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Journal of Bacteriology, October 2005, p. 6982-6990, Vol. 187, No. 20
0021-9193/05/$08.00+0     doi:10.1128/JB.187.20.6982-6990.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Properties of a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase with High Specific Activity (PhaZd) in Wautersia eutropha H16

Tomoko Abe,1 Teruyuki Kobayashi,1 and Terumi Saito1,2*

Laboratory of Molecular Microbiology, Department of Biological Sciences, Faculty of Science, Kanagawa University, 2946 Tsuchiya, Hiratsuka, Kanagawa 259-1293, Japan,1 Research Institute for Integrated Science, Kanagawa University, 2946 Tsuchiya, Hiratsuka, Kanagawa 259-1293, Japan2

Received 14 May 2005/ Accepted 27 July 2005


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ABSTRACT
 
A novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase (PhaZd) of Wautersia eutropha (formerly Ralstonia eutropha) H16 which shows similarity with the catalytic domain of the extracellular PHB depolymerase in Ralstonia pickettii T1 was identified. The positions of the catalytic triad (Ser190-Asp266-His330) and oxyanion hole (His108) in the amino acid sequence of PhaZd deduced from the nucleotide sequence roughly accorded with those of the extracellular PHB depolymerase of R. pickettii T1, but a signal peptide, a linker domain, and a substrate binding domain were missing. The PhaZd gene was cloned and the gene product was purified from Escherichia coli. The specific activity of PhaZd toward artificial amorphous PHB granules was significantly greater than that of other known intracellular PHB depolymerase or 3-hydroxybutyrate (3HB) oligomer hydrolases of W. eutropha H16. The enzyme degraded artificial amorphous PHB granules and mainly released various 3-hydroxybutyrate oligomers. PhaZd distributed nearly equally between PHB inclusion bodies and the cytosolic fraction. The amount of PHB was greater in phaZd deletion mutant cells than the wild-type cells under various culture conditions. These results indicate that PhaZd is a novel intracellular PHB depolymerase which participates in the mobilization of PHB in W. eutropha H16 along with other PHB depolymerases.


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INTRODUCTION
 
Poly(3-hydroxybutyrate) (PHB), a homopolymer of R(–)-3-hydroxybutyrate (3HB), is a storage material produced by a wide variety of bacteria under certain conditions (5). Intracellular PHB, which is accumulated in an amorphous state, is degraded by several hydrolases and the degraded products are used as a source of carbon and energy (36). When PHB-accumulating bacteria die, the PHB released into the environment is hydrolyzed by various microorganisms which secrete extracellular PHB depolymerases (12). In the past few decades, the application of this biopolymer to the production of biodegradable polymers/plastics has been studied extensively (26). The extracellular metabolism of PHB has been clarified in many bacteria including Ralstonia pickettii T1 and Paucimonas lemoignei as well as in some fungi (12).

The intracellular PHB mobilization system is not as well understood as the extracellular PHB degradation system. The intracellular PHB depolymerase system was first reported in Rhodospirillum rubrum in 1964 (27). The system consists of a thermostable activator and a thermolabile esterase and recent reinvestigations have lead to considerable progress being made (7, 8). The amino acid sequence of the intracellular soluble PHB depolymerase in R. rubrum (PhaZ1Rru) (8) shows similarity with the type II catalytic domain of the extracellular PHB depolymerases in bacteria such as Acidovorax sp. strain TP4 (17) which has a lipase box (G-X-S*-X-G) with an active residue at the N-terminal side. Another type of catalytic domain (type I) whose lipase box is located near the middle of the domain has been reported in R. pickettii T1 and other bacteria (14).

In Wautersia eutropha (formerly Ralstonia eutropha) H16, three types of intracellular PHB depolymerase or 3HB oligomer hydrolase genes have been cloned and some features of the gene products have been reported (19, 20, 21, 31, 32). PhaZa1Weu (formerly PhaZ1Reu) exists in PHB inclusion bodies and degrades artificial amorphous PHB (20, 30). The existence of PhaZa1Weu homologs has also been reported in W. eutropha H16 (29, 44). Additionally, enzymes with properties similar to PhaZa1Weu have been studied in other bacteria (6, 18). PhaZbWeu (formerly PhaZ2Reu) (20, 32) and PhaZcWeu (19) mainly degrade 3HB oligomers and the degradation product is 3HB monomer. Another PHB depolymerase or related hydrolase in W. eutropha H16 with different properties from these enzymes has been reported (35), but it has not been fully characterized. PhaZ1Rru of R. rubrum has no similarity with any known intracellular PHB depolymerases or 3HB oligomer hydrolases in W. eutropha H16.

In this report, a fourth novel intracellular PHB depolymerase in W. eutropha H16, PhaZd, whose amino acid sequence is similar to that of the catalytic domain of the extracellular PHB depolymerase of R. pickettii T1 was identified, and some of the properties of the enzyme were examined.


