JOURNAL OF BACTERIOLOGY, May 1992, p. 3282-3289
Vol. 174, No. 10
0021-9193/92/103282-08$02.00/0 Copyright © 1992, American Society for Microbiology
Cloning, Sequencing, and Molecular Analysis of the groESL Operon of Clostridium acetobutylicum FRANZ NARBERHAUS AND HUBERT BAHL*
Institut fiir Mikrobiologie, Georg-August- Universitat Gottingen, Grisebachstrasse 8, W-3400 Gottingen, Germany Received 2 December 1991/Accepted 10 February 1992
The groESL operon of Clostridium acetobutylicum was cloned in Escherichia coli by using a gene probe of E. coli groESL. Sequencing of a positively reacting 2.2-kbp HindIII fragment contained in the recombinant plasmid pFN1 and a 2.5-kbpXbal fragment present in pFN4 revealed that both fragments partially overlapped and together spanned 3,493 bp of the clostridial chromosome. Two complete open reading frames (288 and 1632 bp) were found and identified as the groES- and groEL-homologous genes of C. acetobutylicum, respectively. The 3' end of a third gene (orJZ), which was divergently transcribed, showed no significant homology to other sequences available in the EMBL and GenBank data bases. The length of thegroESL-specific mRNA (2.2 kb), a transcription terminator downstream of groEL, and a transcription start site upstream of groES, identified by primer extension analysis, indicated that groES and groEL of C. acetobutylicum are organized in a bicistronic operon. From the transcription start site, the promoter structure 5'-TTGCTA (17 bp) TATTAT that shows high homology to the consensus promoter sequence of gram-positive bacteria as well as E. coli was deduced. Transcription of the groESL operon was strongly heat inducible, and maximum levels of mRNA were detected 15 min after heat shock from 30 to 42'C. An 11-bp inverted repeat, located between promoter and translation start sites of groES and partially identical with similar structures in front of several heat shock genes of other bacteria, may play an important role in the regulation of heat shock gene expression in this organism. A set of heat shock proteins is clearly induced when cells are exposed to higher temperatures. This phenomenon has been observed in all organisms, from bacteria and fungi to plants and animals (36). In Escherichia coli, about 20 heat shock proteins are known (26), among them the evolutionarily highly conserved DnaK and GroEL proteins. The important role of these two proteins during normal growth has also been recently established. They are involved in the assembly and disassembly of protein complexes (molecular chaperones) and in protein translocation processes (8, 14,
regulatory mechanism for the expression of this heat shock operon in C. acetobutylicum is different from that in E. coli. MATERIALS AND METHODS Bacterial strains, plasmids, and growth conditions. C. acetobutylicum DSM1731 was used as a source of genomic DNA and total RNA. E. coli JM109 [recAl endAl gyrA96 thi hsdR17(rK iMK+)supE44 relAl X-A(lac-proAB)(F' traD36 proAB+ lacIq lacZAM15)] (48) was used as host for the cloning experiments. The plasmid pUC18 (48) served as vector for the construction of genomic libraries. Plasmid pKT200 (9) containing the groESL operon of E. coli on an 8.1-kbp EcoRI fragment was used to isolate a probe for hybridization experiments. C. acetobutylicum was grown anaerobically in CBM medium (27) at 37°C. E. coli was routinely grown at 37°C in Luria-Bertani medium (34) supplemented with ampicillin (50 ,ug/ml), isopropyl-3-thiogalactopyranoside (IPTG, 50 ,ug/ml), or 5-bromo-4-chloro-3-indolyl-p-D-galactopyranoside (X-Gal, 40 p,g/ml) if required. DNA isolation and manipulation. Chromosomal DNA of C. acetobutylicum was isolated by the method of Marmur (21) as modified by Bertram and Durre (2). Plasmid isolation from E. coli was performed by the method of Birnboim and Doly (3) or with the Quiagen Midi Kit (Diagen GmbH, Dusseldorf,
29, 30). The groE genes (groES and groEL) of E. coli were originally defined as genes necessary for the morphogenesis of several bacteriophages (12). They comprise an operon under positive transcriptional control exerted by the heat shock sigma factor, u32 (16, 18). The groEL and groES genes encode abundant proteins with molecular masses of 57,259 and 10,368 Da, respectively (42), which are essential for the growth of E. coli at all temperatures (10). Homologous proteins have been found in other bacteria (24, 43); chloroplasts (18); and mitochondria from plants, fungi, and animals (23). In contrast, very little is known about the nature and regulation of GroE proteins in obligate anaerobic bacteria. In Clostridium acetobutylicum, a gram-positive, spore-forming anaerobe, a GroEL protein is synthesized at elevated rates after a heat shock and during the switch from acid to solvent formation (33). To investigate the molecular structure and regulation of the C. acetobutylicum groE locus, this article describes the molecular characterization of the groEL and groES genes of this organism. Evidence is presented that the
*
Germany). DNA was manipulated by standard methods (34). Restriction enzymes were obtained from GIBCO/BRL GmbH (Eggenstein, Germany) or Pharmacia LKB GmbH (Freiburg, Germany), and calf intestinal phosphatase was from Boehringer GmbH (Mannheim, Germany). The enzymes were used according to the instructions of the manufacturers. DNA restriction fragments were isolated from agarose gels by electroelution in a Biotrap BT1000 (Schleicher & Schuell, Dassel, Germany).
Corresponding author. 3282
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-pUC18-2.2 kbp-
FIG. 1. Hybridization of HindIII-digested plasmids from clones of a partial gene bank of C. acetobutylicum with the 32P-labeled 1.7-kbp SacII-BamHI fragment of pKT200. The partial gene bank was constructed with HindIII-digested chromosomal DNA of C. acetobutylicum (2.2-kbp fragment size) and pUC18. (A) Autoradiogram of plasmids from pools containing 50 clones hybridized to the probe. Lanes 1 to 6, plasmids of six different pools digested with HindIll; lane 7, chromosomal DNA of C. acetobutylicum digested with HindIII; lane 8, X DNA digested with PstI. (B) Autoradiogram of five plasmids from the positive pool (lane 2 in panel A). Lanes 1 and 8, A DNA digested with PstI; lane 2, chromosomal DNA of C. acetobutylicum digested with HindIll; lanes 3 to 7, plasmids of five clones digested with HindIlI. The bands corresponding to pUC18 and the 2.2-kbp HindIII fragment are marked.
