Escherichia coli plasmid ColE1 lacks active partitioning, and copies are distributed randomly to daughter cells at division. The plasmid is maintained stably in the bacterial population as long as its copy number remains high. The accumulation of plasmid dimers and higher multimers depresses copy number, and is an important cause of multicopy plasmid instability. ColE1 dimers are restored to the monomeric state by site-specific recombination, which requires the host-encoded proteins XerCD, ArgR and PepA acting at the plasmid cer site. In addition, a 70 nt RNA expressed from the cer site of plasmid dimers delays the division of dimer-containing cells. Here, we report that the global regulator FIS binds to cer in a sequence-specific manner, close to the Rcd promoter (Pcer). FIS is not required for plasmid dimer resolution, but is essential for repression of Pcer in plasmid monomers. Repression also requires the XerCD recombinase, but not ArgR or PepA. We propose a model for monomer–dimer control of Pcer in which the promoter is repressed in plasmid monomers by the concerted action of FIS and XerCD. Rcd transcription is triggered in plasmid dimers by the lifting of XerCD-mediated repression in the synaptic complex.
BaldingC.,
BlabyI.,
SummersD.2006; A mutational analysis of the ColE1-encoded cell cycle regulator Rcd confirms its role in plasmid stability. Plasmid 56:68–73
BlakelyG.,
MayG.,
McCullochR.,
ArciszewskaL. K.,
BurkeM.,
LovettS. T.,
SherrattD. J.1993; 2 related recombinases are required for site-specific recombination at dif and cer in Escherichia coli K12. Cell 75:351–361
CollomsS. D.,
SykoraP.,
SzatmariG.,
SherrattD. J.1990; Recombination at ColE1 cer requires the Escherichia coli xerC gene product, a member of the lambda integrase family. J Bacteriol 172:6973–6980
GillenJ. R.,
WillisD. K.,
ClarkA. J.1981; Genetic analysis of the RecE pathway of genetic recombination in Escherichia coli K-12. J Bacteriol 145:521–532
HiguchiR.,
KrummelB.,
SaikiR. K.1988; A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res 16:7351–7367
HoS. N.,
HuntH. D.,
HortonR. M.,
PullenJ. K.,
PeaseL. R.1989; Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59
JohnsonR. C.,
BallC. A.,
PfefferD.,
SimonM. I.1988; Isolation of the gene encoding the Hin recombinational enhancer binding protein. Proc Natl Acad Sci U S A 85:3484–3488
KitagawaM.,
AraT.,
ArifuzzamanM.,
Ioka-NakamichiT.,
InamotoE.,
ToyonagaH.,
MoriH.2005; Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. DNA Res 12:291–299
NordströmK.,
IngramL. C.,
LundbackA.1972; Mutations in R-factors of Escherichia coli causing an increased number of R-factor copies per chromosome. J Bacteriol 110:562–569
StirlingC. J.,
SzatmariG.,
StewartG.,
SmithM. C. M.,
SherrattD. J.1988; The arginine repressor is essential for plasmid stabilizing site-specific recombination at the ColE1 cer locus. EMBO J 7:4389–4395
StirlingC. J.,
CollomsS. D.,
CollinsJ. F.,
SzatmariG.,
SherrattD. J.1989; xerB, an Escherichia coli gene required for plasmid ColE1 site-specific recombination is identical to pepA, encoding aminopeptidase A, a protein with substantial similarity to bovine lens leucine aminopeptidase. EMBO J 8:1623–1627
StraterN.,
SherrattD. J.,
CollomsS. D.1999; X-ray structure of aminopeptidase A from Escherichia coli and a model for the nucleoprotein complex in Xer site-specific recombination. EMBO J 18:4513–4522
SummersD. K.,
SherrattD. J.1984; Multimerization of high copy number plasmids causes instability: ColE1 encodes a determinant essential for plasmid monomerization and stability. Cell 36:1097–1103
VieiraJ.,
MessingJ.1982; The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268