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, and culture. The bacterial strains and plasmids used in the present study are listed in Table 1. All Escherichia coli strains were grown in Luria-Bertani (LB) medium. E. coli BLR(DE3)/pLysS was used as a host cell for the recombinant plasmids carrying phaZd. E. coli S17-1/{lambda}pir was used for mobilization of the suicide vector into W. eutropha H16. All W. eutropha strains were grown in a nutrient-rich medium at 30°C as described previously (31). To produce PHB, W. eutropha cells were cultured on a minimum salt medium containing 2% (wt/vol) fructose and 0.1% (wt/vol) ammonium sulfate as described previously (16) (PHB-accumulating conditions). To induce the degradation of PHB, W. eutropha cells grown under PHB-accumulating conditions for 60 h were transferred to a minimum salt medium containing 0.5% (wt/vol) ammonium sulfate (PHB-degrading conditions) (35).


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  		TABLE 1. Bacterial strains and plasmids

Cloning of phaZd from W. eutropha H16. A candidate for the PHB depolymerase gene was identified in a BLAST search using the amino acid sequence of the catalytic domain of the extracellular PHB depolymerase of Ralstonia pickettii T1 (accession number P12625) (34) as a probe. DNA sequences of the putative intracellular PHB depolymerase genes in W. eutropha JMP134, W. metallidurans CH34, and Rhizobium solanacearum GMI1000 (accession numbers ZP_00166687, ZP_00276315, and NP_520270, respectively) were used to design a set of degenerate primers: ZdprobeF (5'-CACRCGCATGAAYGCGCTGGCCG-3') and ZdprobeR (5'-CAGGCSGTTGTAGGCCAGGAACTG-3'). All DNA manipulations were performed using standard procedures (37). Using chromosomal DNA of W. eutropha H16 as a template, these primers were employed to amplify a fragment of about 500 bp. The PCR product was labeled with 32P and used as a probe in Southern hybridization. EcoRI-digested chromosomal DNA fragments of 8 or 12 kbp were ligated to an EcoRI-digested cosmid vector, charomid 9-36 (33), and then packaged. E. coli DH5{alpha} was infected by the packaged charomid. To select a positive clone, colony PCR was performed with the degenerate primers (ZdprobeF and ZdprobeR). The DNA fragment obtained was subcloned into pBluescript II SK(+) phagemid (STRATAGENE) and sequenced.

Construction of an expression vector and purification of PhaZd from E. coli. To express phaZd, the gene was amplified by PCR with a pair of primers: PhaZd1f (5'-AGAATTCCATATGACCAAAAGCTTTGCCGCTGAC-3') and PhaZd1r (5'-TTCTCGAGTCAACGGCGGTGCTGGCTGAAG-3'). The product was digested with NdeI and XhoI, and inserted into the corresponding sites of pET23b (Novagen). The resulting plasmid was designated pE3ReZd1. To express N-terminally histidine-tagged phaZd, the gene was inserted into pET15b (Novagen) from pE3ReZd1 as described above. The resulting plasmid was designated pE3ReZd1NHis. The integrity of the nucleotide sequence of all newly constructed plasmids was confirmed by DNA sequencing.

E. coli BLR(DE3)/pLysS was transformed with pE3ReZd1 or pE3ReZd1NHis. The transformed cells were grown in LB medium with ampicillin (50 µg/ml), tetracycline (12.5 µg/ml), and chloramphenicol (34 µg/ml) at 37°C. At an optical density of 600 nm of 0.6, the culture temperature was reduced, and the gene expression was induced by the addition of isopropyl-ß-D-thiogalactopyranoside (IPTG). The culture temperature and IPTG concentration were 22°C and 1 mM or 20°C and 10 µM for pE3ReZd1 and pE3ReZd1NHis, respectively. The cultures were incubated overnight. Bacteria were harvested by centrifugation, and the cells were suspended in 20 mM Tris-HCl (pH 8.0). The resuspended cells were sonicated on ice for 4 min (20 kHz, 30 W), and centrifuged at 10,000 x g for 30 min. The supernatant of E. coli harboring pE3ReZd1NHis was applied to a Ni-chelating column (1 ml, Amersham Biosciences), and the column was washed with 20 mM sodium phosphate (pH 7.4) containing 0.5 M NaCl. PhaZd was eluted with a linear gradient of imidazole (0.04 to 0.4 M, 25 ml) in 20 mM sodium phosphate (pH 7.4) containing 0.5 M NaCl.

