Purification and properties of two chitinolytic enzymes of Serratia plymuthica HRO-C48.

The chitinolytic rhizobacterium Serratia plymuthica HRO-C48 was previously selected as a biocontrol agent of phytopathogenic fungi. One endochitinase (E.C. 3.2.1.14), CHIT60, and one N-acetyl-beta-1,4- D-hexosaminidase (E.C. 3.2.1.52), CHIT100, were purified and characterized. The endochitinase CHIT60, with an apparent molecular mass of 60.5 kDa, had a N-terminal amino acid sequence highly similar to that of chitinases A from Serratia liquefaciens and Serratia marcescens. The enzyme activity had its peak at 55 degrees C and pH 5.4, and increased by more than 20% in the presence of 10 mM Ca(2+), Co(2+) or Mn(2+). Activity was inhibited by 80% in the presence of 10 mM Cu(2+). CHIT100 appeared to be a monomeric enzyme with a molecular mass of 95.6 kDa and a pI of 6.8. Optimal activity was obtained at 43 degrees C and pH 6.6, and decreased by more than 90 % in the presence of 10 mM Co(2+) or Cu(2+). CHIT100 (100 microg ml(-1)) inhibited spore germination and germ tube elongation of the phytopathogenic fungus Botrytis cinerea by 28 % and 31.6 %, respectively. With CHIT60 (100 microg ml(-1)), the effect was more pronounced: 78 % inhibition of of germination and 63.9 % inhibition of germ tube elongation.

Molecular analysis of the mannitol operon of Clostridium acetobutylicum encoding a phosphotransferase system and a putative PTS-modulated regulator.

Clostridium acetobutylicum DSM 792 accumulates and phosphorylates mannitol via a phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS). PEP-dependent mannitol phosphorylation by extracts of cells grown on mannitol required both soluble and membrane fractions. Neither the soluble nor the membrane fraction could be complemented by the opposite fraction prepared from glucose-grown cells, indicating that the mannitol-specific PTS consists of both a soluble (IIA) and a membrane-bound (IICB) component. The mannitol (mtl) operon of C. acetobutylicum DSM 792 comprises four genes in the order mtlARFD. Sequence analysis of deduced protein products indicated that the mtlA and mtlF genes respectively encode the IICB and IIA components of the mannitol PTS, which is a member of the fructose-mannitol (Fru) family. The mtlD gene product is a mannitol-1-phosphate dehydrogenase, while mtlR encodes a putative transcriptional regulator. MtlR contains two PTS regulatory domains (PRDs), which have been found in a number of DNA-binding transcriptional regulators and in transcriptional antiterminators of the Escherichia coli BglG family. Also, near the C-terminus is a well-conserved signature motif characteristic of members of the IIA(Fru)/IIA(Mtl)/IIA(Ntr) PTS protein family. These regions are probably the sites of PTS-dependent phosphorylation to regulate the activity of the protein. A helix-turn-helix DNA-binding motif was not found in MtlR. Transcriptional analysis of the mtl genes by Northern blotting indicated that the genes were transcribed as a polycistronic operon, expression of which was induced by mannitol and repressed by glucose. Primer extension experiments identified a transcriptional start point 42 bp upstream of the mtlA start codon. Two catabolite-responsive elements (CREs), one of which overlapped the putative -35 region of the promoter, were located within the 100 bp upstream of the start codon. These sequences may be involved in regulation of expression of the operon.

Identification of two genes encoding putative new members of the ECF subfamily of eubacterial RNA polymerase sigma factors in Clostridium acetobutylicum.

Two genes from Clostridium acetobutylicum DSM 792 were identified which are predicted to encode new members of the ECF subfamily of eubacterial RNA polymerase sigma factors. The sigX gene has the potential to encode a 184-amino acid protein with a molecular mass of 21,870 Da and with the highest overall similarity to Fecl of Escherichia coli (27 % identical residues). The second gene, which is predicted to encode an alternative sigma factor of the ECF subfamily, is the previously described orf2 gene (Gerischer and Dürre, 1990) located in the adc gene region of C. acetobutylicum. The deduced protein of orf2 has significant similarity to SigX of C. acetobutylicum (22 % identical residues) and shares structural features with other alternative sigma factors. Therefore, it is proposed to rename orf2 as sigY. Analysis of the phylogenetic relationship revealed that SigX from C. acetobutylicum, together with sigmaE from Streptomyces coelicolor and SigX from Bacillus subtilis, form a gram-positive cluster within the ECF subfamily and that SigY from C. acetobutylicum together with UviA from Clostridium perfringens, form a separate cluster located between the gram-positive cluster and the sporulation sigma factor sigmaH from B. subtilis.

