A review of mini-F plasmid maintenance

Kline, Bruce C.

doi:10.1016/0147-619x(85)90027-7

Cited by 90 publications

(57 citation statements)

References 64 publications

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“…However, overproduction of initiator proteins does not necessarily lead to an increase in copy number. The initiator proteins of plasmids P1, F, R6K, and Rtsl have been shown to directly exert both positive and negative effects on replication initiation (11,14,23,28,32). Although the mechanism of the direct inhibitory action of excess initiator protein is not yet fully understood, recent findings support the hypothesis that two different forms of the initiator protein may be responsible and that proteolytic processing may be involved (17,24).…”

supporting

confidence: 64%

The amount of RepR protein determines the copy number of plasmid pIP501 in Bacillus subtilis

Brantl¹,

Behnke²

1992

J Bacteriol

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To prove the hypothesis that the amount of RepR protein is the rate-limiting factor for replication of plasmid pIP501 in Bacillus subtilis, the repR gene was placed under control of the inducible promoter pspac. The plasmid copy number of the pIP501 derivative pRS9 could be deliberately adjusted between approximately 1 and 50 to 100 molecules per cell by varying the concentration of the inducer isopropyl-beta-D-thiogalactopyranoside. Construction of a repR-lacZ fusion proved that the increase in copy number was due to a proportional increase in the amount of RepR protein.

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supporting

confidence: 64%

The amount of RepR protein determines the copy number of plasmid pIP501 in Bacillus subtilis

Brantl¹,

Behnke²

1992

J Bacteriol

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“…It also binds to the promoter/operator region of repE (19,25), which contains an inverted repeat structure whose half sequence (10 bp) is similar to a part of the cri2 and incC direct repeats (8-bp matches) (21). Binding of RepE protein to these DNA sites is essential for the initiation of replication, control of plasmid copy number, and autogenous repression of repE transcription (15). Thus, RepE protein is directly involved in initiation and control of DNA replication and in repression of its own synthesis at the transcriptional level.…”

mentioning

confidence: 99%

“…It contains a set of genes required for its characteristic mode of replication and partition, including the origin of replication (or2), the repE gene encoding the initiator protein, the incompatibility gene incC, and the partition genes sop (par) (15). The minimum mini-F plasmid that consists of or2 and repE can stably replicate in E. coli but with high copy numbers (10 to 15 per chromosome) as a result of the lack of incC, which negatively regulates the copy number (13).…”

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confidence: 99%

Mini-F plasmid mutants able to replicate in Escherichia coli deficient in the DnaJ heat shock protein

et al. 1992

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A subset of Escherichia coli heat shock proteins, DnaJ, DnaK, and GrpE, is required for mini-F plasmid replication, presumably at the step of functioning of the RepE initiator protein. We have isolated and characterized mini-F plasmid mutants that acquired the ability to replicate in the Escherichia coli dnaJ259. The mutant plasmids were found to replicate in any of dnaJ, dnaK, and grpE mutant hosts tested. In each case, the majority of the mutant plasmids carried a unique amino acid alteration in a localized region of repE coding sequence and showed an increased copy number, whereas the minority contained a common single base change (C to T) in the promoter/operator region and produced an increased amount of RepE. All RepE proteins with altered residues (between 92 and 134) exhibited increased initiator activities (hyperactive), and many showed reduced repressor activities as well, indicating that this region is important for the both major functions of RepE protein. These results together with evidence reported elsewhere indicate that the subset of heat shock proteins serves to activate RepE protein prior to or during its binding to the replication origin and that the mutant RepE proteins are active even in their absence. We also found that a C-terminal lesion (repE602) reduces the initiator activity particularly of some hyperactive mutant RepE proteins but does not affect the repressor activity. This finding suggests a functional interaction between the central and C-terminal regions of RepE in carrying out the initiator function.

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“…Replication of mini-F requires several host factors, including DnaA (14,22,31), HU (34,38), and a subset of heat shock proteins (DnaK, DnaJ, and GrpE) (6,18), besides the plasmid-encoded replication initiator protein, RepE (251 residues, 29 kDa) (see reference 21). A minimal mini-F consists of an origin of replication (ori2), repE, and a drug resistance gene such as bla and exhibits high copy numbers (10 to 15 copies per chromosome) (19) due to the lack of incC, which negatively modulates the copy number.…”

mentioning

confidence: 99%

DNA-binding domain of the RepE initiator protein of mini-F plasmid: involvement of the carboxyl-terminal region

et al. 1995

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The RepE initiator protein (251 residues) is essential for mini-F replication in Escherichia coli and exhibits two major functions: initiation of DNA replication from ori2 and autogenous repression of repE transcription. Whereas the initiation is mediated by RepE monomers that bind to the ori2 iterons (direct repeats), the autogenous repression is mediated by dimers that bind to the repE operator, which contains an inverted repeat sequence related to the iterons. We now report that the binding of RepE to these DNA sites is primarily determined by the C-terminal region of this protein. The mutant RepE proteins lacking either the N-terminal 33 (or more) residues or the C-terminal 7 (or more) residues were first shown to be defective in binding to both the ori2 and the operator DNAs. However, direct screening and analysis of mutant RepEs which are specifically affected in binding to the ori2 iterons revealed that the mutations (mostly amino acid substitutions) occur exclusively in the C-terminal region (residues 168 to 242). These mutant proteins exhibited reduced binding to ori2 and no detectable binding to the operator. Thus, whereas truncation of either end of RepE can destroy the DNA-binding activities, the C-terminal region appears to represent a primary DNA-binding domain of RepE for both ori2 and the operator. Analogous DNA-binding domains seem to be conserved among the initiator proteins of certain related plasmids.The mini-F plasmid, like the parental F factor, is stably maintained in Escherichia coli with one or two copies per host chromosome. Replication of mini-F requires several host factors, including DnaA (14,22,31), HU (34,38), and a subset of heat shock proteins (DnaK, DnaJ, and GrpE) (6, 18), besides the plasmid-encoded replication initiator protein, RepE (251 residues, 29 kDa) (see reference 21). A minimal mini-F consists of an origin of replication (ori2), repE, and a drug resistance gene such as bla and exhibits high copy numbers (10 to 15 copies per chromosome) (19) due to the lack of incC, which negatively modulates the copy number. The ori2 region contains two DnaA boxes, an AT-rich region, and four direct repeats (iterons) of 19 bp, whereas the repE operator contains an inverted repeat whose half sequence (10 bp) resembles the ori2 iterons (8-bp matches) (Fig. 1). The RepE protein, a sequence-specific DNA-binding protein, binds to the ori2 iterons and to the operator (27,36). The binding to these separate but related DNA sequences plays a key role in the regulation of mini-F replication: RepE acts as an initiator of DNA replication through binding to ori2 and as an autogenous repressor of repE transcription through binding to the operator (see references 21, 30, and 37).We recently reported that the two functions of RepE are carried out by structurally distinct forms of the protein. One of the RepE mutants (RepE54), previously selected for their ability to replicate in the dnaJ mutant host (16), produced hyperactive RepE that cannot form dimers (17), unlike the wild-type protein that is fou...

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A review of mini-F plasmid maintenance

Cited by 90 publications

References 64 publications

The amount of RepR protein determines the copy number of plasmid pIP501 in Bacillus subtilis

The amount of RepR protein determines the copy number of plasmid pIP501 in Bacillus subtilis

Mini-F plasmid mutants able to replicate in Escherichia coli deficient in the DnaJ heat shock protein

DNA-binding domain of the RepE initiator protein of mini-F plasmid: involvement of the carboxyl-terminal region

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