Construction of a phaZd deletion mutant. A part of the N-terminal (294 bp) and a part of the C-terminal end (340 bp) of phaZd were amplified by PCR. The two PCR products were connected, and then ligated into a suicide vector, pLO3 (575-bp deletion: from nucleotides 295 to 749 in phaZd) (23). The resulting plasmid was designated pO3DZd1. Using pO3DZd1, a phaZd deletion mutant was constructed as described previously (24, 38). Transconjugants were selected on minimum salt medium plates containing tetracycline (10 µg/ml) and subsequent LB plates containing 15% (wt/vol) sucrose. The mutant was confirmed based on antibiotic susceptibility, PCR, Southern hybridization, and immunological analysis. Strain H16DZd1, the phaZd deletion mutant, gave no immunostained band when the antisera against PhaZd were used.

Preparation of substrates. Semicrystalline PHB was prepared from PHB-rich W. eutropha H16 cells as described previously (39). Artificial amorphous PHB granules were prepared from the purified semicrystalline PHB granules as described by Horowitz and Sanders (11). Briefly, PHB was dissolved in chloroform, and then 0.1% (wt/vol) sodium deoxycholate was added to the dissolved PHB solution. The mixture was sonicated (20 kHz, 100 W) for 2 min. The emulsion was heated to remove chloroform and dialyzed for 24 h against 0.01% (wt/vol) sodium deoxycholate at room temperature. Native PHB was isolated from W. eutropha H16 and purified by glycerol density gradient (10). The 3HB oligomers were prepared as described previously (42). Poly(3-hydroxyoctanoate) was prepared according to previous reports (30).

Enzyme assays. PHB depolymerase activity and 3HB oligomer hydrolase activity were assayed by measuring the amount of 3HB released from PHB and 3HB oligomers, respectively. The reaction mixture (50 µl) was composed of 100 mM Tris-HCl (pH 8.5), PHB granules (0.5 mg/ml as a solid), and enzyme. The reaction was started by the addition of substrate at 30°C (20). To quantify the amount of 3HB oligomer released from PHB by the enzyme, the supernatant of the reaction mixture was treated with 1.1 U (3HB dimer-hydrolyzing activity) of 3HB oligomer hydrolase from W. eutropha H16 (PhaZbWeu) (20, 32). The amount of 3HB was measured by an enzymatic method using 3HB dehydrogenase and hydrazine hydrate as described previously (20). Hydrolytic activity toward p-nitrophenyl esters was determined spectrophotometrically and lipase activity was assayed by titration of the acid released from olive oil as described previously (45). To investigate the effect of reagents, they were added to the reaction mixture containing the enzyme, and then the mixture was incubated for 10 min at room temperature. The reaction was started by adding a substrate. To clarify whether a thermostable activator exists (7, 27), the cytosolic fraction of W. eutropha H16 cells was heated at 100°C for 10 min, and the supernatant was mixed with the assay mixture.

Immunoblot analysis. Samples were subjected to immunoblot analysis according to standard procedures using rabbit antisera against PhaZdWeu as the primary antiserum and alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G (SIGMA-ALDRICH) as the secondary antibody. The immunocomplex was visualized using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate p-toluidine. The rabbit antisera against PhaZdWeu were generated by the purified histidine-tagged protein.

Other methods. The protein concentration was measured by the method of Lowry et al. (25) with bovine serum albumin as the standard. The purity and size of the purified proteins were estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) as described by Laemmli (22). Proteins on the gel were stained with Coomassie brilliant blue R250. Sucrose density gradient centrifugation was performed as reported previously (20). PHB content was quantitated as crotonic acid by high-pressure liquid chromatography as described by Karr et al. (15). The periplasmic fraction was separated by lysozyme-EDTA treatment modified for W. eutropha H16 cells (2, 3). PHB degradation activator (apdA) (accession number AAO00724) of R. rubrum (7) was cloned by PCR. ApdA was expressed as a C-terminally histidine-tagged protein in E. coli and purified by a Ni-chelating column.


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RESULTS
 
Identification of a putative intracellular PHB depolymerase. In a BLAST search using the amino acid sequence of PhaZ1Rru, the periplasmic PHB depolymerase recently purified from R. rubrum, or some extracellular PHB depolymerases which have type II catalytic domain (Acidovorax sp. strain TP4 and Comamonas sp.) (8, 14) as references, no candidate for an intracellular PHB depolymerase from the genus Wautersia or Ralstonia was found. However, a BLAST search using the amino acid sequence of the type I catalytic domain of the extracellular PHB depolymerase in R. pickettii T1 as a probe resulted in several hits. Some bacteria of the genus Burkholderia (B. cepacia R1808, B. capacia R18194, and B, mallei ATCC23344) (43) and both W. eutropha JMP134 and W. metallidurans CH34 contained two putative open reading frames (ORFs) each (accession numbers ZP_00166687 and ZP_00170022 [W. eutropha JMP134], and ZP_00276315 and ZP_00273737 [W. metallidurans CH34]). R. solanacearum GMI1000 (NP_520270) and some other bacteria contained a similar ORF.