In Thermoanaerobacterium thermosulfurigenes EM1 S-layer homology domains do not attach to peptidoglycan.

Three exocellular enzymes of Thermoanaerobacterium thermosulfurigenes EM1 possess a C-terminal triplicated sequence related to a domain of bacterial cell surface proteins (S-layer proteins). At least one copy of this sequence, named the SLH (for S-layer homology) domain, is also present at the N terminus of the S-layer protein of this bacterium. The hypothesis that SLH domains serve to anchor proteins to the cell surface was investigated by using the SLH domain-containing xylanase. This enzyme was isolated from T. thermosulfurigenes EM1, and different forms with and without SLH domains were synthesized in Escherichia coli. The interaction of these proteins with isolated components of the cell envelope was determined to identify the attachment site in the cell wall. In addition, a polypeptide consisting of three SLH domains and the N terminus of the S-layer protein of T. thermosulfurigenes EM1 were included in these studies. The results indicate that SLH domains are necessary for the attachment of these proteins to peptidoglycan-containing sacculi. Extraction of the native sacculi with hydrofluoric acid led to the conclusion that not peptidoglycan but accessory cell wall polymers function as the adhesion component in the cell wall. Our results provide further evidence that attachment of proteins via their SLH domains represents an additional mode to display polypeptides on the cell surfaces of bacteria.

Cell wall of Thermoanaerobacterium thermosulfurigenes EM1: isolation of its components and attachment of the xylanase XynA.

Thermoanaerobacterium thermosulfurigenes EM1 has a gram-positive type cell wall completely covered by a surface layer (S-layer) with hexagonal lattice symmetry. The components of the cell envelope were isolated, and the S-layer protein was purified and characterized. S-layer monomers assembled in vitro into sheets with the same hexagonal symmetry as in vivo. Monosaccharide analysis revealed that the S-layer is associated with fucose, rhamnose, mannosamine, glucosamine, galactose, and glucose. The N-terminal 31 amino acid residues of the S-layer protein showed significant similarity to SLH (S-layer homology) domains found in S-layer proteins of different bacteria and in the exocellular enzymes pullulanase, polygalacturonate hydrolase, and xylanase of T. thermosulfurigenes EM1. The xylanase from T. thermosulfurigenes EM1 was copurified with the S-layer protein during isolation of cell wall components. Since SLH domains of some structural proteins have been shown to anchor these proteins noncovalently to the cell envelope, we propose a common anchoring mechanism for the S-layer protein and exocellular enzymes via their SLH domains in the peptidoglycan-containing layer of T. thermosulfurigenes EM1.

Functional characterisation of the chaperones DnaK, DnaJ, and GrpE from Clostridium acetobutylicum.

The DnaK chaperone system is involved in various cellular processes such as the control of the folded and oligomeric state of proteins under stress and non-stress conditions. In this study we functionally characterised the homologues of the DnaK system from Clostridium acetobutylicum DnaK, DnaJ, GrpE and OrfA were heterologously synthesised in Escherichia coli and affinity purified via a His-tag. By optimising the stoichiometry, we were able to refold guanidinium hydrochloride-denatured firefly luciferase in vitro with 22% of the yield obtained with the E. coli DnaK system. In addition, C. acetobutylicum DnaJ could stimulate the E. coli DnaK ATPase by a factor of 55. Furthermore, the DnaK system from C. acetobutylicum was able to prevent the aggregation of OrfA from C. acetobutylicum, which is similar to the repressor HrcA of CIRCE-regulated heat shock genes in Bacillus subtilis.

Molecular characterization of genes encoding a novel ABC transporter in Thermoanaerobacterium thermosulfurigenes EM1.

The nucleotide sequence of two open reading frames (ORFs) from Thermoanaerobacterium thermosulfurigenes EM1 was determined that encode proteins with similarity to components of ATP-binding cassette (ABC) transport systems. Sequence analysis suggests that the deduced proteins AbcA and AbcB consist of an NH2-terminal membrane-spanning domain and a COOH-terminal ATP-binding domain. The deduced proteins AbcA and AbcB showed highest similarity to proteins of the MsbA subfamily of ABC transporters. AbcA and AbcB probably function as a heterodimer. An ORF predicted to encode the primary sigma factor SigA was identified downstream of abcB.

