-3. Table 2. Relative PHA Synthase Activities of PHA Inclusions Isolated from Pseudomonas sp. 61-3a relative activity (%) substrate
P(3HB) inclusions
P(3HB-co-3HA) inclusions
(R)-3HB-CoA (R)-3HHx-CoA (RS)-3HO-CoA (RS)-3HD-CoA (RS)-3HDD-CoA
100
