Cloning and nucleotide sequence of a hsp70 gene from Streptomyces griseus

JOURNAL OF FERMENTATIONAND BIOENGINEERING Vol. 77, No. 5, 461-467. 1994

Cloning and Nucleotide Sequence of a hsp70 Gene from

Streptomyces griseus YUJI HATADA, H I D E N O R I SHINKAWA,* KAZUYUKI KAWAMOTO, HARUYASU KINASHI, AND OSAMU NIMI

Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, 1-4-1 Kagamiyama, HigashiHiroshima 724, Japan Received 6 December 1993/Accepted 31 January 1994 The cultivation of Streptomyces griseus 2247 at the growth-limited temperature (37°C) or in liquid medium containing 5% ethanol (toxic for growth) revealed the presence of heat-induced proteins in the total cellular proteins. Among them, a 70 kDal protein was isolated and its N-terminal amino acid sequence was determined. The 70 kDal protein possessed a possible ATP-hinding site in the N-terminus, which was conserved among the HSP70 family. A D N A fragment encoding the HSP70 homologue was isolated from a genomic library of S. griseus 2247 strain using an oligonucleotide probe based on the N-terminal amino acid sequence of the 70 kDal protein. D N A sequence analysis of the cloned gene revealed an open reading frame consisting of 618 amino acid residues. The deduced amino acid sequence is highly homologous to the HSP70 family proteins; it is 59.8 % identical to Clostridium perfringens HSPT0, 59.7% to the Bacillus megaterium DnaK protein, 58.4% to the Methanosarcina mazei DnaK protein, 58.1% to Synechocystis HSPT0, 52.8% to the DnaK protein of Escherichia coli, and about 50% to some of the mitochondrial heat shock proteins. The cloned gene could encode the HSP70 of S. griseus.

Induction of heat shock proteins (HSPs) is a principal reaction of heat shock response. Such a phenomenon is observed commonly in a wide spectrum of organisms and is a homeostatic mechanism to protect the ceils from various environmental stresses. The HSPs can be grouped into several families based on their molecular masses. The HSP70 family has been found in many organisms and acts as chaperones in the transport of certain polypeptides (1, 2). Under stress conditions, some of the HSP70 family proteins were also proposed to prevent aggregation of denatured proteins or to reactivate heat-inactivated enzymes, in the presence of ATP (3, 4). An Escherichia coli DnaK protein, a member of the HSP70 family, plays critical roles in regulation of the heat shock response. For example, under the normal growth condition, it was proposed that the major physiological role of DnaK was to regulate the heat shock response negatively (5). At higher temperatures, high levels of DnaK protein are required primarily for the survival of cells (6-8). Since the hsp genes are highly conserved in evolution, their products may exhibit the same kind of important functions in all organisms. Recently 4 major heat shock proteins (94 to 100 kDal, 70 kDal, 56 to 58 kDal and 16 to 18 kDal) were detected in Streptomyces. Among them, HSP18 and HSP56-58 proteins of Streptomyces albus were found to be similar to GroEL-like proteins (9). On the other hand, we found that pleiotropic mutation in Streptomyces griseus 2247 was induced by the incubation of mycelium under two different stress conditions for growth, such as at higher temperatures than that required for optimal growth and in liquid medium containing 5% ethanol, which is toxic for growth (to be published elsewhere). Several kinds of proteins, likely to be the heat shock proteins, were induced in the myce-

lium. The relationship of stress shock response to pleiotropic mutation in S. griseus 2247 strain is still unclear. In this paper, we describe the isolation of HSP70 homologous protein of S. griseus induced commonly by two culture conditions, and the nucleotide sequence analysis of its gene. MATERIALS AND METHODS Bacterial strains, plasmids, and growth conditions

