Replication And Control Of Circular Bacterial Plasmids Pdf
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- Stress responses and replication of plasmids in bacterial cells
- Replicate Once Per Cell Cycle: Replication Control of Secondary Chromosomes
- Replication and Control of Circular Bacterial Plasmids
- Mechanisms of Theta Plasmid Replication.
All genes critical for plasmid replication regulation are located on the plasmid rather than on the host chromosome. In spite of this possibility, low copy number plasmids appear to exist stably in host populations.
Stress responses and replication of plasmids in bacterial cells
All genes critical for plasmid replication regulation are located on the plasmid rather than on the host chromosome. In spite of this possibility, low copy number plasmids appear to exist stably in host populations. We examined this paradox using a multilevel selection model. Simulations showed that, a slightly higher copy number mutant could out-compete the wild type.
Consequently, another mutant with still higher copy number could invade the first invader. However, the realized benefit of increasing intra-host fitness was saturating whereas that of inter-host fitness was exponential. As a result, above a threshold, intra-host selection was overcompensated by inter-host selection and the low copy number wild type plasmid could back invade a very high copy number plasmid.
This led to a rock-paper-scissor RPS like situation that allowed the coexistence of plasmids with varied copy numbers. Furthermore, another type of cheater that had lost the genes required for conjugation but could hitchhike on a conjugal plasmid, could further reduce the advantage of copy-up mutants. These sociobiological interactions may compliment molecular mechanisms of replication regulation in stabilizing the copy numbers.
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Competing interests: The authors have declared that no competing interests exist. Plasmids are extra chromosomal elements of circular DNA in bacteria, which replicate independent of the host genome. Plasmids exploit the machinery of host cell for their replication, but many of them carry useful or conditionally advantageous genes and therefore cannot be generalized as parasites.
Many experiments have shown that the host cell has to bear a cost for carrying a plasmid  , . The cost can be ameliorated by host parasite coevolution  — . Although there are a number of confounding factors, the cost can be generally assumed to increase with copy number and the length of the plasmid  , .
However, plasmids impart a wide range of unique features to the host by contributing to metabolic versatility and resistance to environmental factors  , . Therefore it has been argued that in the presence of positive selection for a plasmid borne gene the plasmid can be stable. However the presence of useful genes does not seem to be central to the evolution and stability of plasmids because a useful gene could ultimately be incorporated in the bacterial chromosome  saving the cost of carrying the plasmid.
Having such genes on plasmids make horizontal gene transfer possible coercing the cheaters to make the extracellular products . Although such systems can be robust to bacterial cheaters, they may not be robust to plasmid cheaters with the gene for the extracellular products deleted. Such plasmids will have an equal chance of horizontal transfer but the shorter plasmids could replicate faster and replace the wild type.
Therefore although many plasmids carry useful genes, that does not appear to be necessary and sufficient cause of plasmid stability. Furthermore, it has been suggested that in the absence of selection for any plasmid borne gene a plasmid free cell should have a selective advantage and the stability of the plasmid is difficult to explain. Factors such as spread of the plasmid by conjugal transfer to compensate for the loss in host fitness, compensatory mutations, co-evolution of host and plasmid to reduce the cost  —  or plasmid addiction  ,  are some of the possible mechanisms responsible for maintenance of a plasmid.
Many mathematical models have focused on the problem of persistence of plasmid in the host cells  ,  , . More perplexing is the problem of stability of copy number of a plasmid in the host cell. This is one of the many paradoxes in the evolution and stability of plasmids .
Most of the plasmids are considerably smaller than the chromosome in terms of length of DNA. Therefore if the same machinery is being used for replication, there can be many replication cycles of the plasmid per single replication of the chromosome. This can result into rapid escalation of plasmid numbers in a cell leading to increased metabolic burden on the host cell and eventually cell death .
This can be prevented only by tight regulation of plasmid replication  ,  , . Ironically, all critical genes involved in the regulation of plasmid replication are on the plasmid. This raises a potential evolutionary problem. A low copy number is optimal for long term survival of the plasmid in a host cell lineage.
However, any mutation that loosens the control and thereby increases the copy number has a short term advantage over the wild type although it may affect long term stability of the system.
Plasmids with copy numbers as high as are observed in natural populations  ,  indicating that the problem is not hypothetical alone. Presence of high copy number plasmids and the stability of low copy number plasmids in spite of the potential threat of invasion is very much a real life paradox.
A number of mechanisms exist for the regulation of plasmid replication and one or more of these mechanisms can control the plasmid replication separately or in combination  ,  — . Paulsson  recognized the conflict between two levels of selection in the control of plasmid copy numbers.
Plasmids that systematically over-replicate relative to their cell mates have a higher chance of fixing in a cell intra-host selection. However cells having such plasmids have to pay a greater cost and therefore are likely to be competed out by hosts with low copy numbers or ones without a plasmid inter-host selection.
