Cloning and Characterization of a Haloarchaeal Heat Shock Protein 70

RESEARCH LETTER Cloning and characterization ofa haloarchaeal heat shock protein 70 functionally expressed in Escherichia coli Hao Zhang, Lu Lin, Chi Zeng, Ping Shen & Yu-Ping Huang

Laboratory of Microbial Genetics, College of Life Sciences, Wuhan University, Wuhan, China Downloaded from https://academic.oup.com/femsle/article/275/1/168/499933 by guest on 01 October 2021 Correspondence: Yu-Ping Huang, Abstract Laboratory of Microbial Genetics, College of Life Sciences, Wuhan University, Wuhan The Hsp70 molecular chaperone machine is constituted by the 70-kDa heat shock 430072, China. Tel.: 186-27-68754533; protein Hsp70 (DnaK), cochaperone protein Hsp40 (DnaJ) and a nucleotide- fax: 186-27-68754833; exchange factor GrpE. Although it is one of the best-characterized molecular e-mail: [email protected] chaperone machines, little is known about it in archaea. A 5.2-kb region containing the hsp70 (dnaK) gene was cloned from Natrinema sp. J7 strain and sequenced. Received 7 May 2007; revised 29 June 2007; It contained the Hsp70 chaperone machine gene locus arranged unidirectionally in accepted 13 July 2007. the order of grpE, hsp70 and hsp40 (dnaJ). The hsp70 gene from Natrinema sp. J7 First published online August 2007. was overexpressed in Escherichia coli BL21 (DE3). The recombinant Hsp70 protein was in a soluble and active form, and its ATPase activity was optimally active in DOI:10.1111/j.1574-6968.2007.00881.x 2.0 M KCl, whereas NaCl had less effect. In vivo, the haloarchaeal hsp70 gene 1 Editor: Marco Moracci allowed an E. coli dnak-null mutant to propagate l phages and grow at 42 C. The results suggested that haloarchaeal Hsp70 should be beneﬁcial for extreme Keywords halophiles survival in low-salt environments. hsp70 ; Hsp70; ATPase activity; haloarchaea.

structural homology, Hsp70s are highly specific and the Introduction basis of this functional specificity is not well understood A variety of environmental stresses induce the synthesis of a (Buchberger et al., 1994; James et al., 1997). set of highly conserved proteins called heat shock proteins Hsp70 proteins in bacteria and eukaryotes have been (Hsps). Some Hsps are also molecular chaperones, and play extensively studied (Mayer & Bukau, 2005). However, little important roles in normal cellular physiology and in cell information is known regarding haloarchaeal Hsp70 pro- survival in the face of stress (Ellis & van der Vies, 1991). The teins. An Hsp70 homologue from Halobacterium cutiru- heat shock Hsp70 system is one of the best-characterized brum was identified and purified (Tokunaga et al., 1999). chaperone machineries, which contains Hsp70 (DnaK), its Nevertheless, the functional specificity of haloarchaeal cochaperone protein Hsp40 (DnaJ) and the nucleotide Hsp70 has not been reported. Here, the hsp70 gene was exchange factor GrpE (Bukau & Horwich, 1998). Hsp70 cloned from an extremely halophilic archaeon Natrinema sp. systems are not only universally distributed in bacteria and J7, which harbors a high copy number plasmid pHH205 (Ye eukaryotes but are also found in some archaea, especially et al., 2003) and possesses extracellular proteolytic activity that they occur in all haoarchaea with no exceptions (Shi et al., 2006). Then, the ATPase activities of recombinant (Macario et al., 2004). Hsp70 protein expressed in Escherichia coli and functional Hsp70 proteins are one of the most conserved proteins complementation of an E. coli dnaK mutant were analyzed. known to date, which form the central part of the Hsp70 system. Hsp70s are composed of a highly homologous N-terminal ATPase domain, a substrate-binding domain Materials and methods and a C-terminal variable domain (Mayer & Bukau, 2005), Strains, plasmids and culture conditions the ATPase activities of which are all stimulated by Hsp40, GrpE and substrate binding (Liberek et al., 1991), and the Natrinema sp. J7 strain was grown in a complex medium at molecular chaperone functions of Hsp70 seem to be based 45 1C(Yeet al., 2003). The E. coli MC4100 dnaK-null mutant on its ATPase activity. Despite the high sequence and BM271 (Paek & Walker, 1987) and plasmid pTrc99a (Wang

