Characterization of FtsZ homolog from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1

JOURNAL OF BIOSCIENCEAND BIOENGINEERING Vol. 89, No. 2, 181-187. 2000

Characterization of FtsZ Homolog from Hyperthermophilic Archaeon Pyrococcus kodakaraensis KOD 1 KEISUKE NAGAHISA,’

TSUYOSHI

NAKAMURA,’ SHINSUKE FUJIWARA,’ TADAYUKI IMANAKA,2 AND MASAHIRO TAKAGI’* Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871’ and Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida-honmachi, Sakyo-ku, Kyoto 606-8501,2 Japan Received 8 October 1999/Accepted 25 November 1999

The gene of bacterial type ftsZ bomolog in bypertbermopbilic archaeon, Pyrococcus kodukuruensis KODl (Pk-ftsz), was identified. The gene product of the Pk-ftsZgene is composed of 380 amino acids with a molecular mass of 41,354 Da. In the deduced amino acid sequence of the Pk-f&Z gene, a glyclne-rich sequence (Gly-GlyGly-Tbr-Gly-Ala-Gly) implicated in GTP binding was well conserved. The Pk-ftsZ gene was overexpressed using Escherichia coli as a host and the recombinant protein was purified. The purified Pk-FtsZ protein exhibited GTPase activity with optimum temperatures higher than 80% However, the protein showed little GTPase activity at 4O”C, indicating that a high reaction temperature is required for the GTPase activity in accordance with the tbermopbillc nature of P. kodukuruensis KODl. The GTP-binding ability of Pk-FtsZ protein could also be detected by UV-induced cross-linking of a protein to [a-32P] GTP. The Pk-ftsZ gene was expressed in E. coli cells with a temperature-sensitive ftsZ mutation, E. coli ftsZ84 (ts), but its mutant phenotype of elongated cell form at a nonpermissive temperature (42°C) could not be compensated, possibly because of the tbermopbilic nature of the Pk-FtsZ. Pk-FtsZ could form protofilaments in a GTP-dependent manner at 90°C. Results of pbylogenetic analysis suggest that there might be additional factors required for formation of the Z ring in P. kodakuruensis KODl. [Key words: hyperthermophile,

cell division,

FtsZ, archaea, Pyrococcus]

The phylogenetic trees based on 16s rRNA and protein sequences showed that all living creatures are divided into three fundamental domains, Eucarya, Bacteria, and Archaea (1,2). In every domain, cell division is one of the most important events for cell propagation. In Escherichia coli cells, FtsZ protein has been reported as an essential protein for cell division (3) and the protein homologs can be found in almost all bacterial strains (4). FtsZ proteins polymerize through self-assembly to form a cytokinetic ring, designated as Z ring, at the division site, constricting at the leading edge of the invaginating septum that eventually separates the two daughter cells (5, 6). Upon completion of division, FtsZ is not retained at the new cell pole, but is released into the cytoplasm. Based on biochemical and amino acid sequence data, it has been proposed that FtsZ is a bacterial version of tubulin (7). Comparison of FtsZ with tubulin revealed that the two proteins share some similarities, with the glycine-rich sequence ((G/A)GGTG(T/S)G) implicated in GTP binding (8-11). Analysis of purified FtsZ revealed that it binds and hydrolyzes GTP (8-11) and can undergo nucleotide-dependent assembly to form protofilaments and larger structures by a mechanism analogous to assembly of tubulin into microtubules in eucaryal cells (12, 13). Although phylogenetic evidence suggests that Archaea are more closely related to Eucarya than to Bacteria (14, 15), bacterial and archaeal cells are generally similar in morphology, lack a nucleus, and normally have relatively small circular genomes (16, 17). Therefore, it is reasonable that some FtsZ homologs of archaea corresponding *Corresponding

to FtsZs of bacteria and tubulins of eucarya have been reported (18-20). However, very little is known about the properties of FtsZs from hyperthermophilic archaea, although it is interesting to understand the mechanism of cell division at elevated temperatures. In the present study, we cloned and sequenced the ftsZ homolog from hyperthermophilic archaeon, Pyrococcus kodakaraensis KODl, and characterized the thermophilic nature of the recombinant protein. MATERIALS

