High-copy bacterial plasmids diffuse in the nucleoid-free space,replicate stochastically and are randomly partitioned at cell division

Bacterial plasmids play important roles in the metabolism, pathogenesis and bacterial evolution and are highly versatile biotechnological tools. Stable inheritance of plasmids depends on their autonomous replication and efficient partition to daughter cells at cell division. Active partition systems have not been identified for high-copy number plasmids, and it has been generally believed that they are partitioned randomly at cell division. Nevertheless, direct evidence for the cellular location of replicating and nonreplicating plasmids, and the partition mechanism has been lacking. We used as model pJHCMW1, a plasmid isolated from Klebsiella pneumoniae that includes two β-lactamase and two aminoglycoside resistance genes. Here we report that individual ColE1-type plasmid molecules are mobile and tend to be excluded from the nucleoid, mainly localizing at the cell poles but occasionally moving between poles along the long axis of the cell. As a consequence, at the moment of cell division, most plasmid molecules are located at the poles, resulting in efficient random partition to the daughter cells. Complete replication of individual molecules occurred stochastically and independently in the nucleoid-free space throughout the cell cycle, with a constant probability of initiation per plasmid.     

The plasmid-maintenance functions of the P7 prophage

The region responsible for the maintenance of the prophage of bacteriophage P7 as a stable, unit-copy plasmid was isolated in a lambda att vector which lysogenizes Escherichia coli as a stable unit-copy plasmid under the control of the P7 replication origin. The P7 plasmid-maintenance region was shown to consist of adjacent replication and partition regions capable of functioning independently. The isolated replication region could support plasmid maintenance but the resulting plasmids were highly unstable unless the partition region was also included. Stable composite plasmids were isolated containing the putative P7 partition region and the origin of replication of the unrelated plasmid F, indicating that P7 encoded an active partition mechanism. The replication regions of P7 and P1 were shown to be highly homologous but the partition regions of the two plasmids appear to be unrelated in sequence. The incompatibility determinants associated with the two replication regions showed the same specificity, whereas the partition-region incompatibility determinants were different, showing no cross-specificity.     

Genetic and physical map of a P1 miniplasmid

The prophage form of bacteriophage P1 is a unit-copy plasmid which is maintained with great fidelity in its Escherichia coli host. The plasmid maintenance functions of P1 are clustered in one region of the genome. An 11.5-kilobase fragment from this region has been cloned into a lambda delta att vector and promotes stable unit-copy plasmid maintenance. The properties of the lambda vector facilitated the isolation of deletion mutants affecting the P1 DNA. Twenty-eight deletion mutants were isolated, and their lesions were mapped by physical techniques. The genetic properties of the mutants with respect to plasmid replication, stability of plasmid maintenance, and ability to exert incompatibility effects against P1 and P7 plasmids were determined. These properties, along with those of several subfragments of the P1 insert cloned into high-copy-number plasmid vectors, allow the construction of an unambiguous genetic and physical map of the maintenance functions. A region of less than 3 kilobases, the rep region, is essential for plasmid replication and contains the incA incompatibility determinant within an 800-base-pair segment. Immediately adjacent to rep is a second region of approximately 3 kilobases which is required for stable plasmid maintenance, but not replication. This region, par, contains a second incompatibility element incB which is approximately 1 kilobase in size. The par region appears to specify equipartition of plasmid copies to daughter cells during cell division.     

A Monte Carlo simulation of plasmid replication during the bacterial division cycle

