Multicopy plasmid suppression of stationary phase chaperone toxicity in Escherichia coli by phosphogluconate dehydratase and the N-terminus of DnaK

David Rockabrand, Paul H Blum

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Overproduction of DnaK in Escherichia coli results in a bacteriocidal effect. This effect is most acute in stationary phase cells. A selection scheme was developed to isolate multicopy suppressors from an E. coli plasmid expression library, which overcome the stationary phase toxicity of excess DnaK. Two suppressor plasmids were recovered which contained inserts of 1.85 kb and 2.69 kb, respectively. Rearranged and deleted plasmid derivatives were constructed and used to further localize the suppressors. DNA sequence analysis demonstrated that one suppressor encoded phosphogluconate dehydratase (Edd) while the other suppressor encoded the N-terminal 237 amino acids of DnaK itself (DnaK′). Strains bearing the suppressor plasmids constitutively overproduced proteins with apparent masses of 66 kDa (Edd) and 37 kDa (DnaK′) as determined by gel electrophoresis. Western blot analysis using polyclonal antisera specific for either Edd or DnaK confirmed the identity of these overproduced proteins. Suppression of DnaK toxicity was eliminated by the introduction of a + 1 frameshift mutation early in the respective coding regions of either of the two suppressors. These results suggest that suppressor gene translation plays a role in the mechanism of DnaK suppression.

Original languageEnglish (US)
Pages (from-to)498-506
Number of pages9
JournalMGG Molecular & General Genetics
Volume249
Issue number5
DOIs
StatePublished - Sep 1 1995

Fingerprint

phosphogluconate dehydratase
Plasmids
Escherichia coli
Suppressor Genes
Frameshift Mutation
DNA Sequence Analysis
Electrophoresis
Immune Sera
Proteins
Western Blotting
Gels
Amino Acids

Keywords

  • Entner-Doudoroff pathway
  • Escherichia coli
  • HSP70
  • Phosphogluconate dehydratase
  • Stationary phase

ASJC Scopus subject areas

  • Genetics

Cite this

@article{b91b98ec79d24c0b83470215ca02f6e8,
title = "Multicopy plasmid suppression of stationary phase chaperone toxicity in Escherichia coli by phosphogluconate dehydratase and the N-terminus of DnaK",
abstract = "Overproduction of DnaK in Escherichia coli results in a bacteriocidal effect. This effect is most acute in stationary phase cells. A selection scheme was developed to isolate multicopy suppressors from an E. coli plasmid expression library, which overcome the stationary phase toxicity of excess DnaK. Two suppressor plasmids were recovered which contained inserts of 1.85 kb and 2.69 kb, respectively. Rearranged and deleted plasmid derivatives were constructed and used to further localize the suppressors. DNA sequence analysis demonstrated that one suppressor encoded phosphogluconate dehydratase (Edd) while the other suppressor encoded the N-terminal 237 amino acids of DnaK itself (DnaK′). Strains bearing the suppressor plasmids constitutively overproduced proteins with apparent masses of 66 kDa (Edd) and 37 kDa (DnaK′) as determined by gel electrophoresis. Western blot analysis using polyclonal antisera specific for either Edd or DnaK confirmed the identity of these overproduced proteins. Suppression of DnaK toxicity was eliminated by the introduction of a + 1 frameshift mutation early in the respective coding regions of either of the two suppressors. These results suggest that suppressor gene translation plays a role in the mechanism of DnaK suppression.",
keywords = "Entner-Doudoroff pathway, Escherichia coli, HSP70, Phosphogluconate dehydratase, Stationary phase",
author = "David Rockabrand and Blum, {Paul H}",
year = "1995",
month = "9",
day = "1",
doi = "10.1007/BF00290575",
language = "English (US)",
volume = "249",
pages = "498--506",
journal = "Molecular Genetics and Genomics",
issn = "1617-4615",
publisher = "Springer Verlag",
number = "5",

}

TY - JOUR

T1 - Multicopy plasmid suppression of stationary phase chaperone toxicity in Escherichia coli by phosphogluconate dehydratase and the N-terminus of DnaK

AU - Rockabrand, David

AU - Blum, Paul H

PY - 1995/9/1

Y1 - 1995/9/1

N2 - Overproduction of DnaK in Escherichia coli results in a bacteriocidal effect. This effect is most acute in stationary phase cells. A selection scheme was developed to isolate multicopy suppressors from an E. coli plasmid expression library, which overcome the stationary phase toxicity of excess DnaK. Two suppressor plasmids were recovered which contained inserts of 1.85 kb and 2.69 kb, respectively. Rearranged and deleted plasmid derivatives were constructed and used to further localize the suppressors. DNA sequence analysis demonstrated that one suppressor encoded phosphogluconate dehydratase (Edd) while the other suppressor encoded the N-terminal 237 amino acids of DnaK itself (DnaK′). Strains bearing the suppressor plasmids constitutively overproduced proteins with apparent masses of 66 kDa (Edd) and 37 kDa (DnaK′) as determined by gel electrophoresis. Western blot analysis using polyclonal antisera specific for either Edd or DnaK confirmed the identity of these overproduced proteins. Suppression of DnaK toxicity was eliminated by the introduction of a + 1 frameshift mutation early in the respective coding regions of either of the two suppressors. These results suggest that suppressor gene translation plays a role in the mechanism of DnaK suppression.

AB - Overproduction of DnaK in Escherichia coli results in a bacteriocidal effect. This effect is most acute in stationary phase cells. A selection scheme was developed to isolate multicopy suppressors from an E. coli plasmid expression library, which overcome the stationary phase toxicity of excess DnaK. Two suppressor plasmids were recovered which contained inserts of 1.85 kb and 2.69 kb, respectively. Rearranged and deleted plasmid derivatives were constructed and used to further localize the suppressors. DNA sequence analysis demonstrated that one suppressor encoded phosphogluconate dehydratase (Edd) while the other suppressor encoded the N-terminal 237 amino acids of DnaK itself (DnaK′). Strains bearing the suppressor plasmids constitutively overproduced proteins with apparent masses of 66 kDa (Edd) and 37 kDa (DnaK′) as determined by gel electrophoresis. Western blot analysis using polyclonal antisera specific for either Edd or DnaK confirmed the identity of these overproduced proteins. Suppression of DnaK toxicity was eliminated by the introduction of a + 1 frameshift mutation early in the respective coding regions of either of the two suppressors. These results suggest that suppressor gene translation plays a role in the mechanism of DnaK suppression.

KW - Entner-Doudoroff pathway

KW - Escherichia coli

KW - HSP70

KW - Phosphogluconate dehydratase

KW - Stationary phase

UR - http://www.scopus.com/inward/record.url?scp=0029622435&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029622435&partnerID=8YFLogxK

U2 - 10.1007/BF00290575

DO - 10.1007/BF00290575

M3 - Article

VL - 249

SP - 498

EP - 506

JO - Molecular Genetics and Genomics

JF - Molecular Genetics and Genomics

SN - 1617-4615

IS - 5

ER -