Genetic studies of F plasmid maintenance genes involved in copy number control, incompatability, and partitioning

Seelke, Ralph; Kline, Bruce C.; Trawick, John D.; Ritts, Graham D.

doi:10.1016/0147-619x(82)90075-0

Cited by 51 publications

(22 citation statements)

References 27 publications

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“…Moreover, we provide direct confirmation of the model with evidence that overproduction of RepA can overcome the inhibitory activity of the repeated sequences and that RepA binds specifically to these sequences. The findings that mutations that result in increased plasmid copy-number are located in the repA locus of P1 (19) and also in the analogous locus of F (20,21) are also consistent with the hypothesis.…”

Section: Discussionsupporting

confidence: 77%

Plasmid P1 replication: negative control by repeated DNA sequences.

Chattoraj¹,

Cordes²,

Abeles³

1984

Proc. Natl. Acad. Sci. U.S.A.

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The incompatibility locus, incA, of the unitcopy plasmid P1 is contained within a fragment that is essentially a set of nine 19-base-pair repeats. One or more copies of the fragment destabilizes the plasmid when present in trans. Here we show that extra copies of incA interfere with plasmid DNA replication and that a deletion of most of incA increases plasmid copy dumber. Thus, incA is not essential for replicatlon but is required for its control. When cloned ih a highcopy-number vector, pieces of the incA fragment that each contain only three repeats destabilize Pi plasmids efficiently.This result makes it unlikely that incA specifies a regulatory product. Our in vivo results suggest that the repeating DNA sequence itself negatively controls replication by titrating a P1-determined protein, RepA, that is essential for replication. Consistent with this hypothesis is the observation that the RepA protein binds to the incA fragment in vitro.

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Section: Discussionsupporting

confidence: 77%

Plasmid P1 replication: negative control by repeated DNA sequences.

Chattoraj¹,

Cordes²,

Abeles³

1984

Proc. Natl. Acad. Sci. U.S.A.

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“…To illustrate the test paper assay, bacterial colonies were grown on TYE agar from a mixture of E. coli CSH50 (ampicillin susceptible) with or without plasmid pMF45, a mini-F derivative that codes for TEM-1 P-lactamase (24) (Table 1). Exceptions to this pattern were seen with strains containing plasmids R46, RIP64, and pUZ8-Rl5lA1, which encode OXA-2, OXA-3, and PSE-2 P-lactamases, respectively.…”

Section: Resultsmentioning

confidence: 99%

“…Levels of TEM ,-lactamases are known to be proportional to P-lactamase gene dosage, a phenomenon commonly used to measure plasmid copy number (28). pMF45, pBK105, and pBK110, three closely related mini-F plasmids each carrying one TEM-1 ,B-lactamase gene, are present in strain CSH50 in copy numbers of 2, 12, and 14 per bacterial chromosome, respectively, as measured by the dye-CsCl technique of Seelke et al (24). When grown on TYE agar, colonies of CSH50 with these plasmids varied in the intensity of yellow color on test paper, with colonies containing the lower-copy-number plasmid pMF45 being much less intense than those with the higher-copynumber plasmids pBK105 and pBK110 (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Rapid Method for Screening Large Numbers of Escherichia coli Colonies for Production of Plasmid-Mediated β-Lactamases

Wolfson

Hooper

Swartz

et al. 1983

Antimicrob Agents Chemother

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“…The active role of RepE protein in the regulation of mini-F replication has been implied by several lines of evidence. First, structural alteration of RepE by a cop mutation affects plasmid copy number (3,12,26). Second, the incC region, containing 19-bp repeated sequences similar to those found at ori2, exhibits a modest affinity to RepE protein (29) and modulates plasmid copy number (26,31).…”

Section: Repe Proteinmentioning

confidence: 99%

“…First, structural alteration of RepE by a cop mutation affects plasmid copy number (3,12,26). Second, the incC region, containing 19-bp repeated sequences similar to those found at ori2, exhibits a modest affinity to RepE protein (29) and modulates plasmid copy number (26,31). Third, the synthesis of RepE protein is autogenously regulated at the transcription level, with RepE serving as a repressor (14,28,34); a structural change in RepE protein (3) or the operator region that binds the repressor (25) can affect repression.…”

Section: Repe Proteinmentioning

confidence: 99%

Mini-F plasmid mutants able to replicate in the absence of sigma 32: mutations in the repE coding region producing hyperactive initiator protein

1991

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Mini-F plasmids cannot replicate in Escherichia coli strains (ArpoH) lacking a32, presumably because transcription of the repE gene encoding the replication initiator protein (RepE protein) depends mostly on RNA polymerase containing 732. We have isolated and characterized mini-F mutants able to replicate in ArpoH cells. Contrary to the initial expectation, five mutants with mutations in the repE coding region that produce altered RepE proteins were obtained. The mutations caused replacement of a single amino acid: the 92nd glutamic acid was replaced by lysine (repE10, repE16, and repE25) or glycine (repE22) or the 109th glutamic acid was replaced by lysine (repE26). These plasmids overproduced RepE protein and exhibited very high copy numbers. Two major activities of mutated RepE proteins have been determined in vivo; the autogenous repressor activity was significantly reduced, whereas the initiator activity was much enhanced in all mutants. These results indicate the importance of a small central region of RepE protein for both initiator and repressor activities. Thus the decreased repE transcription in ArpoH cells can be compensated for by an increased initiator activity and a decreased repressor activity of RepE, resulting in the increased synthesis of hyperactive RepE protein.

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Genetic studies of F plasmid maintenance genes involved in copy number control, incompatability, and partitioning

Cited by 51 publications

References 27 publications

Plasmid P1 replication: negative control by repeated DNA sequences.

Plasmid P1 replication: negative control by repeated DNA sequences.

Rapid Method for Screening Large Numbers of Escherichia coli Colonies for Production of Plasmid-Mediated β-Lactamases

Mini-F plasmid mutants able to replicate in the absence of sigma 32: mutations in the repE coding region producing hyperactive initiator protein

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