Plasmid R1 Is Present as Clusters in the Cells of Escherichia coli

doi:10.1006/plas.1999.1457

Plasmid

Volume 43, Issue 3, May 2000, Pages 200-204

Communicated by D. Helinski

https://doi.org/10.1006/plas.1999.1457 Get rights and content

Abstract

Fluorescence microscopy was used to determine the location(s) of the replication origin of plasmid R1 in exponentially growing cells of Escherichia coli. The number of oriR1 foci per cell was smaller than the number of R1 copies per cell and was found to be the same for a copA mutant of R1 and for the wild-type plasmid. The intensities of individual foci were stronger for the cop mutant than for the wild type. We interpreted these results to imply that the plasmid DNA molecules were localized in small groups/clusters, a result that seems contrary to the earlier observations that plasmid R1 replicates randomly and segregates as a single-copy unit. The implications for the quantitative behavior of plasmid R1 in stability, incompatibility tests, replication, and partition experiments are discussed.

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Cited by (42)

Hitchhiking on chromosomes: A persistence strategy shared by diverse selfish DNA elements
2019, Plasmid
Citation Excerpt :
The problem of clustering and the questions it raises regarding replication and segregation are not unique to the yeast plasmid. Clustering has been observed in bacterial plasmids as well, both low- and high-copy (Ebersbach and Gerdes, 2005; Gordon et al., 2004; Nordstrom and Gerdes, 2003; Pogliano et al., 2001; Weitao et al., 2000). The measured loss rates of low-copy plasmids lacking a par locus would be more consistent with each plasmid molecule, rather than the cluster itself, being the unit of segregation (Nordstrom and Gerdes, 2003).
An underlying theme in the segregation of low-copy bacterial plasmids is the assembly of a ‘segrosome’ by DNA-protein and protein-protein interactions, followed by energy-driven directed movement. Analogous partitioning mechanisms drive the segregation of host chromosomes as well. Eukaryotic extra-chromosomal elements, exemplified by budding yeast plasmids and episomes of certain mammalian viruses, harbor partitioning systems that promote their physical association with chromosomes. In doing so, they indirectly take advantage of the spindle force that directs chromosome movement to opposite cell poles. Molecular-genetic, biochemical and cell biological studies have revealed several unsuspected aspects of ‘chromosome hitchhiking’ by the yeast 2-micron plasmid, including the ability of plasmid sisters to associate symmetrically with sister chromatids. As a result, the plasmid overcomes the ‘mother bias’ experienced by plasmids lacking a partitioning system, and elevates itself to near chromosome status in equal segregation. Chromosome association for stable propagation, without direct energy expenditure, may also be utilized by a small minority of bacterial plasmids–at least one case has been reported. Given the near perfect accuracy of chromosome segregation, it is not surprising that elements residing in evolutionarily distant host organisms have converged upon the common strategy of gaining passage to daughter cells as passengers on chromosomes.
Organization of ribosomes and nucleoids in escherichia coli cells during growth and in quiescence
2014, Journal of Biological Chemistry
Citation Excerpt :
The notion of structureless, homogeneous bacterial cytoplasm through which macromolecules diffuse freely to interact by random collisions has been replaced by a system of macromolecular machines designed for specific functions assembled at specific locations at appropriate times such that growth, replication, and cell division processes function in coordination to maintain remarkably error-free cycles of growth and reproduction for generations (1–3). This was also suspected earlier with the discovery of plasmids clustered at characteristic intracellular positions (4, 5) and the sequential movement of the bacterial chromosomes during replication (6–9). Now it has been revealed that the bacterial interior possesses a highly ordered subcellular architecture comprising dynamic networks of cytoskeletal fibers (10–12), multiprotein complexes constituting replication, transcription, and translation machineries assembled at characteristic locations (13–19), and oscillatory relocalization of protein complexes in defined trajectories resulting in concentration gradients (20, 21).
We have examined the distribution of ribosomes and nucleoids in live Escherichia coli cells under conditions of growth, division, and in quiescence. In exponentially growing cells translating ribosomes are interspersed among and around the nucleoid lobes, appearing as alternative bands under a fluorescence microscope. In contrast, inactive ribosomes either in stationary phase or after treatment with translation inhibitors such as chloramphenicol, tetracycline, and streptomycin gather predominantly at the cell poles and boundaries with concomitant compaction of the nucleoid. However, under all conditions, spatial segregation of the ribosomes and the nucleoids is well maintained. In dividing cells, ribosomes accumulate on both sides of the FtsZ ring at the mid cell. However, the distribution of the ribosomes among the new daughter cells is often unequal. Both the shape of the nucleoid and the pattern of ribosome distribution are also modified when the cells are exposed to rifampicin (transcription inhibitor), nalidixic acid (gyrase inhibitor), or A22 (MreB-cytoskeleton disruptor). Thus we conclude that the intracellular organization of the ribosomes and the nucleoids in bacteria are dynamic and critically dependent on cellular growth processes (replication, transcription, and translation) as well as on the integrity of the MreB cytoskeleton.
