Institut für Mikrobiologie, Westfälische Wilhelms-Universität Münster, Corrensstrasse 3, D-48149 Münster, Germany1
Author for correspondence: A. Steinbüchel. Tel: +49 251 8339821. Fax: +49 251 8338388. e-mail: steinbu{at}uni-muenster.de
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ABSTRACT |
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Keywords: inverse PCR, thermotolerant enzymes, PHB, bioplastic, Anabaena cylindrica, Gloeothece sp.
Abbreviations: CDM, cell dry matter; PHA; poly(3-hydroxyalkanoate); PHB, poly(3-hydroxybutyrate); 3HB, 3-hydroxybutyrate; 3HV, 3-hydroxyvaleric acid
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INTRODUCTION |
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The biochemical and molecular basis of PHA synthesis has been investigated intensively in many micro-organisms (Steinbüchel & Hein, 2001 ). However, in cyanobacteria, studies at the molecular level regarding PHA biosynthesis have been reported only for Synechocystis sp. strain PCC 6803 (Hein et al., 1998
; Taroncher-Oldenburg et al., 2000
). Only from this cyanobacterium has the PHA synthase gene been cloned (Hein et al., 1998
). Interestingly, the PHA synthase of Synechocystis sp. PCC 6803 is composed of two different subunits, encoded by two contiguous, adjacent co-transcribed genes, referred to as phaE and phaC (Hein et al., 1998
). The translational products of these two structural genes showed similarity to the corresponding PHA synthases of the anoxygenic purple sulfur bacteria Allochromatium vinosum (Liebergesell & Steinbüchel, 1992
; Liebergesell et al., 1994
), Thiocystis violacea (Liebergesell & Steinbüchel, 1993
), Thiococcus (formerly Thiocapsa) pfennigii (Liebergesell et al., 2000
) and Ectothiorhodospira shaposhnikovii (Genbank accession no. AAG30259 for phaC and AAG30260 for phaE). These enzymes belong to the type-III PHA synthases characteristic of
-Proteobacteria (Rehm & Steinbüchel, 1999
; Steinbüchel & Hein, 2001
). Expression of functionally active type-III PHA synthases requires the expression of both subunits PhaE and PhaC; PhaE alone was completely inactive and PhaC alone exhibited only negligible activity, if at all (Liebergesell et al., 1994
; Müh et al., 1999
; Jia et al., 2000
). The PhaC proteins exhibit much higher similarities than the PhaE proteins and the latter revealed absolutely no similarities to other PHA synthases, but contained two amino acid stretches at the C-terminal regions like PHA granule binding proteins (phasins), which might serve as binding domains of PHA synthases to the surface of PHA granules (Liebergesell et al., 2000
). However, the function of PhaE has not yet been revealed.
Due to knowledge of only one cyanobacterial PHA synthase, it is still unclear whether the type-III PHA synthase is a unique enzyme of cyanobacteria, or whether other types (Rehm & Steinbüchel, 1999 ) also occur in this group of photosynthetic bacteria. The aim of this study was to investigate the distribution of type-III PHA synthases among cyanobacteria and to clone the pha genes from two thermophilic cyanobacteria. Thermotolerant PHA synthases have so far never been described and corresponding genes are not available. Such enzymes might be useful for biotechnological applications such as in vitro PHA biosynthesis processes (Steinbüchel, 2001
).
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METHODS |
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Triclosan (15 µg ml-1) was added to the second phase when the cells had been transferred into BG11o medium plus 15 mM acetate. For studying the influence of triclosan on PHA accumulation, a concentration of 1·5 µg triclosan ml-1 was applied for all strains and growth was monitored for a further 7 d.
Escherichia coli strains were grown with shaking (150 r.p.m.) at 37 °C in LuriaBertani (LB) medium (Sambrook et al., 1989 ) with or without antibiotics. Competent cells of Escherichia coli were prepared by using the standard CaCl2 method (Sambrook et al., 1989
). For recombinant strains of Escherichia coli harbouring pha genes, PHA accumulation experiments were carried out in liquid LB containing 0·6% (w/v) glucose as carbon source. In addition, 50 µM thiamine and 0·2 mM IPTG were added if Escherichia coli harboured pBluescriptSK- (pSK-) or derivatives of this vector.
Preparation of PHA granules.
PHA granules were isolated from acetate-grown cells of Synechocystis sp. PCC 6803 by centrifugation in a glycerol gradient as described previously (Hein et al., 1998 ). The PHA granules were enriched in the 65% (v/v) glycerol fraction, which was confirmed by GC analysis. The granule-bound proteins were analysed by Western immunoblotting.
