1 Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstrasse 3, 48149 Münster, Germany
2 Genomik Netzwerk Göttingen, Georg-August-Universität Göttingen, Grisebachstrasse 8, 37077 Göttingen, Germany
3 Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, Grisebachstrasse 8, 37077 Göttingen, Germany
4 Institut für Biologie, Humboldt-Universität Berlin, Chausseestrasse 117, 10115 Berlin, Germany
Correspondence
Alexander Steinbüchel
steinbu{at}uni-muenster.de
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ABSTRACT |
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The GenBank accession numbers for the nucleotide sequences reported in this paper are AY489113 (Ralstonia eutropha H16 phaP3), AY489114 (phaP4) and AY489115 (phaZ5).
The following are available as supplementary data with the online version of this paper at http://mic.sgmjournals.org: an alignment of the amino-acid sequences of phasin homologues of R. eutropha H16 PhaP1 in supplementary Fig. I; the regions adjacent to phaP1, phaP2, phaP3 and phaP4 in the R. eutropha H16 genome in supplementary Fig. II; the alignment of multiple Re1052 digestion fragments with the proposed H16 sequence in supplementary Fig. III; the molecular masses of R. eutropha phaP1 knock-out mutant Re1052 granule proteome fragments by MALDI-TOF in supplementary Table I; the similarities of the five phaZ homologues of R. eutropha in supplementary Table II; the similarities of the four phaP homologues of R. eutropha in supplementary Table III.
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INTRODUCTION |
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Since the PHA operon, comprising the genes for -ketothiolase (phaA), acetoacetyl-CoA reductase (phaB) and PHA synthase (phaC), was cloned during the 1980s, many other genes involved in PHA metabolism have been identified, and the functions of the encoded proteins at least partially analysed. Whereas the PHA synthase encoded by phaC is indispensable for PHA and PTE biosynthesis in R. eutropha, PhaA and PhaB can be replaced by isoenzymes. A prominent example is BktB, which is only one of many
-ketothiolases in this bacterium (Slater et al., 1998
). In addition to PHA synthase, PHA depolymerases (PhaZ) are also bound to the PHA granule surface. Only recently, three intracellular PHA depolymerases (PhaZ1, PhaZ2 and PhaZ3) and a 3HB-oligomer hydrolase (previously also designated PhaZ2), which hydrolyse PHAs and the cleavage products produced by PhaZs, were cloned and characterized in R. eutropha (Saegusa et al., 2001
, 2002
; Kobayashi et al., 2003
; York et al., 2003
). However, in contrast to the extracellular degradation of PHAs (Jendrossek et al., 1996
, 2002
; Jendrossek & Handrick, 2002
), relatively little is known about intracellular mobilization and degradation in PHA-accumulating cells (Handrick et al., 2000
; Saito & Kobayashi, 2002
).
The layer at the surface of the PHA granules contains not only the enzymes mentioned above, but also phospholipids and other proteins, with the phasin PhaP1 as the predominant protein. Phasins occur in any PHASCL-accumulating bacterium, and are analogues of oleosins, which are bound to the surface of the oleosome in plants (Wieczorek et al., 1995; Steinbüchel et al., 1995
; Wieczorek & Steinbüchel, 1996
; Mayer & Hoppert, 1997
; Hanley et al., 1999
). Hitherto, only one phasin (PhaP1) has been known to occur in R. eutropha. Recently, a gene encoding a protein exhibiting similarity to PhaP1 was identified on the megaplasmid of R. eutropha H16 (Schwartz et al., 2003
). In this study, we describe the occurrence of two additional paralogous and orthologous phasin genes in R. eutropha. We show that all four genes are expressed, and that the proteins are synthesized when the cells accumulate PHAs. Since all previous models for the structure of PHA granules and metabolism of PHAs in R. eutropha were based on the knowledge of only a single phasin, awareness of at least four phasin proteins in this bacterium will be important for PHA research and to adapt the models for the structure of PHASCL granules (Steinbüchel et al., 1995
; Jurasek et al., 2001
; Jurasek & Marchessault 2002
; Dennis et al., 2003
and references cited therein). Moreover, R. eutropha is not exceptional regarding the occurrence of multiple phasins, and this study will show that other PHASCL-accumulating bacteria also possess multiple copies of phasin genes. Therefore, the data presented in this study are of general relevance.
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METHODS |
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Transfer of DNA.
Competent cells of E. coli were prepared and transformed by the CaCl2 procedure (Hanahan, 1983).
Cloning of phaP2 and purification of recombinant His6-tagged PhaP2 from recombinant E. coli.