Molecular biology of S-layers.

In this chapter we report on the molecular biology of crystalline surface layers of different bacterial groups. The limited information indicates that there are many variations on a common theme. Sequence variety, antigenic diversity, gene expression, rearrangements, influence of environmental factors and applied aspects are addressed. There is considerable variety in the S-layer composition, which was elucidated by sequence analysis of the corresponding genes. In Corynebacterium glutamicum one major cell wall protein is responsible for the formation of a highly ordered, hexagonal array. In contrast, two abundant surface proteins from the S-layer of Bacillus anthracis. Each protein possesses three S-layer homology motifs and one protein could be a virulence factor. The antigenic diversity and ABC transporters are important features, which have been studied in methanogenic archaea. The expression of the S-layer components is controlled by three genes in the case of Thermus thermophilus. One has repressor activity on the S-layer gene promoter, the second codes for the S-layer protein. The rearrangement by reciprocal recombination was investigated in Campylobacter fetus. 7-8 S-layer proteins with a high degree of homology at the 5' and 3' ends were found. Environmental changes influence the surface properties of Bacillus stearothermophilus. Depending on oxygen supply, this species produces different S-layer proteins. Finally, the molecular bases for some applications are discussed. Recombinant S-layer fusion proteins have been designed for biotechnology.

Copurification of ribosomal protein S2 and DNA-dependent RNA polymerase from heat-shocked cells of Bacillus subtilis.

The DNA-dependent RNA polymerases from heat-shocked and vegetatively grown cells of Bacillus subtilis were isolated and compared. The RNA polymerase from non-stressed cells had the well known alpha, beta, beta' and sigma composition of eubacterial RNA polymerases. The RNA polymerase from heat-shocked cells exhibited one additional band shown by SDS-PAGE. N-terminal sequencing of the first 16 amino acids of the associated protein demonstrated its identity with the ribosomal protein S2.

Maltophilin: a new antifungal compound produced by Stenotrophomonas maltophilia R3089.

Strains of Stenotrophomonas maltophilia R3089, isolated from the rhizosphere of rape plants (Brassica napus L.), produced a novel antifungal compound, named maltophilin, which inhibited the growth of various saprophytic, human-pathogenic and phytopathogenic fungi but was inactive against Gram-positive and Gram-negative bacteria. Maltophilin is a novel macrocyclic lactam antibiotic with a molecular mass of 510 mu. The compound was isolated from the culture filtrate by ethyl acetate extraction and gel filtration on Sephadex LH20 and purified by preparative HPLC on reversed phase. The structure of maltophilin was elucidated by electrospray mass spectrometry and 1H and 13C NMR spectroscopy.

Quaternary structure, Mg2+ interactions, and some kinetic properties of the beta-galactosidase from Thermoanaerobacterium thermosulfurigenes EM1.

The beta-galactosidase from Thermoanaerobacterium thermosulfurigenes EM1 was found to be a dimer with a monomer molecular weight of about 85,000. It lacks the alpha-peptide and an important alpha-helix that are both needed for dimer-dimer interaction and there is no homology in other important dimer-dimer interaction areas. These differences in structure probably account for the dimeric (rather than tetrameric) structure. Only 0.19 Mg2+ bound per monomer and Mg2+ had only small effects on the activity and heat stability. The absence of residues equivalent to Glu-416 and His-418 (two of the three ligands to Mg2+ in the beta-galactosidase from Escherichia coli) probably accounts for the low level of Mg2+ binding and the consequent lack of response to Mg2+. Both Na+ and K+ also had no effect on the activity. The enzyme activity with o-nitrophenyl-beta-D-galactopyanoside (ONPG) was very similar to that with p-nitrophenyl-beta-D-beta-D-galactopyranoside (PNPG) and the ONPG pH profile was very similar to the PNPG pH profile. These differences are in contrast to the E.coli beta-galactosidase, which dramatically discriminates between these two substrates. The lack of discrimination by the T. thermosulfurigenes beta-galactosidase could be due to the absence of the sequence equivalent to residues 910-1023 of the E. coli beta-galactosidase. Trp-999 is probably of the most importance. Trp-999 of the E. coli beta-galactosidase is important for aglycone binding and ONPG and PNPG differ only in their aglycones. The suggestion that the aglycone site of the T. thermosulfurigenes beta-galactosidase is different was strengthened by competitive inhibition studies. Compared to E. coli beta-galactosidase, D-galactonolactone was a very good inhibitor of the T. thermosulfurigenes enzyme, while L-ribose inhibited poorly. These are transition-state analogs and the results indicate that T. thermosulfurigenes beta-galactosidase binds the transition state differently than does E. coli beta-galactosidase. Methanol and glucose were good acceptors of galactose, and allolactose was formed when glucose was the acceptor. Allolactose could not, however, be detected by TLC when lactose was the substrate. The differences noted may be due to the thermophilic nature of T. thermosulfurigenes.