DNA isolation and manipulation.
Total DNA from the various cyanobacterial strains was extracted following a previously described protocol (Hein et al., 1998 ). Plasmid isolations were done by standard methods (Sambrook et al., 1989
) or by using commercial kits (Qiagen). Restriction and ligation of DNA molecules were done according to standard protocols (Sambrook et al., 1989
) or according to the instructions of the suppliers of the enzymes.
Southern hybridization experiments.
The phaCSyn gene of Synechocystis sp. PCC 6803 was amplified by PCR, employing the primers Haphapcr1 and Haphapcr2 (Table 2) and Vent polymerase (New England Biolabs), according to the Biochemica PCR applications manual (1995). The PCR products (approx. 560 bp) were purified by using the Nucleotrap kit (Macherey & Nagel), following the instructions of the manufacturer, and were labelled by using the DIG-High Prime kit (Boehringer Mannheim). The hybridization, which was done at 53 °C, and other related procedures followed standard methods (Sambrook et al., 1989
). For visualizing the chemoluminescence, the substrate disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'(5'-chloro)tricyclo[3.3.1.13,7]decal}12-4-yl) phenyl phosphate (CSPD) was used.
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Production and purification of antibodies against PhaESyn.
The His6-tagged PhaESyn was isolated from an SDS-polyacrylamide gel and submitted to Eurogentec for antibody production. The latter was achieved by three subcutaneous injections into a rabbit (animal code SA 6918; Eurogentec) over a period of 3 months following standard procedures. The antibodies were purified from the crude serum by using FPLC on a Protein A-Sepharose CL-4B affinity column (Hjelm et al., 1972 ).
Western blotting and immunodetection employing anti-PhaESyn antibodies.
Crude extracts were obtained from cells of cyanobacteria and recombinant Escherichia coli as follows. The washed cells (approx. 0·2 g wet wt) were dissolved in 1 ml 10 mM Tris/HCl buffer, pH 8·0, containing 5 mM DTT, and subsequently disintegrated by 1 min treatment with a Sonoplus GM200 sonifier (Bandelin Electronic). Three volumes of this crude protein solution were then mixed with 1 vol. SDS-additive solution consisting of 8% (w/v) SDS, 40% (w/v) glycerol, 20% (v/v) 2-mercaptoethanol and 0·004% (w/v) bromophenol blue. The proteins were denatured by incubating at 95 °C for 5 min. The proteins were then separated by SDS-PAGE in 11·5% (w/v) polyacrylamide gels as described by Laemmli (1970) . The protein bands from one gel were stained for proteins with Serva Blue R (Weber & Osborn, 1969
), whereas the proteins from a second gel were blotted onto a nitrocellulose BA83 membrane (Schleicher & Schüll) using a semi-dry transfer blotter (Bio-Rad) and applying a voltage of 24 V for 100 min. The membrane was then equilibrated with 10 mM Tris/HCl buffer, pH 8·0, containing 150 mM NaCl and 0·05% (v/v) Tween 20 (TNT buffer) and blocked as described previously (Hein et al., 1998
). The immunological detection followed a standard method (Sambrook et al., 1989
) with the only modification that 5% (w/v) skimmed milk in TNT buffer was used as blocking agent. The purified antibodies, as eluted from the protein A Sepharose-4B column, were diluted 1:1000 and used as the primary antibody. A solution of an anti-rabbit immunoglobulin G-alkaline phosphatase conjugate (Sigma), diluted 1:30000, was used as the secondary antibody. After removing the excess of the secondary antibody by washing the membrane three times with TNT buffer containing 0·1% (w/v) bovine albumin fraction V and 0·1% (v/v) Nonidet P40, the bound antibodies were visualized with nitroblue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolylphosphate (BCIP) with a commercially available detection kit (Sigma) following the instructions of the manufacturer.
PCR using degenerate primers.
The nucleotide sequences of two primers, Haphapcr1 (sense) and Haphapcr2 (reverse), were deduced from highly conserved regions of superfamily phaC genes (Table 2), including phaC of the anoxygenic phototrophic bacteria Allochromatium vinosum and Thiocystis violacea, and the cyanobacterium Synechocystis sp. PCC 6803. The regions corresponded to amino acid positions 8597 and 275263, respectively, in PhaC of Synechocystis sp. PCC 6803.
PCR was performed with DNA isolated from various cyanobacteria by using Vent DNA polymerase and applying the following temperature programme: 1 cycle of 95 °C for 2 min, 30 cycles of 95 °C for 30 s, 50 °C for 30 s and 72 °C for 80 s. The PCR products were purified by using the Nucleotrap kit (Macherey Nagel) and were subsequently ligated to pSK- DNA, which was linearized by treatment with EcoRV. The ligation products were then transformed into Escherichia coli following standard protocols (Sambrook et al., 1989 ).
