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PMID: 1350776 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Cloning and characterization of the groESL operon from Bacillus subtilis.

Journal of bacteriology ·Vol. 174 ·No. 12 ·1992-06-00 ·Pages 3981-92

Li M, Wong SL

Abstract

The sequence of the 10 N-terminal amino acids of a Bacillus subtilis protein that cross-reacts with antibody to Escherichia coli GroEL was used to design a set of degenerate oligonucleotide probes. These probes identified a clone which carries almost the entire groESL operon from a B. subtilis subgenomic library. By chromosomal walking, an additional fragment carrying the 3' end of groESL and its flanking sequence was isolated. Sequence analysis revealed two open reading frames (ORFs) in the cloned DNA. The upstream ORF encodes a 10-kDa protein which has 47% amino acid identity with E. coli GroES. The downstream ORF encodes a 58-kDa protein which is 62% identical to E. coli GroEL. A 2.1-kb groESL mRNA from B. subtilis was detected independently by Northern (RNA) blot analyses with a groES- and a groEL-specific probe. This demonstrated that groES and groEL are in an operon. The groESL promoter was located by using a promoter-probing plasmid, and the apparent transcription start site was mapped by primer extension analysis. The same promoter is utilized under normal and heat shock conditions. This promoter has the same features as a typical sigma A promoter. A strain in which the groESL operon was under the control of the sucrose-inducible sacB promoter was created. With this strain, it was possible to show that both groES and groEL are essential genes under both normal and heat shock conditions.

Related Genes
groEL groES
MeSH Terms
Amino Acid Sequence Bacillus subtilis/genetics Bacterial Proteins/chemistry,genetics Base Sequence Blotting, Northern Cell Division/genetics Chaperonin 10 Chaperonin 60 Cloning, Molecular Heat-Shock Proteins/chemistry,genetics Molecular Sequence Data Oligodeoxyribonucleotides/genetics Operon/genetics Promoter Regions, Genetic/genetics Recombinant Fusion Proteins/genetics Temperature
Chemicals
Bacterial Proteins Chaperonin 10 Chaperonin 60 Heat-Shock Proteins Oligodeoxyribonucleotides Recombinant Fusion Proteins
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Li M
Department of Biological Sciences, University of Calgary, Alberta, Canada.
Wong S L
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47 references, click to expand
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Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/jb.174.12.3981-3992.1992
Published
1992-06-00
Pages
3981-92
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC206107
Subset
IM
Databases
GENBANK
M79367, M79368, M79369, M79370, M79371, M79372, M81132, M84973, M84974, M84980
External Links
PubMed source DOI: 10.1128/jb.174.12.3981-3992.1992 PMC Full Text View journal details
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