S. griseus 2247 was grown in glucose-meat extract-peptone (GMP) medium (10) containing 1% glucose, 0.2% meat extract, 0.4% peptone, 0.2% yeast extract, 0.5% NaCI, and 0.025% MgSO4.7H20 (pH 7.0). Other Streptomyces strains, which were obtained from stock of Hiroshima University (HUT) and Institute for Fermentation, Osaka (IFO), were cultivated in tryptic soy broth (Difco Laboratories, Detroit, USA). E. coli JM109 (11) was used as a cloning host and pUC19 (12) as a cloning vector. E. coli was grown in Luria-Bertani (LB) medium (13) at 37°C. Ampicillin (Meiji Seika Kaisha Ltd., Tokyo) was added (final concentration of 100/Lg/ml) to the medium as necessary. D N A manipulation and Southern hybridization

Microbiological and recombinant DNA techniques for E. coli were as those described by Sambrook et al. (13). Total DNA of S. griseus, isolated by the method of Hopwood et al. (14), was digested with the restriction endonuclease. After agarose gel electrophoresis, separated DNA fragments were transferred to nitrocellulose filters for Southern hybridization (15). An oligonucleotide probe was synthesized by a Cyclone DNA synthesizer (MilliGen/Biosearch Inc., USA) and labeled with digoxigenin (DIG)-labeled dUTP by using a DIG oligonucleotide tailing kit (Boehringer Mannheim, Germany). The probe was hybridized to the blotted filter in 5 × SSC

* Corresponding author. 461

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HATADA ET AL.

(1 × SSC; 0.15 M NaC1, 0.015 M sodium citrate, pH 7.0) at 65°C. The filter was washed twice with 2× SSC containing 0.1% SDS and then washed twice with 0.1 × SSC containing 0.1% SDS at 65°C. Hybridization patterns were detected with a DIG-detection kit (Boehringer Mannheim). Colony hybridization experiments were carried out using the same probe. Procedures of protein analysis Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to the method of Laemmli (16). Two-dimensional (2D) electrophoresis was performed according to the method of O'Farrell (17). The isoelectric focusing dimension was run in gels containing 4% acrylamide, 2% (v/v) ampholines (Bio-Rad Laboratories, Richmond, USA), 50% urea, 2% Nonidet P-40 (Katayama Chem. Inc., Osaka) at 800 V for 15 h. The second-dimension run was performed using SDS polyacrylamide slab gel electrophoresis. Silver staining was carried out using a Silver Stain Kit (Polysciences Inc., Warrington, USA). Protein concentrations were determined using a Protein Assay Kit (Bio-Rad Laboratories). Purification of HSPT0 and determination of the N-terminal amino acid sequence S. griseus 2247 mycelia were grown at 28°C for 12 h and then at 37°C for 1 h. Cells were harvested by centrifugation and washed once with 0.5% NaC1 and twice with TMDP buffer (50mM Tris-HCl [pH 8.0], 1 mM MgSO4-7H20, 0.5raM dithiothreitol, 0.4mM phenylmethylsulfonyl fluoride). After suspending the mycelium in the same buffer and disrupting the cells by ultrasonication, the cell debris was removed by centrifugation at 30,000 × g for 20 min. The supernatant was loaded onto a CM-Toyopeari 650M (Tosoh Co. Ltd., Tokyo) column previously equilibrated with buffer A (10 mM Tris-HCl [pH 8.0], 1 mM disodium EDTA, 6 mM 2-mercaptoethanol, 1.8 mM phenylmethylsulfonyl fluoride). The eluted fraction was subsequently loaded onto a DEAE-Toyopearl 650S (Tosoh Co. Ltd.) column previously equilibrated with buffer A. After washing the column with two column volumes of buffer A, proteins were eluted with a linear gradient of 0-0.3 M NaCI in buffer A. The fractions were analyzed by SDS-PAGE and then appropriate fractions were analyzed by 2D-PAGE to compare the patterns of the present proteins with those of total proteins from nonheat-shocked cells or 1-h heat-shocked cells. HSP70 protein was eluted at about 200 mM NaC1. The 70 kDai protein in the selected fraction was separated by SDSPAGE and electroblotted onto the Immobilon P membrane (Millipore, Bedford, USA) using a semidry electroblotting instrument (Nippon Eido Inc., Tokyo). The 70 kDal protein band was cut out and the amino terminal sequence was determined by a protein sequencer (model 477A; Applied Biosystems Inc., Foster City, USA). Cloning of the hsp70 gene S. griseus 2247 genomic DNA was digested with BamHI and DNA fragments ligated to BamHI-digested pUC19 to create a genomic library. After transformation into E. coli, the library was screened by colony hybridization with a DIG-labeled oligonucleotide probe. Consequently, the 4.5-kbp fragment containing hsp70 gene was obtained. D N A sequence analysis The DNA sequences of both strands were determined by the dideoxy chain-terminator method of Sanger et al. (18) with fluorescent primers, and analyzed on a model 373A automated DNA sequencing system (Applied Biosystems Inc., Foster City, USA). The DNA sequence data were ana-