For cloning of phaP2 into E. coli, PCR was done by using phaP2His_NdeI_fw as sense and phaP2His_XbaI_rv as reverse primers (Table 1). They were deduced from the upstream and downstream regions, respectively, of phaP2 of R. eutropha H16 (Schwartz et al., 2003
). The phaP2His6 PCR product obtained was purified and ligated into pMa/c5-914 (Table 1
), which harbours the cI857ts gene encoding the temperature-sensitive
repressor. The recombinant His6-tagged PhaP2 (N-terminal fusion) was purified from E. coli TOP10 harbouring pMa/c5-914 : : phaP2His6. Protein purification was carried out under non-denaturing conditions, employing Ni2+-NTA-Spin columns (Qiagen), as described by the manufacturer.
PCR amplification.
All PCR amplifications of DNA were carried out as described by Sambrook et al. (1989), employing Pfx-DNA-polymerase (Invitrogen) and an Omnigene HBTR3CM DNA thermal cycler (Hybaid). All PCR primers used in this study are listed in Table 1
.
Isolation of total RNA and RT-PCR.
Total RNA was isolated from 1x109 cells in the stationary growth phase, which had been cultivated for 72 h in MM under storage conditions (0·02 %, w/v, ammonium chloride). Cells were lysed by lysozyme treatment, and lysates were subjected to the RNeasy RNA purification kit (Qiagen). DNA-free total RNA was obtained after on-column DNase I treatment and elution, as described by the manufacturer. Total RNA was analysed by agarose gel electrophoresis to confirm RNA digestion. In order to qualitatively analyse the phaP2-, phaP3- and phaP4-derived mRNA, RT-PCR was applied, using the oligonucleotides shown in Table 1 as primers. One-step RT-PCR (One Step RT-PCR kit; Qiagen) was carried out according to the manufacturer's protocol, employing 0·5 ng RNA as template. In order to exclude any contaminating DNA that might have served as a template for PCR, template RNA was added in a control experiment, after inactivating the reverse transcriptase and activating Taq-polymerase. Absence of PCR products in the control indicated that the RT-PCR products were not derived from contaminating DNA. The PCR products were digested with suitable restriction endonucleases to verify the occurrence of the correct fragments. Amplified PCR products were resolved on a 2 % (w/v) agarose gel and stained with ethidium bromide.
Isolation of native PHA granules.
Poly(3HB) granules were isolated by a modification of the method of Preusting et al. (1993) from cells of R. eutropha, which had been grown in MM under storage conditions. After 72 h incubation, cells of 200 ml cultures were harvested by centrifugation (20 min, 6000 g, 4 °C). The cells were washed and resuspended in 10 ml potassium phosphate (KP) buffer (100 mM, pH 7·5) and, after threefold French press passage (100x106 Pa), 5 ml of the lysate was loaded on the top of a linear glycerol gradient. This gradient was obtained from a discontinuous gradient prepared from 1 ml 90 % (v/v) and 4 ml 50 % (v/v) glycerol in KP buffer. After centrifugation (2 h, 210 000 g, 4 °C), a granule layer was obtained at about 90 % (v/v) glycerol. The granules were isolated and washed with KP buffer by centrifugation (10 min, 100 000 g, 4 °C). The granules were resuspended in KP buffer and subsequently loaded on the top of a second linear glycerol gradient, prepared from 1 ml 90 %, (v/v), 2 ml 80 % (v/v), 1 ml 60 % (v/v) and 1 ml 50 % glycerol in KP buffer. After centrifugation (2 h, 210 000 g, 4 °C), the granules again sedimented at about 90 % (v/v) glycerol. These granules were washed twice with KP buffer and then stored at 20 °C.
Preparation of crystalline PHA granules.
Crystalline poly(3HB) granules were isolated from R. eutropha H16 cells grown in sodium gluconate, by employing the hypochlorite treatment described previously (Jendrossek et al., 1993).
SDS-PAGE, blotting and N-terminal sequence analysis.
Protein samples were resuspended in gel loading buffer (0·6 %, w/v, SDS; 1·25 %, w/v, -mercaptoethanol; 0·25 mM EDTA; 10 %, v/v, glycerol; 0·001 %, w/v, bromophenol blue; 12·5 mM Tris/HCl; pH 6·8) and were separated in 12·5 % (w/v) SDS polyacrylamide gels, as described by Laemmli (1970)
. Proteins were stained with Coomassie brilliant blue R-250 (Weber & Osborn, 1969
) or with silver (Heukeshofen & Dernick, 1985
). For N-terminal sequencing, the protein was blotted from an SDS-polyacrylamide gel onto a PVDF membrane, as described by Towbin et al. (1979)
. Sequence analysis was performed by automated Edman degradation.