AND METHODS

Strains, plasmids and growth conditions P. kodakaraensis KODI was cultured as previously described (21). E. coli strain JM109 was used as a host for cloning and manipulation of the DNA fragments. E. coli strain XLl-Blue MRA (P2) (STRATAGENE, La Jolla, CA, USA) was used for infection of lambda phages. pBR322 (22), pUC18, pUCl9 (23) and PET-8c (24) were used as vectors for cloning and expression. DNA manipulation General DNA manipulations, including plasmid preparations, restriction enzyme digestion, ligation, transformation of E. co/i, and gel electrophoresis were performed following standard protocols (25). Plasmid purification for DNA sequencing was performed using the Wizard plus Minipreps DNA purification system (Promega, Madison, WI, USA). A GeneClean II kit (Bio 101 Inc., La Jolla, CA, USA) was used for extraction of DNA fragments from agarose gel. Polymerase chain reaction (PCR) (26) was performed on a GeneAmp PCR System 2400 (Perkin Elmer). DNA sequencing was performed by the dideoxy-chain termination method (27) with CyS-labeled primers (ALF Read Sequencing Kit, Amersham Pharmacia Biotech.,

author. 181

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Uppsala, Sweden) using an automatic sequencer (ALF Express DNA sequencer, Amersham Pharmacia Biotech.). The sequence was analyzed using the DNASIS software (Hitachi Software, Yokohama). The phylogenetic tree was constructed by the neighbor-joining method (28) using CLUSTAL W (29). Expression and purification of f&Z from P. kodukuraensis KODl The ftsZ gene was amplified by PCR using two primers, 5’-CGTGCCTTGATCATCGGCGTT GGCCAGTGC-3’ and 5’-GAAGA TCZ’CATCGGGATAC CTCCCG-3’. The latter primer contains an additional BgflI site as shown in italics. The PCR product was cleaved with BglII and inserted between NcoI (filled in) and BarnHI sites of PET-8c. The resultant plasmid was designated as PET-ftsZ. Expression of the ftsZ gene cloned into the PET vector was induced by isopropyl-P-n( -)-thiogalactopyranoside (IPTG) using the T7 RNA polymerase expression system (30). Cells were washed with buffer A (50 mM Tris-Cl (pH 8.0), 50 mM NaCl, and 1 mM EDTA) and disrupted by sonication, and the extract was centrifuged at 15,000 x g for 30 min. The supernatant was then heattreated at 80°C for 20min and centrifuged at 15,OOOxg for 30min to remove denatured proteins. The resulting supernatant was collected and ammonium sulfate was slowly added to 50% saturation (31.4 g per 100 ml of supernatant). After stirring overnight, the suspension was centrifuged at 15,OOOxg for 30min. The pellet was dissolved in buffer A and dialyzed overnight against buffer A. The dialyzed crude sample was loaded onto a HiTrap Q (Amersham Pharmacia Biotech.) column (bed volume of 5 ml) previously equilibrated with buffer A, washed with the same buffer, and eluted with a 150-450mM gradient of NaCl at a flow rate of 5 ml. min l. Fractions of 1 ml were collected and the peak fractions for FtsZ protein were eluted at around 250-350mM NaCl. The peak fractions of FtsZ protein recovered from the HiTrap Q column were applied to a Mono Q (Amersham Pharmacia Biotech.) column and eluted with a 150-250 mM gradient of NaCl at a flow rate of 0.5 ml. mini. Additional purification using a Superdex 75 HR lo/30 column (Amersham Pharmacia Biotech.) was performed. The eluted fractions were dialyzed against a buffer containing 50 mM HEPES-NaOH (pH 7.2), 0.1 mM EDTA, and 10% glycerol. GTPase and GTP-binding assays GTPase activity was assayed by detecting the conversion of [u-~~P] GTP to [n-32P] GDP by thin-layer chromatography (TLC). The reaction mixture (20 ~1) contained 50mM HEPESNaOH (pH 7.2), 10mM MgCL, 200mM KCI, 0.5 mM [a-32P] GTP (0.01 &i.,&t), and IO pg of purified protein. The mixtures were incubated at various temperatures for 20min. An aliquot of the reaction mixture (5 ,ul) was spotted onto polyethyleneimine cellulose TLC plate POLYGRAM CEL 300 PEI (Macherey-Nagel, Duren, Germany). The spots on the plate were air-dried and then the plate was developed vertically with 0.75 M KH2P04 (pH 3.4) in a closed chamber at room temperature as described previously (31). The development was terminated when the leading edge of the solvent reached the top of the plate. The plate was then exposed to an X-ray film. GTP-binding ability was examined essentially according to the procedure described previously (32). The measurement of GTP-binding ability was performed by crosslinking the protein to [a-32P] GTP by UV irradiation. A