Table 1. The oligodeoxynucleotide PCR primers used in this paper with the DNA probes. The inserted fragments obtained from Primer Nucleotide sequence Reference the selected positive plasmid were sequenced. For the construction of the expression vector, the haloarch- Primer63 50-GGAAGGCAGCGACCCCGAAATC-30 hsp70 Primer964 50-CCTGTGGCATTCTCGTGGACCC-30 primers for aeal hsp70 ORF was amplified by PCR with the primer HKF- Halobacterium NdeI and HKR-HindIII. The PCR fragment was digested with sp. NRC-1 the NdeI and HindIII, and then ligated into NdeI/HindIII- HKF-NdeI 50-GGGCATATGGCGAGCAACAAAATTC-30 hsp70 primers digested pET26b1 to construct plasmid pHK2. HKR-HindIII 50-GGGAAGCTTCTCCTCGTCGTCCTCGTC-30 for Natrinema Another expression plasmid, named pTrcK, was con- sp. J7 structed by the introduction of the NcoI–HindIII full-length KP-RT 50-GTCGGCTCGTTGAT-30 hsp70 specific KPF-Out 50-TACTTCTCCGACCGACAGC-30 primers for ORF of hsp70 into a plasmid pTrc99a previously digested KPR-Out 50-GCGTTTGATCTTCTGGAGGA-30 RLM-RACE PCR with the same enzymes. KPF-Inner 50-CGCATCATCAACGAGCCGA-30 Downloaded from https://academic.oup.com/femsle/article/275/1/168/499933 by guest on 01 October 2021 0 0 KPR-Inner 5 -ATCGTCTTTTCGGGGTTCTG-3 Overexpression, purification and ATPase activity assay of the haloarchaeal Hsp70 The expression of Hsp70 was achieved by induction of the et al., 2004) were kindly gifted by Prof. B. Lipinska˜ (Gdansk˜ cultures with 1 mM isopropyl-b-D-thiogalactopyranoside University, Poland) and Dr M.R. Volkert (University of (IPTG) at A 0.6, followed by an incubation of 12 h at Massachusetts Medicine School), respectively. Plasmid 600 nm 30 1C. Then, cultures were harvested and resuspended in pDNAJ (Yoshimune et al., 2005), an expression plasmid for 20 mM Tris-Cl buffer (pH 7.2). Following cell lysis by E. coli DnaJ (EcoDnaJ), was kindly provided by Dr N. Esaki sonication (3–6 pulses of 1 min, interspersed with cooling (Kyoto University, Japan). on ice), cell debris was pelleted by centrifugation at 12 000 g for 20 min, and clear supernatants were obtained. Molecular genetic methods Purifications of Hsp70 were carried out with the Ni-NTA HisBind affinity chromatography kit according to the The genome DNA and total RNA were isolated and purified method of QIAGEN (2002). The protein concentrations according to protocols described in The Halohandbook: were determined using the Bradford method (Bradford, Protocols for Halobacterial Genetics, version 4.9 (http:// 1976) with bovine serum albumin as a standard, and the www.microbiol.unimelb.edu.au/people/dyallsmith/resources/ homogeneity of the final Hsp70 preparation was confirmed halohandbook/index.html). Molecular cloning techniques by 10% sodium dodecylsulfate-polyacrylamide gel were performed using standard procedures (Sambrook & electrophoresis (SDS-PAGE). Russell, 2001). RNA ligase-mediated rapid amplification of ATPase activity of Hsp70 protein was determined by cDNA ends by PCR (RLM-RACE PCR) was performed using measuring the free phosphate released from the ATP hydro- the 50-full RACE Core Set (Takara Biotech, China) according lysis according to the malachite green method (Lanzetta et al., to the manufacturer’s instructions. The oligodeoxynucleo- 1979). All reactions were performed in buffer M (50 mM Tris- tide primers used in this study are shown in Table 1. Cl, pH 7.6; 10 mM MgCl2;5mMATP),and2MKClwas added when the effects of temperature on the ATPase Gene cloning and plasmids construction activities were studied. The salt dependence of the ATPase activity was measured between 0 and 3 M NaCl or KCl, For Southern blot analysis, the genomic DNA was digested respectively. After a 30-min incubation of Hsp70 with buffer with restriction enzymes and separated by electrophoresis in M, the reaction was stopped by addition of 20% tricarboxylic 1% agarose gel and then transferred to a nylon membrane acid. The standard curve was obtained using phosphate buffer (Hybond-N1, Amersham, Buckinghamshire, UK). The 900- (pH 6.8). The enzymatic activity was calculated as nanomole bp PCR-amplified fragment of hsp70 (positions 63–964) of ATP per milligram of DnaK per minute. from Halobacterium sp. NRC-1 was used as a probe. The digoxigenin-labeled probes were hybridized with the digested Immunological identification of the fragments at 42 1C overnight. The membrane was washed, haloarchaeal recombinant Hsp70 and the hybridized signals were detected with a digoxigenin detection kit (Boehringer Mannheim, Germany) as described In order to exclude the contamination of E. coli endogenous in the supplier’s instructions. The DNA fragments whose chaperones in archaeal Hsp70 preparation, Western blotting sizes were similar to those of the hybridized fragments were was performed by transferring the proteins, after electro- ligated into the plasmid vector pUC18, and the resultant phoresis on 10% acrylamide gels, to a nitrocellulose mem- constructs were introduced into E. coli JM109. The plasmids brane using the Transblot cell from BioRad, and then carrying the hsp70 genes were identified by hybridization incubating the membrane with polyclonal anti-DnaKEc,

