Cloning and sequencing of the dnaK operon of Bacillus stearothermophilus

Gene, 170 (1996) 81-84 © 1996 Elsevier Science B.V. All rights reserved. 0378-1119/96/$15.00

81

GENE 09561

Cloning and sequencing of the dnaK operon of Bacillus stearothermophilus (Recombinant DNA; grpE; dnaJ; heat-shock protein; molecular chaperone)

Markus Herbort, Ulrike SchOn, Karin Angermann, Jens Lang and Wolfgang Schumann Institute of Genetics, University of Bayreuth, D-95440 Bayreuth, Germany Received by J. Wild: 4 August 1995; Revised/Accepted: 27 November 1995; Received at publishers: 30 November 1995

SUMMARY

Here, we report the cloning of a 5.8-kb PstI fragment of chromosomal DNA from Bacillus stearothermophilus (Bt) carrying the three genes, grpE, dnaK and dnaJ. This fragment contains, in addition, the 3'-end of an open reading flame which has been shown to be part of the dnaK operon in three bacterial species. The dnaJ gene could complement an Escherichia coli dnaJts mutant for growth at high temperature. Sequencing and hybridization data strongly suggest that the Bt chromosome contains an analog of dnaJ.

INTRODUCTION

Heat-shock proteins (HSP) are synthesized when cells are exposed to elevated temperatures (Morimoto et al., 1994). The heat-shock response is highly conserved and presumably allows organisms to adapt to stressful environments. An important group of HSP are the molecular chaperones which assist in the folding of native and misfolded proteins and in the assembly/disassembly of protein complexes (Georgopoulos and Welch, 1993). In Bacillus stearothermophilus (Bt ), temperature upshift is accompanied by the synthesis of at least four HSP (Wu and Welker, 1991). To study the function of chaperones from a thermophilic organism, we started the cloning and sequencing of genes encoding chaperones, and purification and biophysical characterization of their proteins. Recently, we published cloning and sequencing of the Correspondence to: Dr. W. Schumann, Institute of Genetics, University of Bayreuth, D-95440 Bayreuth, Germany. Tel. (49-921) 552-708; Fax (49-921) 552-710; e-mail: [email protected] Abbreviations: aa, amino acid(s); bp, base pair(s); Bs, Bacillus subtilis; Bt, B. stearothermophilus; dnaK, gene encoding DnaK; dnaJ, gene encoding DnaJ; Ec, Escherichia coli; grpE, gene encoding GrpE; HSP, heat-shock protein(s); kb, kilobase(s) or 1000 bp; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; ts, temperature sensitive; [], denotes plasmid-carrier state.

SSD1 0378-1119(95)00859-4

groESL operon ofBt (SchOn and Schumann, 1993). Here, we describe cloning and sequencing of the Bt dnaK operon.

EXPERIMENTAL AND DISCUSSION

(a) Cloning of the Bt dnaK operon To identify the dnaK operon of Bt NUB36, an internal 0.8-kb BgllI fragment of the dnaK gene of B. subtilis (Bs) was isolated from plasmid pMWB25 (Wetzstein et al., 1992) labelled with DIG-11-dUTP (Zuber and Schumann, 1991) and hybridized against Bt chromosomal DNA digested with HindlII or PstI. The luminogram of a Southern-blot revealed a hybridizing 8-kb HindlII and a 5.8-kb PstI fragment (data not shown). The PstI fragment was chosen for cloning and sequencing. Chromosomal DNA was digested with PstI, sizefractionated through a 10-40% sucrose gradient, and those fractions containing the 5.8-kb fragment were identified by Southern blot hybridization. Positive fractions were pooled, dialyzed and ligated into PstI-linearized pACYC177 (Chang and Cohen, 1978). After transformation a positive clone could be identified by Southern hybridization (pMHMS01). It contained the complete 5.8-kb PstI fragment.