Background: We studied ribosome and nucleoid distribution in Escherichia coli under growth and quiescence.
Results: Spatially segregated ribosomes and nucleoids show drastically altered distribution in stationary phase or when treated with drugs affecting translation, transcription, nucleoid-topology, or cytoskeleton. Ribosome inheritance in daughter cells is frequently unequal.
Conclusion: Cellular growth processes modulate ribosome and nucleoid distribution.
Significance: This provides insight into subcellular organization of molecular machines.
Eclipse period of R1 plasmids during downshift from elevated copy number: Nonrandom selection of copies for replication
2012, Plasmid
Citation Excerpt :
Plasmid R1, like many other plasmids, forms clusters containing more than one plasmid copies (Eliasson et al., 1992; Weitao et al., 2000; Pogliano, 2002). The average number of clusters for the wild type and a cop mutant of R1 with about fourfold higher copy number was found to be the same; the clusters for the copy number mutants appearing brighter due to higher plasmid DNA content (Weitao et al., 2000). The large number of plasmids from runaway replication would therefore be expected to form clusters of different sizes with varying localized concentrations of the inhibitor CopA.
The classical Meselson-Stahl density-shift method was used to study replication of pOU71, a runaway-replication derivative of plasmid R1 in Escherichia coli. The miniplasmid maintained the normal low copy number of R1 during steady growth at 30 °C, but as growth temperatures were raised above 34 °C, the copy number of the plasmid increased to higher levels, and at 42 °C, it replicated without control in a runaway replication mode with lethal consequences for the host. The eclipse periods (minimum time between successive replication of the same DNA) of the plasmid shortened with rising copy numbers at increasing growth temperatures (Olsson et al., 2003). In this work, eclipse periods were measured during downshifts in copy number of pOU71 after it had replicated at 39 and 42 °C, resulting in 7- and 50-fold higher than normal plasmid copy number per cell, respectively. Eclipse periods for plasmid replication, measured during copy number downshift, suggested that plasmid R1, normally selected randomly for replication, showed a bias such that a newly replicated DNA had a higher probability of replication compared to the bulk of the R1 population. However, even the unexpected nonrandom replication followed the copy number kinetics such that every generation, the plasmids underwent the normal inherited number of replication, n, independent of the actual number of plasmid copies in a newborn cell.
Evaluating quantitative methods for measuring plasmid copy numbers in single cells
2012, Plasmid
The life of plasmids is a constant battle against fluctuations: failing to correct copy number fluctuations can increase the plasmid loss rate by many orders of magnitude, as can a failure to more evenly divide the copies between daughters at cell division. Plasmids are therefore long-standing model systems for stochastic processes in cells, much thanks to the efforts of Kurt Nordström to whose memory this issue is dedicated. Here we analyze a range of experimental methods for measuring plasmid copy numbers in single cells, focusing on challenges, trade-offs, and necessary experimental controls. In particular we analyze published and unpublished strategies to infer copy numbers from expression of plasmid-encoded reporters, direct labeling of plasmids with fluorescent probes or DNA binding proteins fused to fluorescent reporters, PCR based methods applied to single cell lysates, and plasmid-specific replication arrest. We conclude that no method currently exists to measure plasmid copy numbers in single cells, and that most methods are overwhelmed by various types of experimental noise. We also discuss how accurate methods can be developed.
New pSC101-derivative cloning vectors with elevated copy numbers
2008, Plasmid
Mutations that increase the copy number of the pSC101 replicon have been used for construction of new cloning vectors. Replacement of glutamate at position 93 in RepA yields plasmids that replicate at medium (27 copies/cell) and high (∼240 copies/cell) copy numbers. Based on the crystal structure of RepE, a structurally similar replication initiator protein from the F factor, the pSC101 repA mutants are predicted to be defective in dimerization. The cloning vectors permit increased expression of gene products along with the advantages of pSC101-derivative plasmids, including stable maintenance and compatibility with ColE1 plasmids. The plasmids also allow blue/white screening for DNA inserts and impart resistance to ampicillin, chloramphenicol and kanamycin. The vectors were used in a genetic assay to suppress temperature-sensitive mutants of ffh, encoding the protein component of the Escherichia coli signal recognition particle, by overproduction of 4.5S RNA. While expression of 4.5S RNA from a wild type pSC101-derivative plasmid was not sufficient for suppression, use of the new vectors did suppress the temperature-sensitive phenotype.
Plasmid Segregation: Is a Total Understanding within Reach?
2008, Current Biology
Citation Excerpt :
And how does the interaction with ParR-parC stabilize ParM filaments? How can one spindle consist of multiple ParM filaments, as Campbell and Mullins [4] demonstrated, and are the observed ‘plasmids’ actually clusters of multiple plasmids [12]? Moving up in complexity, it is not obvious that the proposed search-and-capture mechanism can account for all the observed interactions between plasmids.