Construction and screening of gene libraries. The gene libraries were constructed with completely digested chromosomal DNA of C. acetobutylicum. For partial gene banks, 100 ,ug of completely digested DNA was centrifuged in a sucrose density gradient from 10 to 40% (wt/vol). Fractions containing the desired fragments were dialyzed against TrisEDTA buffer for 24 h and used for ligation. For total gene banks, digested chromosomal DNA was used after extraction with phenol-chloroform and chloroform and precipitation with ethanol. The DNA fragments were ligated to the appropriately digested and dephosphorylated vector. E. coli was transformed with the ligation mixture by electroporation in a Gene Pulser (Bio-Rad Laboratories GmbH, Munich, Germany). White colonies containing inserts were screened by colony hybridization with the respective probe. Positive clones were tested by restriction endonuclease digestion and were sequenced. Preparation of RNA. Total RNA was isolated from C. acetobutylicum by the hot phenol-chloroform procedure described by Oelmuller et al. (28). To obtain higher yields of
3283
RNA, cells were not washed in the acetate-EDTA buffer and the extraction in 60°C hot phenol-chloroform was extended to 10 min. Hybridization. Total chromosomal DNA of C. acetobutylicum was digested to completion with the desired restriction enzymes and separated on agarose gels. For hybridization with digoxigenin-labeled probes, nitrocellulose membranes (Biotrace; Gelman Sciences, Ann Arbor, Mich.) were used. Southern blots and hybridization experiments were performed according to the manufacturer's instructions. DNA probes were labeled with a DNA labeling and detection kit (Boehringer) and hybridization was performed at 37°C with 30% formamide. For hybridization with 32P-labeled probes, nylon membranes (GeneScreen Plus; Dupont, NEN Research Products, Dreieich, Germany) were used. DNA probes were labeled with [a-32P]dCTP (Amersham Buchler GmbH, Braunschweig, Germany) by using a nick translation kit (GIBCO/BRL). The labeled fragments were purified by column chromatography on Sephadex G-25, and hybridization was done in 0.2% (wt/vol) polyvinylpyrrolidone-0.2% (wt/vol) Ficoll-0.2% (wt/vol) bovine serum albumin-50 mM Tris hydrochloride (pH 7.5)-l M NaCI-0.1% (wt/vol) sodium pyrophosphate-1% (wt/vol) sodium dodecyl sulfate-10% (wt/vol) dextran sulfate at 60°C for 2 to 6 h. After hybridization at 60°C for 15 to 20 h, the membranes were washed twice in 2x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate) for 5 min at room temperature and subjected to autoradiography. If necessary, the blot was washed again twice in 2x SSC-1% (wt/vol) SDS for 30 min at 60°C and twice in 0.1% (wtlvol) SDS for 30 min at room temperature. Colony hybridization was performed with BA85 nitrocellulose filters (Schleicher & Schuell). Colonies were picked directly on a master plate and on a plate containing the filter. Filters were prepared for hybridization as described by Grunstein and Wallis (17). Hybridization and washing were performed as described above for radioactive Southern blots. RNA for Northern blots was run in formaldehyde gels and transferred to nylon membranes (GeneScreen Plus). A size determination was done by using an RNA ladder (0.24, 1.4, 2.4, 4.4, 7.5, and 9.5 kb; GIBCO/BRL) as a standard. Hybridization and washing was done as described above for the DNA hybridization procedure. DNA sequencing. DNA was sequenced by the dideoxychain termination method of Sanger et al. (35), by using [35S]dATP and a T7 sequencing kit from Pharmacia LKB. Single-stranded templates were prepared from both strands of the plasmids. Sequencing was started with commercially HindlII
HindlII
pFN1 IL l 2224
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TT TGCAGC TTAC TTT TCTTC TTTAGCTTACTTACATATAGTGCAAGATTAACACT GGTAT TAAAT TCTGC TAT GT TGCCATAAACACCGGCAATGGAAAT R Q L K S K K K L K S V Y L A L N V S T N F E A I N G Y V G A I S I
2 401
GGAAGTTAGATTAAATTTGTCCTCAACTCCTTTTCTGTCTAAGCCCACTATGTATTTATTTTTACTGTCTATTTCATTAAAAAATTTAAGAATACCACTG
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TCAAATC TATCAAT TATAAGT TGACTTACT TTGTAACATTCT T CTAGAGAGGCT T TTATAATACAAATAAAATCATCACCACC TATAT GACCTACAAAAT D F R D I I L Q S V K Y C E E L S A K I I C I F D D G G I H G V F
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TTTTAAAAGTAAACAGTTCTAAAAGGCAGTCCTTTATTGTATTTGCAGTGAATTTTAATATTTTATCTCCATTTTCAAAGCCATAAACATCATTGTAAAT K
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TTTAAAGTTATCAAGATCTAGATAGAGTACACAGAAAGACTCGTTTGAATTTAAAATATCTGATAAGGTCTTTTCTATAAGGATGTTTCCAGGAAGCCCT
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GTAAGTGGATTTAATTCTTTAGCATAGTTTTTTTCTATAGTTACTGTAAATTCAAGAAGATTTTTAATTGTAACTATACCGTAATACGTGTTGTTTTTAG
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TAACTATAACATAATCATATATTTTATCAGTATCTCTTTGCATTGCCATTTTAGCAGCACTGTTTATACTTGTATTATAATCTACAACGAGGGCATTCTT
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GTCCATTACAAGCTCTACAGGGCGTTTGGAAAATATAGCTACGCCGTATTGTGTTGCAAGGACTGAATCAAGAGTATGCTTCATAACAAGGCCTACGGGT
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CTGCAATAGTCCCAATTTTATATTTGCGAAAATTAAAATAATCTTTTTTTAGTTTATTATAGGTTATGATGATTTTTTTAACAGATTCAGGTATTTCACA
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FIG. 3. Nucleotide sequence of the 3,493-bpgroESL operon of C. acetobutylicum. Only the antisense strand is shown. The deduced amino acid sequences of groES, groEL, and or/Z are shown below the DNA sequence (single-letter code). Translation stop signals are marked by asterisks below the codons. The putative ribosome-binding sites are underlined. Arrowheads below the DNA sequence show regions of dyad symmetry. The transcription start site (S) identified by primer extension analysis and the corresponding promoter are marked by bold letters in the -10 and -35 regions.
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CLOSTRIDIUM ACETOBUTYLICUM groESL OPERON
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G A T C 1 2
FIG. 4. Northern blot analysis of total RNA of C. acetobutylicum isolated before and at different time points after a heat shock from 30 to 42°C. Each lane contains 5 ,ug of RNA and was probed with the 32P-labeled XbaI fragment of pFN1. Lane 1, before heat shock; lanes 2 to 8: 5, 7.5, 10, 15, 20, 30, and 60 min, respectively, after the temperature upshift. The signal corresponded to a transcript of 2.2 kbp.
available M13/pUC universal sequencing forward primer and reverse primer. Synthetic oligonucleotides (17-mers) complementary to the ends of already sequenced templates were prepared by a Gene Assembler Plus (Pharmacia LKB) according to the instructions of the manufacturers and were used as primers for continued sequencing. The dideoxyterminated fragments were separated on 55-cm wedgeshaped thickness gradient gels (0.2 to 0.4 mm, 7% [wt/vol] polyacrylamide) with a Macrophor sequencing unit (Pharmacia LKB) as recommended by the manufacturer. Determination of the transcription start site. For the determination of the transcription start site, primer extension analysis was used. Two picomoles of oligonucleotide (17mer) was labeled with 10 ,uCi of [y-32P]ATP (Amersham Buchler) and 7 U of polynucleotide kinase (GIBCO/BRL) in PNK buffer (100 mM Tris [pH 7.6], 20 mM MgCI2, 30 mM dithiothreitol) for 3 h at 37°C (10-,u reaction volume). The reaction was stopped by heating for 2 min at 95°C. Ten micrograms of total RNA was incubated together with 12.5 U of RNasin (GIBCO/BRL) and 0.2 pmol of kinased oligonucleotide in 500 mM KCl-20 mM Tris (pH 7.9) buffer for 3 h at 30°C (final volume, 10 RI). For the primer extension reaction, 6 RI of 5 x PE buffer (250 mM Tris [pH 8.3], 125 mM KCI, 15 mM MgCI2, 50 mM dithiothreitol), 500 p,M dNTPs of each nucleotide, 2.5 ,g of actinomycin D and 200 U of SuperScript RNase H- reverse transcriptase (GIBCO/ BRL) per 10 ,ug of RNA and 30 ,u of H20 were combined in the annealing assay and incubated at 37°C for 1 h. The RNA
primer-extended hybrid was phenol-chloroform extracted, ethanol precipitated, and analyzed on 7% polyacrylamideurea sequencing gels. The lengths of the primer extension products were calculated by running a sequencing reaction with the same primer on the same gel. Computer programs. The DNA region sequenced and the deduced proteins were analyzed with the DNA Strider program (20) on a Macintosh SE computer (Apple Computer, Inc., Cupertino, Calif.). Sequence comparisons were done by using the Wisconsin Genetics Computer Group sequence analysis software package, version 6.0 (University of Wisconsin Biotechnology Center, Madison) (7). Nucleotide sequence accession number. The sequence data reported here (see Fig. 3) have been submitted to GenBank and assigned accession no. M74572.
FIG. 5. Mapping of the 5' end of mRNA of the groESL operon by primer extension analysis. A 32P-radiolabeled primer complementary to the 5' end of groES was hybridized to 10 pLg each of total RNA from C. acetobutylicum isolated before (1) and 7.5 min after a heat shock from 30 to 42°C (2). The primer extension products were analyzed on a sequence gel. G, A, T, and C are products of the sequencing reaction obtained by using the same oligonucleotide as the primer. The depicted sequence represents the antisense strand. Broken arrows mark regions of dyad symmetry; the arrow at S points to the base representing the 5' end of the mRNA. -35 and -10 regions are also shown (boldface).
