Natural selection, infectious transfer and the existence conditions for bacterial plasmids

Despite the near-ubiquity of plasmids in bacterial populations and the profound contribution of infectious gene transfer to the adaptation and evolution of bacteria, the mechanisms responsible for the maintenance of plasmids in bacterial populations are poorly understood. In this article, we address the question of how plasmids manage to persist over evolutionary time. Empirical studies suggest that plasmids are not infectiously transmitted at a rate high enough to be maintained as genetic parasites. In part i, we present a general mathematical proof that if this is the case, then plasmids will not be able to persist indefinitely solely by carrying genes that are beneficial or sometimes beneficial to their host bacteria. Instead, such genes should, in the long run, be incorporated into the bacterial chromosome. If the mobility of host-adaptive genes imposes a cost, that mobility will eventually be lost. In part ii, we illustrate a pair of mechanisms by which plasmids can be maintained indefinitely even when their rates of transmission are too low for them to be genetic parasites. First, plasmids may persist because they can transfer locally adapted genes to newly arriving strains bearing evolutionary innovations, and thereby preserve the local adaptations in the face of background selective sweeps. Second, plasmids may persist because of their ability to shuttle intermittently favored genes back and forth between various (noncompeting) bacterial strains, ecotypes, or even species.     

Borrelia genomes in the year 2000

All analyzed members of the spirochete genus Borrelia contain a linear chromosome about 910 kbp long. The complete sequence of the B. burgdorferi B31 genome predicts that its chromosome carries essentially all of this organism's housekeeping genes. In accordance with these bacterial species' obligatory parasitic lifestyle, its genes encode enzymes that are capable of only a minimal metabolism, in which all nucleotides, amino acids, fatty acids and enzyme cofactors must be scavenged from the host. In addition to the chromosome, all Borrelia isolates examined carry multiple linear and circular plasmids with lengths between 5 and 200 kbp. The plasmids, which account for over 600 kbp in isolate B31, carry very few genes with homology to genes outside of the Borrelia genus. But they do carry numerous predicted lipoprotein genes, many of which are have been shown to be or are expected to be outer surface proteins. Ten of the linear plasmids have strikingly low protein coding potential for bacterial DNA. These plasmids have enjoyed numerous past duplicative rearrangements, which have resulted in the presence of a substantial fraction of the DNA that appears to be currently undergoing mutational decay, presumably because it is no longer under selection for function.     

Plasmids foster diversification and adaptation of bacterial populations in soil

It is increasingly being recognized that the transfer of conjugative plasmids across species boundaries plays a vital role in the adaptability of bacterial populations in soil. There are specific driving forces and constraints of plasmid transfer within bacterial communities in soils. Plasmid-mediated genetic variation allows bacteria to respond rapidly with adaptive responses to challenges such as irregular antibiotic or metal concentrations, or opportunities such as the utilization of xenobiotic compounds. Cultivation-independent detection and capture of plasmids from soil bacteria, and complete sequencing have provided new insights into the role and ecology of plasmids. Broad host range plasmids such as those belonging to IncP-1 transfer a wealth of accessory functions which are carried by similar plasmid backbones. Plasmids with a narrower host range can be more specifically adapted to particular species and often transfer genes which complement chromosomally encoded functions. Plasmids seem to be an ancient and successful strategy to ensure survival of a soil population in spatial and temporal heterogeneous conditions with various environmental stresses or opportunities that occur irregularly or as a novel challenge in soil.     

Rule-based modelling of conjugative plasmid transfer and incompatibility

COSMIC-rules, an individual-based model for bacterial adaptation and evolution, has been used to study virtual transmission of plasmids within bacterial populations, in an environment varying between supportive and inhibitory. The simulations demonstrate spread of antibiotic resistance (R) plasmids, both compatible and incompatible, by the bacterial gene transfer process of conjugation. This paper describes the behaviour of virtual plasmids, their modes of exchange within bacterial populations and the impact of antibiotics, together with the rules governing plasmid transfer. Three case studies are examined: transfer of an R plasmid within an antibiotic-susceptible population, transfer of two incompatible R plasmids and transfer of two compatible R plasmids. R plasmid transfer confers antibiotic resistance on recipients. For incompatible plasmids, one or other plasmid could be maintained in bacterial cells and only that portion of the population acquiring the appropriate plasmid-encoded resistance survives exposure to the antibiotics. By contrast, the compatible plasmids transfer and mix freely within the bacterial population that survives in its entirety in the presence of the antibiotics. These studies are intended to inform models for examining adaptive evolution in bacteria. They provide proof of principle in simple systems as a platform for predicting the behaviour of bacterial populations in more complex situations, for example in response to changing environments or in multi-species bacterial assemblages.     