Analysis of granule-associated proteins by 2D gel electrophoresis and MALDI-TOF.
Proteins from native granules (25 mg wet weight) were solubilized in 300 µl solubilization buffer (9 M urea; 4 %, w/v, CHAPS; 50 mM DTT; 2·5 %, w/v, Triton X-114) by stirring for 2 h at room temperature, and the solubilized proteins were separated from the granules by centrifugation (16 100 g, 20 °C). For the first dimension, the sample was mixed with 100 µl of the same buffer, additionally containing carrier ampholytes (covering pH 310; Serva) and bromophenol blue. Electrophoresis was performed using immobilized pH gradient (IPG) strips (Bio-Rad Laboratories). The IPG strips (18 cm, pH 58) were rehydrated with the entire granule-protein sample overnight at room temperature under mineral oil. After rehydration, isoelectric focusing in the IPG strip was carried out for a total of 100 kVh (with a maximum of 6000 V) at 20 °C under mineral oil. The focused strip was reduced in 5 ml 50 mM Tris buffer containing 6 M urea, 30 % (v/v) glycerol, 5 % (w/v) SDS and 15 mM DTT for 20 min at room temperature. It was then alkylated in the same Tris buffer containing 150 mM iodoacetamide for 20 min at room temperature. The strip was then run in a 20 cmx20 cm, 12 % SDS polyacrylamide gel to separate the proteins in the second dimension according to molecular mass. The current was limited to 40 mA per gel. Proteins were detected by Coomassie brilliant blue staining. MALDITOF analysis was done by the method of Shevchenko et al. (1996).
Analysis of nucleotide and amino-acid sequences.
All available sequence data of the National Center for Biotechnology Information (NCBI) at http://www.ncbi.nlm.nih.gov were searched for fragments of high similarity to the translational product of phaP1 from R. eutropha strain H16 using the BLAST program on the BLAST server of the website. The incomplete genome sequence of R. eutropha H16 was searched by the ERGO database (Integrated Genomics) for homologous genes of high similarity to phaP1. The presented search results represent the data available at the above-mentioned databases as of February 19 2004. Contigs containing fragments of highest similarity were analysed using the evaluation version of DNA Tools 5.1. Sequences were aligned using CLUSTAL X 1.8, and phylogenetic trees were constructed using TREE 1.6.5.
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RESULTS |
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Comparison of the primary structures of R. eutropha PhaP1 and the 20 PhaP1 homologues shown in supplementary Fig. I revealed a few highly conserved positions in these proteins. Leu-38 in R. eutropha PhaP1 was the only position which was identical in the 23 proteins, with the exception of PhaP of Rhodospirillum rubrum, where this residue was replaced with Val. Several other positions, such as Leu-18, 24, 40, 47, 68, 71, 86, 92; Glu-33, 41, 72, 102, 108, 161; Gln-41, 161; Ile-95, 145; Lys-34, 44, 84; Pro-78, 129; Ala-128; Arg-90; Asn-39; Gly 131; Thr-25 and Val-43, 121 were in only a few phasins replaced by very similar amino acids. The alignment of the PhaP homologues also revealed alanine-rich areas at the C-terminal regions of the phasins (amino acids 120202).
Analysis of phaP2, phaP3 and phaP4 transcription in R. eutropha H16
The occurrence of four genes for phasin homologues in R. eutropha raised the question whether the three additionally detected genes are intact and active, as previously shown for phaP1 (Wieczorek et al., 1995). Therefore, one-step RT-PCR was employed in order to investigate the transcription of phaP2, phaP3 and phaP4 in R. eutropha H16 qualitatively under conditions permissive for poly(3HB) biosynthesis and accumulation. Cells were cultivated in MM containing 1·5 % (w/v) sodium gluconate as sole carbon source. Total RNA was isolated from these cells in the stationary growth phase, and RT-PCR with primers specific for the megaplasmid-encoded phaP2 as well as the chromosomally-encoded phaP3 and phaP4 was carried out (Table 1
). RT-PCR analysis clearly demonstrated that phaP2, phaP3 and phaP4 were transcribed under storage conditions (Fig. 2
). Control PCR experiments with the isolated RNA as template did not result in PCR-product formation, confirming that DNA contamination did not contribute to RT-PCR product formation.