Plasmids have cell cycle replication patterns that need to be considered in models of their replication dynamics. To compare current theories for control of plasmid replication with experimental data for timing of plasmid replication with the cell cycle, a Monte Carlo simulation of plasmid replication and partition was developed. High-copy plasmid replication was simulated by incorporating equations previously developed from the known molecular biology of ColE1-type plasmids into the cell-cycle simulation. Two types of molecular mechanisms for low-copy plasmid replication were tested: accumulation of an initiator protein in proportion to cell mass and binding of the plasmid origin to the cell membrane. The low-copy plasmids were partitioned actively, with a specific mechanism to mediate the transfer from mother to daughter cells, whereas the high-copy plasmids were partitioned passively with cell mass.The simulation results and experimental data demonstrate cell-cycle-specific replication for the low-copy F plasmid and cell-cycle-independent replication for the high-copy pBR322, ColBM, and R6K plasmids. The simulation results indicate that synchronous replication at multiple plasmid origins is critical for the cell-cycle-specific pattern observed in rapidly growing cells. Variability in the synchrony of initiation of multiple plasmid origins give rise to a cell-cycle-independent pattern and is offered as a plausible explanation for the controversy surrounding the replication pattern of the low-copy plasmids. A comparison of experimental data and simulation results for the low-copy F plasmid at several growth rates indicates that either initiation mechanism would be sufficient to explain the timing of replication with the cell cycle. The simulation results also demonstrate that, although cell-cycle-specific and cell-cycle independent replication patterns give rise to very different gene-expression patterns during short induction periods in age-selected populations, long-term expression of genes encoded on low-copy and high-copy plasmids in exponentially growing cells have nearly the same patterns. These results may be important for the future use of low-copy plasmids as expression vectors and validate the use of simpler models for high-copy plasmids that do not consider cell-cycle phenomena. (c) 1996 John Wiley & Sons, Inc.     

Host controlled plasmid replication: Escherichia coli minichromosomes

Escherichia coli minichromosomes are plasmids replicating exclusively from a cloned copy of oriC, the chromosomal origin of replication. They are therefore subject to the same types of replication control as imposed on the chromosome. Unlike natural plasmid replicons, minichromosomes do not adjust their replication rate to the cellular copy number and they do not contain information for active partitioning at cell division. Analysis of mutant strains where minichromosomes cannot be established suggest that their mere existence is dependent on the factors that ensure timely once per cell cycle initiation of replication. These observations indicate that replication initiation in E. coli is normally controlled in such a way that all copies of oriC contained within the cell, chromosomal and minichromosomal, are initiated within a fairly short time interval of the cell cycle. Furthermore, both replication and segregation of the bacterial chromosome seem to be controlled by sequences outside the origin itself.     

Cell-cycle-specific F plasmid replication: regulation by cell size control of initiation.

F plasmid replication during the Escherichia coli division cycle was investigated by using the membrane-elution technique to produce cells labeled at different times during the division cycle and scintillation counting for quantitative analysis of radioactive plasmid DNA. The F plasmid replicated, like the minichromosome, during a restricted portion of the bacterial division cycle; i.e., F plasmid replication is cell-cycle specific. The F plasmid replicated at a different time during the division cycle than a minichromosome present in the same cell. F plasmid replication coincided with doubling in the rate of enzyme synthesis from a plasmid-encoded gene. When the cell cycle age of replication of the F plasmid was determined over a range of growth rates, the cell size at which the F plasmid replicated followed the same rules as did replication of the bacterial chromosome--initiation occurred when a constant mass per origin was achieved--except that the initiation mass per origin for the F plasmid was different from that for the chromosome origin. In contrast, the high-copy mini-R6K plasmid replicated throughout the division cycle.     

Replication and segregation of a miniF plasmid during the division cycle of Escherichia coli.

Replication of the miniF plasmid pML31 was examined during the division cycle of Escherichia coli growing with doubling times between 40 and 90 min at 37 degrees C and compared to the replication of plasmid pBR322 and the minichromosome pAL70. The replication pattern of pML31 was indistinguishable from that of pBR322 at all growth rates and very different from the cell-cycle-specific replication of the minichromosome. It is concluded that both pML31 and pBR322 plasmids can replicate at all stages of the division cycle, with a probability of replication that increases gradually, but perhaps not exponentially, during the cycle. In contrast, the modes of segregation of pML31 and pBR322 plasmids into daughter cells at division appeared to differ, raising the possibility that pML31 may segregate in a nonrandom fashion similar to that of chromosomes and minichromosomes.     

Clustering versus random segregation of plasmids lacking a partitioning function: a plasmid paradox?