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(b) Sequencing of the dnaK operon First, a restriction map of the genomic insert was constructed and is presented in Fig. 1. Then, the nt sequence of part of the insert was determined. A total of 4141 bp were sequenced and the nt and its deduced aa sequence is presented in Fig. 2. The nt sequence revealed three complete ORFs and the 3' end of an ORF. Alignment of the deduced aa sequences exhibited significant homology to the four genes of the Bs dnaK operon (orf39-grpEdnaK-dnaJ; Wetzstein et al., 1992). The aa sequence of the incomplete ORF exhibits 61.5% identity and 84.6% similarity with Bs ORF39, a negative regulator of the heat-shock response of class-I heat-shock Bs genes

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Fig. 1. Partial restriction map of the Bt 5.8-kb PstI genomic insert present on pMHMS01. The location of the 3' end of off39, and the three genes grpE, dnaK and dnaJ within the PstI fragment are shown below. The arrow indicates the direction of transcription.

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Fig. 2. Nucleotide sequence of the dnaK operon and deduced aa sequence. Indicated are the putative Shine-Dalgarno sequences (asterisks above the sequence), one potential vegetative promoter (bold letters) and one potential terminator (arrowheads below the sequence). The sequence data have been submitted to the EMBL library and assigned accession No. X90709.

83 (Schulz et al., 1995). The aa sequence of the second O R F shows 59% identity and 70.7°,/0 similarity with Bs GrpE. The next two ORFs encode D n a K and DnaJ which exhibit 83.3% identity and 91% homology and 62.8% identity and 77.9% homology, respectively, with their Bs homologs. (c) The Bt dnaK operon can complement a ts phenotype of Escherichia coli dnaJ, but not of grpE or dnaK

Because the Bs groESL operon allows growth of Escherichia coli (Ec) having its own groESL genes deleted (U.S. and W.S., data not shown), we asked whether the dnaK operon could also complement appropriate Ec mutants. Ec mutants BB1752 (grpEts), BB1048 (dnaKts), and BB1458 (dnaJts) carry ts mutations in grpE, dnaK and dnaJ, respectively, and they do not grow at 40°C. Plasmid pMHMS01 was introduced into these mutant strains, and transformants were analyzed for complementation of the ts phenotype. Whereas BB1458 [pMHMS01 ] was able to form colonies at high temperature, the other two strains were not (data not shown). Subsequently, we found that BB1458[pMHMS01] was also able to complement phage X for growth (data not shown). The ability to grow at high temperature could be explained if there is a potential vegetative promoter preceding dnaJ (see Fig. 2) as it is the case for Bs (A. Mogk, unpublished results), but no promoter is present in front of grpE and dnaK. Therefore, complementation of the dnaJ ts phenotype indicates that the DnaJ proteins of Bs and of Ec are functionally interchangeable. (d) Bt chromosome contains a second copy of dnaJ During our analysis of the PstI fragment, T. Henkin sent us a recombinant M13mpl8 phage containing about 1-kb Sau3AI Bt insert together with about 100-bp partial sequence exhibiting homology to dnaJ of Bs. This sequence was extended to about 400 bp, and to our surprise, it was different from that given in Fig. 2, but its translation product exhibited significant homology to DnaJ suggesting that either there are two copies of dnaff within the Bt chromosome or, though rather unlikely, these differences are strain specific (data not shown). To distinguish between these two possibilities, Southern hybridizations were carried out using two different oligos corresponding to an internal part of the two dnaJ genes. When ON1 was used (internal part of dnaJ), one single band was observed hybridizing with chromosomal DNA cut with three different restriction enzymes (Fig. 3A). When ON2 (putative second dnaJ) was used, hybridizing bands which differed in size from those obtained with ON1 were revealed (Fig. 3B). These data exclude the possibility of strain-specific differences and thereby confirm

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Fig. 3. Identificationof two Bt dnaJ genes. Southern blot analysis of chromosomal DNA digested with three different restriction enzymes and hybridizedwith ON1 (5' GTCACGGGGCAGGACAA;dnaJ) (A) and ON2 (5'-GCTTCCGCCTGAGAGGG;putative seconddnaJ) (B). Chromosomal DNA digested with EcoRI (lane 1), HindIII (lane 2), or PstI (lane 3). ST, molecular weight standard (SPP1 DNA cut with EcoRI).