J. FER~mr~X.BIOEr~6., lyzed with the GENETYX program (SDC Software Development Co. Ltd., Tokyo). The sequence data reported in this paper have been submitted to DDBJ, EMBL and GenBank and assigned the accession number D14499. RESULTS AND DISCUSSION Expression of HSPs in S. griseus 2247 The temperature for culture was shifted to 37°C after growth of S. griseus 2247 in GMP medium at 28°C for 12 h. Following incubation for various periods of time at 37°C, mycelia were collected and cell-free extracts were prepared. Some over-existing proteins (approximately 70kDal, 60 kDal, 55 kDal, etc.) were observed in the heat-shocked samples compared with total proteins from the nonheat-shocked cells on the SDS-PAGE gels stained with Coomassie brilliant blue (Fig. 1). Furthermore, after two hours' incubation at 37°C, mycelia were incubated again at 28°C for various periods of time and then cell-free extracts were prepared from these cells. The SDS-PAGE analysis of these samples showed that the heat-inducible bands decreased and existed at a normal level four hours after the temperature decrease to 28°C (data not shown). On the other hand, S. griseus was incubated at 28°C for various periods of time after addition of ethanol to give a final concentration of 5% (v/v). The result showed that a larger band assigned to 70 kDal protein was observed compared with other heat-inducible protein bands (shown in Fig. 1). These phenomena might be caused by the stress shock response in Streptomyces, Partial purification and amino acid sequence of HSPT0 As described above, a protein band of approximately 70 kDal seems to be induced significantly by the stress. This protein was, therefore, characterized further. The 70 kDal protein was separated by a series of ion-exchange chromatographies. A fraction of DEAE anion-exchange chromatography was analyzed by 2DPAGE. Results of 2D-PAGE analysis showed that the 70 kDal protein was sufficiently purified to isolate by SDS-PAGE as a unique band. After blotting the band of 70kDal protein on the SDS-PAGE gel to a membrane, the N-terminal amino acid sequence of this protein was determined by the Edman degradation method. The amino acid sequence from the N-terminus was identified as follows: A-R-A-V-G-I-D-L-G-T-T-N. The motif of the ATP-binding site, which was conserved among the HSP70 proteins, was observed in the determined sequence. Therefore, the 70 kDal protein of S. griseus is likely to be a member of the HSP70 family. The ATPbinding sites of this protein will be described in a later section. Isolation and DNA sequence analysis of the hsp70 gene from S. griseus 2247 To characterize the HSP70 of S. griseus further, we attempted to isolate the gene for HSP70. A 36-met oligonucleotide was designed based on the determined amino acid sequence and by considering the GC-rich genome of Streptomyces (Fig. 2A). Total DNA of S. griseus 2247 was digested with BamHI and analyzed by Southern hybridization using the synthetic 36-mer probe. The probe hybridized to a 4.5-kbp band (Fig. 2B). Subsequently, the fragment was isolated by colony hybridization experiments and sequenced by the dideoxy sequencing method. The nucleotide sequence and the deduced amino acid sequence are shown in Fig. 3. DNA sequence analysis revealed an

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1

2

3

4

5

6

A Determined amino acid sequence

A-R-A-V-G-I-D-L-G-T-T-N 97.4=iii~i!~ii!!ii!!i! ~CC-AAC

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OUgonucleotide sequence ii~iiii!~iiiiiiiiiii~il