Although the BLAST search revealed the putative intracellular PHB depolymerase, phaZd, in several bacteria, the incidence of two putative phaZd genes in a single bacterium was limited. The identity of the deduced amino acid sequences of these ORFs with the catalytic domain of the extracellular PHB depolymerase of R. pickettii T1 was 24.1% (similarity, 52.5%) (ZP_00166687) or 27.1% (55.8%) (ZP_00170022) in W. eutropha JMP134, 24.1% (54.2%) (ZP_00276315) or 24.6% (52.3%) (ZP_00273737) in W. metallidurans CH34, and 29.6% (63.4%) (NP_520270) in R. solanacearum GMI1000. All of these bacteria had PHB synthase genes. Because these species belong to the genus Wautersia or Ralstonia, W. eutropha H16 may have a similar putative intracellular PHB depolymerase.

Among these ORFs, ZP_00166687 of W. eutropha JMP134, ZP_00276315 of W. metallidurans CH34, and NP_520270 of R. solanacearum GMI1000 showed high levels of identity and similarity. Therefore, the DNA sequences of these putative intracellular PHB depolymerase genes were used to design the degenerate primers for PCR. With these primers and a template of W. eutropha H16 genomic DNA, a product of the predicted size (about 500 bp) was amplified by PCR. The DNA sequence of the amplified fragment closely resembled that of the putative intracellular PHB depolymerase genes of the genus Wautersia (data not shown). The PCR product was used as a probe for Southern hybridization.

Cloning and sequencing of phaZd in W. eutropha H16. Using Southern hybridization and colony PCR, two positive clones, designated CharomidZdR108 and CharomidZdR112, were obtained. The different restriction maps of the two charomids indicated that these fragments were different.

One of two positive clones, CharomidZdRI08, was subcloned and 2 kbp of the PstI DNA fragment containing phaZd was sequenced. The fragment included an ORF (phaZd) of 1,089 bp and the putative gene product deduced from the ORF was composed of 362 amino acids (accession number AB206256). A typical catalytic center, a lipase box containing a putative essential serine residue (Ser190), was located in the middle of the amino acid sequence of PhaZd. The positions of the catalytic triad (Asp266 and His330) and putative oxyanion hole (His108) accorded with those in type I catalytic domain of the extracellular PHB depolymerase of R. pickettii T1 and some other extracellular PHB depolymerases such as PhaZ1 in Paucimonas lemoignei (13) (Fig. 1). No linker domain or substrate binding domain was found in the deduced amino acid sequences of the phaZd product. In addition, no signal peptide was identified by the SignalP 3.0 server (28). The other positive clone, CharomidZdR112, is still under investigation.



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  		FIG. 1. Alignment of amino acid sequence for PhaZd in W. eutropha H16 and other putative PhaZds and the catalytic domain (type I) of extracellular PHB depolymerase. PhaZdWe, PhaZd of W. eutropha H16; PhaZdJMP, putative PhaZd of W. eutropha JMP134; PhaZdWm, putative PhaZd of W. metallidurans CH34; PhaZ1Ple, catalytic domain of extracellular PHB depolymerase PhaZ1 in Paucimonas lemoignei; eDepRpT1, catalytic domain of extracellular PHB depolymerase in R. pickettii T1. The alignment was performed with ClustalW 1.8.3. The lipase box and catalytic triad are indicated by an underline and reversed characters, respectively. Boxed H and C show the histidine of the oxyanion hole and cysteines. *, conserved in all proteins; :, substitution of the amino acid residue with one of high similarity; ., substitution of the amino acid residue with one of slightly lower similarity.

Expression and purification of recombinant phaZd. E. coli harboring pE3ReZd1 (phaZd) showed strong activity to degrade artificial PHB granules, though the crude cell supernatant fraction from E. coli harboring pET23b (control) showed no activity. The proteins of the crude cell supernatant and the insoluble fraction (inclusion bodies) were analyzed by SDS-PAGE and immunological staining. Stained bands with the same molecular mass were detected in both fractions (Fig. 2B, lanes 2 and 3). The chemically determined N-terminal amino acid sequence of PhaZdWeu from inclusion bodies corresponded to the deduced amino acid sequence from phaZd, having methionine at the first position (MTKSFAADWH). The immunostained band of PhaZdWeu in the total cell extract of W. eutropha H16 was the same size as that of PhaZdWeu from E. coli harboring pE3ReZd1 (Fig. 2B, lane 1, 2, and 3). These results indicate that phaZd in W. eutropha H16 is expressed intact, as deduced from its nucleotide sequence.