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Analysis of the R. eutropha H16 poly(3HB) granule proteome by 2D PAGE and identification of proteins by MALDI-TOF
One-dimensional SDS-PAGE was for several reasons not suitable to detect all phasin proteins in crude cell extracts or poly(3HB) granules. Firstly, the molecular masses calculated for the four phasin homologues were nearly identical (Table 2). Secondly, large amounts of PhaP1 occurred in the cells and in the granule preparations. Thirdly, PhaP2 and PhaP3 occurred only at low amounts, and even in a phaP1 mutant, these two phasins could not be clearly identified in one-dimensional SDS-polyacrylamide gels. Therefore, the poly(3HB) granule proteome was analysed by two-dimensional PAGE, and the proteins were subsequently identified by MALDI-TOF.
For this, poly(3HB) granules were isolated from the wild-type strain H16 and from the phaP1 knock-out mutant Re1052. For these experiments, to guarantee the correct phenotype, we used the deletion mutant Re1052 instead of the Tn5-induced mutant H2275, which harbours Tn5 inserted into the promoter region. This procedure was necessary, because PhaP1 is the dominant protein at the surface of wild-type granules, completely or partially masking other phasins occurring in minor amounts. The granule-bound proteins were solubilized as described above, and separated by 2D PAGE, as described in Methods. Three different forms of PhaP1 were observed as predominant granule-associated proteins of R. eutropha H16 (Fig. 4A). We also analysed additional polypeptide spots by MALDI-TOF, which were identified as PhaP3 and PhaP4, respectively (Fig. 4B
). Veith et al. (2001)
and Lutter et al. (2001)
suggested that pI heterogeneity can in some instances be related to conformational equilibria, and not posttranslational modifications.
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The granule proteome of the mutant comprised about six different protein species, which were isolated from the gel and digested with trypsin. The molecular masses of the resulting fragments were subsequently determined by MALDI-TOF (supplementary Table I, available as supplementary data with the online version of this paper at http://mic.sgmjournals.org). Definitive evidence of the granule-associated proteins required the alignment of multiple digestion fragments with the proposed sequence (supplementary Fig. III, available as supplementary data with the online version of this paper at http://mic.sgmjournals.org). These masses were in complete agreement with the amino-acid sequences deduced from the putative genes for PhaP1, PhaP3, PhaP4, BktB and GroEL from R. eutropha H16. An additional polypeptide spot close to that of BktB could not be identified by MALDI-TOF analysis. Only PhaP2 was not identified in the 2D gels. Additional proteins detected by SDS-PAGE of isolated PHA granules from the Tn5-mutant R. eutropha H2275 could not be identified in 2D gels, because the IPG strips employed covered a pH range from 5 to 8 only. These studies clearly demonstrated that PhaP1, PhaP3 and PhaP4 are bound to the poly(3HB) granules in R. eutropha. However, an N-terminal amino-acid sequence obtained by other workers for a protein occurring in the total proteome of a glucose-utilizing R. eutropha mutant has been clearly identified as the translational product of phaP2 (Srinivasan et al., 2002). Therefore, all four phasin proteins are synthesized in R. eutropha.
Studies on the binding of PhaP2 to crystalline poly(3HB) granules
To reveal the capability of PhaP2 to bind to poly(3HB) granules, a His6-PhaP2 fusion protein was isolated from a recombinant strain of E. coli. An emulsion of crystalline poly(3HB) granules in water (1·5 %, w/v) was then incubated with the partially purified His6-PhaP2 fusion protein for 90 min on ice (Wieczorek et al., 1995). After this incubation, the granules were collected by centrifugation, washed twice with 1 ml 10 mM Tris/HCl (pH 7·0), and suspended in denaturating buffer. The granule sediments and supernatants of each washing step were analysed by SDS-PAGE (Fig. 5
). The His6-PhaP2 fusion protein exhibited a very high affinity for poly(3HB) granules (Fig. 5
, lanes 1 and 4). Repeated washing with 1 ml 10 mM Tris/HCl (pH 7·0) did not remove the fusion protein from the poly(3HB) granules (Fig. 5
, lanes 2 and 3). To demonstrate that PhaP2 did not precipitate during the assay, PhaP2 was incubated without granules, under the same conditions described above. During the time course of this control experiment, PhaP2 was never detected in the sediment after centrifugation. The results of these experiments indicate that the putative location of PhaP2 is the poly(3HB) granules in R. eutropha; however, for several possible reasons it could not be identified during analysis of the native granule proteome.