45.0,,, FIG. 1. SDS-PAGE analysis of heat-induced proteins. S. griseus was grown at 28°C and then incubated at 37°C for 0 h (lane 1) as control, 1 h (lane 2), 3 h (lane 3), and 5 h (lane 4). Alternatively mycelia were incubated at 28°C after the addition of ethanol (final concentration: 5%) for 1 h (lane 5), and 3 h (lane 6). The band indicated by an arrowhead was isolated. Molecular weight standards (kDal) are shown on the left. open reading frame (ORF) coding for a protein that was highly homologous to HSP70 family. The ORF consisted 1857bp. A consensus Shine-Dalgarno sequence (19) was found in the nucleotide sequence 6 bp upstream o f the translational initiation codon (ATG), while the probe-hybridized sequence was also detected 3 bp downstream o f it. A putative transcriptional termination structure began at 71 bp after the stop codon (positions 20512079) (shown in Fig. 3, by a pair o f horizontal arrows). The deduced amino acid sequence at the N-terminus was identical to the determined sequence o f 70 kDal protein (shown in Fig. 3 by the symbol e ) . The cloned gene seemed to be a hsp70 gene of S. griseus. The hsp70 gene product The hsp70 gene o f S. griseus could encode the 67 kDal protein with 618 amino acid residues. As might be expected for a Streptomyces gene (20), a high G + C content is reflected in a high proportion (94%) o f codons that have a G or C in the third position. Comparison of the deduced amino acid sequence with entries in the S W I S S - P R O T revealed that high homologies to the members o f HSP70 family were recognized, whereas a low level o f homology to glucoseregulated protein precursors (21, 22) was also observed. In particular, the homologies to the HSP70 family o f prokaryotes were remarkable, for example, 59.8% identical with that of Clostridium (23), 59.7% with that o f Bacillus (24), 59.4% with that o f Methanosarcina (25), 58.1% with that of Synechocystis (26), and 52.8% with E. coli DnaK protein (27). Moreover, it is approximately 50% identical with mitochondrial heat shock proteins (28, 29). Some highly conserved domains in all prokaryotic HSP70 proteins were found in the S. griseus HSP70 protein (Fig. 4). The six proteins shown in Fig. 4 could be classified into two groups based on their molecular masses. The excess amino acid residues o f the larger group were observed at the same positions. Some of the HSP70 family have been found to bind to A T P (30). Our preliminary result showed that S. griseus HSP70 protein was also likely to bind to A T P (data not shown). The N-terminal ATP-binding domain, having the following consensus: (I/L/V)X(I/L/V/C)DXG(T/S/G)(T/S/G)

ii!i~!ii!!iii!!~iii!i~il FIG. 2. The N-terminal amino acid sequence of HSP70 and Southern blot analysis of genomic DNA of S. griseus. (A) The Nterminal amino acid sequence determined by the Edman degradation method is shown by the one-letter symbols and the sequence of synthetic oligonucleotide is also shown. (B) S. griseusgenomic DNA was digested with BamHI and resulting fragments were electrophoresed and transferred to a membrane. Hybridization was done using synthetic oligonucleotide as a probe. Size markers (in kb) are indicated on the right. X X ( R / K / C ) (31), was found in all prokaryotic HSP70 proteins. Another ATP-binding domain (32), containing the threonine residue which is autophosphorylated, was found in all proteins (in the case of S. griseus HSP70: - I L V F D L G G G T F D V - ) . Moreover, ATPase and autophosphorylation activities of E. coli DnaK protein were proposed to be effected by Ca 2÷ (33), and the Ca2÷/ calmodulin binding consensus sequence (34) was found to be highly conserved among HSP70 proteins (S. griseus HSP70" - K M A L Q R L R E A A E K A K I E L S S S - ) . These domains may act as a modulator o f ATPase or autophosphorylation activities in prokaryotic cells. The Cterminal sequences o f the HSP70 proteins, which were required for their autophosphorylation activities (33), were also highly conserved among prokaryotic HSP70 proteins. From the above findings, we have cloned a hspTO gene from S. griseus; however, the function of HSP70 in Streptomyces and its relationship to pleiotropic mutation in S. griseus 2247 strain remain to be investigated. D N A probe containing a coding region of hspTO gene was used for Southern analysis of the total DNA o f several Streptomyces strains. As shown in Fig. 5, the sequence of hspTO gene was distributed with significant homology among all Streptomyces strains tested. The result suggests an important role of HSP70 in Streptomyces similarly to other organisms. We also observed the difference in homology among Streptomyces strains (various intensities o f hybridized signals in Fig. 5). Comparison o f the hspTO gene and its product among Streptomyces strains might be useful to estimate evolutional distance of each Streptomyces strain. Both a heat shock specific promoter and a conserved Ea 7° promoter were identified in tandem 5" to the O R F o f DnaK of E. coli (35), and some hsp genes were proposed to have two promoters (36). Similarly to the case o f E. coli, the stress-inducible promoter o f Streptomyces might be identified in the hsp70 gene of S. griseus. Moreover, some useful proteins might be expressed at optional time by using such inducible promoters. On the other hand, a variety o f R N A poly-

