Rate of plasmid transfer among Escherichia coli strains isolated from natural populations.

The conjugative plasmid R1 was introduced into ten strains of Escherichia coli isolated from natural populations. Spontaneous nalidixic-acid-resistant mutants of the ten strains served as recipients. The ten donor and recipient strains were mated in all combinations and the rate at which R1 transferred between the strains was determined. The rate of transfer ranged from 5.2 x 10(-11)-1.1 x 10(-18) ml per cell h-1, and averaged 1.3 x 10(-15) ml per cell h-1. The results of these experiments suggest that the rates of conjugative transfer are far too low for plasmids to be maintained as parasites in their host populations. Infectious transfer is insufficient; plasmids must confer a selective advantage to their host to be maintained.     

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.     

Plasmids spread very fast in heterogeneous bacterial communities

Conjugative plasmids can mediate gene transfer between bacterial taxa in diverse environments. The ability to donate the F-type conjugative plasmid R1 greatly varies among enteric bacteria due to the interaction of the system that represses sex-pili formations (products of finOP) of plasmids already harbored by a bacterial strain with those of the R1 plasmid. The presence of efficient donors in heterogeneous bacterial populations can accelerate plasmid transfer and can spread by several orders of magnitude. Such donors allow millions of other bacteria to acquire the plasmid in a matter of days whereas, in the absence of such strains, plasmid dissemination would take years. This "amplification effect" could have an impact on the evolution of bacterial pathogens that exist in heterogeneous bacterial communities because conjugative plasmids can carry virulence or antibiotic-resistance genes.     

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.     

Amelioration of the cost of conjugative plasmid carriage in Eschericha coli K12

Although plasmids can provide beneficial functions to their host bacteria, they might confer a physiological or energetic cost. This study examines how natural selection may reduce the cost of carrying conjugative plasmids with drug-resistance markers in the absence of antibiotic selection. We studied two plasmids, R1 and RP4, both of which carry multiple drug resistance genes and were shown to impose an initial fitness cost on Escherichia coli. To determine if and how the cost could be reduced, we subjected plasmid-containing bacteria to 1100 generations of evolution in batch cultures. Analysis of the evolved populations revealed that plasmid loss never occurred, but that the cost was reduced through genetic changes in both the plasmids and the bacteria. Changes in the plasmids were inferred by the demonstration that evolved plasmids no longer imposed a cost on their hosts when transferred to a plasmid-free clone of the ancestral E. coli. Changes in the bacteria were shown by the lowered cost when the ancestral plasmids were introduced into evolved bacteria that had been cured of their (evolved) plasmids. Additionally, changes in the bacteria were inferred because conjugative transfer rates of evolved R1 plasmids were lower in the evolved host than in the ancestral host. Our results suggest that once a conjugative bacterial plasmid has invaded a bacterial population it will remain even if the original selection is discontinued.     

Stability and dynamics of R-plasmids in Escherichia coli populations in continuous cultivation

The stability of the conjugative plasmid RP4 and the nonconjugative plasmid pBS94 in Escherichia coli C600 cells containing both plasmids was studied in continuous cultivation under chemostat and pH-stat conditions. The plasmids remained stable in the cells of the bacterial population for 100 generations, and no cells were found without the plasmids. The competition between strains with and without the plasmids in a mixed culture resulted in the removal of the plasmid-free strain from the population. In these experiments, conjugative transfer of plasmids into the plasmid-free strain was observed, and co-transfer of both plasmids was more effective under the pH-stat conditions.     

The segregation of the 2 mu-based yeast plasmid pJDB248 breaks down under conditions of slow, glucose-limited growth

The stability of the 2 mu-based yeast plasmid pJDB248 in Saccharomyces cerevisiae S150-2B(cir0) was investigated in glucose-limited chemostat culture. Plasmid-free cells were detected by loss of (plasmid-encoded) leucine prototrophy and confirmed by colony hybridization. The plasmid was considerably more stable at a high dilution rate (0.12 h-1) than at a lower dilution rate (0.05 h-1). The average plasmid copy number in the cells retaining the plasmid remained constant at approximately 50 in the high dilution rate culture whereas it rose to almost 600 in the slow dilution rate culture. However, in both cultures the overall plasmid level in the total population remained constant, indicating that plasmid segregation breaks down at the low growth rate. Similar experiments on the native 2 mu plasmid demonstrated high stability and no significant differences between the high and low growth rate cultures. It is postulated that the difference in behaviour between the native and chimeric plasmids is related to an interaction between the growth conditions and the loss of the D gene product.     

Mobilization of R387 Inc K conjugative plasmid by the conjugative plasmid R64 Inc I

Plasmid aggregate (R387, R64) was constructed in E. coli K12 strain. Plasmid R387 Inc K was stimulated to conjugational transfer by plasmid R64 Inc I. This stimulation was caused neither by recombination between both plasmids nor by trans-complementation of R387 conjugational systems by gene(s) product(s) of R64 plasmid. The observed phenomenon resembled rather mobilization of nonconjugative plasmids by conjugative ones. As in mobilization, the observed increase in R387 transfer frequency could take place only when both interacting plasmids were present in donor cells. Moreover, the entry exclusion system functioning in recipient cells, toward stimulating R64 plasmid affected strongly the conjugational transfer of stimulated R387 plasmid. Analogous phenomenon was observed during mobilization of nonconjugative plasmids by conjugative ones.     