anti-DnaJEc or anti-GrpEEc antibodies (a generous gift from Nucleotide sequence accession number Axel Mogk, Guenter Kramer and Bernd Bukau, University The nucleotide sequence for fragment p22 sequence from Heidelberg, Germany) diluted 1 : 10 000. Horseradish per- Natrinema sp. J7 has been deposited in the GenBank database oxidase-conjugated goat anti-rabbit IgG (ImmuClub) was (accession no. DQ874621). used at 1 : 5000 dilution as a secondary antibody. The immunoblots were developed with SuperSignal West Pico Substrate (Pierce, UK). Results and discussion

Suppression of the E. coli mutant Isolation of the Hsp70 system genes from thermosensitive phenotype Natrinema sp. J7

The thermosensitive mutant of E. coli, BM271DdnaK, was Southern blot hybridization of Natrinema sp. J7 chromoso- Downloaded from https://academic.oup.com/femsle/article/275/1/168/499933 by guest on 01 October 2021 transformed with pTrcK and pDNAJ. The resultant trans- mal DNA with the Halobacterium NRC-1 PCR probe formants were grown overnight at 30 1C in Luria–Bertani revealed that KpnI digests produced single hybridizing (LB) medium supplemented with ampicillin, and then bands of about 5.5 kb. KpnI-digested fragments of 5–6 kb inoculated into fresh medium with 1 mM IPTG. After were isolated and cloned into pUC18 vectors. The cloned P22 fragment was found to contain a full-length copy of the cultivation at 30 1CtoA600 nm 0.6, aliquots of 2 mL of this sample and successive 10-fold dilutions from 101 to 106 of hsp70 gene. The entire physical map of this region is shown cultures were spread onto selective LB plates in duplicate. in Fig. 1. The nucleotide sequence has been deposited within Test plates were incubated for 18 h at either 30 or 42 1C. GenBank (accession no. DQ874621). The length of P22 Escherichia coli B178 wild-type (wt) and BM271DdnaK were fragment is 5276 bp; the G1C content is 64.3%. Computer used as controls. database searches revealed that the sequence contained three ORFs (NnmGrpE, NnmHsp70 and NnmHsp40), which showed a strong similarity to the Hsp70 chaperone machine Bacteriophage k growth test proteins. Similar to other haloarchaeal proteins, many The transformants were grown in selective LB medium additional acidic residues were present in these three pro- containing 10 mM MgSO4, 0.2% maltose and 1 mM IPTG teins (Madern et al., 2000). The acidic residues percentage of at 30 1C for 12 h. One hundred microliters of the culture was NnmHsp70, NnmHsp40 and NnmGrpE protein is 24.7%, mixed with l phage (10 mL, 103 PFU) and incubated at 30 1C 20.3% and 31.2% (mol/mol), respectively. for 15 min. The mixture was then mixed with 3 mL of l top agar containing 1 mM IPTG and poured onto selective LB Sequence comparisons and analyses plates. After incubation at 30 1C for about 12 h, plaques were counted, and the efﬁciency of plating (e.o.p.) was calculated Pairwise alignment of the Hsp70 sequences using the as the ratio of PFU on the tested strain to the PFU on the wt CLUSTALW program revealed that the NnmHsp70 showed reference strain (E. coli B178). between 77.9% and 82.7% amino acid identity over its All in vivo and in vitro tests were repeated at least three entire length to other halophilic counterparts. The amino times, and the mean values and SDs were calculated. acids identity of haloarchaeal Hsp40 was also high (76.4%),