THE EVOLUTION OF TRANSPOSABLE ELEMENTS: CONDITIONS FOR ESTABLISHMENT IN BACTERIAL POPULATIONS

Previous theoretical studies have shown that bacterial transposons can become established in populations by infectious transfer, even if they reduce the fitness of their host cells. Conditions for the persistence of “parasitic” transposons are, however, restrictive: i) transposition must be replicative, rather than conservative; ii) the rate of transposition must be greater than the loss in host fitness caused by the transposon; and iii) cells must exchange plasmids at rates greater than the fitness cost of the transposon. I sought to test the validity of the model underlying this theory by performing experiments with laboratory populations of the bacterium Escherichia coli, the conjugative plasmid R100, and the transposons Tn3 and Tn5. A plasmid-borne transposon was introduced at low frequency into a population of bacteria carrying the same plasmid without the transposon in a habitat where the transposon offered no benefit to its host. The fate of the invading transposon was followed by tracking the various bacterial populations appearing in the cultures. Using independent estimates of the parameters of the model, predicted population changes were generated with numerical solutions of the model, and these were compared to experimental results. Plasmids transferred into new hosts as predicted by the model, and the resulting transconjugant populations either maintained a steady low density or rose slowly in abundance. Transposition appeared to play no role in population changes. Abundance of all cell types fit theoretical predictions of a system with no transposition, despite evidence that transposition was taking place. This is exactly what the model predicted. It thus appears unlikely that deleterious or neutral transposons have much impact on the genetics of bacterial populations. This is consistent with the hypothesis that most bacterial transposons are not parasitic DNA, but rather invade and persist in populations by providing a fitness advantage to cells carrying them.     

The Population Biology of Bacterial Plasmids: A PRIORI Conditions for the Existence of Conjugationally Transmitted Factors

A mathematical model for the population dynamics of conjugationally transmitted plasmids in bacterial populations is presented and its properties analyzed. Consideration is given to nonbacteriocinogenic factors that are incapable of incorporation into the chromosome of their host cells, and to bacterial populations maintained in either continuous (chemostat) or discrete (serial transfer) culture. The conditions for the establishment and maintenance of these infectious extrachromosomal elements and equilibrium frequencies of cells carrying them are presented for different values of the biological parameters: population growth functions, conjugational transfer and segregation rate constants. With these parameters in a biologically realistic range, the theory predicts a broad set of physical conditions, resource concentrations and dilution rates, where conjugationally transmitted plasmids can become established and where cells carrying them will maintain high frequencies in bacterial populations. This can occur even when plasmid-bearing cells are much less fit (i.e., have substantially lower growth rates) than cells free of these factors. The implications of these results and the reality and limitations of the model are discussed and the values of its parameters in natural populations speculated upon.     

Transitory Derepression and the Maintenance of Conjugative Plasmids

It has been proposed that bacterial plasmids cannot be maintained by infectious transfer alone and that their persistence requires positive selection for plasmid-borne genes. To test this hypothesis, the population dynamics of two laboratory and five naturally occurring conjugative plasmids were examined in chemostat cultures of E. coli K-12. Both laboratory plasmids and three of the five wild plasmids failed to increase in frequency when introduced at low frequencies. However, two of the naturally occurring plasmids rapidly increased in frequency, and bacteria carrying them achieved dominance in the absence of selection for known plasmid-borne genes. Three hypotheses for the invasion and persistence of these two plasmids were examined. It is concluded that although these two extrachromosomal genetic elements are repressed for conjugative pili synthesis, as a consequence of high rates of transfer during periods of transitory derepression in newly formed transconjugants, they become established and are maintained by infectious transfer alone. The implications of these observations to the theory of plasmid maintenance and the evolution of repressible conjugative pili synthesis are discussed.     