Characterization of genes from Thermoanaerobacterium thermosulfurigenes EM1 that encode two glycosyl hydrolases with conserved S-layer-like domains.

Two genes from Thermoanaerobacterium thermosulfurigenes EM1 were identified which are predicted to encode a xylanase (XynA) and a polygalacturonate hydrolase (Pg1A). The xynA gene has the potential to encode a 1234-amino acid product consisting of a signal peptide followed by a repeated domain, a xylanase family F domain, two cellulose-binding domains and a triplicated sequence at its C-terminus. The gene pglA is predicted to encode a product of 1148 amino acids consisting of a signal sequence followed by a fibronectin type III-like domain (Fn3 domain), the catalytic domain, a Gly/Thr/Ser/Asn-rich segment and a triplicated domain. The triplicated segments at the C-termini of deduced XynA and Pg1A are about 95% identical to each other and to the S-layer-like domains of the previously characterized pullulanase (AmyB) from the same organism. In contrast, sequence comparisons revealed only distant amino acid sequence similarities between the fibronectin type III-like domains of Pg1A and AmyB from T. thermosulfurigenes EM1.

Cloning, sequencing, and expression of the gene encoding a large S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 in Escherichia coli.

The gene (xynA) encoding a surface-exposed, S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 was cloned and expressed in Escherichia coli. A 3.8-kb fragment was amplified from chromosomal DNA by using primers directed against conserved sequences of endoxylanases isolated from other thermophilic bacteria. This PCR product was used as a probe in Southern hybridizations to identify a 4.6-kb EcoRI fragment containing the complete xynA gene. This fragment was cloned into E. coli, and recombinant clones expressed significant levels of xylanase activity. The purified recombinant protein had an estimated molecular mass (150 kDa), temperature maximum (80 degrees C), pH optimum (pH 6.3), and isoelectric point (pH 4.5) that were similar to those of the endoxylanase isolated from strain JW/SL-YS 485. The entire insert was sequenced and analysis revealed a 4,044-bp open reading frame encoding a protein containing 1,348 amino acid residues (estimated molecular mass of 148 kDa).xynA was preceded by a putative promoter at -35 (TTAAT) and -10 (TATATT) and a potential ribosome binding site (AGGGAG) and was expressed constitutively in E. coli. The deduced amino acid sequence showed 30 to 96% similarity to sequences of family F beta-glycanases. A putative 32-amino-acid signal peptide was identified, and the C-terminal end of the protein contained three repeating sequences 59, 64, and 57 amino acids) that showed 46 to 68% similarity to repeating sequences at the N-terminal end of S-layer and S-layer-associated proteins from other gram-positive bacteria. These repeats could permit an interaction of the enzyme with the S-layer and tether it to the cell surface.

Molecular analysis of the amy gene locus of Thermoanaerobacterium thermosulfurigenes EM1 encoding starch-degrading enzymes and a binding protein-dependent maltose transport system.

A gene of Thermoanaerobacterium thermosulfurigenes EM1 encoding a protein with similarity to the maltose-binding protein of Escherichia coli was cloned and sequenced. It was located in the amy gene region of the chromosome downstream of the pullulanase-encoding amyB gene and upstream of amyDC, encoding membrane components of an ABC transport system, and the alpha-amylase gene amyA. The gene was designated amyE. Analysis of mRNA by Northern (RNA) blotting revealed that expression of the amy gene region is repressed during growth on glucose. Maximum levels of mRNA were detected with maltose as a substrate. An operon which was transcribed in the order amyBEDC was identified. However, an additional transcription start point was found in front of amyE. The amyA gene represented a monocistronic operon. Putative -35 and -10 promoter sites were deduced from the three transcription start sites of the amy gene region, and possible regulatory regions mediating induction by maltose and catabolite repression by glucose were identified by sequence analysis and comparison. The biochemical characterization of maltose uptake in T. thermosulfurigenes EM1 revealed two transport systems with Km values of 7 microM (high affinity) and 400 microM (low affinity). We conclude that the high-affinity system, which is specific for maltose and maltotriose, is a binding-protein-dependent transporter encoded by amyEDC. The gene for the putative ATP-binding protein has not yet been identified, and in contrast to similar systems in other bacteria, it is not located in the immediate vicinity of the chromosome.