.I. BIOSCI.

BIOEN( ?..

reaction mixture (20 /ll) containing a buffer (50 mM HEPES-NaOH (pH 7.2) 1 mM dithiothreitol, 50 mM KCl, and 10 mM MgC12), 4 &i of [(w-~~P]GTP, and 5 ;lg of purified protein was incubated at 37°C for 5 min. Then, the mixtures were immediately irradiated with LJV light before SDS-gel electrophoresis and autoradiography were performed. Polymerization assay The polymerization assay was performed based on the procedure described previously (33). The reaction mixtures (75 ~1) contained 50mM HEPES-NaOH (pH 7.2), 10mM MgC12, 200mM KCl, and 15 /jg of purified Pk-FtsZ protein. The polymerization reaction was performed at 90°C. At the time indicated by the arrow, GTP was added to the final concentration of 1 mM. Nucleotide sequence accession number The sequence data for the Pk-ftsZ gene from P. kodakaraensis KODl has been assigned the DDBJ accession number, AB031743. RESULTS AND DISCUSSION Isolation of f&Z bomolog from P. kodukuruensis KODl The genomic DNA from P. kodakaraensis KODl was prepared as previously described (34). The total genomic DNA was cleaved into small fragments and these fragments were randomly cloned into a plasmid vector to construct a gene library. The obtained clones were subjected to automated sequencing followed by a homology search. A cloned DNA fragment containing a part of the ftsZ homolog was selected from the collected data. Using the cloned DNA fragment as a probe, the entire coding region of the fts.Z homolog was screened from the previously constructed lambda phage library of KODl chromosomal DNA (35). The ftsZ homolog (Pk-ftsz) from P. kodukaraensis KODl was sequenced and the nucleotide and deduced amino acid sequences of the gene are shown in Fig. 1. Sequence analysis of the gene revealed an open reading frame (ORF) encoding 380 amino acids initiated from the triplet codon of TTG. Utilization of the TTG codon as a translation start has been described previously (36). Moreover, the putative ribosome-binding site (RBS), GGGTG, could be identified as indicated in Fig. 1. Although there is another possible initiation codon (ATG: 20-22) further upstream from the above-mentioned TTG codon, no putative RBS could be found. Therefore, we concluded that this TTG is the most likely putative translation initiation codon. In FtsZs of bacteria and tubulins of eucarya, glycine-rich sequences ((G/A)GGTG(T/S)G) implicated in GTP binding (8-11) are well conserved. Indeed, Pk-FtsZ has glycine-rich sequence GGGTGAG at the amino acid position 101 to 107. This result suggests that the Pk-ftsZ gene product would have GTPase activity as well as the ability for GTP binding. The calculated molecular mass of the Pk-ftsZ gene product (Pk-FtsZ) from the deduced amino acid sequence (380 amino acids) is 41,354 Da. Expression and purification of recombinant Pk-FtsZ protein High-level expression of Pk-ftsZ and purification of the expressed recombinant protein (Pk-FtsZ) were attempted. It was previously reported that for the purpose of high-level expression of heterologous genes in E. coli, the expression level is influenced by codon usage around the translation initiation codon (37). If there are codons which are not efficiently utilized in E. coli in the

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amino acid sequences of Pk-ftsZ gene from P. kodakuruensis KODI. codon at 1196 to 1198. The putative ribosome-binding site is underlined.