RESULTS Cloning and sequencing of the groESL operon of C. acetobutylicum. A digoxigenin-labeled 1.7-kbp SacII-BamHI fragment of the E. coli groESL operon was used to detect homologous sequences in the DNA of C. acetobutylicum. Hybridization signals were obtained with C. acetobutylicum DNA digested with HindIII (2.2- and 4.8-kbp fragments), AccI (2.9 kbp), EcoRI (4.3 kbp), EcoRV (5.2 kbp), and XbaI (2.5 kbp) (data not shown). Since it could be expected that, in the attempt to clone the clostridial homolog in E. coli using agroESL probe, the signal from the lysed E. coli DNA in plaques or colonies swamps out the weak cross-hybridization signals, direct screening of such gene banks was not possible. Therefore, the procedure applied to clone the dnaK
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TABLE 1. Comparison of promoter regions in C. acetobutylicum identified by primer extension analysis Sequence Gene
Glutamine synthetase
Reference
Promoter
-35 region
-10 region
+1
or source
groESL operon
TAAAAAAATCAC TGAGCAAAGGCG TTTTTAATTTTT ATGGTGTAAAAA AAATAAGAAAAG AATAGACTAATT GAACTATAGGAA TAAAATTGATGT
TTGATT TCTTAAAAAAAAGGGAAG TATAAT TTAGTTA TTCGAT AGAAGTTTATACTTGTC TATTGT GCGC TTGCTA TCTCAACATTGCTTAATGC TATAAT AAGT TTTACT TAAAAAAACAATATGTGT TATAAT GTAAATA TTGACA AAGATAATGTCAGGTGA TATTTT ATAAACA TTATGA AATGT AAATAAGAAAAGTTGAC AAAGAT TTATAG AAAGT GACCGACAAGGATACCT TATTCA TTGCTA ATATATTCAGGATTATT TATTAT AATAATTG
This study
Consensus, gram-positive bacteriaa
Ta AAAAA
TTGAcA
15
Acetoacetate decarboxylase dnaK operon
P1 P2 P3
P1 P2 P3
a
A
a T TG
TATAAT AAtAt
19
19 19
13 25 25
25
a Boldface type indicates highly conserved bases.
locus of C. acetobutylicum (25) was chosen and the cloned fragments were separated from host and vector DNAs before hybridization. A partial gene bank was constructed with HindIlI-digested chromosomal DNA of C. acetobutylicum in the size range of 2.2 kbp and the pUC18 cloning vector. Pools of 50 clones were grown in Luria-Bertani medium, and the plasmids were isolated, digested with HindIII, transferred to a nylon membrane, and hybridized with the radioactively labeled probe. One positive pool was identified, and the respective clone carrying the recombinant plasmid pFN1 with a 2.2-kbp HindIll fragment was isolated (Fig. 1). Also evident are problems encountered with background signals, which occurred because of the required nonstringent washing conditions (Fig. 1). Even the vector pUC18 produced hybridization signals with the groESL probe under these conditions. DNA sequencing of the 2.2-kbp HindIll fragment revealed the presence of two open reading frames (ORFs), both truncated at the 5' end and divergently transcribed. The deduced amino acid sequence of one ORF showed high homology to the carboxy terminus of the E. coli GroEL protein. With a 1.3-kbp HindIII-XbaI fragment of that gene as a probe, a second clone was isolated by colony hybridization of a gene library of C. acetobutylicum constructed with XbaI-digested chromosomal DNA. The 2.5-kbp XbaI fragment of the resulting positively reacting plasmid, designated pFN4, partially overlapped with the previously cloned HindlIl fragment and contained the complete groEL gene of C. acetobutylicum and an additional ORF immediately upstream of groEL, as shown by DNA sequencing. Figure 2 shows the cloned fragments of pFN1 and pFN4, their positions in relation to each other, and the ORFs found. The combined nucleotide sequence of the cloned fragments in pFN1 and pFN4 stretches over 3,493 bp and is shown in Fig. 3. The gene upstream of groEL was identified as the groES homolog of C. acetobutylicum (48.4% identity and 68.4% similarity at the amino acid level to the E. coli protein). The values for the GroEL protein were 60.3% identity and 75.8% similarity. The groES gene coded for a protein of 10,419 Da with a pl of 4.83, and the groEL gene coded for a protein of 58,037 Da with a pl of 4.72, as calculated from the deduced amino acid sequences. The divergently transcribed ORF (orJZ) showed no homology to other sequences available in the data bases. At the 3' end of this gene, 1,231 bp coding for 410 amino acids with a molecular mass of 46,411 Da were sequenced. Typical ribosome-binding sites (38) could be identified in front of the groES and groEL genes. A stem-
loop structure resembling a procaryotic rho-independent transcription terminator with a free energy of -78.3 kJ/mol (calculated with the Fold [50] computer program according to the determination of Freier et al. [11]) and then a stretch of Us and As (Fig. 3) are located downstream from the groEL gene. Another hairpin-loop structure without the features of a typical transcription terminator and with a free energy of -61.5 kJ/mol was located in front of the groES gene. Further upstream, a consensus promoter sequence (15, 22) was found. mRNA analysis of the groEL gene region. The synthesis rate of the GroEL protein of C. acetobutylicum is transiently increased after a heat shock (33, 41). To determine whether this fact is consistent with corresponding mRNA levels, a Northern blot was prepared with RNA of C. acetobutylicum isolated before and at different time points after a heat shock from 30 to 42°C. It was hybridized with a radioactively labeled 1.3-kbp XbaI fragment of the groEL gene, and the resulting autoradiogram is shown in Fig. 4. On the blot, one band corresponding to an mRNA of 2.2 kb is visible. Thus, it represents a transcript of a bicistronic operon including groES and groEL since the calculated size of a transcript from the putative promoter region in front of groES to the transcription terminator downstream of groEL is in the same size range (2,150 bp). Furthermore, it was obvious that transcription of this operon was induced after heat shock and that the induction was transient. However, the decrease in the mRNA levels after the maximum, which was observed 15 min after the shock, was slower compared with that of the dnaK region of C. acetobutylicum (25). Determination of the transcription start point of the groESL operon. To prove that the promoter identified upstream of groES by sequence comparison is indeed the recognition site of the RNA polymerase of C. acetobutylicum, the transcription start point of the groESL operon was determined by primer extension analysis using an oligonucleotide complementary to the 5' end of groES. Total RNA was isolated from C. acetobutylicum cells before and 7.5 min after a heat shock from 30 to 42°C. The results are shown in Fig. 5. A strong signal was obtained with RNA prepared from heatshocked cells, and the transcription start point was located 109 bases upstream of groES with a G as the 5' end of the mRNA. Thus, the above proposed promoter (5'-TTGCTA [17 bp]TATTAT) could be confirmed. The second strong signal located in the hairpin-loop structure does not represent a transcription start point and is probably caused by interference of the reverse transcriptase with this secondary structure or by specific processing of the
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B
A
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TGTTAGCACTCAAGATTAACGAGTGCTAACA ATTAGvCACTCAA-AGAGAGTGAGTGCTAAT C. acetobutylicum dnaK locus (25) TTAGCACTCGCTTATTGAGAGTGCTAA B. subtilis dnaK locus (45) TTAGCACTCTTTAGTGCTGAGTGCTAA B. subtilis groESL (37) ATTAGCACTCAGGTACTGGGAGTGCTAAT Synechococcus groESL (44) TTAGCACTCCACTGCCAAGAGTGCTAA Synechocystis cpn60 (4) CTAGCACTCTCATGTATAGAGTGCTAG Mycobacteriurn bovis inuogenic protein MB57 (46) Mycobacteriumn tuberculosis BCG-a heatshock protein (40) CTAGCACTCTCATGTATAGAGTGCTAG CAG7CACTCTCATGTATAGAGTGCT1A Mycobacteriumn tuberculosis 10 kDa antigen (1) TTGCACTCGCCTTAGGGGAGGCTAA Mycobacterium leprae 65 kDa anigen (24) TTGCACTCGGCATAGGCGAGTGCTAA Mycobacterium tuberculosis 65 kDa antgen (39) C. acetobutylicumn groESL (this study)
dnaK-containing operon (B) of C. FIG. 6. Comparison of hairpin-loop structures located 5' to the groESL operon (A) andofthe heat shock genes of different bacteria in similar upstream with sequences are compared DNA sequences Corresponding acetobutylicum. in brackets are references. panel C. Bold letters represent regions of dyad symmetry, and double points mark identical bases. Numbers