464

H A T A D A ET AL.

J. FEIUd]~NT. BIOENO.,

Cc~GGTGTGCTC~CGTAGGCAcT~AAG~GAG~TAAGGGACTCAGG~G~GA~T~GCG~CGG~cA~CATTC~GAG~CA

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GATCCACTCAAG~GTCAGTTGG~qQ~TTGAAATG~A~TGCGGT~GCATCGAC~G~G~A~CTAA~TcCGTCG~CAGcG~TCTc 180 M k R k V G l D L G T T N S V V S V L Q O 6 O I O O O Q O Q O GAAGG~GG~GAG~Ac~T~T~A~AA~cAGAGGG~c~GGAC~CGCCGTCCGTcGTCGccTTCGcCAAGAAcGGCGAGGTG~c 270 E G G E P T V [ T N A E G A R T T P S V V A F A K N G E V L GTCGGCG~GGT~CGAAGCGCCAGGcGGT~CCAA~T~A~GGA~AT~T~GTCAAG~GC~TGGGCACTGAC~GAAGATC V G E V A K R O A V T N V D R T l R S V K R H M G T D W K l

360

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AAGGTGAC~AcGCGGTCATCA~T~ccGG~GTAC~cAA~AcTC~AG~T~AGG~A~AAGGAGGCCGGcGAGATCG~GGGcCTG K V T n A V 1T V P A Y F N D S E R Q A T K E A G E l A G L

640

AA~T~CTGCG~AT~TCAACGAGC~GA~CCGC~G~G~GGCGTA~G~CTCGA~AAGGA~GA~AGACGAT~T~GT~CGAC~T~ N V L R 1V N E P T A A A L A Y G L D K D O Q T l L V F O L

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1260

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G~GACGGCTGCCCCGGC~GGGACCT~ACCCGAC~TCTGACC2114 FIG. 3. The nucleotide sequence of the S. ~ u s h ~ gene and the deduced ~ i n o acid sequence. The N-terminal a ~ n o acid sequence of HSP70 from S. griseus, d~ermined by the Edman degradmion method, is shown by symbols ( ~ ) . A putative ribosome b i n ~ n g sffe is underlined. The potentiM stem-loop structure is indicated by a pair of horizontal arrows.

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[BA]

......