Paulsson  suggested that intra-host selection should favor evolution of cis acting activators while inter-host selection favors evolution of retaliation by trans acting inhibitors of replication. However, such refinements in the mechanisms of replication control and reorganization of genes involved do not rule out the possibility as well as selective advantage of copy-up mutants. The demonstration of copy-up mutants in spite of multiple mechanisms of replication regulation  implies that the molecular mechanisms of replication regulation are not infallible and therefore unable to explain the evolutionary stability of copy numbers.
There can be multiple mechanisms by which a copy-up mutant can outcompete a low copy wild type. The effective growth rate of the mutant in terms of the number of replications per host cell cycle is greater than the wild type resulting into progressive dilution of the wild type. Since the wild type replication gets inhibited at a lower copy number and if the copy number control mechanism operates in response to the total copy number, the mutant can suppress the replication of the wild type.
Also if we assume that a random copy is transferred in a conjugational event, the probability of getting transferred will be higher for a copy-up mutant. Despite a multiplicity of potential advantages of high copy numbers, plasmids with low copy numbers are surprisingly common. We therefore need to look at other mechanisms for the stability of low copy number plasmids.
It is known that host cells already bearing a plasmid are at least partially immune to conjugal intake of another plasmid . This is a likely mechanism by which horizontal invasion by copy up mutants can be arrested.
It has not been critically examined theoretically or experimentally whether this mechanism is necessary and sufficient to prevent high copy number cheaters. Although a host cell lineage having a low copy number plasmid may be immune to conjugational invasion by another plasmid, mutants can nevertheless arise within this population and get transmitted vertically in the cell lineage as well as horizontally to plasmid free cells. In order to test whether this mechanism is necessary to prevent invasion by high copy number mutants, the best approach would be to start with a model without any such mechanism and see whether stability of low copy numbers is possible.
We explore here the possibility that copy numbers are selected by complex sociobiological interactions and are always in a dynamic steady state emerging out of conflicting levels of selection.
We use a multilevel selection approach similar to Paulsson  but instead of looking at the evolution of molecular mechanisms we focus on the long term population dynamics of different types of plasmid and host populations in a competitive environment.
Apart from copy numbers another type of social cheating is possible in plasmid populations. Plasmids spread by conjugal transfers and the genes for conjugal mechanisms are borne by plasmids. There is likely to be a cost to the plasmid in terms of increased replication time in carrying the conjugal genes and making the conjugal machinery would be a cost to the host cell. Non-conjugal plasmids can arise as cheaters in such a system and hitch-hike on conjugal plasmids during conjugal transfer.
Using our model system we also examine how the two types of cheaters would interact. We assumed four different variants or mutants of a plasmid. The wild type or lc is a low copy number plasmid with stringent control over replication and having conjugal abilities Table 1. Plasmid ln is a mutant with deletion of the gene cluster required for conjugation such as the tra gene complex but retains constraint on copy number.
Plasmid hc is a mutant with less constrained replication regulation resulting into higher copy number, but retaining the tra gene complex. Plasmid hn is a mutant of hc that has lost the tra gene complex. The host cells can be free of plasmid x 0 or have one of the 4 types of plasmids x lc , x ln , x hc , x hn respectively or can have multiple infections x m , see below.
The cost of carrying a plasmid which is assumed to be directly proportional to the copy number g lc , g ln , g hc , g hn affects the intrinsic growth rates of the five types of host populations.
The cost of carrying the tra gene complex is also assumed to affect the rate of replication of the plasmids as well as the host cells carrying them. Therefore the intrinsic growth rate of a host cell type r lc , r ln , r hc , r hn is calculated as the baseline fitness r 0 , assumed to be 1 minus the cost of carrying a particular plasmid and the cost of conjugation if the plasmid bears the conjugal machinery.
The growth rates g i reflect the competitive advantages of different types of plasmids. In the base line model we do not assume any plasmid incompatibility and multiple infections are permitted. The four types of plasmids could result in many possible combinations of plasmids in cells with multiple infections. However, for simplicity of the model we considered only a single category x m that represented the pooled population of all cells with multiple infections having different proportions p lc , p ln , p hc , p hn of the four plasmids.
For example if we make a limiting assumption that all types of plasmids co-occur in a single cell, the probability that a non-conjugal ln type of plasmid will get transferred by hitch-hiking on the conjugal plasmid lc can be assumed to be a function of the proportion of lc and that of ln.
If on the other hand we assume that plasmid types co-occur only in pairs then a non-conjugal plasmid can get hitch hiked only when it pairs with a conjugal plasmid. Thus both the limiting assumptions give rise to very similar mathematical forms. Therefore we believe that treating multiple infections as a single category is unlikely to bias the results. Treating every possible combination of plasmids in mixed infection will add 11 more population types making the model highly complex and therefore to avoid the complexity we merged them into a single category.