Plasmids lacking a functional partition system are randomly distributed to the daughter cells; plasmid-free daughter cells are formed with a frequency of (1/2)2n per cell and cell generation where 2n is the (average) copy number at cell division. Hence, the unit of segregation is one plasmid copy. However, plasmids form clusters in the cells. A putative solution to this potential paradox is presented: one plasmid copy at a time is recruited from the plasmid clusters to the replication factories that are located in the cell centres. Hence, replication offers the means of declustering that is necessary in a growing host population. The daughter copies diffuse freely and each copy may with equal probability end up in either of the two cell halves. In this way, the random segregation of the plasmids is coupled to replication and occurs continuously during the cell cycle, and is not linked to cell division. The unit of segregation is the plasmid copy and not the plasmid clusters. In contrast, the two daughters of a Par+ plasmid are directed in opposite directions by the plasmid-encoded partition system, thereby assuring that each daughter cell receives the plasmid.     

Cell cycle analysis of F''lac replication in Escherichia coli B/r.

The timing and control of replication of an F'lac plasmid was investigated in two substrains of Escherichia coli B/r lac/F'lac growing at a variety of rates. The cellular content of covalently closed circular F'lac deoxyribonucleic acid and the cellular mass at the time of F'lac replication both increased as a function of growth rate. The timing of plasmid replication during the division cycle was determined by measuring the inducibility of beta-galactosidase in cells of different ages in exponentially growing cultures. At all growth rates, the rate of induced beta-galactosidase synthesis increased in a step-wise fashion during the division cycle, indicating that the F'lac plasmid replicated at a discrete time in the cycle. At growth rates greater than one doubling per h, the cell age at F'lac replication was indistinguishable from the cell age at chromosomal lac+ replication in an isogenic F- parent. The ratio of plasmids to chromosomal origins decreased from about 0.7 to 0.4 between growth rates of 1.0 to 2.5 doublings per h. These observations are all consistent with replication of F'lac at about the same time in the division cycle as replication of the homologous chromosomal region at these growth rates. This similarity in timing of replication of homologous deoxyribonucleic acid regions was not evident in slower-growing cells.     

P1 and NR1 Plasmid Replication during the Cell Cycle of Escherichia coli

Replication patterns of the miniP1 plasmid pZC176, the miniNR1 plasmid pRR933, and the high-copy miniNR1 derivative pRR942 were examined during the Escherichia coli cell division cycle and compared to the cycle-specific replication pattern of a minichromosome and the cycle nonspecific pattern of pBR322. In E. coli cells growing with doubling times of 40 and 60 min, the miniP1 plasmid was found to replicate with a slight periodicity during the division cycle. The periodicity was not nearly as pronounced as that of the minichromosome, was not affected by the presence of a minichromosome, and was not evident in cells growing more rapidly with a doubling time of 25 min. Both miniNR1 plasmids, pRR933 and pRR942, replicated with patterns indistinguishable from that of pBR322 and clearly different from that of the minichromosome. It is concluded that both P1 and NR1 plasmids can replicate at all stages of the cell cycle but that P1 displays a slight periodicity in replication probability in the cycle of slower growing cells. This periodicity does not appear to be coupled to a specific age in the cycle, but could be associated with the achievement of a specific cell mass per plasmid. During temperature shifts of a dnaC(Ts) mutant, the miniP1 plasmid and pBR322 replicated with similar patterns that differed from that of the minichromosome, but were consistent with a brief eclipse between rounds of replication.     

Cell cycle analysis of plasmid-containing Escherichia coli HB101 populations with flow cytometry

The relationship between cell mass and cell number dynamics for bacteria such as Escherichia coli depends on the cell cycle parameters C and D. Effects of plasmid copy number on these cell cycle parameters have been studied for Escherichia coli HB101 containing pMB1 plasmids propagated at different copy numbers ranging from 12 to 122. Determination of cell cycle and cell size parameters was accomplished using flow cytometry data on single-cell light scattering and DNA content frequency functions in conjunction with a mathematical model of cell population statistics. Two independent methods for estimating C and D intervals based on flow cytometry were developed and applied with essentially identical results. The presence of plasmids decreases the C and D periods, mean cell sizes, and initiation masses for chromosome replication by 14, 24, 38, and 18%, respectively, relative to corresponding values for plasmid-free host cells. Plasmid copy number has a negligible influence on these parameters, suggesting that host-plasmid inter actions which determine these properties are centered on the single plasmid selected for replication according to the random selection model established for ColE1-type plasmids.     