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  		FIG. 2. SDS-PAGE of total protein of W. eutropha H16 and PhaZds expressed in E. coli. Coomassie brilliant blue stain (A) and immunostain (B). Lane 1, total cell extract of W. eutropha H16 cultured in PHB-accumulating conditions for 48 h (A, 5.0 µg; B, 25 µg); lane 2, crude cell supernatant of E. coli harboring pE3ReZd1 (A, 10 µg; B, 0.14 µg); lane 3, inclusion bodies of E. coli harboring pE3ReZd1 (A, 2.0 µg; B, 0.040 µg); lane 4, purified N-terminally histidine-tagged PhaZd (A, 0.22 µg; B, 3.0 ng).

To purify PhaZdWeu, histidine-tagged forms of the protein were constructed. The purified N-terminally histidine-tagged PhaZdWeu showed a single band with an apparent molecular mass of 41 kDa on SDS-PAGE (Fig. 2A and B, lane 4). The size corresponded to the molecular mass (41,349 Da) deduced from the nucleotide sequence. The purified N-terminally histidine-tagged PhaZdWeu was used for the characterization of PhaZdWeu.

Properties of PhaZdWeu. Table 2 shows the substrate specificity of the purified N-terminally histidine-tagged PhaZdWeu. Although purified PhaZdWeu degraded both artificial amorphous PHB granules and native PHB inclusion bodies, the depolymerase activity was much stronger for the former. The specific PHB-degrading activity of PhaZdWeu was 40- to 150-fold that of PhaZa1Weu, PhaZbWeu, or PhaZcWeu. The enzyme like other intracellular PHB depolymerases did not degrade semicrystalline PHB. Poly(3-hydroxyoctanoate), medium-chain-length poly(3-hydroxyalkanoate), was not degraded. Although its 3HB oligomer-hydrolyzing activity was relatively weak compared with that of PhaZbWeu or PhaZcWeu, PhaZdWeu degraded the 3HB dimer and 3HB trimer. The products of the degradation of amorphous PHB by PhaZdWeu were mainly the 3HB dimer and 3HB trimer as detected by high-pressure liquid chromatography (date not shown). The monomer was rarely detected in the hydrolyzed products of PHB and 3HB oligomers.


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  		TABLE 2. Substrate specificity of PhaZd and other intracellular PHB depolymerases in W. eutropha H16

To estimate the kinds of 3HB oligomers among the hydrolytic products of amorphous PHB generated by PhaZdWeu, a 3HB oligomer hydrolase of Acidovorax sp. strain SA1 (PhaZcAsp), which hydrolyzes mainly the 3HB dimer and 3HB trimer (41), and PhaZbWeu, which hydrolyzes 3HB oligomers from the 3HB dimer to 3HB pentamer at a similar rate (20), were used. Judging from the difference in the amount of 3HB produced by the two hydrolases, nearly 50% of the products of the degradation by PhaZdWeu were 3HB tetramers and higher 3HB oligomers, 45% were 3HB dimers and trimers, and 5% were monomers. The hydrolyzing activity of PhaZdWeu, PhaZa1Weu, PhaZbWeu, and PhaZcWeu for p-nitrophenyl esters or olive oil was examined (Table 2). PhaZdWeu hydrolyzed p-nitrophenyl acetate or p-nitrophenyl butyrate faster than other depolymerases and 3HB oligomer hydrolases. p-Nitrophenyl palmitate was not hydrolyzed. Only PhaZbWeu slightly hydrolyzed olive oil.

The effect of various reagents on the activity of PhaZdWeu was determined (Table 3). The PHB-degrading activity of PhaZdWeu was completely inhibited by 100 µM of diisopropylfluorophosphate. which is a serine hydrolase inhibitor (1), but not by 1 mM of phenylmethylsulfonyl fluoride. 1,4-Dithiothreitol (DTT) (1 mM) inhibited the depolymerase activity. The activity of PhaZdWeu inhibited by DTT was restored completely by oxidation with hydrogen peroxide (2 mM) (Fig. 3). SDS (0.1% [wt/vol]) inhibited the activity completely, but Triton X-100 (1% [wt/vol]) did not fully inhibit it. MgCl2 and CaCl2 partially inhibited the activity. Chelating by adding EDTA in an equimolar amount to these metals restored the activity completely.


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  		TABLE 3. Effect of agents on PHB depolymerase activity of PhaZd



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  		FIG. 3. Effect of DTT and hydrogen peroxide on PHB-degrading activity of PhaZd. The reaction mixture (50 µl) contained purified PhaZd (0.18 µg), 100 mM Tris-HCl (pH 8.5), and 2 mM DTT. The mixture was incubated at room temperature and then assayed for remaining activity. After 20 min, 2 mM hydrogen peroxide was added to the solution; 100% activity was equal to 110 µmol/min/mg.

The optimal pH of PhaZdWeu was 8.5 to 9.0 in Tris-HCl buffer. The optimal temperature of the enzyme was 20 to 30°C. PhaZdWeu was stable at 30°C for 10 min, but after 10 min at 40°C, more than 80% of the activity was lost. At 50°C or above, PhaZdWeu lost all activity. On long-term storage, at least 3 months, PhaZdWeu was stable in 20 mM Tris-HCl buffer (pH 8.0) containing 50% glycerol at –20°C.