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A Search for phaZ homologues putatively encoding PHA depolymerases involved in the intracellular mobilization of PHAs revealed five genes. Four of them encoded the recently detected PHA depolymerase genes phaZ1, phaZ2 and phaZ3 (Saegusa et al., 2001; York et al., 2003
), which are localized on chromosome 1 (phaZ1 and phaZ2) and chromosome 2 (phaZ3), and also the PHA depolymerase gene phaZ4, which is localized on megaplasmid pHG1 (Schwartz et al., 2003
), whereas the fifth gene was hitherto unknown and was localized on chromosome 1 (phaZ5, Genbank accession no. AY489115). Supplementary Table II (available as supplementary data with the online version of this paper at http://mic.sgmjournals.org) shows the similarities of the five phaZ homologues of R. eutropha.
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DISCUSSION |
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Since R. eutropha strain H16 and mutants derived from this strain are currently employed for the production of PHASCL, and since the PHASCL biosynthesis genes of this bacterium are currently used to establish heterologous production systems for PHASCL, this bacterium serves as a model organism to reveal all aspects of PHASCL metabolism. This includes PHA biosynthesis and mobilization, as well as the biogenesis and structure of PHA granules and the regulation of these processes. Biosynthesis of the coenzyme A thioesters of the various hydroxyalkanoic acids and mercaptoalkanoic acids, which serve as substrates of the PHA synthase, is achieved by several enzymes: from acetyl-CoA, in the case of poly(3HB) biosynthesis, and by pathways linking the catabolism of carbon sources used as precursor substrates for biosynthesis of all other PHASCL and PTESCL in R. eutropha (Steinbüchel, 2001; Steinbüchel & Lütke-Eversloh, 2003). Obviously, only one PHA synthase exists in R. eutropha, and this enzyme is responsible for the biosynthesis of a wide range of different PHASCL and PTESCL.
In contrast to PHA biosynthesis, more than one PHA depolymerase gene is present that may be involved in PHA mobilization. Three different PHA depolymerases were recently detected as functionally active enzymes in R. eutropha H16 (Saegusa et al., 2001; York et al., 2003
). Furthermore, Schwartz et al. (2003)
recently detected a fourth PHA depolymerase gene (phaZ4) on megaplasmid pHG1. The analysis of the R. eutropha H16 genome in this study revealed an additional gene putatively encoding a fifth PHA depolymerase. Why this bacterium has five PHA depolymerase genes, whereas for biosynthesis of PHAs only one PHA synthase is sufficient, is an enigma. At least three of these PHA depolymerase genes are expressed (York et al., 2003
). In addition, a putative poly(3HB)-dimer hydrolase was recently detected, which further degrades the cleavage products formed by the PHA depolymerases (Saegusa et al., 2002
; Kobayashi et al., 2003
).
Another enigma is the presence of four genes encoding highly homologous phasin proteins (Table 3). Whereas PhaP1, PhaP3 and PhaP4 are bound in vivo to the poly(3HB) granules, the in vivo location of PhaP2, which has the capability to bind crystalline poly(3HB) granules, remains unclear. PhaP2 may be in vivo only loosely bound to the granules and washed off during the purification of the granules, or it may in vivo only bind to granules under specific cultivation conditions or at a specific stage of PHA accumulation which were not met in this study. PhaP1 is certainly the major phasin protein in R. eutropha, and phaP1 mutants exhibit a distinct phenotype. Such mutants accumulate less poly(3HB) than the wild-type and possess only one single large granule per cell, whereas cells of the wild-type possess several poly(3HB) granules. In a well-regulated process employing the autoregulator and transcriptional repressor protein PhaR, PhaP1 is synthesized in large amounts, contributing 35 % of the total cellular protein. PhaP1 is obviously not efficiently compensated by any of the other three phasins (Wieczorek et al., 1995
; Pötter et al., 2002
). Otherwise, phaP1 mutants of R. eutropha would not exhibit the phenotype PHA-leaky, and the PHA granules would not coalesce to a single granule in the cells of this mutant.
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These findings are new and unexpected, and should affect our current models of the structures of PHA granules (Steinbüchel et al., 1995; Jurasek et al., 2001
; Jurasek & Marchessault 2002
; Dennis et al., 2003
). Furthermore, these findings may also have a considerable impact on the optimization of production strains of R. eutropha and on the establishment of heterologous production systems for PHAs, such as genetically modified bacteria and transgenic plants, particularly if the modified organisms rely on R. eutropha PHA-biosynthesis genes. Extensive further studies are now required, to reveal the functions of the multiple phasins in R. eutropha and other bacteria.
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ACKNOWLEDGEMENTS |
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Received 8 December 2003;
revised 9 February 2004;
accepted 26 March 2004.
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