F plasmid replication and the division cycle of Escherichia coli B/r

A terminal stage in the duplication of many bacterial plasmids involves the transient formation of catenated molecules containing two interlocked monomeric plasmid units. This property of plasmid replication was exploited to examine the relationship between F replication and the division cycle of Escherichia coli B/r cells growing in undisturbed, exponential-phase cultures. Various cultures of F′lac- or FKm^r-containing cells were briefly exposed to [³H]thymidine, and then the transfer of radioactivity into, and out of, a catenated dimer consisting of two closed circular monomers was measured during a chase period. The fraction of plasmid molecules present in this dimer form was determined by separating cellular DNA in alkaline sucrose gradients. In addition, plasmid replication was studied in synchronously growing cultures by measuring both [³H]thymidine incorporation into covalently closed circular DNA and β-galactosidase inducibility. The results suggest that replication of F plasmids can take place throughout the cell division cycle, with the probability of replication increasing toward the end of the cycle. The presence of DNA homologous to the chromosome on the F′lac did not alter the replication pattern of the plasmid during the division cycle.     

Delta dnaK52 mutants of Escherichia coli have defects in chromosome segregation and plasmid maintenance at normal growth temperatures.

Major heat shock proteins, such as the Escherichia coli DnaK protein, not only are required for cell growth after heat shock but seem to possess important functions in cellular metabolism at normal growth temperatures as well. E. coli delta dnaK52 mutants have severe cellular defects at 30 degrees C, one of which is in cell division (B. Bukau and G. C. Walker, J. Bacteriol, 171:2337-2346, 1989). Here we show that at 30 degrees C, delta dnaK52 mutants have defects in chromosome segregation and in maintenance of low-copy-number plasmids. Fluorescence microscopic analysis revealed that chromosomes were frequently lacking at peripheries of cell filaments of delta dnaK52 mutants and clustered at other locations. In other parts of the cell filaments, chromosomes were apparently normally distributed and they were also present in most of the small cells found in populations of delta dnaK52 cells. These defects might be at the level of DNA replication, since delta dnaK52 mutants have a threshold lower rate of DNA synthesis than wild-type cells. Chromosome segregation defects of delta dnaK52 mutants were also observed in an rnh dnaA mutant background, in which initiation of DNA replication is DnaA-oriC independent. We also found that low-copy-number P1 miniplasmids could not be stably maintained in delta dnaK52 mutants at 30 degrees C. delta par P1 miniplasmids that carry the P1-encoded rep functions required for their replication but lack the P1-encoded par functions required for faithful partitioning of the plasmids during cell division were also unstable in delta dnaK52 mutants. Taken together, our results indicate important, although not absolutely essential, functions for DnaK at 30 degrees C in one or more processes necessary for correct replication and/or partitioning of chromosomes and P1 miniplasmids. Furthermore, we found that P1 miniplasmids were also highly unstable in dnaJ259 mutants, indicating a role for the DnaJ heat shock protein in maintenance of these plasmids.     

Origin inactivation in bacterial DNA replication control

Initiation of DNA replication is a highly regulated process in all organisms. Proteins that are required to recruit DNA polymerase - initiator proteins - are often used to regulate the timing or frequency of initiation in the cell cycle by limiting either their own synthesis or availability. Studies of the Escherichia coli chromosome and of bacterial plasmids with iterated initiator binding sites (iterons) have revealed that, in addition to initiator limitation, replication origin inactivation is used to prevent replication that is untimely or excessive. Our recent studies of plasmid P1 revealed that this additional mode of control becomes a requirement when initiator availability is limited only by autoregulation. Thus, although initiator limitation appears to be a well-conserved and central mode of replication control, optimal replication might require additional control mechanisms. This review gives examples of how the multiple mechanisms can act synergistically, antagonistically or be partially redundant to guarantee low frequency events. The lessons learned are likely to help understand many other regulatory systems in the bacterial cell.     

