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PMID: 8631720 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Plasmid RK2 toxin protein ParE: purification and interaction with the ParD antitoxin protein.

Journal of bacteriology ·Vol. 178 ·No. 5 ·1996-03-00 ·Pages 1420-9

Johnson EP, Strom AR, Helinski DR

Abstract

The parDE operon, located within the 3.2-kb stabilization region of plasmid RK2, encodes antitoxin (ParD) and toxin (ParE) proteins that stabilize the maintenance of this broad-host-range plasmid via a postsegregational killing mechanism. A ParE protein derivative, designated ParE', was purified by construction of a fusion protein, GST-ParE, followed by glutathione-agarose binding and cleavage of the fusion protein. ParE' has three additional amino acids on the N terminus and a methionine residue in place of the native leucine residue. The results of glutathione-agarose affinity binding and glutaraldehyde cross-linking indicate that ParE' exists as a dimer in solution and that it binds to the dimeric form of ParD to form a tetrameric complex. The formation of this complex is presumably responsible for the ability of ParD to neutralize ParE toxin activity. Previous studies demonstrated that the parDE operon is autoregulated as a result of the binding of the ParD protein to the parDE promoter. ParE' also binds to the parDE promoter but only in the presence of the autoregulatory ParD protein. ParE', in the presence or absence of the ParD protein, does not bind to any other part of the 3.2-kb stabilization region. The binding of the ParE' protein to ParD did not alter the DNase I footprint pattern obtained as a result of ParD binding to the parDE promoter. The role of ParE in binding along with ParD to the promoter, if any, remains unclear.

MeSH Terms
Bacterial Proteins/genetics,isolation & purification,metabolism Bacterial Toxins/genetics,isolation & purification,metabolism DNA Topoisomerase IV DNA, Bacterial/metabolism DNA-Binding Proteins/genetics,isolation & purification,metabolism Escherichia coli/genetics,metabolism Escherichia coli Proteins Gene Expression Regulation, Bacterial Glutathione Transferase/biosynthesis,genetics Plasmids/genetics Promoter Regions, Genetic Protein Binding Protein Conformation Recombinant Fusion Proteins/isolation & purification,metabolism Restriction Mapping Transformation, Genetic
Chemicals
Bacterial Proteins Bacterial Toxins DNA, Bacterial DNA-Binding Proteins Escherichia coli Proteins ParD protein, E coli ParD protein, Plasmid RK2 Recombinant Fusion Proteins parD protein, Bacteria Glutathione Transferase DNA Topoisomerase IV
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Johnson E P
Department of Biology, University of California at San Diego, La Jolla 92037-0634, USA.
Strom A R
Helinski D R
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Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/jb.178.5.1420-1429.1996
Published
1996-03-00
Pages
1420-9
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC177817
Subset
IM
Grants
NIGMS NIH HHS · 5T32GM07317 · United States
NIAID NIH HHS · AIO7194 · United States
External Links
PubMed source DOI: 10.1128/jb.178.5.1420-1429.1996 PMC Full Text View journal details
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