region, efficient expression of the cloned gene cannot be expected. Therefore, when the forward primer for PCR amplification of the Pk-f&Z gene was designed, rare codons were substituted with codons frequently used in E. coli (38). The Pk-ftsZ gene, amplified by PCR with this forward primer and a reverse primer, was cloned into T7 RNA polymerase expression vector PET-8c, and the resultant plasmid was designated as PET-&Z. The recombinant Pk-FtsZ protein was purified to homogene-

The Pk-f&Z gene begins at positions The glycine-rich sequence is boxed.

ity from E. coli BL21 (DE3) harboring PET-ftsZ grown in NZCYM medium containing ampicillin (50 ,ug . mll l) which had been induced with 1 mM IPTG for 3 h. The Pk-FtsZ protein was purified according to the procedure described in Materials and Methods. As shown in Fig. 2, the sample was purified to homogeneity as indicated by the presence of a single band on a SDS-PAGE gel stained with Coomassie brilliant blue. The molecular weight of the protein band

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A 94 67

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20.1 k FIG. 2. Purification of the recombinant Pk-FtsZ protein expressed in E. coli cells. The Pk-FtsZ protein from P. kodakuraensis KODl was purified from BL21 (DE3) harboring PET&Z. Lane 1, Molecular weight markers (from the top: phosphorylase b, 94 k; albumin, 67 k; ovalbumin, 43 k; carbonic anhydrase, 30 k; and trypsin inhibitor, 20.1 k). Lane 2, Pk-FtsZ protein purified as described in Materials and Methods.

(43 k) was slightly larger than that calculated from the deduced amino acid sequence (41.4 k) but the difference was within an allowable deviation. GTPase and GTP-binding assays Bacterial and archaeal FtsZ proteins from E. coli, Bacillus subtilis, Mycoplasma pulmonis and Haloferax volcanii were reported to bind and/or hydrolyze GTP like eucaryal tubulins (8-11, 20, 39). To determine whether the biochemical properties were also conserved in Pk-FtsZ, purified recombinant protein of Pk-FtsZ was examined for GTPase activity and GTP-binding ability. GTPase activity was measured by TLC to assess the conversion of [IY-~~P]GTP to [a-32P] GDP at various reaction temperatures. As shown in Fig. 3, Pk-FtsZ possesses apparent GTPase activity and the optimum GTPase activity was at temperatures higher than 8O”C, however, at temperatures lower than 4O”C, little GTPase activity could be detected. This result indicated that a high reaction temperature was required for the GTPase activity of the Pk-FtsZ protein in accordance with the thermophilic nature of P. kodakaraensis KODl. This observation was similar to the report that FtsZ from H. volcanii exhibited GTPase activity only at high salt concentrations, consistent with its halophilic nature (20). The Pk-ftsZ gene from P. kodakaraensis KODl could not complement an E. coli ftsZ84 (ts) mutant (40). Moreover, the expression of the Pk-ftsZ gene in E. coli cells (strain JM109) did not lead to any striking changes in cell morphology (data not shown), although previous reports mentioned that heterologous ftsZ genes expressed in E. coli could cause significant morphological alteration (39, 41, 42). These results might be due to the fact that Pk-FtsZ requires extremely high temperatures for its function and the culture temperature for E. coli was too low for Pk-FtsZ to compensate or influence the cell division system of E. co/i. GTP-binding ability was examined by cross-linking the protein to [WEEP] GTP by UV irradiation. The band labeled by cross-linking would appear at the position corresponding to the size of the protein on the SDS-PAGE

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gel when the protein could be cross-linked to [(u-~~P] GTP. As shown in Fig. 4, Pk-FtsZ protein bound [WEEP]GTP efficiently at 37”C, while BSA as the negative control could not, suggesting that a high reaction temperature was not required for GTP binding to PkFtsZ, in contrast to the case of its GTPase activity. Thermostabie polymerization of Pk-FtsZ protein The conservation of biochemical properties such as GTPase activity and GTP-binding ability in Pk-FtsZ indicates the possibility of polymerization (protofilament formation) of Pk-FtsZ protein as in E. coli FtsZ. Furthernature of P. more, considering the thermophilic kodakaraensis KODl , the protofilament formation of Pk-FtsZ protein could be observed at elevated temperatures. Accordingly, we examined Pk-FtsZ polymerization