Cell division in Escherichia coli after changes in the velocity of DNA replication

A method of computer analysis was developed to evaluate the kinetic changes in the rate of cell division in non-synchronous cultures of E. coli resulting from changes in the velocity or initiation of chromosome replication. This method takes into account that the cell division pathway in E. coli includes a reaction of indeterminate length described by a probability function that applies to the cell population. The analysis yields a hypothetical cell number kinetics as it would be observed if the stochastic element in the division pathway were absent. Since this derived cell number curve responds to experimentally induced perturbations of replication at defined times whereas the actual cell number curve reflects these perturbations only in a blurred fashion, replication and division events can be precisely correlated with this method. The method was applied to the evaluation of thymine starvation experiments with two Thy- derivatives of E. coli B/r; one of the strains has a mutationally altered (60% increased) cell mass at initiation of chromosome replication. In both strains, the stochastic phase of the cell cycle had the same half-life value of 10 min and began 18 min after each termination of replication. This suggests that the time of cell division is linked to replication, not to cell mass or length. This interpretation is supported by results of experiments in which the rate of cell growth was altered at the time of thymine starvation.     

Replication of plasmids in gram-negative bacteria.

Replication of plasmid deoxyribonucleic acid (DNA) is dependent on three stages: initiation, elongation, and termination. The first stage, initiation, depends on plasmid-encoded properties such as the replication origin and, in most cases, the replication initiation protein (Rep protein). In recent years the understanding of initiation and regulation of plasmid replication in Escherichia coli has increased considerably, but it is only for the ColE1-type plasmids that significant biochemical data about the initial priming reaction of DNA synthesis exist. Detailed models have been developed for the initiation and regulation of ColE1 replication. For other plasmids, such as pSC101, some hypotheses for priming mechanisms and replication initiation are presented. These hypotheses are based on experimental evidence and speculative comparisons with other systems, e.g., the chromosomal origin of E. coli. In most cases, knowledge concerning plasmid replication is limited to regulation mechanisms. These mechanisms coordinate plasmid replication to the host cell cycle, and they also seem to determine the host range of a plasmid. Most plasmids studied exhibit a narrow host range, limited to E. coli and related bacteria. In contrast, some others, such as the IncP plasmid RK2 and the IncQ plasmid RSF1010, are able to replicate in nearly all gram-negative bacteria. This broad host range may depend on the correct expression of the essential rep genes, which may be mediated by a complex regulatory mechanism (RK2) or by the use of different promoters (RSF1010). Alternatively or additionally, owing to the structure of their origin and/or to different forms of their replication initiation proteins, broad-host-range plasmids may adapt better to the host enzymes that participate in initiation. Furthermore, a broad host range can result when replication initiation is independent of host proteins, as is found in the priming reaction of RSF1010.     

The cell cycle in Escherichia coli B/r

Cell division and DNA synthesis were measured in synchronous cultures of E. coll B/r growing in glucose minimal medium at 37 °. The kinetic curves were analysed in order to find the variability of replication initiation, termination, and cell division events during the cell cycle. It is inferred that under the conditions used, cells begin to divide 17 min (D₀ = minimum D-period) after each termination of chromosome replication with a constant probability per unit of time (half-life = 4·5–6 min). This randomness produces an asymmetric frequency distribution of D-periods, similar but mirror-symmetric frequency distributions of initiation and termination periods, a symmetric, non-Gaussian distribution of interdivision intervals, and complex kinetic changes in the rate of DNA synthesis as a function of cell age. The results suggest that replication and division are precisely controlled with respect to mass accumulation, and the apparent variability of cell cycle events would only result from the use of the time of cell separation as a reference point for the definition of cell age rather than initiation or termination of replication.