145 TDWKIDLDGKSFNPQQMSAFILQKLKRDAESYLGEKVTDAVITVPAYFND*ERQATKEAGE *AN**V**N**OY***El**M******A***A****T*KQ******************O**A . . . . . . . . . . . . . . . . . . . . ************************************************************** .................... * * * * * * * * * * D ,* * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * t O * * * •*T•E•TE••••VV•D•N•••KLD•P•Q**•*•*•••**•V*R**VD**•K****T**•************•*****•**K E••RDV••MP•K•••••••--*•••••K*••••*P*I**•V*K*•*KT**•****P**•***********A•*****D**R 210 ]AGLNVLRIVNEPTAAALAYGLDKDD--QTILVFDLGGGTFDVSLLEI . . . . GDGVVEVKATNGDNHLGGDDWUQRVVDVL * * * * * * * * * * * * * * * * * * * * * * * * 6 * * * ** * * * * * * * * * * * * * * * * * *. . . . *6*************************** * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *. . . . ***************************** * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *. . . . ***************************** *********[******S**********NE*******************. . . . ***************************** ********************************************************************************* ....................... 290 V••••••••••L••••••L*•••••••••K••L•••T••T••L•••••••••••LH••*••LT•••••••••••L•••C•TP• ********************************************************************************* ****************************************************-**************************** ****************************************************-***************************** ••E**••E****•***•*****************•••**E****•***•*•***•****•*•*••*•E*••******•**• ****************************************************-**************************** 371 •N•••D••••L•••DHVVLV•••TR•P•V•EL•••LT••••••••••PDEV•••*••L•••VL••E••DVLLLDVTPL•L• *************************************-******************************************* *************************************-******************************************* •E••*•••*•*•***•*************•*•**••*•***••*******•**•**•********•*************** *************************************-******************************************* •*••*****••*•**•******••***•*•K•*•*•*•*•*••*******•*****••*•***•***************** 452 •ETK•••MT•L••R•TT•PT•••••FTT•••N•P•V•••V•*•E•••••YN••L••••LT•LPP•P•••P•••••FD•DA• ********************************************************************************** ********************************************************************************* ********************************************************************************* •*****•**************•****•****•*••*E***•*****•*••**•****•*•*•******************* ********************************************************************************* 533 •••••AA•DL•T•KEQ••T•T••••LP•D•••R••••••••••EDHA•R•••••R•••••L•••T••FL•D•••••P•••• **L•*•*•***•**•*••••••P••*•••*••*•••D*•••**••R•*••EV••**N•*A*•••A**••*E••*L•T•*•* **•N*R******•***••**•••TG*•D**••**••***E•*DA*••*K*•V*L**•••***FT***T***L*•**•E•EV **•K*•*T*•A****AN•*•*••TN*•D•*•D•AV•***•F****•K*•**•*V•*NA**T******T*••LG***•*E•* ********************************************************************************* **L**•***K••***•*•*••••*•*••**••K*•R•**••**•*R••E*LV•T****•••*LH•*•*•V•••G**L***D* 598 TEVETAVGELKEKLKGEDSAEIKTATEKVRGRLPRIW-ARRCT---AN-A-QA---, . . . . . GAAPGA--DAPGDAQAKA* SK*NA*IED**KA*E*K*AED*KAK**ALQESVYP*ST*MYOK---*QQ*Q**AGG* .... ****G---T**** ..... P* *KANE*KDA**AAIEKN*LE**KAKKDELQEIVQALTVKLYEQ---*QO*Q**-G-* ....... , - * * ....... ON. . . . •*••E*••*•*KVK•*•*•••••K*•*•LT••FYK••••LY•Q••G*•-••---•••P••M•**••**•••*•--T•NN•* IKA*GLIKD***AVAO**D*K*Q*VMPELOQV*YS*OSNMYQ, .......... AOA*---AGVG***tGPE*-*-TS*GG* **ItS*LTA*ETA*****K*A*EAKMOELAQVSOKLMEIAQQQI[--*QO-QTt-GAO. . . . . . **A . . . . . . . . . NN**-*

[ST] . . . . . . . . . . . . . . . . . . . . [ME] . . . . . . . . . . . . . . . . . . . [BA] [CL]

[SY] [EC] [**]

*ME* [BA]

[CL] IS** [EC]

*ST*

*ME* [B*] [CL] [SY]

[EC] [ST]

*ME* [BA] [CL] [S*] [EC]

[ST]

[ME] [BA] [CL] IS*]

[EC] [ST]

[ME] [BA] [CL] [SY] [*C] [S'l']