Diverse Broad-Host-Range Plasmids from Freshwater Carry Few Accessory Genes

Broad-host-range self-transferable plasmids are known to facilitate bacterial adaptation by spreading genes between phylogenetically distinct hosts. These plasmids typically have a conserved backbone region and a variable accessory region that encodes host-beneficial traits. We do not know, however, how well plasmids that do not encode accessory functions can survive in nature. The goal of this study was to characterize the backbone and accessory gene content of plasmids that were captured from freshwater sources without selecting for a particular phenotype or cultivating their host. To do this, triparental matings were used such that the only required phenotype was the plasmid''s ability to mobilize a nonconjugative plasmid. Based on complete genome sequences of 10 plasmids, only 5 carried identifiable accessory gene regions, and none carried antibiotic resistance genes. The plasmids belong to four known incompatibility groups (IncN, IncP-1, IncU, and IncW) and two potentially new groups. Eight of the plasmids were shown to have a broad host range, being able to transfer into alpha-, beta-, and gammaproteobacteria. Because of the absence of antibiotic resistance genes, we resampled one of the sites and compared the proportion of captured plasmids that conferred antibiotic resistance to their hosts with the proportion of such plasmids captured from the effluent of a local wastewater treatment plant. Few of the captured plasmids from either site encoded antibiotic resistance. A high diversity of plasmids that encode no or unknown accessory functions is thus readily found in freshwater habitats. The question remains how the plasmids persist in these microbial communities.     

Coexistence of incompatible plasmids in a bacterial population living under a feast and famine regime

A model is formulated to examine the possibility of (co)existence of plasmids of the same incompatibility and surface exclusion group in a bacterial population living under a feast-and-famine regime. The condition is given under which a growth rate decreasing plasmid can invade a bacterial population. It appears that in case only one plasmid type is present, the frequency of plasmid bearers will tend to a stable equilibrium if the food supply at each growth site gets exhausted and if both plasmid-free and plasmid-bearing bacteria need an equal quantity of food per cell division. If these two conditions are not satisfied, the frequency of plasmid-bearers might oscillate. Two plasmids will sometimes be able to coexist, but only if they follow different survival strategies; one with a high conjugational transfer rate and a lower fitness of its host, and the other with a low transfer rate and a higher host fitness. Coexistence of three plasmids of the same surface exclusion group is impossible.     

Modeling the ecology of parasitic plasmids

Plasmids are autonomous genetic elements that can be exchanged between microorganisms via horizontal gene transfer (HGT). Despite the central role they play in antibiotic resistance and modern biotechnology, our understanding of plasmids’ natural ecology is limited. Recent experiments have shown that plasmids can spread even when they are a burden to the cell, suggesting that natural plasmids may exist as parasites. Here, we use mathematical modeling to explore the ecology of such parasitic plasmids. We first develop models of single plasmids and find that a plasmid’s population dynamics and optimal infection strategy are strongly determined by the plasmid’s HGT mechanism. We then analyze models of co-infecting plasmids and show that parasitic plasmids are prone to a “tragedy of the commons” in which runaway plasmid invasion severely reduces host fitness. We propose that this tragedy of the commons is averted by selection between competing populations and demonstrate this effect in a metapopulation model. We derive predicted distributions of unique plasmid types in genomes—comparison to the distribution of plasmids in a collection of 17,725 genomes supports a model of parasitic plasmids with positive plasmid–plasmid interactions that ameliorate plasmid fitness costs or promote the invasion of new plasmids.Subject terms: Theoretical ecology, Microbial ecology     

The Population Biology of Bacterial Plasmids: A PRIORI Conditions for the Existence of Mobilizable Nonconjugative Factors

A mathematical model for the population dynamics of nonconjugative plasmids that can be mobilized by conjugative factors is presented. In the analysis of the properties of this model, primary consideration is given to the conditions under which these nonself-transmissible extrachromosomal elements could become established and would be maintained in bacterial populations. The results of this analysis demonstrate the existence of conditions where, as a consequence of infectious transmission via mobilization, nonconjugative plasmids could become established and be maintained even when the bacteria carrying them have lower reproductive fitnesses than plasmid-free members of the population. However, these existence conditions are stringent and suggest therefore, that it is highly unlikely that plasmids of this type would become established and maintained without some direct selection favoring their carriage. The general implications of these results and limitations of the model are discussed. Brief consideration is also given to the implications of these theoretical findings to the problems of the spread of multiple antibiotic resistance plasmids (R-factors) and the risk of contaminating natural populations of bacteria with chimeric plasmids produced by work with recombinant DNA.     