Inverse PCR to clone the 5' and 3' regions of phaC and phaE from Synechococcus sp. MA19 and Chlorogloeopsis fritschii PCC 6912.
To clone the 5' and 3' regions adjacent to the PCR products, inverse PCR (Triglia et al., 1988 ) was applied. Primers MAphaCL and MAphaCR (Table 2
), corresponding to base positions 735759 and 385360 in phaCMA19 (accession no. AY030295), respectively, were used for Synechococcus sp. MA19 DNA. Similarly, PhaC69R and PhaC69L (Table 2
), corresponding to base positions 643666 and 302323 in phaCCf (accession no. AF371369), respectively, were used for the Chlorogloeopsis fritschii PCC 6912 template. The sequences of these primers were deduced from those of the 5' and 3' regions of the corresponding PCR products. Genomic DNA was first restricted with PstI, cyclic products were then obtained by incubation with T4 DNA ligase and these were linearized by digestion with SspI. Using Vent polymerase, the following temperature programme was applied to both DNA templates: 1 cycle of 95 °C for 2 min, 32 cycles of successive programming of 95 °C for 30 s, 57 °C for 30 s and 72 °C for 2 min. The PCR products were then precipitated by adding 2·5 vols ethanol, washed with 70% (v/v) ethanol, dried at room temperature and dissolved in 20 µl 5 mM Tris/HCl, pH 8·0, containing 0·5 mM EDTA. About 400 ng PCR products were ligated at room temperature overnight with 400 ng EcoRV-linearized pSK- DNA and were then transformed into Escherichia coli XL-1 Blue (see Fig. 3
).
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Construction of hybrid plasmids.
To study PHA synthase activity of PhaC subunits some hybrid plasmids were constructed. The expression vectors were derived from pSK- as follows. (i) A PCR product containing slr1993, encoding ß-ketothiolase (phaASyn), together with slr1994, encoding acetoacetyl CoA reductase (phaBSyn), from Synechocystis sp. PCC 6803 was amplified by using primers PhaABsense and PhaABreverse, which included degenerate NdeI and BamHI restriction sites, respectively (Table 2). The purified PCR product (2·45 kbp) was cloned to obtain pSKABSyn (see Fig. 7
). After digestion with BamHI, the purified phaABSyn fragment was ligated to the BamHI-treated PCR product of phaCMA19 giving the phaABC fragment. (ii) The purified phaABC was ligated to EcoRV-digested pSK-. The hybrid plasmid obtained, pSKABCMA19, was transformed into Escherichia coli XL-1 Blue. Similarly, other hybrid plasmids were constructed, such as pSKABCCf and pSKABECSyn containing fragment phaECSyn, which was obtained in a previous study (Hein et al., 1998
). The hybrid plasmid pSKABECSyn was also transformed into Escherichia coli S17-1 and served in this study as a positive control.
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The nucleic acid sequences obtained from both strands were analysed with a computer program available from the Heidelberg Unix Sequence Analysis Resources (HUSAR, release 4.0). Sequence comparisons were performed by using the network service programs BLAST provided by the National Center for Biotechnology Information (NCBI). The sequences corresponding to C-terminal amino acids of PhaE and PhaC, as well as the 120 aa of regions surrounding the substrate-binding sites (Cys-149 in PhaC of Allochromatium vinosum) and the first 355 aa of PhaC, were aligned using the CLUSTAL W and also CLUSTAL X programs provided by the European Bioinformatics Institute. The phylogenetic distance tree was reconstructed by using PROTDIST and PROTPARS of PHYLIP (Phylogeny Inference Package; Felsenstein, 1989 ), which are available on the internet (http://www.es.embnet.org/Sevices/).
Electrophoresis of proteins and nucleic acids.
SDS-PAGE of proteins was performed in 11·5% (w/v) gels according to Laemmli (1970) . Molecular mass marker proteins were purchased from Bio-Rad. Protein staining was done with Serva Blue R. Protein concentrations were estimated following the method of Bradford (1976)
. For analysis of nucleic acids, electrophoresis was done in 0·8 and 1·2% (w/v) agarose gels according to standard methods (Sambrook et al., 1989
).
Analysis of PHA.