*ME* [BA] [CL] [S*] [EC]

618 [ST] DDVVDA--EIV-DDEKDTKGGAA [ME] ET****DY*V*-****R-*

[BA]

******EF*E*N*-**

*N****DF*-*Q**K* [SY] * * * I * * E F S E P - - * - * [C*]

[EC]

*******F*E*-K*K*

FIG. 4. Comparison of the predicted amino acid sequence of S. griseus HSP70 [ST] with those of Methanosarcina [ME] (25), Bacillus [BA] (24), Clostridium [CL] (23), Synechocystis [SY] (26) and E. coil [EC] (27) HSP70. Asterisks indicate the presence of the same amino acid residue as that of S. griseus HSP70 and gaps are indicated by a dash. The putative ATP-binding sites (bold line on top) and a calmodulin-binding domain (broken line on top) are noted.

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J. FERMENT. BIOENG.,

3 4 5 6

7

8

FIG. 5. Distribution of the hspTO gene among Streptomyces strains. Total DNA from S. griseus 2247 (lane 1), S. fradiae HUT6018 (lane 2), S. venezuelae HUT6024 (lane 3), S. scabies HUT6027 (lane 4), S. aureofaciens HUT6113 (lane 5), S. cattleya IFO14057 (lane 6), S. viridochromogenes IFO14061 (lane 7) and S. coelicolor A3(2) M130 (lane 8) was digested with BamHI. Restriction fragment of a hsp70 gene of S. griseus (2-kb PstI fragment) was isolated and used as a probe. Size markers (in kb) are shown on the left. merase h o l o e n z y m e o f Streptomyces was r e p o r t e d (37, 38). F u r t h e r studies are r e q u i r e d to clarify the relationship b e t w e e n R N A p o l y m e r a s e h e t e r o g e n e i t y a n d such inducible p r o m o t e r s in Streptomyces. ACKNOWLEDGMENT This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan. REFERENCES 1. Chirico, W . J . , Waters, M. G., and Biobel, G.: 70K heat shock related proteins stimulate protein translocation into microsomes. Nature, 332, 805-810 (1988). 2. Deshaies, R.J., Koch, B.D., Werner-Washburne, M., Craig, E.A., and Schekman, R.: A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides. Nature, 332, 800-805 (1988). 3. Gaitanaris, G.A., Papavassiliou, A.G., Ruboek, P., Silverstein, S.J., and Gottesman, M.E.: Renaturation of denatured ,~ repressor requires heat shock proteins. Cell, 61, 1013-1020 (1990). 4. Skowyra, D., Georgopoulos, C., and Zyliez, M.: The E. coli dnaK gene product, the hsp70 homolog, can reactivate heatinactivated RNA polymerase in an ATP hydrolysis-dependent manner. Cell, 62, 939-944 (1990). 5. Bukau, B. and Walker, G.C.: Mutations altering heat shock specific subunit of RNA polymerase suppress major cellular defects of E. coil mutants lacking the DnaK chaperone. EMBO J., 9, 4027-4036 (1990). 6. Kusukawa, N. and Yura, T.: Heat shock protein GroE of Escherichia coil: key protective roles against thermal stress. Genes Dev., 2, 874-882 (1988). 7. Miyazaki, T., Tanaka, S., Fujita, H., and Itikawa, H.: DNA sequence analysis of the dnaK gene of Escherichia coli B and of two dnaK genes carrying the temperature-sensitive mutations dnaK7 (Ts) and dnaK756 (Ts). J. Bacteriol., 174, 3715-3722 (1992). 8. Sherman, M.Y. and Goldberg, A.L.: Involvement of the chaperonin dnaK in the rapid degradation of a mutant protein in Escherichia coll. EMBO J., 11, 71-77 (1992). 9. Guglielmi, G., Mazodler, P., Thompson, C. J., and Davies, J.: A survey of the heat shock response in four Streptomyces species reveals two groEL-like genes and three GroEL-like proteins in Streptomyces albus. J. Bacteriol., 173, 7374-7381

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