Expression of heat shock genes in Clostridium acetobutylicum.

Characterization of the heat shock response in Clostridium acetobutylicum has indicated that at least 15 proteins are induced by a temperature upshift from 30 to 42 degrees C. These so-called heat shock proteins include DnaK and GroEL, two highly conserved molecular chaperones. Several genes encoding heat shock proteins of C. acetobutylicum have been cloned and analysed. The dnaK operon includes the genes orfA (a heat shock gene with an unknown function), grpE, dnaK, and dnaJ; and the groE operon the genes groES and groEL. The hsp18 gene coding for a member of the small heat shock protein family constitutes a monocistronic operon. Interestingly, the heat shock response in this bacterium is regulated by a mechanism, which is obviously different from that found in Escherichia coli. So far, no evidence for a heat shock-specific sigma factor of the RNA polymerase in C. acetobutylicum has been found. In this bacterium, like in many Gram-positive and several Gram-negative bacteria, a conserved inverted repeat is located upstream of chaperone/chaperonin-encoding stress genes such as dnaK and groEL and may be implicated as a cis-acting regulatory site. The inverted repeat is not present in the promoter region of hsp18. Therefore, in C. acetobutylicum there are at least two classes of heat shock genes with respect to the type of regulation. Evidence has been found that a repressor is involved in the regulation of the heat shock response in C. acetobutylicum. However, this regulation seems to be independent of the inverted repeat motif, and the mechanism by which the inverted repeat motif mediates regulation remains to be elucidated.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclodextrin formation by the thermostable alpha-amylase of Thermoanaerobacterium thermosulfurigenes EM1 and reclassification of the enzyme as a cyclodextrin glycosyltransferase.

Extensive characterization of the thermostable alpha-amylase of Clostridium thermosulfurogenes EM1, recently reclassified as Thermoanaerobacterium thermosulfurigenes, clearly demonstrated that the enzyme is a cyclodextrin glycosyltransferase (CGTase). Product analysis after incubation of the enzyme with starch revealed formation of alpha-, beta-, and gamma-cyclodextrins, as well as linear sugars. The specific activity for cyclization of this CGTase was similar to those of other CGTases, whereas the specific activity for hydrolysis was relatively high in comparison with other CGTases. Alignment of the amino acid sequence of the T. thermosulfurigenes enzyme with sequences from known bacterial CGTases showed high homology. The four consensus regions of carbohydrate-converting enzymes, as well as a C-terminal raw-starch binding motif, could be identified in the sequence.

Synthesis of heat shock proteins in Thermoanaerobacterium thermosulfurigenes EM1 (Clostridium thermosulfurogenes EM1).

The response to heat stress was examined in Thermoanaerobacterium thermosulfurigenes EM1. Upon a temperature shift-up from 50 degrees to 62 degrees C, four heat shock proteins (hsps) were synthesized at an elevated level. Two proteins were found to be immunologically related to the Escherichia coli GroEL protein and the Mycobacterium tuberculosis hsp71 (DnaK similar protein), and the corresponding groE and dnaK homologous sequences were detected in the chromosome of T. thermosulfurigenes EM1. The heat shock response in this thermophile was transient, with a maximum synthesis of hsps between 10 and 15 min after the shock. The enhanced synthesis of DnaK and GroEL was consistent with increased mRNA levels of the genes, which reached a maximum 15 min after heat treatment.

Pullulanase of Thermoanaerobacterium thermosulfurigenes EM1 (Clostridium thermosulfurogenes): molecular analysis of the gene, composite structure of the enzyme, and a common model for its attachment to the cell surface.