For quantitative and qualitative analysis of PHA, 57 mg lyophilized cells were subjected to methyl esterification (methanolysis) in a 1:1 (v/v) chloroform/methanol solvent mixture containing 15% (v/v) sulfuric acid. The resulting hydroxyacyl methylesters were analysed by GC as described by Timm et al. (1990) . The retention times of authentic 3-hydroxy fatty acids were used for identification of PHA constituents.
Determination of PHA synthase activity.
Determination of PHA synthase activity was done by a spectrometric assay with a 500 µl assay mixture containing 25 mM Tris/HCl, pH 7·4, 1 mM 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), 20 mM MgCl2, 100 µM D(-)-3-hydroxybutyryl-CoA and approximately 30 µg protein from the soluble protein fraction of cells of Escherichia coli. The enzyme reactions were carried out at 30 °C and the activity estimations followed the method described by Valentin & Steinbüchel (1994) .
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RESULTS AND DISCUSSION |
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We therefore purified the PHA synthase PhaE from Synechocystis sp. PCC 6803 (PhaESyn). A His6-tagged phaESyn (1·1 kbp) construct was made and expressed in Escherichia coli TOP10 under the control of PL and PR of the promoter of plasmid pMa/c5-914, the construction of which is shown in Fig. 1
. The fusion protein was purified by affinity chromatography on an Ni-NTA column followed by SDS-PAGE. PhaESyn, which was eluted from the SDS-polyacrylamide gel, was verified by N-terminal amino acid sequence analysis and was submitted for antibody production. The polyclonal antibodies raised against PhaESyn were purified from rabbit serum as described in Methods.
Western blotting using the anti-PhaESyn antibodies was performed with protein crude extracts from 12 strains of cyanobacteria, including Synechocystis sp. PCC 6803. As reference samples a crude extract of recombinant Escherichia coli expressing PhaESyn and proteins solubilized from poly(3-hydroxybutyrate) (PHB) granules of Synechocystis sp. PCC 6803 (Hein et al., 1998 ) were applied. Specific cross-reactions occurred with a 37 kDa protein in the crude protein fraction of Synechocystis sp. PCC 6803, in the PHB granule protein extract and in the recombinant Escherichia coli extract. In addition, crude extracts from Synechoccocus sp. MA19, Chlorogloeopsis fritschii PCC 6912 and Cyanothece PCC 8303 gave weak signals, each corresponding to a 40 kDa protein band. Moreover, two signals occurred at 36 and 40 kDa in Cyanothece sp. strains PCC 7424 and PCC 8801, and also in Synechococcus sp. PCC 6715 (Table 4
). It remains to be analysed whether these three latter cyanobacteria possess two PhaE subunits of different molecular masses, or whether the 36 kDa protein is a proteolysis product of the 40 kDa protein. Crude extracts from the following cyanobacteria did not reveal any protein cross-reaction with the anti-PhaESyn antibodies: Anabaena cylindrica SAG 1403-2, Cyanothece sp. PCC 8955, Gloeothece sp. PCC 6501, Gloeocapsa sp. PCC 7428 and Stanieria sp. PCC 7437 (Table 4
). It is remarkable that Anabaena cylindrica SAG 1403-2 obviously contains phaC according to hybridization with the phaC-specific probe, but seems to lack PhaE according to the result of the Western blotting experiment. Since this strain is similar to Anabaena cylindrica 10C (Lama et al., 1996
), as indicated by the accumulation of poly(3HB-co-3HV) (Vincenzini & De Philippis, 1999
), PhaE or a homologous protein might be more diverse among the strains studied.
In conclusion, these immunological studies provide further evidence for the widespread distribution of type-III PHA synthases in the cyanobacterial strains investigated in this study. As mentioned above, the antibodies against PhaCRe and PhaCAv did not reveal cross-reaction with PhaCSyn. Therefore, it is clearly shown that antibodies raised against the more specific and typical components are suitable to screen for the distribution of type-III PHA synthases among cyanobacteria and probably also in other bacteria.
Screening cyanobacterial genomes for phaC by using PCR
A third approach used to investigate the occurrence and distribution of type-III PHA synthases in cyanobacteria employed PCR using genomic DNA of the various cyanobacteria as template and oligonucleotides specific for the phaC gene as primers. For this, the nucleotide sequences of the oligonucleotides were designed according to a highly conserved region of the PhaC superfamily. PCR products were obtained for Chlorogloeopsis fritschii PCC 6912 (555 bp), Synechococcus sp. MA19 (565 bp), Cyanothece sp. PCC 8303 (550 bp), Anabaena cylindrica SAG 1403-2 (approx. 600 bp) and Synechococcus sp. PCC 6715 (approx. 600 bp). The sizes of the first three PCR products were verified by DNA sequencing, whereas the sizes of the last two products were determined by 1·2% (w/v) agarose gel electrophoresis only. A fragment of the expected size of 560 bp was obtained for Synechocystis sp. PCC 6803, which again served in this study as a positive control. The minor variations in the sizes of the PCR products were not only caused by the individual nucleotide sequences of templates, but also by the positions where the degenerate primers hybridized.