The existence of a thermostable activator (7, 27) for PhaZdWeu was investigated using the boiled cytosolic fraction of W. eutropha H16. No activation of PhaZdWeu was found when native PHB inclusions or artificial PHB granules was used as a substrate. Additionally, the activator of R. rubrum (7) did not activate the PHB-degrading activity of PhaZdWeu (data not shown).

Subcellular localization of PhaZd. Since PhaZdWeu was not secreted out of the cell (data not shown), the subcellular localization of PhaZdWeu in W. eutropha H16 was examined by sucrose density gradient centrifugation (Fig. 4). PhaZa1Weu is known to localize solely in PHB inclusion bodies (20, 31) and was used as a marker of the protein in PHB inclusion bodies. 3HB dehydrogenase (3HBDH) was used as a marker of the protein in the cytosol of the cell (20). As judged from the intensity of the immunostained bands, PhaZdWeu seemed to be distributed nearly equally between the PHB inclusion bodies and the cytosolic fraction. We also separated the periplasmic fraction from the cells by lysozyme-EDTA treatment (2, 3). An immunostained band was detected in the PHB inclusion bodies and the cytosolic fraction, but not in the periplasmic fraction (data not shown).



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  		FIG. 4. Subcellular localization of PhaZd determined by sucrose density gradient centrifugation. Ten microliters of each fraction (1.1 ml) was analyzed by Western blotting and immunostaining with antiserum against PhaZdWeu, PhaZa1Weu, or 3HB dehydrogenase (3HBDH). Columns and circles show the amount of PHB and protein, respectively.

Accumulation of PHB in the phaZd deletion mutant Since PhaZdWeu efficiently degrades amorphous PHB, it probably participates in the mobilization of PHB. We constructed a phaZd deletion ({Delta}phaZd) mutant, strain H16DZd1, and compared it with the wild type in terms of the accumulation of PHB. When cultured in nutrient-rich medium (Fig. 5A), a temporary accumulation of PHB in the logarithmic phase with a rapid mobilization of PHB in the wild-type and {Delta}phaZd mutant strains was observed as reported earlier (20, 31). Although the amount of PHB reached a larger maximum and a rapid PHB accumulation was observed in the {Delta}phaZd mutant than the wild-type strain, it decreased at a similar rate in both forms (Fig. 5A).



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  		FIG. 5. Growth, PHB accumulation, and expression of PhaZd. A and C: dry cell weight (upper) and accumulation of PHB (lower). The wild-type and {Delta}phaZd mutant cells were cultured in nutrient-rich medium (A) or PHB-accumulating (C, solid line) or PHB-degrading (C, dotted line) conditions. PHB accumulation: cells were grown in minimal salt medium containing 2% (wt/vol) fructose and 0.1% (wt/vol) ammonium sulfate. PHB degradation; 60-h-cultured cells that had accumulated PHB were transferred to medium containing 0.5% (wt/vol) ammonium sulfate (arrows). Solid circle, wild type; open circle, H16DZd1 ({Delta}phaZd mutant). Results are the means for three independent measurements. The range of error bars was within ±5% of each value in the wild-type and ±10% in H16DZd1. B and D: time-dependent immunostaining analysis using the antiserum against PhaZdWeu. Asterisks show the time after the transfer. Each lane contained 50 µg of protein.

Under PHB-accumulating conditions, the {Delta}phaZd mutant accumulated about 10% (wt/dry cell wt) more PHB than the wild-type strain in the stationary phase (Fig. 5C, full line). When the cells were transferred to PHB-degrading conditions, a rapid decrease of PHB was found in both the wild-type and {Delta}phaZd mutant cells (Fig. 5C, dotted line).

PHB accumulation-dependent expression of phaZd in W. eutropha H16. The total cell extract of W. eutropha H16 grown under several sets of conditions was analyzed by Western blotting and immunostaining using antiserum against PhaZdWeu (Fig. 5B, D). In nutrient-rich medium, immunostaining analysis showed a temporary expression of phaZd in the logarithmic phase and immunostained bands of PhaZdWeu were only found until the 19th h. Under PHB-accumulating conditions, immunostained bands were found in all samples. The amount of PhaZdWeu reached a maximum after 48 h. From the intensity of the immunostained bands, the molecular ratio of PhaZa1Weu, PhaZbWeu, PhaZcWeu, and PhaZdWeu in the cell at 48 h was estimated to be 7.2:0.4:20:1.0. When the cells were transferred to PHB-degrading conditions, a very faint immunostained band was found at 72 h (12 h after the cells were transferred). No band was found after 84 h.