Fig. 1. (a) Physical map of the cloned 5.3-kb region of genomic DNA from Natrinema sp. J7. Orientations and lengths of identiﬁed ORFs are shown by arrows. Restriction sites are indicated above and a scale bar (in bp) is shown below. (b) RLM-RACE PCR analysis of the transcription start site of the hsp70 gene. The transcription site point is indicated by the bent arrows. The promoter motif is indicated by a solid line under the sequence.

c 2007 Federation of European Microbiological Societies FEMS Microbiol Lett 275 (2007) 168–174 Published by Blackwell Publishing Ltd. All rights reserved A haloarchaeal Hsp70 functionally expressed in E. coli 171 whereas haloarchaeal GrpEs had a relatively low sequence homology (40.6%). The Nnmhsp70 ORF consists of 1929 bp and encodes a polypeptide consisting of 642 amino acid residues with a deduced molecular mass of 68 602 Da. The Nnmhsp40 ORF consists of 1170 bp and encodes a polypeptide consisting of 389 amino acid residues. The NnmgrpE ORF consists of 1089 bp and encodes a polypeptide consisting of 362 amino acid residues. The NnmgrpE gene was located 176 bp upstream of the Nnmhsp70 gene, while the Nnmhsp40 gene was located 807 bp downstream of the Nnmhsp70 gene. This Hsp70 chaperone machine gene locus was arranged unidir- Downloaded from https://academic.oup.com/femsle/article/275/1/168/499933 by guest on 01 October 2021 ectionally. However, in all known hsp70 loci of haloarchaea, there is only one cochaperone gene (hsp40 or grpE) near the hsp70 gene; the others are far away (Macario et al., 2006).

Identification of the transcription start sites for hsp70 In order to localize the promoter, the transcription start site Fig. 2. Identification of the recombinant Hsp70 from Escherichia coli. of Nnmhsp70 was determined by 50 RLM-RACE PCR (a) Different soluble cytoplasmic fractions of the Hsp70-expressed recombinants are shown in SDS-PAGE. Lane M, molecular mass markers; analysis. One clear ending sequence, corresponding to lanes 1, 2, the soluble cytoplasmic fractions of E. coli BL21(DE3)/ position 4 bp upstream from the A of the first ATG, was pET26b1 and BL21(DE3)/pHK-2, respectively; lane 3, the purified re- obtained by DNA sequencing of the PCR product (Fig. 1b). combinant Hsp70; lanes 4–6, the soluble cytoplasmic fractions of E. coli As a result, the Nnmhsp70 mRNA is among those that would BM271DdnaK, BM271DdnaK/pTrcK and BM271DdnaK/pTrcK1pDNAJ, be considered a leaderless mRNA. Natural leaderless mRNAs respectively. (b) Western blotting analysis of the purified NnmHsp70 have been identified in all kingdoms of life and are quite (lane Nnm) and E. coli chaperones (lane Ec) using anti-DnaKEc, anti- common in archaea (Torarinsson et al., 2005). DnaJEc or anti-GrpEEc polyclonal antibodies, respectively. The hsp70 mRNA start point suggested a putative pro- protein was overproduced in a soluble form in E. coli BL21 moter sequence at 24 bp upstream of the start site (Fig. 1b). (DE3) recombinants (Fig. 2a), the apparent molecular mass This TATA-box motif was in good agreement with the of which was much higher than that calculated from the archaeal consensus promoter element (Reeve, 2003). The amino acids’ composition (68.7 kDa). The discrepancy of purine-rich region as transcription factor B recognition the molecular mass has been frequently observed for halo- element (BRE) was also detected upstream of the philic proteins, the reason for which is attributed to the TATA-box. Northern hybridizations of the RNA with an strong excess of negatively charged acidic amino acids, hsp70 probe showed a single transcript of 2.0 kb (data not which decreases the amount of bound SDS on Hsp70 and shown). This mRNA was inconsistent with the size of the affects the mobility of the protein on SDS-PAGE gels. It was hsp70 gene (1922 bp) and indicated that Nnmhsp70 was a reported that in some cases, the apparent molecular mass of monocistronic gene based on the location of the transcrip- halophilic proteins on the gel could be overestimated by as tion start site. In previous works, it was demonstrated that much as 50% (Madern et al., 2000). The recombinant the three genes of Hsp70 system in an archaeon (Methano- NnmHsp70 that existed in the cytoplasmic fractions of the sarcina mazei) are transcribed individually from a different cells was purified by affinity chromatography on a Ni-NTA promoter for each gene (Hickey et al., 2002). Hence, it HisBind column. The purified protein was apparently appears likely that the same mode of transcription will occur homogenous as judged by denaturing SDS-PAGE (Fig. 2a, in Natrinema sp. J7. lane 3). In order to exclude the contamination of E. coli endogenous chaperones in haloarchaeal Hsp70 preparation, Expression, purification and properties of Western blotting was performed with polyclonal anti-Dna- haloarchaeal Hsp70 KEc, anti-DnaJEc or anti-GrpEEc antibodies (Fig. 2b). The Hsp70 protein is found in all haloarchaea with no excep- results indicate that there is no trace of DnaK in NnmHsp70 tions. To ascertain the functional properties of haloarchaeal preparation, consistent with the observation that DnaK is Hsp70, the recombinant plasmid pHK-2 was constructed to expressed at low levels in the absence of heat shock and express NnmHsp70 protein in E. coli BL21 (DE3). After should not contribute by more than 1% to the total proteins induction with IPTG, a polypeptide of about 97-kDa (Wickner et al., 1992).

