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PMID: 1577695 Published · ppublish English Comparative Study Journal Article Research Support, Non-U.S. Gov't

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

Journal of bacteriology ·Vol. 174 ·No. 10 ·1992-05-00 ·Pages 3290-9

Narberhaus F, Giebeler K, Bahl H

Abstract

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.

MeSH Terms
Amino Acid Sequence Bacterial Proteins/genetics Base Sequence Chromosome Walking Cloning, Molecular Clostridium/genetics Escherichia coli Proteins HSP40 Heat-Shock Proteins HSP70 Heat-Shock Proteins Heat-Shock Proteins/genetics Molecular Sequence Data Promoter Regions, Genetic/genetics RNA, Messenger/analysis Reading Frames Sequence Homology, Nucleic Acid Transcription, Genetic
Chemicals
Bacterial Proteins DnaJ protein, E coli Escherichia coli Proteins GrpE protein, Bacteria GrpE protein, E coli HSP40 Heat-Shock Proteins HSP70 Heat-Shock Proteins Heat-Shock Proteins RNA, Messenger dnaK protein, E coli
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Narberhaus F
Institut für Mikrobiologie, Georg-August-Universität Göttingen, Germany.
Giebeler K
Bahl H
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Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/jb.174.10.3290-3299.1992
Published
1992-05-00
Pages
3290-9
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC205998
Subset
IM
Databases
GENBANK
M74569, M79367, M79368, M79369, M79370, M79371, M79372, M87491, M87492, M87836
External Links
PubMed source DOI: 10.1128/jb.174.10.3290-3299.1992 PMC Full Text View journal details
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