that Bt codes for at least two different dnaJ genes which can be distinguished by using specific oligos as probes. A second dnaJ gene which is not part of the cr32-dependent heat-shock regulon has already been described for Ec (Ueguchi et al., 1994). (e) Conclusions

(I) We have cloned and sequenced the grpE, dnaK and dnaJ genes of Bt. (2) These genes, together with off39, form one operon in Bs (Wetzstein et al., 1992), C. acetobutylicum (Narberhaus et al., 1992) and S. aureus (Ohta et al., 1994). (3) The Bt dnaJ gene was able to complement an Ec dnaJts mutant for growth at high temperature and phage growth at all temperatures. (4) Sequencing and hybridization experiments strongly suggest the existence of two copies of dnaJ within the Bt chromosome. (5) Experiments are in progress to overproduce, purify and crystallize the three Bt HSP, GrpE, DnaK and DnaJ.

ACKNOWLEDGEMENTS We thank T. Henkin for the M13mpl8 phage carrying part of a putative second dnaJ gene of Bt, B. Bukau for providing Ec strains, and the Bacillus Genetic Stock Center for sending us Bt NUB36. This work was supported by the Deutsche Forschungsgemeinschaft and in part by a CEC Biotech Grant BIO2 CT 920254.

REFERENCES Chang, A.C.Y. and Cohen, S.N.: Construction and characterizationof amplifyablemulticopyDNA cloningvehiclesderivedfrom the P 15A cryptic miniplasmid. J. Bacteriol. 134 (1978) 1141-1156. Georgopoulos, C. and Welch, W.J.: Role of the major heat shock

84 proteins as molecular chaperones. Annu. Rev. Cell Biol. 9 (1993) 601-634. Morimoto, R.I., Tissi~res, A. and Georgopoulos, C.: Progress and perspectives on the biology of heat shock proteins and molecular chaperones. In: Morimoto, R.I., Tissi~res, A. and Georgopoulos, C. (Eds.), The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1994, pp. 1-30. Narberhaus, F., Giebeler, K. and Bahl, H.: Molecular characterization of the dnaK gene region of Clostridium acetobutylicum, including grpE, dnaJ, and a new heat shock gene. J. Bacteriol. 174 (1992) 3290-3299. Ohta, T., Saito, K., Kuroda, M., Honda, K., Hirata, H. and Hayashi, H.: Molecular cloning of two new heat shock genes related to the hsp70 genes in Staphylococcus aureus. J. Bacteriol. 176 (1994) 4779-4783. Sch6n, U. and Schumann, W.: Molecular cloning, sequencing, and

transcriptional analysis of the groESL operon from Bacillus stearothermophilus. J. Bacteriol. 175 (1993) 2465-2469. Schulz, A., Tzschaschel, B. and Schumann, W.: Isolation and analysis of mutants of the dnaK operon of Bacillus subtilis. Mol. Microbiol. 15 (1995) 421-429. Ueguchi, C., Kakeda, M., Yamada, H. and Mizuno, T.: An analogue of the DnaJ molecular chaperone in Escherichia coli. Proc. Natl. Acad. Sci. USA 91 (1994) 1054-1058. Wetzstein, M., V/)lker, U., Dedio, J., L6bau, S., Zuber, U., Schiesswohl, M., Herget, C., Hecker, M. and Schumann, W.: Cloning, sequencing, and molecular analysis of the dnaK locus from Bacillus subtilis. J. Bacteriol. 174 (1992) 3300 3310. Wu, L. and Welker, N.E.: Temperature-induced protein synthesis in Bacillus stearothermophilus NUB36. J. Bacteriol. 173 (1991) 4889-4892. Zuber, U. and Schumann, W.: Tn5cos: a useful tool for restriction mapping of large plasmids. Gene 103 (1991) 69-72.