One of the most important distinctions between 2.5 M, respectively. When KCl was replaced by NaCl in NnmHsp70 and other halophilic proteins is the higher concentrations from 0.5 to 3 M, only 38.1% of the maximal solubility of overexpressed NnmHsp70 in E. coli. While activity was found. Moreover, it was striking that 11.2% previous reports showed that the overexpressed haloarchaeal remaining activity was observed when there was no KCl and proteins were usually in the form of an inclusion body, and NaClinthebuffer.TheoptimaltemperatureforHsp70ATPase needed to be reactivated in a buffer containing a high activity was 45 1C in the presence of 2.0 M KCl (Fig. 4b). concentration of NaCl or KCl, very little recombinant NnmHsp70 maintains ATPase activities in solutions with proteins, such as citrate synthase from Haloferax volcanii, low ionic strength, which is unusual in the halophilic produced in a soluble but inactive form, were reactivated by proteins studied so far. The growth of haloarchaea abso- incubating with 2 M KCl (Connaris et al., 1999). lutely requires a concentration of NaCl as high as 5.2 M (saturated NaCl). Therefore, most of the haloarchaeal pro- Downloaded from https://academic.oup.com/femsle/article/275/1/168/499933 by guest on 01 October 2021 Effect of salts and temperatures on the ATPase teins have a requirement of a high salt concentration in activities of haloarchaeal Hsp70 order to maintain their activities. Some of them denature upon decreasing the NaCl concentration to 1–2 M (Madern TheeffectofKClandNaClontheATPaseactivityof et al., 2000), while NnmHsp70 shows low ATPase activity recombinant NnmHsp70 was tested between 0 and 3 M in the even without NaCl and KCl. The solubility and ATPase presence of buffer M (Fig. 3a). The recombinant NnmHsp70 activity of NnmHsp70 over a wide range of salt concentra- showed high activity at 2 M KCl; 31.3% and 44.8% remaining tions suggest that the NnmHsp70 should function against activities were observed at KCl concentrations of 1.0 and osmotic stress.