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DISCUSSION
 
PhaZdWeu showed unique properties different from those of known intracellular PHB depolymerases or 3HB oligomer hydrolases PhaZa1Weu, PhaZbWeu, and PhaZcWeu. Table 4 presents the properties of PhaZdWeu and other PhaZs in W. eutropha H16. Although the properties of each enzyme are different, PhaZbWeu and PhaZcWeu have an overall similarity in substrate specificity. The strong PHB-hydrolyzing activity of PhaZdWeu is a remarkable feature that is not found in other intracellular PHB depolymerases except for PhaZ1Rru in R. rubrum (8). Although PhaZdWeu showed relatively intense esterase activity toward p-nitrophenyl esters (Table 2), the ratio of PHB-hydrolyzing activity to esterase activity is about 10:1. Since no lipase activity was detected, PhaZdWeu seems to be a specific enzyme for the mobilization of PHB. The degradation product was mainly 3HB oligomers when artificial amorphous PHB granules were used as a substrate. Since only a small amount of 3HB monomer was detected in the degradation product, PhaZdWeu probably works as an endo-type depolymerase.


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  		TABLE 4. Properties of PhaZd and other intracellular PHB depolymerases in W. eutropha H16

PhaZdWeu is the second PHB depolymerase to show similarity to the extracellular PHB depolymerases. Bacterial extracellular PHB depolymerases are known to have a composite domain structure. Most of these depolymerases consist of a signal peptide, a catalytic domain, a linker domain, and a substrate binding domain from the N terminus to C terminus (14). Three types of linker domain and two types of substrate binding domain are known. In regard to the order of the active amino acids in the catalytic domain, two types of catalytic domain of extracellular PHB depolymerase are known (14). In type I (R. pickettii T1, Paucimonas lemoignei, etc.), the sequential order of these active amino acids is histidine (oxyanion hole)-serine-aspartate-histidine from the N terminus to the C terminus. Therefore, the active center (lipase box) serine is located in the middle of the catalytic domain. In type II (Acidovorax sp. strain TP4, Comamonas sp., etc.), the order is serine-aspartate-histidine-histidine (oxyanion hole). The active center is located at the N-terminal side. There is no significant similarity in amino acid sequence between type I and type II catalytic domains.

PhaZdWeu is similar to the type I catalytic domain of the extracellular PHB depolymerase of R. pickettii T1 in the positions of its catalytic triad and putative oxyanion hole (Fig. 1). On the other hand, PhaZ1Rru is similar to the extracellular PHB depolymerase having a type II catalytic domain. Another structural difference between PhaZdWeu and PhaZ1Rru is that PhaZ1Rru has a signal peptide, but PhaZdWeu does not. In R. rubrum, PhaZ1Rru localizes to the periplasm in a form lacking the signal peptide (8). PhaZdWeu from W. eutropha H16 is distributed nearly equally between the PHB inclusion bodies and the cytosolic fraction (Fig. 4), and was not found in the periplasmic fraction. In Fig. 2, the size of an immunologically stained band of PhaZdWeu in W. eutropha H16 was same to the size of PhaZdWeu from E. coli whose N-terminal portion was not cleaved.

Recently, amorphous PHB-specific extracellular PHB depolymerase was found from Paucimonas lemoignei (PhaZ7ple) (9). Although PhaZ7ple show a high PHB-hydrolyzing specific activity, the amino acid sequence was not similar to either the type I or type II catalytic domain. These structural differences are summarized in Fig. 6. Furthermore, PhaZ1Rru has been reported to be activated by a thermostable activator within the cell (7, 27). However, there seems to be no thermostable activator for PhaZdWeu in the cytosolic fraction of W. eutropha H16. PhaZdWeu was strongly inhibited by diisopropylfluorophosphate, suggesting that the active center of the enzyme is a serine residue in the lipase box (Ser190). Furthermore, DTT inhibited the activity of PhaZdWeu. These effects were similar in PhaZdWeu and PhaZ1Rru. However, PhaZ1Rru is inhibited by EDTA and slightly activated by monovalent cations such as Na+ or K+, but these effects were not found in PhaZdWeu. The temperature optimum is 50°C in PhaZ1Rru and 20 to 30°C in PhaZdWeu. Although PhaZ1Rru has strong hydrolyzing activity for both PHB and 3HB oligomers, the 3HB oligomer-hydrolyzing activity of PhaZdWeu was relatively weak. As a whole, PhaZdWeu seems to be a novel type of intracellular PHB depolymerase which is apparently different from PhaZ1Rru in R. rubrum.



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  		FIG. 6. Comparison of structures of PhaZdWeu, PhaZ1Rru, and extracellular PHB depolymerases. Reversed characters (S, D, H) show amino acid residues in the catalytic triad. An asterisk indicates the histidine of the supposed oxyanion hole. SP, signal peptide; SBD, substrate binding domain.