In situ monitoring of IncF plasmid transfer on semi-solid agar surfaces reveals a limited invasion of plasmids in recipient colonies

Most natural conjugative IncF plasmids encode a fertility inhibition system that represses transfer gene expression in the majority of plasmid-carrying cells. The successful spread of these plasmids in clinically relevant bacteria has been suggested to be supported by a transitory derepression of transfer gene expression in newly formed transconjugants. In this study, we aimed to monitor the extent of transitory derepression during agar surface matings in situ by comparing plasmid spread of the IncF plasmid R1 and its derepressed mutant R1drd19 at low initial cell densities. A zygotic induction strategy was used to visualize the spatial distribution of fluorescent transconjugants within the heterogeneous environment. Epifluorescence and confocal microscopy revealed different transfer patterns for both plasmids, however, spread beyond the first five recipient cell layers adjacent to the donor cells was not observed. Similar results were observed for other prototypical conjugative plasmids. These results cannot rule out that transitory derepression contributes to the limited R1 plasmid invasion, but other factors like nutrient availability or spatial structure seem to limit plasmid spread.     

Conjugation is necessary for a bacterial plasmid to survive under protozoan predation

Horizontal gene transfer by conjugative plasmids plays a critical role in the evolution of antibiotic resistance. Interactions between bacteria and other organisms can affect the persistence and spread of conjugative plasmids. Here we show that protozoan predation increased the persistence and spread of the antibiotic resistance plasmid RP4 in populations of the opportunist bacterial pathogen Serratia marcescens. A conjugation-defective mutant plasmid was unable to survive under predation, suggesting that conjugative transfer is required for plasmid persistence under the realistic condition of predation. These results indicate that multi-trophic interactions can affect the maintenance of conjugative plasmids with implications for bacterial evolution and the spread of antibiotic resistance genes.     

Genetic properties of the Salmonella enteritidis R404 plasmid aggregate. II. Separation of plasmids by transformation.

Transformational separation of plasmids from R404 plasmid aggregate found in Salmonella enteritidis strain was performed. Three classes of transformants differing in their resistance patterns were isolated. Genetic properties of the transformants suggest that their resistance is determined by single plasmids. Plasmid pCK3 (Tra-ApCbCrSuSm) and pCK4 (Tra-ApCbCrCm) are nonconjugative while plasmid pCG1 (TraApCbCrSuSmTcKmNm) is conjugative. Separation of all plasmids of R404 plasmid aggregate allowed to determine their genetic properties and the manner of conjugational transfer of R404 plasmid aggregate R-determinants.     

Chromosomally integrated conjugative plasmids are common in antibiotic-resistant Haemophilus influenzae.

Twenty-three highly antibiotic-resistant strains of Haemophilus influenzae and two of Haemophilus parainfluenzae without detectable large plasmids were examined for conjugative transfer of their resistance to H. influenzae strain Rd or to other strains. Very inefficient transfer was observed for 18 H. influenzae strains and 1 H. parainfluenzae strain. All H. influenzae transcipients carried a large plasmid, and they were in turn efficient donors of their resistances in standard conjugation crosses with isogenic recipients. This was not seen for the H. parainfluenzae transcipients. It is concluded that most of the original antibiotic-resistant cultures carried an integrated conjugative R plasmid which had been excised in a few cells in each population. It was these cells which transferred resistance in the primary crosses.     

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.     

TraM of plasmid R1 controls transfer gene expression as an integrated control element in a complex regulatory network

Site-directed mutagenesis was used to investigate the functions of the traM gene in plasmid R1-mediated bacterial conjugation. Three mutant alleles, a null mutation, a sense mutation and a stop mutation, were recombined back into the R1-16 plasmid, a transfer-derepressed ( finO  ⁻) variant of plasmid R1. The frequency of conjugative transfer of the traM null mutant derivative of R1-16 was 10⁷-fold lower than that of the isogenic parent plasmid, showing the absolute requirement for this gene in conjugative transfer of plasmid R1. Measurements of the abundance of plasmid specified traJ , traA and traM mRNAs, TraM protein levels, and complementation studies indicated that the traM gene of plasmid R1 has at least two functions in conjugation: (i) positive control of transfer gene expression; and (ii) a function in a process distinct from gene expression. Since expression of the negatively autoregulated traM gene is itself affected positively by the expression of the transfer operon genes, this gene constitutes a decisive element within a regulatory circuit that co-ordinates expression of the genes necessary for horizontal DNA transfer. Based on our studies, we present a novel model for the regulation of the transfer genes of plasmid R1 that might also be applicable to other IncF plasmids.     