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primary transcript. The same phenomenon has been observed upstream of the orfA heat shock gene of C. acetobutylicum, where a similar structure exists (25). Since the same (however weak) signals were also obtained with RNA from non-heat-shock cells after long exposure times, no evidence for additional transcription start sites after heat shock was found. Furthermore, no additional transcription start site was located upstream of groEL (data not shown). Thus, these results also clearly indicate that groES and groEL of C. acetobutylicum are organized in a bicistronic operon. The expression of both genes is regulated by a heat-inducible promoter upstream of groES. DISCUSSION The C. acetobutylicum structural genes for the molecular chaperones and heat shock proteins GroES and GroEL have been identified and sequenced. As in E. coli, the genes are organized in one operon. However, the regulatory mechanism for the expression of these genes seems to be different in some aspect. Whereas the huge but transient increase in the transcription of these genes corresponds to a similar expression pattern in E. coli (47), no evidence for an alternate sigma factor of the RNA polymerase was found. The primer extension experiments clearly indicate that only one identical transcription initiation site is used for groESL expression during growth at low temperatures and after heat shock. Furthermore, the deduced promoter region showed no striking differences to the consensus promoter of grampositive bacteria as well as to the few already identified promoters of the non-heat shock genes adc (acetoacetate decarboxylase) (13) and glnA (glutamine synthetase) (19) of C. acetobutylicum (Table 1). Although promoter regions of quite a number of other cloned genes from C. acetobutylicum have been proposed because of sequence similarity to the consensus sequence (for a review, see reference 49), further experiments (S1 nuclease mapping and primer extension analysis) are necessary to prove that these regions function as recognition sites for the RNA polymerase of C. acetobutylicum. This organism has a G+C content of 28 mol% (6), and thus, AT-rich regions which resemble consensus promoter regions are very frequent, especially in the noncoding sites of the chromosome. The fact that the identified promoter regions of the inducible genes adc, ginA, dnaK, and groESL of C. acetobutylicum are similar (Table 1) makes it evident that additional unknown structures must exist, which makes discrimination of these promoters for differential expression possible, depending on the different inducing conditions. We propose that the 11-bp inverted repeat located between the transcription and translation start sites is involved in the regulation of groESL expression. Such a structure with an almost identical sequence is also present in front of the dnaK-containing heat shock operon of C. acetobutylicum (25) and several heat shock genes of other bacteria (Fig. 6). In E. coli, where a consensus sequence of heat shock promoters was found (5), a comparable region preceding known heat shock genes does not exist. It has yet to be shown whether this putative regulatory region is an activator binding site, e.g., to increase the binding affinity of RNA polymerase to the respective promoter. It is also possible that, after the initiation of transcription, this structure causes pausing of the RNA polymerase to a certain extent. This effect might then be abolished after heat shock by the action of a protein, which opens the mRNA secondary structure and enables RNA polymerase to continue at a higher rate. In this respect, it is of interest that
J. BACTERIOL.
the RNA polymerase from heat-shocked cells of C. acetobutylicum contains an additional protein which reacts to polyclonal antibodies raised against (r2 of E. coli but does not function as a sigma factor (31, 32). Experiments are in progress to determine the nature of this protein and its possible function in the expression of C. acetobutylicum heat shock genes. ACKNOWLEDGMENTS We thank C. Georgopoulos for providing plasmid pKT200, P. Durre for stimulating discussions, and G. Gottschalk for continuous support. We are obliged to W. Schumann and M. Hecker for sharing their results on the groESL locus of B. subtilis with us prior to publication. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to H.B. and a fellowship of the Graduiertenforderung of the University of Gottingen to F.N. REFERENCES 1. Baird, P. N., L. M. C. Hall, and A. R. M. Coates. 1989. Cloning and sequence analysis of the 10 kDa antigen gene of Mycobacterium tuberculosis. J. Gen. Microbiol. 135:931-939. 2. Bertram, J., and P. Durre. 1989. Conjugal transfer and expression of streptococcal transposons in Clostridium acetobutylicum. Arch. Microbiol. 151:551-557. 3. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. 4. Chitnis, P. R., and N. Nelson. 1991. Molecular cloning of the genes encoding two chaperone proteins of the cyanobacterium Synechocystis sp. PCC 6803. J. Biol. Chem. 266:58-65. 5. Cowing, D. W., J. C. A. Bardwell, E. A. Craig, C. Woolford, R. W. Hendrix, and C. A. Gross. 1985. Consensus sequence for Escherichia coli heat shock gene promoters. Proc. Natl. Acad. Sci. USA 80:2679-2683. 6. Cummins, C. S., and J. L. Johnson. 1971. Taxonomy of clostridia: wall composition and DNA homologies in Clostridium butyricum and other butyric acid-producing clostridia. J. Gen. Microbiol. 67:33-46. 7. Devereux, J., P. Haeberli, and 0. Smithies. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12:387-395. 8. Ellis, J. 1987. Proteins as molecular chaperones. Nature (London) 328:378-379. 9. Fayet, O., J.-M. Louarn, and C. Georgopoulos. 1986. Suppression of the Escherichia coli dnaA46 mutation by amplification of the groES and groEL genes. Mol. Gen. Genet. 202:435-445. 10. Fayet, O., T. Ziegelhoffer, and C. Georgopoulos. 1989. The groES and groEL heat shock gene products of Escherichia coli are essential for bacterial growth at all temperatures. J. Bacteriol. 171:1379-1385. 11. Freier, S. M., R. Kierzek, J. A. Jaeger, N. Sugimoto, M. H. Caruthers, T. Neilson, and D. H. Turner. 1986. Improved free-energy parameters for predictions of RNA duplex stability. Proc. Natl. Acad. Sci. USA 83:9373-9377. 12. Friedman, D. I., E. R. Olson, K. Tilly, C. Georgopoulos, I. Herskowitz, and F. Banuett. 1984. Interactions of bacteriophage and host macromolecules in the growth of bacteriophage lambda. Microbiol. Rev. 48:299-325. 13. Gerischer, U., and P. Durre. 1991. mRNA analysis of the adc gene region of Clostridium acetobutylicum during the shift to solventogenesis. J. Bacteriol. 174:426-433. 14. Goloubinoff, P., A. A. Gatenby, and G. H. Lorimer. 1988. GroE heat-shock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Escherichia coli. Nature (London) 337:44-47. 15. Graves, M. C., and J. C. Rabinowitz. 1986. In vivo and in vitro transcription of the Clostridium pasteurianum ferredoxin gene. J. Biol. Chem. 261:11409-11415. 16. Grossman, A. D., J. W. Erickson, and C. A. Gross. 1984. The htpR gene product of E. coli is a sigma factor for heat-shock
VOL. 174, 1992 promoters. Cell 38:383-390. 17. Grunstein, M., and J. Wallis. 1979. Colony hybridization. Methods Enzymol. 68:379-389. 18. Hemmingsen, S. M., C. Woolford, S. M. van der Vies, K. Tilly, D. T. Dennis, C. P. Georgopoulos, R. W. Hendrix, and R. J. Ellis. 1988. Homologous plant and bacterial proteins chaperone oligomeric protein assembly. Nature (London) 333:330-334. 19. Janssen, P. J., D. T. Jones, and D. R. Woods. 1990. Studies on Clostnidium acetobutylicum gInA promoters and antisense RNA. Mol. Microbiol. 4:1575-1583. 20. Marck, C. 1988. 'DNA Strider': a 'C' program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers. Nucleic Acids Res. 16:1829-1836. 21. Marmur, J. 1960. A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. Mol. Biol. 3:208-218. 22. McClure, W. R. 1985. Mechanism and control of transcription initiation in prokaryotes. Annu. Rev. Biochem. 54:171-204. 23. McMullin, T. W., and R. Hallberg. 1988. A highly evolutionarily conserved mitochondrial protein is structurally related to the protein encoded by the Eschenichia coli groEL gene. Mol. Cell. Biol. 8:371-380. 24. Mehra, V., D. Sweetser, and R. Young. 1986. Efficient mapping of protein antigenic determinants. Proc. Natl. Acad. Sci. USA 83:7013-7017. 25. Narberhaus, F., K. Giebeler, and H. Bahl. 1992. Molecular characterization of the dnaK gene region of Clostridium acetobutylicum, including grpE, dnaJ, and a new heat shock gene. J. Bacteriol. 174:3290-3299. 26. Neidhardt, F. C., R. A. VanBogelen, and V. Vaughn. 1984. The genetics and regulation of heat-shock proteins. Annu. Rev. Genet. 18:295-329. 27. O'Brien, R. W., and J. G. Morris. 1971. Oxygen and growth and metabolism of Clostridium acetobutylicum. J. Gen. Microbiol. 68:307-318. 28. Oelmuller, U., N. Kriiger, A. Steinbuchel, and C. Friedrich. 1990. Isolation of prokaryotic RNA and detection of specific mRNA with biotinylated probes. J. Microbiol. Methods 11:7381. 29. Parsell, D. A., and R. T. Sauer. 1989. Induction of a heat shock-like response by unfolded protein in Escherichia coli: dependence on protein level not protein degradation. Genes Dev. 3:1226-1232. 30. Pelham, H. R. B. 1986. Speculations on the functions of the major heat shock and glucose-regulated proteins. Cell 46:959961. 31. Pich, A., and H. Bahl. 1991. Purification and characterization of the DNA-dependent RNA polymerase from Clostndium acetobutylicum. J. Bacteriol. 173:2120-2124. 32. Pich, A., and H. Bahl. 1991. Unpublished results. 33. Pich, A., F. Narberhaus, and H. Bahl. 1990. Induction of heat shock proteins during solvent formation in Clostridium acetobutylicum. Appl. Microbiol. Biotechnol. 33:697-704. 34. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor
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Laboratory, Cold Spring Harbor, N.Y. 35. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467. 36. Schlesinger, M. J., M. Ashburner, and A. Tissieres. 1982. Heat shock from bacteria to man. Cold Spring Harbor Laboratory. Cold Spring Harbor, N.Y. 37. Schumann, W. Personal communication. 38. Shine, J., and L. Dalgarno. 1974. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementary to nonsense triplets and ribosome-binding sites. Proc. Natl. Acad. Sci. USA 71:1342-1346. 39. Shinnick, T. M. 1987. The 65-kilodalton antigen of Mycobacterium tuberculosis. J. Bacteriol. 169:1080-1088. 40. Shinnick, T. M., B. B. Plikaytis, A. D. Hyche, R. M. van Landingham, and L. L. Walker. 1989. The Mycobacterium tuberculosis BCG-a protein has homology with the Escherichia coli GroES protein. Nucleic Acids Res. 17:1254. 41. Terracciano, J. S., E. Rapaport, and E. R. Kashket. 1988. Stress and growth-phase associated proteins of Clostridium acetobutylicum. Appl. Environ. Microbiol. 54:1989-1995. 42. Tilly, K., H. Murialdo, and C. Georgopoulos. 1981. Identification of a second E. coli groE gene whose product is necessary for bacteriophage morphogenesis. Proc. Natl. Acad. Sci. USA 78:1629-1633. 43. Vodkin, M. H., and J. C. Williams. 1988. A heat shock operon in Coxiella bumnetii produces a major antigen homologous to a protein in both mycobacteria and Escherichia coli. J. Bacteriol. 170:1227-1234. 44. Webb, R., K. J. Reddy, and L. A. Sherman. 1990. Regulation and sequence of the Synechocystis sp. strain PCC 7942 groESL operon, encoding a cyanobacterial chaperonin. J. Bacteriol. 172:5079-5088. 45. Wetzstein, M., U. Volker, J. Dedio, S. LBbau, U. Zuber, M. Schiesswohl, C. Herget, M. Hecker, and W. Schumann. 1992. Cloning, sequencing, and molecular analysis of the dnaK locus from Bacillus subtilis. J. Bacteriol. 174:3300-3310. 46. Yamaguchi, R., K. Matsuo, A. Yamazaki, S. Nagai, K. Terasaka, and T. Yamada. 1988. Immunogenic protein MPB57 from Mycobacterium bovis BCG: molecular cloning, nucleotide sequence and expression. FEBS Lett. 240:115-117. 47. Yamamori, T., and T. Yura. 1982. Genetic control of heat-shock protein synthesis and its bearing on growth and thermal resistance in Eschenichia coli K12. Proc. Natl. Acad. Sci. USA 79:860-864. 48. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mpl8 and pUC19 vectors. Gene 33:103-119. 49. Young, M., N. P. Minton, and W. L. Staudenbauer. 1989. Recent advances in the genetics of clostridia. FEMS Microbiol. Rev. 63:301-326. 50. Zuker, M., and P. Stiegler. 1981. Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res. 9:133-148.
Vol. 174, No. 10
0021-9193/92/103282-08$02.00/0 Copyright © 1992, American Society for Microbiology
Cloning, Sequencing, and Molecular Analysis of the groESL Operon of Clostridium acetobutylicum FRANZ NARBERHAUS AND HUBERT BAHL*
Institut fiir Mikrobiologie, Georg-August- Universitat Gottingen, Grisebachstrasse 8, W-3400 Gottingen, Germany Received 2 December 1991/Accepted 10 February 1992
The groESL operon of Clostridium acetobutylicum was cloned in Escherichia coli by using a gene probe of E. coli groESL. Sequencing of a positively reacting 2.2-kbp HindIII fragment contained in the recombinant plasmid pFN1 and a 2.5-kbpXbal fragment present in pFN4 revealed that both fragments partially overlapped and together spanned 3,493 bp of the clostridial chromosome. Two complete open reading frames (288 and 1632 bp) were found and identified as the groES- and groEL-homologous genes of C. acetobutylicum, respectively. The 3' end of a third gene (orJZ), which was divergently transcribed, showed no significant homology to other sequences available in the EMBL and GenBank data bases. The length of thegroESL-specific mRNA (2.2 kb), a transcription terminator downstream of groEL, and a transcription start site upstream of groES, identified by primer extension analysis, indicated that groES and groEL of C. acetobutylicum are organized in a bicistronic operon. From the transcription start site, the promoter structure 5'-TTGCTA (17 bp) TATTAT that shows high homology to the consensus promoter sequence of gram-positive bacteria as well as E. coli was deduced. Transcription of the groESL operon was strongly heat inducible, and maximum levels of mRNA were detected 15 min after heat shock from 30 to 42'C. An 11-bp inverted repeat, located between promoter and translation start sites of groES and partially identical with similar structures in front of several heat shock genes of other bacteria, may play an important role in the regulation of heat shock gene expression in this organism. A set of heat shock proteins is clearly induced when cells are exposed to higher temperatures. This phenomenon has been observed in all organisms, from bacteria and fungi to plants and animals (36). In Escherichia coli, about 20 heat shock proteins are known (26), among them the evolutionarily highly conserved DnaK and GroEL proteins. The important role of these two proteins during normal growth has also been recently established. They are involved in the assembly and disassembly of protein complexes (molecular chaperones) and in protein translocation processes (8, 14,
regulatory mechanism for the expression of this heat shock operon in C. acetobutylicum is different from that in E. coli. MATERIALS AND METHODS Bacterial strains, plasmids, and growth conditions. C. acetobutylicum DSM1731 was used as a source of genomic DNA and total RNA. E. coli JM109 [recAl endAl gyrA96 thi hsdR17(rK iMK+)supE44 relAl X-A(lac-proAB)(F' traD36 proAB+ lacIq lacZAM15)] (48) was used as host for the cloning experiments. The plasmid pUC18 (48) served as vector for the construction of genomic libraries. Plasmid pKT200 (9) containing the groESL operon of E. coli on an 8.1-kbp EcoRI fragment was used to isolate a probe for hybridization experiments. C. acetobutylicum was grown anaerobically in CBM medium (27) at 37°C. E. coli was routinely grown at 37°C in Luria-Bertani medium (34) supplemented with ampicillin (50 ,ug/ml), isopropyl-3-thiogalactopyranoside (IPTG, 50 ,ug/ml), or 5-bromo-4-chloro-3-indolyl-p-D-galactopyranoside (X-Gal, 40 p,g/ml) if required. DNA isolation and manipulation. Chromosomal DNA of C. acetobutylicum was isolated by the method of Marmur (21) as modified by Bertram and Durre (2). Plasmid isolation from E. coli was performed by the method of Birnboim and Doly (3) or with the Quiagen Midi Kit (Diagen GmbH, Dusseldorf,
29, 30). The groE genes (groES and groEL) of E. coli were originally defined as genes necessary for the morphogenesis of several bacteriophages (12). They comprise an operon under positive transcriptional control exerted by the heat shock sigma factor, u32 (16, 18). The groEL and groES genes encode abundant proteins with molecular masses of 57,259 and 10,368 Da, respectively (42), which are essential for the growth of E. coli at all temperatures (10). Homologous proteins have been found in other bacteria (24, 43); chloroplasts (18); and mitochondria from plants, fungi, and animals (23). In contrast, very little is known about the nature and regulation of GroE proteins in obligate anaerobic bacteria. In Clostridium acetobutylicum, a gram-positive, spore-forming anaerobe, a GroEL protein is synthesized at elevated rates after a heat shock and during the switch from acid to solvent formation (33). To investigate the molecular structure and regulation of the C. acetobutylicum groE locus, this article describes the molecular characterization of the groEL and groES genes of this organism. Evidence is presented that the
*
Germany). DNA was manipulated by standard methods (34). Restriction enzymes were obtained from GIBCO/BRL GmbH (Eggenstein, Germany) or Pharmacia LKB GmbH (Freiburg, Germany), and calf intestinal phosphatase was from Boehringer GmbH (Mannheim, Germany). The enzymes were used according to the instructions of the manufacturers. DNA restriction fragments were isolated from agarose gels by electroelution in a Biotrap BT1000 (Schleicher & Schuell, Dassel, Germany).