Although PhaZdWeu was located in the cell, it was inhibited completely by DTT. Only two cysteines (Cys110 and Cys148) exist in the amino acid sequence of PhaZdWeu. These two cysteines probably form an essential disulfide bond. DTT inhibits all extracellular PHB depolymerases as far as we know (12). From the results of experiments with PhaZdWeu, we may specify an essential disulfide bond in the extracellular PHB depolymerase of R. pickettii T1 which has 4 cysteine residues in the catalytic domain (Fig. 1). Cytoplasmic proteins do not generally contain structural disulfide bonds, although certain cytoplasmic enzymes form such bonds as part of their catalysis (4, 40). The activation or inactivation of PhaZdWeu may be regulated by oxidation-reduction in vivo. Alternatively, the disulfide bond of PhaZdWeu may be required for the association of PhaZdWeu with highly hydrophobic PHB inclusion bodies to maintain the structure of PhaZdWeu, or the structure of the type I catalytic domain of the extracellular PHB depolymerase may be simply conserved.

Immunostaining showed that there was less PhaZdWeu in the cell than other intracellular PHB depolymerases or 3HB oligomer hydrolase except for PhaZbWeu in W. eutropha H16. Although PhaZdWeu distributed nearly equally between PHB inclusion bodies and the cytosolic fraction, the enzyme in the cytosol may not participate in the mobilization of PHB considering its relatively weak 3HB oligomer hydrolase activity. More detailed study is necessary to elucidate why the enzyme is also found in the cytosol. A comparison of PHB content in the cell revealed a clear difference between the wild-type and {Delta}phaZd mutant in the PHB-accumulating period. In nutrient-rich medium, the difference was also observed during the temporary accumulation of PHB. Since the rate of decrease after the accumulation of PHB is almost the same in both the wild-type and {Delta}phaZd mutant, PhaZdWeu seems not to participate in the rapid degradation of PHB (Fig. 5A). As a rapid decrease in the amount of PHB was also found in both the wild-type and {Delta}phaZd mutant cells under PHB-degrading conditions (Fig. 5C, dotted line), again the enzyme seems not to participate in the rapid degradation induced by the addition of ammonium sulfate.

The difference in PHB content between the wild-type and {Delta}phaZd mutant well matched the time-dependent analysis of PhaZdWeu (Fig. 5B), in which PhaZdWeu was not detected in the degradation phase. Both the accumulation and degradation of PHB have been reported to occur at the same time under certain conditions in vivo (16). Expression of PhaZdWeu was observed in PHB-accumulating conditions. These results may indicate that PhaZdWeu controls the amount of PHB in the cell.

PhaZa1Weu, PhaZbWeu, PhaZcWeu, and PhaZdWeu differed in substrate specificity and subcellular localization (Table 4). PhaZa1Weu and PhaZdWeu were found in the PHB inclusion bodies. They may participate in the degradation of PHB in the cell. PhaZbWeu probably participates also in the degradation of PHB, because a synergistic effect for PhaZa1Weu has been reported (20). Although PhaZcWeu also has weak PHB-hydrolyzing activity, most PhaZcWeu occurs in the cytosolic fraction (19). Therefore, PhaZcWeu probably mainly hydrolyzes 3HB oligomers produced by other depolymerases during the mobilization of PHB together with PhaZbWeu. Since PhaZdWeu had high specific activity for PHB and hardly hydrolyzed 3HB oligomers at all, the enzyme may specifically degrade the PHB within the cell. PHB depolymerase activity in the cytosolic fraction in W. eutropha H16, which is different from PhaZbWeu and PhaZcWeu, has been suggested (35). PhaZdWeu probably contributes to the PHB depolymerase activity in the cytosolic fraction in W. eutropha H16.

A BLAST search indicated the existence of another phaZd-like hydrolase (PhaZd homolog) in W. eutropha JMP 134 and in W. metallidurans CH34. In this study, two positive fragments from W. eutropha H16 were obtained by Southern hybridization. Clarification of the other PhaZdWeu is required. Analysis of the other positive clone obtained by Southern hybridization is in progress.


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ACKNOWLEDGMENTS
 
This study was supported in part by a grant-in-aid for the High-Tech Research Center Project (2002) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

We thank M. Shiraki and M. Takanashi for their advice and A. Sugiyama for 3HB oligomers.


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratory of Molecular Microbiology, Department of Biological Sciences, Faculty of Science, Kanagawa University, 2946 Tsuchiya, Hiratsuka, Kanagawa 259-1293, Japan. Phone and fax: 81-463-59-8673. E-mail: 43saito-bio{at}kanagawa-u.ac.jp. Back


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Journal of Bacteriology, October 2005, p. 6982-6990, Vol. 187, No. 20
0021-9193/05/$08.00+0     doi:10.1128/JB.187.20.6982-6990.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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