Abstract
The P1 plasmid addiction operon increases the apparent stability of a plasmid that carries it by killing plasmid-free (cured) segregants. The operon consists of a gene encoding an endotoxin responsible for death on curing (doc), preceded by a gene encoding a relatively unstable antidote that can prevent host death (phd). When the copy number of the operon was increased, expression of a lacZ reporter fused to the promoter of the operon decreased, indicating that expression of the operon was stabilized by an autoregulatory circuit. Transcription of the lacZ reporter was repressed about 10-fold when phd, without doc, was expressed from an exogenous promoter. DNase I footprinting showed that Phd binds a perfect 10-base pair palindromic DNA sequence and, at higher concentrations, an adjacent, imperfect palindrome. The palindromic sites are located between the -10 region of the putative promoter and the start codon of phd. Electrophoretic mobility of DNA containing the promoter region was retarded in the presence of Phd and further retarded in the presence of Phd and Doc. When doc was co-expressed with phd, repression of the lacZ fusion was enhanced more than 100-fold. Thus, both products of the addiction operon participate in its autoregulation.
Highlights
We examine the role of Phd and Doc in the autoregulation of the P1 addiction operon and discuss the possible similarity of antidote proteins to each other and to well-studied DNA-binding proteins
Transcriptional Regulation of the P1 Addiction Operon—The promoter of the P1 addiction operon was fused to lacZYA, and a single copy of the fusion was integrated into the chromosome
A chromosomally integrated, transcriptional lacZ fusion to the putative promoter of the P1 addiction operon was repressed about 400-fold when the addiction operon was furnished in trans on a moderate copy plasmid (Table II, pG3), indicating that transcription of the operon is negatively regulated by one or more products of the operon
Summary
Vol 271, No 31, Issue of August 2, pp. 18705–18710, 1996 Printed in U.S.A. Roy Magnuson‡§, Hansjorg Lehnherr§¶, Gauranga Mukhopadhyayʈ, and Michael B. When doc was co-expressed with phd, repression of the lacZ fusion was enhanced more than 100-fold Both products of the addiction operon participate in its autoregulation. ** To whom correspondence should be addressed: Laboratory of Biochemistry, NCI, National Institutes of Health, Bldg. Plasmid addiction elements functionally analogous to Phd/ Doc include CcdA/CcdB of F, the PemI/PemK of R100 (identical to Kis/Kid of R1), and ParD/ParE of RK2 (and RP4). Their toxin targets may differ and homology among the analogous proteins is weak, the structure of the operons and the details of autoregulation are strikingly similar [7]. We examine the role of Phd and Doc in the autoregulation of the P1 addiction operon and discuss the possible similarity of antidote proteins to each other and to well-studied DNA-binding proteins
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