The complete pullulanase gene (amyB) from Thermoanaerobacterium thermosulfurigenes EM1 was cloned in Escherichia coli, and the nucleotide sequence was determined. The reading frame of amyB consisted of 5,586 bp encoding an exceptionally large enzyme of 205,991 Da. Sequence analysis revealed a composite structure of the pullulanase consisting of catalytic and noncatalytic domains. The N-terminal half of the protein contained a leader peptide of 35 amino acid residues and the catalytic domain, which included the four consensus regions of amylases. Comparison of the consensus regions of several pullulanases suggested that enzymes like pullulanase type II from T. thermosulfurigenes EM1 which hydrolyze alpha-1,4- and alpha-1,6-glycosidic linkages have specific amino acid sequences in the consensus regions. These are different from those of pullulanases type I which only cleave alpha-1,6 linkages. The C-terminal half, which is not necessary for enzymatic function, consisted of at least two different segments. One segment of about 70 kDa contained two copies of a fibronectin type III-like domain and was followed by a linker region rich in glycine, serine, and threonine residues. At the C terminus, we found three repeats of about 50 amino acids which are also present at the N-termini of surface layer (S-layer) proteins of, e.g., Thermus thermophilus and Acetogenium kivui. Since the pullulanase of T. thermosulfurigenes EM1 is known to be cell bound, our results suggest that this segment serves as an S-layer anchor to keep the pullulanase attached to the cell surface. Thus, a general model for the attachment of extracellular enzymes to the cell surface is proposed which assigns the S-layer a new function and might be widespread among bacteria with S-layers. The triplicated S-layer-like segment is present in several enzymes of different bacteria. Upstream of amyB, another open reading frame, coding for a hypothetical protein of 35.6 kDa, was identified. No significant similarity to other sequences available in DNA and protein data bases was found.

Cloning, nucleotide sequence and structural analysis of the Clostridium acetobutylicum dnaJ gene.

The complete dnaJ gene of Clostridium acetobutylicum was isolated by chromosome walking using the previously cloned 5' end of the gene as a probe. Nucleotide sequencing of a positively reacting 2.2-kb HincII fragment, contained in the recombinant plasmid pKG4, revealed that the reading frame of the dnaJ gene of C. acetobutylicum consists of 1125 bp, encoding a protein of 374 amino acids with a calculated M(r) of 40376 and an isoelectric point of 9.54. The deduced amino acid sequence showed high similarity to the DnaJ proteins of other bacteria (e.g. Escherichia coli, Bacillus subtilis) as well as of an archaeon (Methanosarcina mazei) and to the corresponding proteins of eukaryotes (Saccharomyces cerevisiae, Homo sapiens). The areas of similarity included a conserved N-terminal domain of about 70 amino acids, a glycine-rich region of about 30 residues, and a central domain containing four repeats of a CXXCXGXG motif, whereas the C-terminal domain was less conserved. Northern (RNA) blot analysis indicated that dnaJ is induced by heat shock and that it is part of the dnaK operon of C. acetobutylicum. The 5' end (901 bp) of another gene (orfB), downstream of dnaJ and not heat-inducible, showed no significant similarity to other sequences available in EMBL and GenBank databases.

Molecular characterization of the dnaK gene region of Clostridium acetobutylicum, including grpE, dnaJ, and a new heat shock gene.

The dnaK gene region of Clostridium acetobutylicum was cloned in Escherichia coli by using the pBluescript SK+ and pUC18 vectors. By using the E. coli dnaK gene as a probe and by in vivo chromosome walking, three positive clones harboring the recombinant plasmids pKG1, pKG2, and pKG3 containing 1.2-kbp HindIII, 3.55-kbp EcoRV, and 1.2-kbp PstI fragments of the chromosome of C. acetobutylicum, respectively, were isolated. The cloned fragments partially overlapped, and together they spanned 4,083 bp of the clostridial genome that were completely sequenced. On one strand, four open reading frames of which the last was obviously truncated were identified. The last three genes showed high homology to the grpE, dnaK, and dnaJ heat shock genes of E. coli, respectively. They were preceded by an open reading frame (orfA) without any homology to sequences available in the EMBL or GenBank data bases. Typical translational start sites could be found in front of all four genes. Northern (RNA) blot analysis revealed transcripts of this region with a maximum length of 5.0 kb. Thus, these genes are probably organized in an operon. A transcription terminator could be found between the dnaK and dnaJ genes. By primer extension analysis, a major heat-inducible transcription start site was identified 49 bases upstream of orfA. This site was preceded by a region (5'-TTGACA[17 bp]TATTTT) that exhibited high homology to the consensus promoter sequences of gram-positive bacteria as well as sigma 70-dependent E. coli. Between this promoter and the initiation codon of orfA, a hairpin-loop structure with a possible regulatory role in the expression of these genes was found. Additional heat-inducible transcription start sites were located 69 bases upstream of orfA and 87 bases upstream of grpE; the corresponding promoter regions showed less similarity to other known promoter sequences. Maximum mRNA levels of this heat shock operon were found about 15 min after a heat shock from 30 to 42 degrees C. Our results indicate that orfA codes for an unknown heat shock protein.