Broad host range plasmids can invade an unexpectedly diverse fraction of a soil bacterial community

Conjugal plasmids can provide microbes with full complements of new genes and constitute potent vehicles for horizontal gene transfer. Conjugal plasmid transfer is deemed responsible for the rapid spread of antibiotic resistance among microbes. While broad host range plasmids are known to transfer to diverse hosts in pure culture, the extent of their ability to transfer in the complex bacterial communities present in most habitats has not been comprehensively studied. Here, we isolated and characterized transconjugants with a degree of sensitivity not previously realized to investigate the transfer range of IncP- and IncPromA-type broad host range plasmids from three proteobacterial donors to a soil bacterial community. We identified transfer to many different recipients belonging to 11 different bacterial phyla. The prevalence of transconjugants belonging to diverse Gram-positive Firmicutes and Actinobacteria suggests that inter-Gram plasmid transfer of IncP-1 and IncPromA-type plasmids is a frequent phenomenon. While the plasmid receiving fractions of the community were both plasmid- and donor- dependent, we identified a core super-permissive fraction that could take up different plasmids from diverse donor strains. This fraction, comprising 80% of the identified transconjugants, thus has the potential to dominate IncP- and IncPromA-type plasmid transfer in soil. Our results demonstrate that these broad host range plasmids have a hitherto unrecognized potential to transfer readily to very diverse bacteria and can, therefore, directly connect large proportions of the soil bacterial gene pool. This finding reinforces the evolutionary and medical significances of these plasmids.     

Investigating the impact of bisphosphonates and structurally related compounds on bacteria containing conjugative plasmids

Bacterial plasmids propagate through microbial populations via the directed process of conjugative plasmid transfer (CPT). Because conjugative plasmids often encode antibiotic resistance genes and virulence factors, several approaches to inhibit CPT have been described. Bisphosphonates and structurally related compounds (BSRCs) were previously reported to disrupt conjugative transfer of the F (fertility) plasmid in Escherichia coli. We have further investigated the effect of these compounds on the transfer of two additional conjugative plasmids, pCU1 and R100, between E. coli cells. The impact of BSRCs on E. coli survival and plasmid transfer was found to be dependent on the plasmid type, the length of time the E. coli were exposed to the compounds, and the ratio of plasmid donor to plasmid recipient cells. Therefore, these data indicate that BSRCs produce a range of effects on the conjugative transfer of bacterial plasmids in E. coli. Since their impact appears to be plasmid type-dependent, BSRCs are unlikely to be applicable as broad inhibitors of antibiotic resistance propagation.     

Species differences in plasmid carriage in the Enterobacteriaceae

Modern concerns about the spread of antibiotic resistance raise questions about the effect of bacterial species on plasmid evolution and maintenance. We studied 223 Enterobacteriaceae isolated from wild mammals and determined the number of plasmids per isolate, the size of those plasmids, and the distribution of plasmid incompatibility groups N, P, W, FII, and A/C. All of these variables were non-randomly distributed with respect to bacterial species, suggesting that host-cell factors constrain the plasmids that a strain will carry. The implication for the evolution of multiple-resistance plasmids in a clinical setting is that although inter-generic plasmid transfer may introduce a novel resistance plasmid into a bacterial genus, it is likely to be modified to suit the requirements of the new host cell. This then further suggests that resistance plasmids will evolve independent lineages within bacterial species although the genes incorporated in them may have come from the same original source.     

Combinational variation of restriction modification specificities in Lactococcus lactis

Three genes coding for a type I R-M system related to the class C enzymes have been identified on the chromosome of Lactococcus lactis strain IL1403. In addition, plasmids were found that encode only the HsdS subunit that directs R-M specificity. The presence of these plasmids in IL1403 conferred a new R-M phenotype on the host, indicating that the plasmid-encoded HsdS is able to interact with the chromosomally encoded HsdR and HsdM subunits. Such combinational variation of type I R-M systems may facilitate the evolution of their specificity and thus reinforce bacterial resistance against invasive foreign unmethylated DNA.     

