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Sequence and molecular characterization of a DNA region encoding a small heat shock protein of Clostridium acetobutylicum.

ABSTRACT: A DNA region of Clostridium acetobutylicum containing a gene (hsp 18) with significant homology to a family of small eukaryotic heat shock proteins was cloned and sequenced. It is the second reported sequence of a low-molecular-weight heat shock protein from gram-positive bacteria and is induced not only by heat shock but also at the onset of solventogenesis, as determined by Northern (RNA) blot analysis, thus confirming the results of an earlier study performed at the protein level (A. Pich, F. Narberhaus, and H. Bahl, Appl. Microbiol. Biotechnol. 33:697-704, 1990). By primer extension analysis, a transcriptional start site was identified 149 bp upstream of hsp18. This site was preceded by a region that exhibits high homology to the consensus promoter sequences of gram-positive bacteria, as well as sigma 70-dependent Escherichia coli. A direct repeat structure was detected in the -35 region. The promoter is located 196 bp from the start of a potential regulatory tRNA(Thr)ACG gene involved in the shift to solventogenesis which is transcribed in the opposite direction. A putative rho-independent transcription termination structure was identified at the 3' end of hsp18.

SUBMITTER: Sauer U

PROVIDER: S-EPMC204737 | biostudies-other | 1993 Jun

REPOSITORIES: biostudies-other

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Sequence and molecular characterization of a DNA region encoding a small heat shock protein of Clostridium acetobutylicum.

Sauer U U Dürre P P

Journal of bacteriology 19930601 11

A DNA region of Clostridium acetobutylicum containing a gene (hsp 18) with significant homology to a family of small eukaryotic heat shock proteins was cloned and sequenced. It is the second reported sequence of a low-molecular-weight heat shock protein from gram-positive bacteria and is induced not only by heat shock but also at the onset of solventogenesis, as determined by Northern (RNA) blot analysis, thus confirming the results of an earlier study performed at the protein level (A. Pich, F. ...[more]

PMID: 8501044

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Project description: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.

Project description:Acetoacetate decarboxylase (ADC) (EC4.1.1.4) of Clostridium acetobutylicum DSM 792 was purified to homogeneity, and its first 25 N-terminal amino acids were determined. Oligonucleotide probes deduced from this sequence were used to detect positive clones in partial gene banks derived from Sau3A and HaeIII digests with following ligation into the vector pUC9. In Escherichia coli, the 2.1-kbp HaeIII clones expressed high levels of ADC activity. The expression was independent of the orientation of the insert with respect to the lac promoter of the vector and also of the addition of isopropyl-beta-D-thiogalactopyranoside, thus indicating that sequences located on the clostridial DNA controlled transcription and translation. From the E. coli clone with the recombinant plasmid pUG93 containing the 2.1-kbp HaeIII fragment, the ADC protein was purified and compared with the native enzyme. Both were indistinguishable with respect to the molecular mass of subunits and native protein as well as to activity stain. The 2.9-kbp Sau3A fragment could be shown to contain the amino terminus of the acetoacetate decarboxylase (adc) gene but did not express enzyme activity. It partially overlapped with the HaeIII fragment, spanning together 4,053 bp of the clostridial genome that were completely sequenced. Four open reading frames (ORFs) could be detected, one of which was unambiguously assigned to the acetoacetate decarboxylase (adc) gene. Amino acid sequences of the N terminus and the catalytic center as deduced from the nucleotide sequence were identical to sequences obtained from direct analysis of the protein. Typical procaryotic transcriptional and translational start and stop signals could be found in the DNA sequence. Together with these regulatory sequences, the adc gene formed a single operon. The carboxyl terminus of the enzyme proved to be rather hydrophobic. In vitro transcription-translation assays resulted in formation of ADC and ORF3 gene product; the other two ORFs were not expressed. Whereas no homology of the adc gene and ORF2 could be detected with sequences available in the EMBL or GenBank data bases, the obviously truncated ORF1 showed significant similarity to alpha-amylase of Bacillus subtilis. The restriction pattern and N-terminal amino acid sequence (as deduced from the nucleotide sequence) of ORF3 proved to be identical to those of the large subunit of acetoacetyl coenzyme A:acetate/butyrate:coenzyme A transferase.

Dataset Information

Sequence and molecular characterization of a DNA region encoding a small heat shock protein of Clostridium acetobutylicum.

Publications

Sequence and molecular characterization of a DNA region encoding a small heat shock protein of Clostridium acetobutylicum.

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