Corresponding author. 3282
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FIG. 1. Hybridization of HindIII-digested plasmids from clones of a partial gene bank of C. acetobutylicum with the 32P-labeled 1.7-kbp SacII-BamHI fragment of pKT200. The partial gene bank was constructed with HindIII-digested chromosomal DNA of C. acetobutylicum (2.2-kbp fragment size) and pUC18. (A) Autoradiogram of plasmids from pools containing 50 clones hybridized to the probe. Lanes 1 to 6, plasmids of six different pools digested with HindIll; lane 7, chromosomal DNA of C. acetobutylicum digested with HindIII; lane 8, X DNA digested with PstI. (B) Autoradiogram of five plasmids from the positive pool (lane 2 in panel A). Lanes 1 and 8, A DNA digested with PstI; lane 2, chromosomal DNA of C. acetobutylicum digested with HindIll; lanes 3 to 7, plasmids of five clones digested with HindIlI. The bands corresponding to pUC18 and the 2.2-kbp HindIII fragment are marked.
Construction and screening of gene libraries. The gene libraries were constructed with completely digested chromosomal DNA of C. acetobutylicum. For partial gene banks, 100 ,ug of completely digested DNA was centrifuged in a sucrose density gradient from 10 to 40% (wt/vol). Fractions containing the desired fragments were dialyzed against TrisEDTA buffer for 24 h and used for ligation. For total gene banks, digested chromosomal DNA was used after extraction with phenol-chloroform and chloroform and precipitation with ethanol. The DNA fragments were ligated to the appropriately digested and dephosphorylated vector. E. coli was transformed with the ligation mixture by electroporation in a Gene Pulser (Bio-Rad Laboratories GmbH, Munich, Germany). White colonies containing inserts were screened by colony hybridization with the respective probe. Positive clones were tested by restriction endonuclease digestion and were sequenced. Preparation of RNA. Total RNA was isolated from C. acetobutylicum by the hot phenol-chloroform procedure described by Oelmuller et al. (28). To obtain higher yields of
3283
RNA, cells were not washed in the acetate-EDTA buffer and the extraction in 60°C hot phenol-chloroform was extended to 10 min. Hybridization. Total chromosomal DNA of C. acetobutylicum was digested to completion with the desired restriction enzymes and separated on agarose gels. For hybridization with digoxigenin-labeled probes, nitrocellulose membranes (Biotrace; Gelman Sciences, Ann Arbor, Mich.) were used. Southern blots and hybridization experiments were performed according to the manufacturer's instructions. DNA probes were labeled with a DNA labeling and detection kit (Boehringer) and hybridization was performed at 37°C with 30% formamide. For hybridization with 32P-labeled probes, nylon membranes (GeneScreen Plus; Dupont, NEN Research Products, Dreieich, Germany) were used. DNA probes were labeled with [a-32P]dCTP (Amersham Buchler GmbH, Braunschweig, Germany) by using a nick translation kit (GIBCO/BRL). The labeled fragments were purified by column chromatography on Sephadex G-25, and hybridization was done in 0.2% (wt/vol) polyvinylpyrrolidone-0.2% (wt/vol) Ficoll-0.2% (wt/vol) bovine serum albumin-50 mM Tris hydrochloride (pH 7.5)-l M NaCI-0.1% (wt/vol) sodium pyrophosphate-1% (wt/vol) sodium dodecyl sulfate-10% (wt/vol) dextran sulfate at 60°C for 2 to 6 h. After hybridization at 60°C for 15 to 20 h, the membranes were washed twice in 2x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate) for 5 min at room temperature and subjected to autoradiography. If necessary, the blot was washed again twice in 2x SSC-1% (wt/vol) SDS for 30 min at 60°C and twice in 0.1% (wtlvol) SDS for 30 min at room temperature. Colony hybridization was performed with BA85 nitrocellulose filters (Schleicher & Schuell). Colonies were picked directly on a master plate and on a plate containing the filter. Filters were prepared for hybridization as described by Grunstein and Wallis (17). Hybridization and washing were performed as described above for radioactive Southern blots. RNA for Northern blots was run in formaldehyde gels and transferred to nylon membranes (GeneScreen Plus). A size determination was done by using an RNA ladder (0.24, 1.4, 2.4, 4.4, 7.5, and 9.5 kb; GIBCO/BRL) as a standard. Hybridization and washing was done as described above for the DNA hybridization procedure. DNA sequencing. DNA was sequenced by the dideoxychain termination method of Sanger et al. (35), by using [35S]dATP and a T7 sequencing kit from Pharmacia LKB. Single-stranded templates were prepared from both strands of the plasmids. Sequencing was started with commercially HindlII
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FIG. 3. Nucleotide sequence of the 3,493-bpgroESL operon of C. acetobutylicum. Only the antisense strand is shown. The deduced amino acid sequences of groES, groEL, and or/Z are shown below the DNA sequence (single-letter code). Translation stop signals are marked by asterisks below the codons. The putative ribosome-binding sites are underlined. Arrowheads below the DNA sequence show regions of dyad symmetry. The transcription start site (S) identified by primer extension analysis and the corresponding promoter are marked by bold letters in the -10 and -35 regions.
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5
6
7
8
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FIG. 4. Northern blot analysis of total RNA of C. acetobutylicum isolated before and at different time points after a heat shock from 30 to 42°C. Each lane contains 5 ,ug of RNA and was probed with the 32P-labeled XbaI fragment of pFN1. Lane 1, before heat shock; lanes 2 to 8: 5, 7.5, 10, 15, 20, 30, and 60 min, respectively, after the temperature upshift. The signal corresponded to a transcript of 2.2 kbp.
available M13/pUC universal sequencing forward primer and reverse primer. Synthetic oligonucleotides (17-mers) complementary to the ends of already sequenced templates were prepared by a Gene Assembler Plus (Pharmacia LKB) according to the instructions of the manufacturers and were used as primers for continued sequencing. The dideoxyterminated fragments were separated on 55-cm wedgeshaped thickness gradient gels (0.2 to 0.4 mm, 7% [wt/vol] polyacrylamide) with a Macrophor sequencing unit (Pharmacia LKB) as recommended by the manufacturer. Determination of the transcription start site. For the determination of the transcription start site, primer extension analysis was used. Two picomoles of oligonucleotide (17mer) was labeled with 10 ,uCi of [y-32P]ATP (Amersham Buchler) and 7 U of polynucleotide kinase (GIBCO/BRL) in PNK buffer (100 mM Tris [pH 7.6], 20 mM MgCI2, 30 mM dithiothreitol) for 3 h at 37°C (10-,u reaction volume). The reaction was stopped by heating for 2 min at 95°C. Ten micrograms of total RNA was incubated together with 12.5 U of RNasin (GIBCO/BRL) and 0.2 pmol of kinased oligonucleotide in 500 mM KCl-20 mM Tris (pH 7.9) buffer for 3 h at 30°C (final volume, 10 RI). For the primer extension reaction, 6 RI of 5 x PE buffer (250 mM Tris [pH 8.3], 125 mM KCI, 15 mM MgCI2, 50 mM dithiothreitol), 500 p,M dNTPs of each nucleotide, 2.5 ,g of actinomycin D and 200 U of SuperScript RNase H- reverse transcriptase (GIBCO/ BRL) per 10 ,ug of RNA and 30 ,u of H20 were combined in the annealing assay and incubated at 37°C for 1 h. The RNA
primer-extended hybrid was phenol-chloroform extracted, ethanol precipitated, and analyzed on 7% polyacrylamideurea sequencing gels. The lengths of the primer extension products were calculated by running a sequencing reaction with the same primer on the same gel. Computer programs. The DNA region sequenced and the deduced proteins were analyzed with the DNA Strider program (20) on a Macintosh SE computer (Apple Computer, Inc., Cupertino, Calif.). Sequence comparisons were done by using the Wisconsin Genetics Computer Group sequence analysis software package, version 6.0 (University of Wisconsin Biotechnology Center, Madison) (7). Nucleotide sequence accession number. The sequence data reported here (see Fig. 3) have been submitted to GenBank and assigned accession no. M74572.
