Definition of a minimal plasmid stabilization system from the broad-host-range plasmid RK2

Roberts, Richard C.; Helinski, Donald R.

doi:10.1128/jb.174.24.8119-8132.1992

Cited by 85 publications

(83 citation statements)

References 41 publications

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“…However, several lines of evidence indicate that resolution by ParA does not account for the stabilizing effect of the parCBA operon. In studies with a miniplasmid, use of a different resolution system in place of the parCBA operon did not enhance plasmid stability even though multimers were resolved (41). In this study, the ⌬parCBA mutation reduced the stability of pRK2526 in three hosts despite the presence of another resolvase (TnpR) from Tn1 (36), which should compensate for any loss of resolution by ParA.…”

Section: Discussionmentioning

confidence: 84%

“…One region likely to be important is the 3.3-kb par region, which is able to stabilize mini-RK2 plasmids and other replicons in Escherichia coli, Pseudomonas aeruginosa, Azotobacter vinelandii, and Agrobacterium tumefaciens (40). The par region contains five genes (parABCDE) transcribed in two divergent, autoregulated operons (7,9,15,41). The parCBA operon expresses a site-specific recombinase (ParA) (10,18,40) that also functions as a repressor to autoregulate transcription of the operon (7,9).…”

mentioning

confidence: 99%

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Different relative importances of the par operons and the effect of conjugal transfer on the maintenance of intact promiscuous plasmid RK2

et al. 1995

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The par region of the broad-host-range, IncP␣ plasmid RK2 has been implicated as a stability determinant by its ability to enhance the maintenance of mini-RK2 plasmids or heterologous replicons in a growing population of host cells. The region consists of two operons: parCBA, which encodes a multimer resolution system, and parDE, which specifies a postsegregational response mechanism that is toxic to plasmidless segregants. To assess the importance of this region to the stable maintenance of the complete RK2 plasmid in different hosts, we used the vector-mediated excision (VEX) deletion system to specifically remove the entire par region or each operon separately from an otherwise intact RK2 plasmid carrying a lacZ marker. The par region was found to be important to stable maintenance of RK2lac (pRK2526) in Escherichia coli and five other gram-negative hosts (Agrobacterium tumefaciens, Azotobacter vinelandii, Acinetobacter calcoaceticus, Caulobacter crescentus, and Pseudomonas aeruginosa). However, the relative importance of the parCBA and parDE operons varied from host to host. Deletion of parDE had no effect on the maintenance of pRK2526 in A. calcoaceticus, but it severely reduced pRK2526 maintenance in A. vinelandii and resulted in significant instability in the other hosts. Deletion of parCBA did not alter pRK2526 stability in E. coli, A. tumefaciens, or A. vinelandii but severely reduced plasmid maintenance in A. calcoaceticus and P. aeruginosa. In the latter two hosts and C. crescentus, the ⌬parCBA mutant caused a notable reduction in growth rate in the absence of selection for the plasmid, indicating that instability resulting from the absence of parCBA may trigger the postsegregational response mediated by parDE. We also examined the effect of the conjugal transfer system on RK2 maintenance in E. coli. Transfer-defective traJ and traG mutants of pRK2526 were stably maintained in rapidly growing broth cultures. On solid medium, which should be optimal for IncP-mediated conjugation, colonies from cells containing the pRK2526 tra mutants displayed significant numbers of white (Lac ؊ ) sectors on X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside) plates, whereas sectors appeared rarely in colonies from tra ؉ plasmid-containing cells. Both the traJ and traG mutations further reduced the maintenance of the already unstable ⌬par derivative. Thus, these experiments with defined mutations in an intact RK2 plasmid have revealed (i) that the par region allows RK2 to adapt to the different requirements for stable maintenance in various hosts and (ii) that conjugal transfer can contribute to the maintenance of RK2 in a growing population, particularly under conditions that are favorable to RK2 transfer.The extraordinary success of plasmids in nature relies largely on plasmid-specified functions that promote their dissemination and ensure their persistence in growing populations of bacteria. The highly promiscuous, self-transmissible plasmids of incompatibility group P (IncP␣ and IncP␤) are particularly remarka...

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Section: Discussionmentioning

confidence: 84%

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confidence: 99%

Different relative importances of the par operons and the effect of conjugal transfer on the maintenance of intact promiscuous plasmid RK2

et al. 1995

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“…Suitable dilutions of the resulting cultures were plated to give separate colonies on TSB-Congo Red plates at 37°C, and the proportion of red to white colonies was determined after overnight growth. The rate of accumulation of Crb Ϫ cells (i.e., the presumed rate of loss of the virulence plasmid) during the approximately 20 generations of growth in liquid medium was calculated from the initial and final ratios of red to white colonies according to the formula of Roberts et al (13).…”

Section: Methodsmentioning

confidence: 99%

Surprising Dependence on Postsegregational Killing of Host Cells for Maintenance of the Large Virulence Plasmid of Shigella flexneri

et al. 2005

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Low-copy-number plasmids all encode multiple systems to ensure their propagation, including replication, partition (active segregation), and postsegregational killing (PSK) systems. PSK systems kill those rare cells that lose the plasmid due to replication or segregation errors. PSK systems should not be used as the principle means of maintaining the plasmid. The metabolic cost of killing the many cured cells that would arise from random plasmid segregation is far too high. Here we describe an interesting exception to this rule. Maintenance of the large virulence plasmid of Shigella flexneri is highly dependent on one of its PSK systems, mvp, at 37°C, the temperature experienced during pathogenesis. At 37°C, the plasmid is very unstable and mvp efficiently kills the resulting cured bacterial cells. This imposes a major growth disadvantage on the virulent bacterial population. The systems that normally ensure accurate plasmid replication and segregation are attenuated or overridden at 37°C. At 30°C, a temperature encountered by Shigella in the outside environment, the maintenance systems function normally and the plasmid is no longer dependent on mvp. We discuss why the virulent pathogen tolerates this self-destructive method of propagation at the temperature of infection.

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“…In all of the analyses done, no evidence of DNA rearrangement or deletion was detected. The segregational stability of pHS17 was examined in the transformant strain KH21, maintained in liquid cultures without antibiotic selection (23). After 100 generations, the percentage loss of plasmid per generation was 0.02, with an average of 98% of the viable cells demonstrating the clindamycin resistance phenotype.…”

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confidence: 99%

Native Plasmids of Fusobacterium nucleatum : Characterization and Use in Development of Genetic Systems

Haake

Yoder²,

Attarian³

et al. 2000

J Bacteriol

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Three native plasmids of Fusobacterium nucleatum were characterized, including DNA sequence analysis of one plasmid, pFN1. A shuttle plasmid, pHS17, capable of transforming Escherichia coli and F. nucleatum ATCC 10953 was constructed with pFN1. pHS17 was stably maintained in the F. nucleatum transformants, and differences in the transformation efficiencies suggested the presence of a restriction-modification system in F. nucleatum.

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Definition of a minimal plasmid stabilization system from the broad-host-range plasmid RK2

Cited by 85 publications

References 41 publications

Different relative importances of the par operons and the effect of conjugal transfer on the maintenance of intact promiscuous plasmid RK2

Different relative importances of the par operons and the effect of conjugal transfer on the maintenance of intact promiscuous plasmid RK2

Surprising Dependence on Postsegregational Killing of Host Cells for Maintenance of the Large Virulence Plasmid of Shigella flexneri

Native Plasmids of Fusobacterium nucleatum : Characterization and Use in Development of Genetic Systems

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