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¹: To whom correspondence should be addressed. Fax: (46) 18 53 03 96. E-mail: [email protected].

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Abstract

References (20)

Chromosome and low copy plasmid segregation in E. coli: Visual evidence for distinct mechanisms

Cell

Selection and timing of replication of plasmids R1drd-19 and F′lac in Escherichia coli

Plasmid

Subcellular distribution of actively partitioning F plasmid during the cell division cycle in E. coli

Cell

Kinetic aspects of control of plasmid replication by antisense RNA

TIBS

Partitioning of plasmid R1 in Escherichia coli. I. Kinetics of loss of plasmid derivatives deleted of the par region

Plasmid

Partitioning of plasmid R1 in Escherichia coli. II. Incompatibility properties of the partitioning system

Plasmid

Plasmid R1 in Salmonella typhimurium: Molecular instability and gene dosage effects

Plasmid

R plasmid gene dosage effects in Escherichia coli K12: Copy mutants of the R plasmid R1drd-19

Plasmid

Cell division in Escherichia coli minB mutants

Mol. Microbiol.

Cited by (42)

Hitchhiking on chromosomes: A persistence strategy shared by diverse selfish DNA elements

Organization of ribosomes and nucleoids in escherichia coli cells during growth and in quiescence

Eclipse period of R1 plasmids during downshift from elevated copy number: Nonrandom selection of copies for replication

Evaluating quantitative methods for measuring plasmid copy numbers in single cells

New pSC101-derivative cloning vectors with elevated copy numbers

Plasmid Segregation: Is a Total Understanding within Reach?

Regular ArticlePlasmid R1 Is Present as Clusters in the Cells of Escherichia coli

Abstract

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Cell division in Escherichia coli minB mutants

Mol. Microbiol.

Regular Article
Plasmid R1 Is Present as Clusters in the Cells of Escherichia coli