The fitness of multiple infected cell r m was calculated by taking the weighted average cost of all plasmids. The assumption that plasmid curing occurred at a constant rate was not completely arbitrary. For plasmids with higher copy number the probability of curing due to stochastic unequal segregation is low by chance alone while for plasmids with low copy number selection favors evolution of efficient partition system reducing the chances of spontaneous curing .
As a result, the plasmid curing rate need not scale with copy number. Conjugation and curing is assumed to result in the transformation of one type of cell to another as given in Figure 1. We assume that only one type of plasmid is transferred or cured at a time so that a cell does not go from an uninfected state to a multiple infection state in a single step. Also, a multiple infected cell does not become completely cured of plasmids in single time step. Only one plasmid is assumed to get transferred or get cured at a time.
Therefore x 0 cannot be directly transformed into x m and vice versa. Black arrows indicate conjugation and grey straight arrow indicates curing of plasmid.
Curved grey arrows are intrinsic growth rates of respective cell types. Symbols used for the model are explained in Table 1. The dynamics of transfer of one host to another host can be realized from Table 2. Based on Table 2 we can write the differential equations which govern the change in the frequency of different types of cells by adding all the terms that contribute to the change in the cell type.
For instance, the differential equation for a plasmid free cell can be given as, Similarly, the dynamics of change in the proportions of plasmids p lc , p ln , p hc , p hn in the case of multiple infections can be written based on Table 3.
Analytical solutions were possible when only one type of plasmid or two types of host cells were present. Considering all plasmid types and cell types was difficult to study analytically owing to the inherent complexity of the model.
Replicate Once Per Cell Cycle: Replication Control of Secondary Chromosomes
A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria ; however, plasmids are sometimes present in archaea and eukaryotic organisms. In nature, plasmids often carry genes that benefit the survival of the organism and confer selective advantage such as antibiotic resistance. While chromosomes are large and contain all the essential genetic information for living under normal conditions, plasmids are usually very small and contain only additional genes that may be useful in certain situations or conditions. Artificial plasmids are widely used as vectors in molecular cloning , serving to drive the replication of recombinant DNA sequences within host organisms. In the laboratory, plasmids may be introduced into a cell via transformation.
Faithful vertical transmission of genetic information, especially of essential core genes, is a prerequisite for bacterial survival. Hence, replication of all the replicons is tightly controlled to ensure that all daughter cells get the same genome copy as their mother cell. Essential core genes are very often carried by the main chromosome. However they can occasionally be found on secondary chromosomes, recently renamed chromids. Chromids have evolved from non-essential megaplasmids, and further acquired essential core genes and a genomic signature closed to that of the main chromosome. All chromids carry a plasmidic replication origin, belonging so far to either the iterons or repABC type. Based on these differences, two categories of chromids have been distinguished.
Metrics details. Plasmids, DNA or rarely RNA molecules which replicate in cells autonomously independently of chromosomes as non-essential genetic elements, play important roles for microbes grown under specific environmental conditions as well as in scientific laboratories and in biotechnology. For example, bacterial plasmids are excellent models in studies on regulation of DNA replication, and their derivatives are the most commonly used vectors in genetic engineering. Detailed mechanisms of replication initiation, which is the crucial process for efficient maintenance of plasmids in cells, have been elucidated for several plasmids. However, to understand plasmid biology, it is necessary to understand regulation of plasmid DNA replication in response to different environmental conditions in which host cells exist. Knowledge of such regulatory processes is also very important for those who use plasmids as expression vectors to produce large amounts of recombinant proteins.
This is solved, in circular plasmids, by two main strategies: (i) opening of the strands followed by RNA priming (theta and strand displacement replication) or (ii).
Replication and Control of Circular Bacterial Plasmids
DoriC, a database of replication origins, was initially created to present the bacterial oriC s predicted by Ori-Finder or determined by experiments in DoriC 5. In the current release, the database of DoriC has made significant improvements compared with version 5. Now, DoriC becomes the most complete and scalable database of replication origins in prokaryotic genomes, and facilitates the studies in large-scale oriC data mining, strand-biased analyses and replication origin predictions.
Mechanisms of Theta Plasmid Replication.
An essential feature of bacterial plasmids is their ability to replicate as autonomous genetic elements in a controlled way within the host. Therefore, they can be used to explore the mechanisms involved in DNA replication and to analyze the different strategies that couple DNA replication to other critical events in the cell cycle. In this review, we focus on replication and its control in circular plasmids.
Skip to search form Skip to main content You are currently offline. Some features of the site may not work correctly. DOI: Giraldo and M. Espinosa and R. SUMMARY An essential feature of bacterial plasmids is their ability to replicate as autonomous genetic elements in a controlled way within the host.
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