Complementation of the dnaK-null mutant of E. coli with the haloarchaeal Hsp70 The dnaK-null mutant of E. coli, BM271DdnaK, lacks the ability to grow at elevated temperatures and propagate l phages even at moderate temperatures (Paek & Walker, 1987; Alfano & McMacken, 1989). However, both defects were rescued by transformation of recombinant plasmid encoding the EcoDnaK gene (Suppini et al., 2004). To ascertain whether the NnmHsp70 can function in bacterium, the suppression of the E. coli dnaK-null mutant by a cloned Nnmhsp70 gene was examined. Mogk et al. (1999) showed that the essential level of the functional EcoDnaJ was reduced due to a polar effect of the chloramphenicol-resistant marker downstream of the EcoDnaJ gene. Therefore, the plasmid pDNAJ carrying the EcoDnaJ gene was transformed into BM271DdnaK, accom- panied by NnmHsp70 expression plasmid pTrcK. A highly expressed NnmHsp70 protein was conﬁrmed on the SDS-PAGE gel (Fig. 2, lanes 5 and 6). As shown in Fig. 4, NnmHsp70 rescued the thermosensitive growth defect of the E. coli dnaK-null mutant at 42 1C, whether it was with the assistance of EcoDnaJ or not. Moreover, l phage- resistant tests with the coexpression of EcoDnaJ or not demonstrated that NnmHsp70 were able to support the propagation of l phage in the E. coli dnaK-null mutant, with an e.o.p. of 0.32 and 0.19, respectively. Complementation of the dnaK-null mutant of E. coli with the Nnmhsp70 in vivo indicates a functional similarity

Fig. 3. Effect of salts and temperatures on the ATPase activity of between Natrinema sp. J7 and E. coli Hsp70 (DnaK). To the NnmHsp70. The purified NnmHsp70 were assayed for ATPase activity at authors’ knowledge, this is the first report of archaeal hsp70 various concentrations (a) of KCl(’) or NaCl() and various tempera- complementation in a dnaK-deficient bacterium. Among tures (b) in the range from 37 to 55 1C. the archaea with hsp70 gene, the hsp70 from the methanogen

Fig. 4. Complementation experiments of the Escherichia coli dnaK-null mutant with the Nmhsp70 gene at 30 1C(a)or421C (b). From left to right are spots of serial dilutions from 101 to 106 of the bacterial suspensions. B178, E. coli B178 (wild type); DK, E. coli dnaK-null mutant strain BM271DdnaK; DK1pTrcK, E. coli BM271DdnaK carrying plasmid pTrcK; DK1pTrcK1pJ, E. coli BM271DdnaK carrying plasmid pTrcK and pDNAJ. Downloaded from https://academic.oup.com/femsle/article/275/1/168/499933 by guest on 01 October 2021

M. mazeii is the only one that has been extensively investi- Bukau B & Horwich AL (1998) The Hsp70 and Hsp60 chaperone gated. Functional analyses showed that the M. mazeii Hsp70 machines. Cell 92: 351–366. in vitro is similar to that of the bacterium E. coli used for Connaris H, Chaudhuri JB, Danson MJ & Hough DW (1999) comparison; however, in vivo analyses indicate significant Expression, reactivation, and purification of enzymes from differences (Zmijewski et al., 2004). Haloferax volcanii in Escherichia coli. Biotechnol Bioeng 64: In summary, the data presented in this study demonstrate 38–45. that in vivo the Hsp70 protein from Natrinema sp. J7 is able to Ellis RJ & van der Vies SM (1991) Molecular chaperones. complement an E. coli dnaK mutant strain for growth at high Annu Rev Biochem 60: 321–347. temperatures and l phage propagation. The functional com- Hickey AJ, Conway de Macario E & Macario AJ (2002) plementation and characterization of the haloarchaeal Hsp70 Transcription in the archaea: basal factors, regulation, and implied that this unique chaperone protein would be bene- stress-gene expression. Crit Rev Biochem Mol Biol 37: 537–599. ficial for extreme halophile survival in low-salt environments. James P, Pfund C & Craig EA (1997) Functional specificity among Hsp70 molecular chaperones. Science 275: 387–389. Lanzetta PA, Alvarez LJ, Reinach PS & Candia OA (1979) An improved assay for nanomole amounts of inorganic Acknowledgements phosphate. Anal Biochem 100: 95–97. The authors thank Nobuyoshi Esaki for the plasmid pDNAJ, Liberek K, Marszalek J, Ang D, Georgopoulos C & Zylicz M Michaeal R. Volkert for plasmid pTrc99a and Barbara (1991) Escherichia coli DnaJ and GrpE heat shock proteins Lipinska˜ for E. coli MC4100 strain and dnaK-null mutant jointly stimulate ATPase activity of DnaK. Proc Natl Acad Sci BM271 strain. The authors also thank Axel Mogk, Guenter USA 88: 2874–2878. Kramer and Bernd Bukau for the generous gifts of poly- Macario AJ, Malz M & Conway de Macario E (2004) Evolution of assisted protein folding: the distribution of the main clonal anti-DnaKEc, anti-DnaJEc and anti-GrpEEc antibodies. This work was supported by the National Grand chaperoning systems within the phylogenetic domain archaea. Fundamental Research Program (973) of China (No. Front Biosci 9: 1318–1332. 2004CB719600) and the National Natural Science Founda- Macario AJ, Brocchieri L, Shenoy AR & Conway de Macario E (2006) Evolution of a protein-folding machine: genomic tion of China (No. 30470033, 30570026). and evolutionary analyses reveal three lineages of the archaeal hsp70(dnaK) gene. J Mol Evol 63: 74–86. Madern D, Ebel C & Zaccai G (2000) Halophilic adaptation of References enzymes. Extremophiles 4: 91–98. Alfano C & McMacken R (1989) Heat shock protein-mediated Mayer MP & Bukau B (2005) Hsp70 chaperones: cellular disassembly of nucleoprotein structures is required for the functions and molecular mechanism. Cell Mol Life Sci 62: initiation of bacteriophage lambda DNA replication. J Biol 670–684. Chem 264: 10709–10718. Mogk A, Bukau B, Lutz R & Schumann W (1999) Construction Bradford MM (1976) A rapid and sensitive method for and analysis of hybrid Escherichia coli–Bacillus subtilis dnaK quantition of proteins utilizing the principles of protein–dye genes. J Bacteriol 181: 1971–1974. binding. Anal Biochem 72: 248–254. Paek KH & Walker GC (1987) Escherichia coli dnaK-null mutants Buchberger A, Valencia A, McMacken R, Sander C & Bukau B are inviable at high temperature. J Bacteriol 169: 283–290. (1994) The chaperone function of DnaK requires the coupling QIAGEN (2002) A Handbook for High-Level Expression and of ATPase activity with substrate binding through residue Purification of 6xHis-Tagged Proteins. QIAGEN GmbH, E171. EMBO J 13: 1687–1695. Hilden.