FtsZ HOMOLOG

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at 90°C. Polymerization assay based on light scattering (43) was performed and the optical density at 340 nm (OD& was monitored at 90°C. In the absence of GTP, Pk-FtsZ protein did not scatter light, indicating that the protofilament was not formed (Fig. 5). After addition of GTP (final concentration 1 mM) as indicated by the arrow in

FIG. 5. Time course of Pk-FtsZ polymerization at 90°C. The arrow indicates the time when GTP was added to the final concentration of 1 mM. The reaction mixture contained 50 mM HEPES-NaOH @H 7.2), 10 mM MgCIZ, 200 mM KCl, and 15 pg of Pk-FtsZ protein. ODX4,,was recorded every 1 s.

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FIG. 6. Phylogenetic analysis of Pk-FtsZ with eight archaeal and three bacterial FtsZ homologs and nine eucaryal tubulins. The phylogenetic tree was constructed using CLUSTAL W. The scale bar represents the estimated number of amino acid substitutions per site. The bootstrap percentage values from 1000 resamplings of the data set are given at each node. Swissprot accession numbers for FtsZs are as follows: Escherichia coli, P06138; Bacillus subtilis, P17865; Pseudomonas aeruginosa, P47204; Halobacterium salinarum, 448290; Haloferax volcanii, 448327; Methanococcusjannaxhii MJ0370,Q57816; M. jannaschii MJO622,Q58039; Pyrococcus woesei, Q52630; Pyrococcus horikoshii PHOO03, DDBJ accession no. BAA29071; P. horikoshii PH0769, DDBJ accession no. BAA29860; and P. horikoshii PH1335, DDBJ accession no. BAA30441. Swissprot accession numbers for tubulins are as follows: a-tubulin of Drosophila melanogaster, PO6604; P-tubulin of D. melanogaster, PO8840; r-tubulin of D. melanogaster, P42271; a-tubulin of Homo sapiens, P04687; /3-tubulin of H. sapiens, P07437; r-tubulin of H. sapiens, P23258; a-tubulin of Schizosaccharomycespombe, P04689; ,!%tubulin of S. pombe, P05219; and r-tubulin of S. pombe, P25295.

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Fig. 5, Pk-FtsZ protein scattered light. This result indicates that the increase in OD34,, was not due to the heat denaturation of Pk-FtsZ, but due to the polymerization of Pk-FtsZ using CTP. Therefore, Pk-FtsZ can polymerize to form protofilaments at 90°C. The polymerization of Pk-FtsZ was induced by addition of GTP as in E. co/i FtsZ (33). Phylogenetic analysis Pk-FtsZ was analyzed phylogenetically with other archaeal FtsZs, bacterial FtsZs, and eucaryal tubulins. The phylogenetic tree was constructed by the neighbor-joining method as shown in Fig. 6. The tree is mainly divided into two distinct branches. One is composed of eucaryal tubulins and the other group includes bacterial FtsZ proteins and archaeal homologs. In the branch for FtsZs, bacterial FtsZ proteins form an independent group from archaeal FtsZs, which are clearly divided into three groups, paralogs 1, 2, and 3, as shown in Fig. 6. Paralog 3 with PkFtsZ is located outside the grouping of the bacterial and two other archaeal groups (paralogs 1 and 2). Therefore, Pk-FtsZ is considered to be a novel FtsZ protein which is phylogenetically distinct from other FtsZs, although our experimental results such as GTPase activity, GTPbinding ability, and GTP-dependent polymerization indicated that the hyperthermophilic archaeon P. kodakaraensis KODI possesses a bacterial type ftsZ homolog which might be important for Z ring formation during cell division. It is possible that the FtsZ homologs corresponding to paralogs 1 and 2 could exist in P. kodakaraensis KODl and that these proteins might interact with the Pk-FtsZ as additional factors. Screening of other FtsZ homologs from P. kodakaraensis is now in progress.

12. 13. 14.

15.

16. 17. 18. 19.

20. 21.

22. ACKNOWLEDGMENT This work was supported by a grant from CREST Research for Evolutional Science and Technology, Japan).

(Core

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