Mobilizable narrow host range plasmids as natural suicide vectors enabling horizontal gene transfer among distantly related bacterial species

Klebsiella pneumoniae 287-w carries three small narrow host range (NHR) plasmids (pIGMS31, pIGMS32, and pIGRK), which could be maintained in several closely related species of Gammaproteobacteria, but not in Alphaproteobacteria. The plasmids contain different mobilization systems (MOB), whose activity in Escherichia coli was demonstrated in the presence of the helper transfer system originating from plasmid RK2. The MOBs of pIGMS31 and pIGMS32 are highly conserved in many bacterial plasmids (members of the MOB family), while the predicted MOB of pIGRK has a unique structure, encoding a protein similar to phage-related integrases. The MOBs of pIGMS31 and pIGMS32 enabled the transfer of heterologous replicons from E. coli into both gammaproteobacterial and alphaproteobacterial hosts, which suggests that these NHR plasmids contain broad host range MOB systems. Such plasmids therefore represent efficient carrier molecules, which may act as natural suicide vectors promoting the spread of diverse genetic information (including other types of mobile elements, e.g. resistance transposons) among evolutionarily distinct bacterial species. Thus, mobilizable NHR plasmids may play a much more important role in horizontal gene transfer than previously thought.     

Origin and evolution of plasmids

Studies on the origin and evolution of plasmids may provide valuable insights on the promiscuous nature of DNA. The first examples of the selfish nature of nucleic acids are exemplified by primordial oligoribonucleotides which evolved into primitive replicons. The propagation of these molecules were likely patterned after the current viral RNA ribozymes, which have been recently shown to possess RNA synthesizing and template mediated polymerizing capabilities in the absence of proteins. The parasitic nature of nucleic acids is depicted by satellite nucleic acid molecules associated with viruses. The satellites of adenovirus and tobacco ringspot virus serve as established examples: they contain no open reading frames. Comparative analysis of the replication origins of virions and plasmids show them to be conserved, originating from the simplest autocatalytic replicon to highly complex and evolved plasmids, replicating by a rolling circle mechanism. The eventual association of proteins with nucleic acids provided added efficiency and protective advantages for molecular perpetuation. The promiscuous and selfish nature of plasmids is demonstrated by their ability to genetically engineer their host so that the host cell is best able to cope and survive in hostile environments. Survival of the host ensures survival of the plasmid. Sequestering of genes by plasmids occurs when the environmental conditions negatively affect the host. The sequestering mechanism is fundamental and forms the outreach mechanisms to generate and propagate macromolecules of increasing size when necessary for survival. The level of sophistication of plasmids increases with the addition of new genes such as those that allow the host to occupy a specific environment normally inhospitable to the host cell. The vast range of plasmid types which have obtained genes interchangeably reflect the levels of sophistication achieved by these macromolecules. The Ti plasmid in Agrobacterium tumefaciens and the pSym and accessory plasmids in Rhizobium illustrate the level of complexity attained by replicons.     

Five genes involved in self-transmission of pSN22, a Streptomyces plasmid.

An 11-kbp multicopy plasmid, pSN22, was isolated from Streptomyces nigrifaciens SN22. pSN22 is self-transmissible (conjugative), is maintained stably in S. lividans, and forms pocks in a wide range of Streptomyces strains. Mutational analyses showed that a fragment of pSN22 contained five genes involved in plasmid transfer and pock formation. traB was essential for plasmid transfer. traA was required for pock formation, but not for plasmid transfer. spdA or spdB were concerned with pock size; mutations in these genes decreased pock size. The fifth gene, traR, could be deleted together with other genes to give nontransmissible plasmids, but plasmids with insertions or deletions only within traR became nonviable. traR is probably needed to counterbalance the lethal effects of another plasmid gene. Transfer of pSN22 promoted the cotransfer of nontransmissible plasmids and enhanced chromosome recombination between the host and recipient strains, suggesting that plasmid transfer accompanies cytoplasmic mixing.