Reeve JN (2003) Archaeal chromatin and transcription. Wang JY, Sarker AH, Cooper PK & Volkert MR (2004) The Mol Microbiol 48: 587–598. single-strand DNA binding activity of human PC4 prevents Sambrook J & Russell DW (2001) Molecular Cloning: mutagenesis and killing by oxidative DNA damage. Mol Cell A Laboratory Manual, 3rd edn. Cold Spring Harbor Biol 24: 6084–6093. Laboratory Press, New York. Wickner S, Skowyra D, Hoskins J & McKenney K (1992) DnaJ, Shi W, Tang XF, Huang Y, Gan F, Tang B & Shen P (2006) An DnaK, and GrpE heat shock proteins are required in oriP1 extracellular halophilic protease SptA from a halophilic DNA replication solely at the RepA monomerization step. archaeon Natrinema sp. J7: gene cloning, expression and Proc Natl Acad Sci USA 89: 10345–10349. Ye X, Ou J, Ni L, Shi W & Shen P (2003) Characterization of a novel characterization. Extremophiles 10: 599–606. plasmid from extremely halophilic Archaea: nucleotide Suppini JP, Amor M, Alix JH & Ladjimi MM (2004) sequence and function analysis. FEMS Microbiol Lett 221:53–57. Complementation of an Escherichia coli DnaK defect by

Yoshimune K, Galkin A, Kulakova L, Yoshimura T & Esaki N Downloaded from https://academic.oup.com/femsle/article/275/1/168/499933 by guest on 01 October 2021 Hsc70–DnaK chimeric proteins. J Bacteriol 186: 6248–6253. (2005) Cold-active DnaK of an Antarctic psychrotroph Tokunaga H, Hara S, Arakawa T, Ishibashi M, Gupta RS & Shewanella sp. Ac10 supporting the growth of dnaK-null Tokunaga M (1999) Identiﬁcation and partial puriﬁcation of mutant of Escherichia coli at cold temperatures. Extremophiles DnaK homologue from extremely halophilic archaebacteria, 9: 145–150. Halobacterium cutirubrum. J Protein Chem 18: 837–844. Zmijewski MA, Macario AJ & Lipinska B (2004) Functional Torarinsson E, Klenk HP & Garrett RA (2005) Divergent similarities and differences of an archaeal Hsp70(DnaK) stress transcriptional and translational signals in Archaea. Environ protein compared with its homologue from the bacterium Microbiol 7: 47–54. Escherichia coli. J Mol Biol 336: 539–549.