FIG. 5. Mapping of the 5' end of mRNA of the groESL operon by primer extension analysis. A 32P-radiolabeled primer complementary to the 5' end of groES was hybridized to 10 pLg each of total RNA from C. acetobutylicum isolated before (1) and 7.5 min after a heat shock from 30 to 42°C (2). The primer extension products were analyzed on a sequence gel. G, A, T, and C are products of the sequencing reaction obtained by using the same oligonucleotide as the primer. The depicted sequence represents the antisense strand. Broken arrows mark regions of dyad symmetry; the arrow at S points to the base representing the 5' end of the mRNA. -35 and -10 regions are also shown (boldface).
RESULTS Cloning and sequencing of the groESL operon of C. acetobutylicum. A digoxigenin-labeled 1.7-kbp SacII-BamHI fragment of the E. coli groESL operon was used to detect homologous sequences in the DNA of C. acetobutylicum. Hybridization signals were obtained with C. acetobutylicum DNA digested with HindIII (2.2- and 4.8-kbp fragments), AccI (2.9 kbp), EcoRI (4.3 kbp), EcoRV (5.2 kbp), and XbaI (2.5 kbp) (data not shown). Since it could be expected that, in the attempt to clone the clostridial homolog in E. coli using agroESL probe, the signal from the lysed E. coli DNA in plaques or colonies swamps out the weak cross-hybridization signals, direct screening of such gene banks was not possible. Therefore, the procedure applied to clone the dnaK
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TABLE 1. Comparison of promoter regions in C. acetobutylicum identified by primer extension analysis Sequence Gene
Glutamine synthetase
Reference
Promoter
-35 region
-10 region
+1
or source
groESL operon
TAAAAAAATCAC TGAGCAAAGGCG TTTTTAATTTTT ATGGTGTAAAAA AAATAAGAAAAG AATAGACTAATT GAACTATAGGAA TAAAATTGATGT
TTGATT TCTTAAAAAAAAGGGAAG TATAAT TTAGTTA TTCGAT AGAAGTTTATACTTGTC TATTGT GCGC TTGCTA TCTCAACATTGCTTAATGC TATAAT AAGT TTTACT TAAAAAAACAATATGTGT TATAAT GTAAATA TTGACA AAGATAATGTCAGGTGA TATTTT ATAAACA TTATGA AATGT AAATAAGAAAAGTTGAC AAAGAT TTATAG AAAGT GACCGACAAGGATACCT TATTCA TTGCTA ATATATTCAGGATTATT TATTAT AATAATTG
This study
Consensus, gram-positive bacteriaa
Ta AAAAA
TTGAcA
15
Acetoacetate decarboxylase dnaK operon
P1 P2 P3
P1 P2 P3
a
A
a T TG
TATAAT AAtAt
19
19 19
13 25 25
25
a Boldface type indicates highly conserved bases.
locus of C. acetobutylicum (25) was chosen and the cloned fragments were separated from host and vector DNAs before hybridization. A partial gene bank was constructed with HindIlI-digested chromosomal DNA of C. acetobutylicum in the size range of 2.2 kbp and the pUC18 cloning vector. Pools of 50 clones were grown in Luria-Bertani medium, and the plasmids were isolated, digested with HindIII, transferred to a nylon membrane, and hybridized with the radioactively labeled probe. One positive pool was identified, and the respective clone carrying the recombinant plasmid pFN1 with a 2.2-kbp HindIll fragment was isolated (Fig. 1). Also evident are problems encountered with background signals, which occurred because of the required nonstringent washing conditions (Fig. 1). Even the vector pUC18 produced hybridization signals with the groESL probe under these conditions. DNA sequencing of the 2.2-kbp HindIll fragment revealed the presence of two open reading frames (ORFs), both truncated at the 5' end and divergently transcribed. The deduced amino acid sequence of one ORF showed high homology to the carboxy terminus of the E. coli GroEL protein. With a 1.3-kbp HindIII-XbaI fragment of that gene as a probe, a second clone was isolated by colony hybridization of a gene library of C. acetobutylicum constructed with XbaI-digested chromosomal DNA. The 2.5-kbp XbaI fragment of the resulting positively reacting plasmid, designated pFN4, partially overlapped with the previously cloned HindlIl fragment and contained the complete groEL gene of C. acetobutylicum and an additional ORF immediately upstream of groEL, as shown by DNA sequencing. Figure 2 shows the cloned fragments of pFN1 and pFN4, their positions in relation to each other, and the ORFs found. The combined nucleotide sequence of the cloned fragments in pFN1 and pFN4 stretches over 3,493 bp and is shown in Fig. 3. The gene upstream of groEL was identified as the groES homolog of C. acetobutylicum (48.4% identity and 68.4% similarity at the amino acid level to the E. coli protein). The values for the GroEL protein were 60.3% identity and 75.8% similarity. The groES gene coded for a protein of 10,419 Da with a pl of 4.83, and the groEL gene coded for a protein of 58,037 Da with a pl of 4.72, as calculated from the deduced amino acid sequences. The divergently transcribed ORF (orJZ) showed no homology to other sequences available in the data bases. At the 3' end of this gene, 1,231 bp coding for 410 amino acids with a molecular mass of 46,411 Da were sequenced. Typical ribosome-binding sites (38) could be identified in front of the groES and groEL genes. A stem-
loop structure resembling a procaryotic rho-independent transcription terminator with a free energy of -78.3 kJ/mol (calculated with the Fold [50] computer program according to the determination of Freier et al. [11]) and then a stretch of Us and As (Fig. 3) are located downstream from the groEL gene. Another hairpin-loop structure without the features of a typical transcription terminator and with a free energy of -61.5 kJ/mol was located in front of the groES gene. Further upstream, a consensus promoter sequence (15, 22) was found. mRNA analysis of the groEL gene region. The synthesis rate of the GroEL protein of C. acetobutylicum is transiently increased after a heat shock (33, 41). To determine whether this fact is consistent with corresponding mRNA levels, a Northern blot was prepared with RNA of C. acetobutylicum isolated before and at different time points after a heat shock from 30 to 42°C. It was hybridized with a radioactively labeled 1.3-kbp XbaI fragment of the groEL gene, and the resulting autoradiogram is shown in Fig. 4. On the blot, one band corresponding to an mRNA of 2.2 kb is visible. Thus, it represents a transcript of a bicistronic operon including groES and groEL since the calculated size of a transcript from the putative promoter region in front of groES to the transcription terminator downstream of groEL is in the same size range (2,150 bp). Furthermore, it was obvious that transcription of this operon was induced after heat shock and that the induction was transient. However, the decrease in the mRNA levels after the maximum, which was observed 15 min after the shock, was slower compared with that of the dnaK region of C. acetobutylicum (25). Determination of the transcription start point of the groESL operon. To prove that the promoter identified upstream of groES by sequence comparison is indeed the recognition site of the RNA polymerase of C. acetobutylicum, the transcription start point of the groESL operon was determined by primer extension analysis using an oligonucleotide complementary to the 5' end of groES. Total RNA was isolated from C. acetobutylicum cells before and 7.5 min after a heat shock from 30 to 42°C. The results are shown in Fig. 5. A strong signal was obtained with RNA prepared from heatshocked cells, and the transcription start point was located 109 bases upstream of groES with a G as the 5' end of the mRNA. Thus, the above proposed promoter (5'-TTGCTA [17 bp]TATTAT) could be confirmed. The second strong signal located in the hairpin-loop structure does not represent a transcription start point and is probably caused by interference of the reverse transcriptase with this secondary structure or by specific processing of the
CLOSTRIDIUM ACETOBU7YLICUM groESL OPERON
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B
A
061v~~~~~~~_
6'~~~~~~~~~~~~0
A 0G
_
6~~~~~~~~~~~~v--
0
.3 --
__ A~~~~~~~~~~~~~~~ n C
'.3~~~~~~~~~~~~~~~~~~~~~~~~~
-
.