The amino acid sequences deduced from the nucleotide sequences of the PCR products of Chlorogloeopsis fritschii PCC 6912, Cyanothece sp. PCC 8303 and Synechococcus sp. MA19 exhibited 76, 83 and 75% identity, respectively, with the corresponding regions of PhaCSyn. Alignment of these sequences, using CLUSTAL W (1.81) software of the European Bioinformatics Institute (http://www2.ebi.ac.uk), showed striking homologies not only to amino acid sequences of the PhaC proteins of other type-III PHA synthases, but also to those of type-I and type-II PHA synthases, such as PhaC of R. eutropha and PhaC1 of P. aeruginosa, respectively. These regions are considered as the covalent substrate binding regions, containing conserved cysteine residues corresponding to positions Cys-130 and Cys-149 in PhaC from Allochromatium vinosum, as shown recently by Jia et al. (2000) . It is remarkable that these regions also contain a highly conserved Cys-157 residue and a stretch of amino acids typical for cyanobacteria that does not occur in the type-III PhaC proteins of anoxygenic photosynthetic bacteria. We have therefore termed this the cyanobacterial box (see Fig. 2
). This region is different from the corresponding sequences of all other type-III PHA synthases and also from type-I and type-II PHA synthases.
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Two ORFs, corresponding to slr1830 (phaCSyn) and slr1829 (phaESyn), were identified in the inverse PCR products obtained with Chlorogloeopsis fritschii PCC 6912 and Synechococcus sp. MA19 template DNA. Both genes were separated by intragenic regions of 165 and 93 bp in the genomes of strains PCC 6912 and MA19, respectively. Unfortunately, both 5' regions of the phaE genes of strains MA19 (phaEMA19) and PCC 6912 (phaECf) possessed a restriction site for PstI. Therefore, approximately 100 aa of the PhaE protein were missing in both strains. The amino acids deduced from the previously sequenced DNA fragments of phaECf and phaEMA19 exhibited higher similarities to the PhaE proteins of anoxygenic photosynthetic bacteria than did PhaESyn to the PhaE proteins of other type-III PHA synthases. However, their C-terminal regions showed no homology to the Gln-Val-Ala-Ala-Leu-Ala-Gly stretch, which is typical of the C-terminal regions of PhaE proteins of the photosynthetic -Proteobacteria (Fig. 4
). This stretch has been suggested to act as the binding site of the PHA synthase complex to the PHA granule surface of these bacteria (Liebergesell et al., 2000
). It is remarkable that the C-terminal region of PhaECf exhibited a unique Ala-Pro-Ala-Pro-Ala-Thr stretch, which is identical to that of Thiocystis violacea (Fig. 4
) and is related to other binding sites of phasin proteins (Liebergesell et al., 2000
).
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The high level of similarity among these sequences confirms the high level of conservation of these PhaC regions, from which the universal phaC screening primers, Haphapcr1 and Haphapcr2, were deduced and degenerated. However, the amino acid sequences deduced from PhaCMA19 and PhaCCf also exhibited highly conserved regions adjacent to the positions corresponding to Cys-149, Asp-302, His-303 and His-331 in the sequence of Allochromatium vinosum. These residues are important for activation of the 3-hydroxylalkyl moiety of 3-hydroxybutyryl-CoA (Asp-302, His-303) and nucleophilic attack (His-331) (Fig. 5), as well as for covalent catalysis carried out at Cys-149 (Jia et al., 2000
; see also Fig. 2
). In addition to the results shown above, about 180 aa could be deduced from the phaC-specific PCR product obtained from Cyanothece sp. PCC 8303 (PhaCC.sp). This partial gene sequence was also aligned with all PhaC proteins of type-III PHA synthases using the CLUSTAL W program. The amino acid sequences at the covalent catalysis site of PhaC of both thermophilic cyanobacteria and the mesophilic cyanobacterium Cyanothece sp. PCC 8303 also reveal striking similarities to the active centres of lipases, which exhibit a mechanism of catalysis similar to that of PHA synthases (Liebergesell & Steinbüchel, 1993
; Jia et al., 2000
).
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ACKNOWLEDGEMENTS |
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Received 26 April 2001;
revised 6 July 2001;
accepted 25 July 2001.
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