..
..
..
....
TGTTAGCACTCAAGATTAACGAGTGCTAACA ATTAGvCACTCAA-AGAGAGTGAGTGCTAAT C. acetobutylicum dnaK locus (25) TTAGCACTCGCTTATTGAGAGTGCTAA B. subtilis dnaK locus (45) TTAGCACTCTTTAGTGCTGAGTGCTAA B. subtilis groESL (37) ATTAGCACTCAGGTACTGGGAGTGCTAAT Synechococcus groESL (44) TTAGCACTCCACTGCCAAGAGTGCTAA Synechocystis cpn60 (4) CTAGCACTCTCATGTATAGAGTGCTAG Mycobacteriurn bovis inuogenic protein MB57 (46) Mycobacteriumn tuberculosis BCG-a heatshock protein (40) CTAGCACTCTCATGTATAGAGTGCTAG CAG7CACTCTCATGTATAGAGTGCT1A Mycobacteriumn tuberculosis 10 kDa antigen (1) TTGCACTCGCCTTAGGGGAGGCTAA Mycobacterium leprae 65 kDa anigen (24) TTGCACTCGGCATAGGCGAGTGCTAA Mycobacterium tuberculosis 65 kDa antgen (39) C. acetobutylicumn groESL (this study)
dnaK-containing operon (B) of C. FIG. 6. Comparison of hairpin-loop structures located 5' to the groESL operon (A) andofthe heat shock genes of different bacteria in similar upstream with sequences are compared DNA sequences Corresponding acetobutylicum. in brackets are references. panel C. Bold letters represent regions of dyad symmetry, and double points mark identical bases. Numbers
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primary transcript. The same phenomenon has been observed upstream of the orfA heat shock gene of C. acetobutylicum, where a similar structure exists (25). Since the same (however weak) signals were also obtained with RNA from non-heat-shock cells after long exposure times, no evidence for additional transcription start sites after heat shock was found. Furthermore, no additional transcription start site was located upstream of groEL (data not shown). Thus, these results also clearly indicate that groES and groEL of C. acetobutylicum are organized in a bicistronic operon. The expression of both genes is regulated by a heat-inducible promoter upstream of groES. DISCUSSION The C. acetobutylicum structural genes for the molecular chaperones and heat shock proteins GroES and GroEL have been identified and sequenced. As in E. coli, the genes are organized in one operon. However, the regulatory mechanism for the expression of these genes seems to be different in some aspect. Whereas the huge but transient increase in the transcription of these genes corresponds to a similar expression pattern in E. coli (47), no evidence for an alternate sigma factor of the RNA polymerase was found. The primer extension experiments clearly indicate that only one identical transcription initiation site is used for groESL expression during growth at low temperatures and after heat shock. Furthermore, the deduced promoter region showed no striking differences to the consensus promoter of grampositive bacteria as well as to the few already identified promoters of the non-heat shock genes adc (acetoacetate decarboxylase) (13) and glnA (glutamine synthetase) (19) of C. acetobutylicum (Table 1). Although promoter regions of quite a number of other cloned genes from C. acetobutylicum have been proposed because of sequence similarity to the consensus sequence (for a review, see reference 49), further experiments (S1 nuclease mapping and primer extension analysis) are necessary to prove that these regions function as recognition sites for the RNA polymerase of C. acetobutylicum. This organism has a G+C content of 28 mol% (6), and thus, AT-rich regions which resemble consensus promoter regions are very frequent, especially in the noncoding sites of the chromosome. The fact that the identified promoter regions of the inducible genes adc, ginA, dnaK, and groESL of C. acetobutylicum are similar (Table 1) makes it evident that additional unknown structures must exist, which makes discrimination of these promoters for differential expression possible, depending on the different inducing conditions. We propose that the 11-bp inverted repeat located between the transcription and translation start sites is involved in the regulation of groESL expression. Such a structure with an almost identical sequence is also present in front of the dnaK-containing heat shock operon of C. acetobutylicum (25) and several heat shock genes of other bacteria (Fig. 6). In E. coli, where a consensus sequence of heat shock promoters was found (5), a comparable region preceding known heat shock genes does not exist. It has yet to be shown whether this putative regulatory region is an activator binding site, e.g., to increase the binding affinity of RNA polymerase to the respective promoter. It is also possible that, after the initiation of transcription, this structure causes pausing of the RNA polymerase to a certain extent. This effect might then be abolished after heat shock by the action of a protein, which opens the mRNA secondary structure and enables RNA polymerase to continue at a higher rate. In this respect, it is of interest that
J. BACTERIOL.
the RNA polymerase from heat-shocked cells of C. acetobutylicum contains an additional protein which reacts to polyclonal antibodies raised against (r2 of E. coli but does not function as a sigma factor (31, 32). Experiments are in progress to determine the nature of this protein and its possible function in the expression of C. acetobutylicum heat shock genes. ACKNOWLEDGMENTS We thank C. Georgopoulos for providing plasmid pKT200, P. Durre for stimulating discussions, and G. Gottschalk for continuous support. We are obliged to W. Schumann and M. Hecker for sharing their results on the groESL locus of B. subtilis with us prior to publication. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to H.B. and a fellowship of the Graduiertenforderung of the University of Gottingen to F.N. REFERENCES 1. Baird, P. N., L. M. C. Hall, and A. R. M. Coates. 1989. Cloning and sequence analysis of the 10 kDa antigen gene of Mycobacterium tuberculosis. J. Gen. Microbiol. 135:931-939. 2. Bertram, J., and P. Durre. 1989. Conjugal transfer and expression of streptococcal transposons in Clostridium acetobutylicum. Arch. Microbiol. 151:551-557. 3. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. 4. Chitnis, P. R., and N. Nelson. 1991. Molecular cloning of the genes encoding two chaperone proteins of the cyanobacterium Synechocystis sp. PCC 6803. J. Biol. Chem. 266:58-65. 5. Cowing, D. W., J. C. A. Bardwell, E. A. Craig, C. Woolford, R. W. Hendrix, and C. A. Gross. 1985. Consensus sequence for Escherichia coli heat shock gene promoters. Proc. Natl. Acad. Sci. USA 80:2679-2683. 6. Cummins, C. S., and J. L. Johnson. 1971. Taxonomy of clostridia: wall composition and DNA homologies in Clostridium butyricum and other butyric acid-producing clostridia. J. Gen. Microbiol. 67:33-46. 7. Devereux, J., P. Haeberli, and 0. Smithies. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12:387-395. 8. Ellis, J. 1987. Proteins as molecular chaperones. Nature (London) 328:378-379. 9. Fayet, O., J.-M. Louarn, and C. Georgopoulos. 1986. Suppression of the Escherichia coli dnaA46 mutation by amplification of the groES and groEL genes. Mol. Gen. Genet. 202:435-445. 10. Fayet, O., T. Ziegelhoffer, and C. Georgopoulos. 1989. The groES and groEL heat shock gene products of Escherichia coli are essential for bacterial growth at all temperatures. J. Bacteriol. 171:1379-1385. 11. Freier, S. M., R. Kierzek, J. A. Jaeger, N. Sugimoto, M. H. Caruthers, T. Neilson, and D. H. Turner. 1986. Improved free-energy parameters for predictions of RNA duplex stability. Proc. Natl. Acad. Sci. USA 83:9373-9377. 12. Friedman, D. I., E. R. Olson, K. Tilly, C. Georgopoulos, I. Herskowitz, and F. Banuett. 1984. Interactions of bacteriophage and host macromolecules in the growth of bacteriophage lambda. Microbiol. Rev. 48:299-325. 13. Gerischer, U., and P. Durre. 1991. mRNA analysis of the adc gene region of Clostridium acetobutylicum during the shift to solventogenesis. J. Bacteriol. 174:426-433. 14. Goloubinoff, P., A. A. Gatenby, and G. H. Lorimer. 1988. GroE heat-shock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Escherichia coli. Nature (London) 337:44-47. 15. Graves, M. C., and J. C. Rabinowitz. 1986. In vivo and in vitro transcription of the Clostridium pasteurianum ferredoxin gene. J. Biol. Chem. 261:11409-11415. 16. Grossman, A. D., J. W. Erickson, and C. A. Gross. 1984. The htpR gene product of E. coli is a sigma factor for heat-shock
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