Mathematical modeling of the evolution of bacterial populations in continuous cultures with regard to the nonmutagenic variability of genome

A mathematical model of the evolution of the genetic structure of the bacterial population during prolonged cultivation in a chemostat has been constructed. In addition to genetic mutations, some factors of the nonmutagenic variability of genome were taken into account, namely, the structural reorganization of plasmid and virus DNA, the DNA loss due to cellular division, the conjugative transfer of plasmids, and the plasmid replication. The general model also takes into account the formation of cellular aggregates during conjugation. The results of numerical and analytical investigation of the special cases of the general model were treated. Simplified mathematical models are considered, which can be used to explain the experimentally observed evolutionary variations resulting from the plurality of evolution attractors, multi-stage microevolutionary transitions, the semi-stable states of the bacterial population genome, and the undamped oscillations of the genetic structure of the population during prolonged cultivation in the chemostat.     

Impact of antibiotics on conjugational resistance gene transfer in Staphylococcus aureus in sewage

The growing rate of microbial pathogens becoming resistant to standard antibiotics is an important threat to public health. In order to assess the role of antibiotics in the environment on the spread of resistance factors, the impact of subinhibitory concentrations of antibiotics in sewage on gene transfer was investigated using conjugative gentamicin resistance (aacA-aphD) plasmids of Staphylococcus aureus. Furthermore, the concentration of antibiotics in hospital sewage was measured by high-performance liquid chromatography (HPLC)-electrospray tandem mass spectrometry. Several antibiotics were found to be present in sewage, e.g. ciprofloxacin up to 0.051 mgl(-1) and erythromycin up to 0.027 mgl(-1). Resistance plasmid transfer occurred both on solidified (dewatered) sewage and in liquid sewage in a bioreactor with a frequency of 1.1x10(-5)-5.0x10(-8). However, low-level concentrations of antibiotics measured in sewage are below concentrations that can increase plasmid transfer frequencies of gentamicin resistance plasmids of staphylococci.     

The evolution of a conjugative plasmid and its ability to increase bacterial fitness

Conjugative plasmids are extra-chromosomal DNA elements that are capable of horizontal transmission and are found in many natural isolated bacteria. Although plasmids may carry beneficial genes to their bacterial host, they may also cause a fitness cost. In this work, we studied the evolution of the R1 plasmid and we found that, in spite of the R1 plasmid conferring an initial cost to its host, after 420 generations the cost disappeared in all five independent evolution experiments. In fact, in two of these five experiments evolved conjugative plasmids actually conferred a fitness advantage to their hosts. Furthermore, the relative fitness of the ancestral clone bearing one of the evolved plasmids is significantly higher than both the plasmid-free ancestral cells and the evolved cells carrying the evolved plasmid. Given that the R1 plasmid may spread among different species of enterobacteria, we wondered what the effect of the evolved plasmid would be inside Salmonella enterica cells. We found that the evolved plasmid is also able to dramatically increase the relative fitness of these cells. Our results suggest that even if general usage of antibiotics is halted, conjugative plasmids that have been selected with antibiotics in previous years can still persist among bacterial populations or even invade new strains.     

Interaction between the RP4 coupling protein TraG and the pBHR1 mobilization protein Mob

It is currently believed that interaction between the relaxosome of a mobilizable plasmid and the transfer machinery of the helper conjugative plasmid is mediated by a TraG family coupling protein. The coupling proteins appear as an essential determinant of mobilization specificity and efficiency. Using a two-hybrid system, we demonstrated for the first time the direct in vivo interaction between the coupling protein of a conjugative plasmid (the TraG protein of RP4) and the relaxase of a mobilizable plasmid (the Mob protein of pBHR1, a derivative of the broad host range plasmid pBBR1). This interaction was confirmed in vitro by an overlay assay and was shown to occur even in the absence of the transfer origin of pBHR1. We showed that, among 11 conjugative plasmids tested, pBHR1 is efficiently mobilized only by plasmids encoding an IncP-type transfer system. We also showed that the RP4 TraG coupling protein is essential for mobilization of a pBBR1 derivative and is the element that allows its mobilization by R388 plasmid (IncW) at a detectable frequency.     

Determination of the mechanism of retrotransfer by mechanistic mathematical modeling.

Two mathematical models to elucidate the mechanism of retromobilization (or retrotransfer), that is, the ability of conjugative plasmids to mobilize genes into the cell containing the conjugative plasmid, were developed. This study deals with retromobilization of nonconjugative plasmids (Tra-Mob+). Plasmid transfer was modeled by two mass action models. The first is based on the hypothesis that retromobilization of the Tra-Mob+ vector occurs in one step, by means of the pilus formed by the Tra+ plasmid in the original host. In the second model, retromobilization is considered to be a two-step process involving two transfer events. The first step involves the transfer of the Tra+ plasmid from the recipient cell to the donor of the nonconjugative vector, and during the second encounter the nonconjugative vector is mobilized toward the recipient. Since the relationships between the number of transconjugants and the number of recipients for the two models are different, filter matings were performed for short time periods with different initial densities of the recipient population. Comparison of the numbers of transconjugants with the results of the mathematical equations confirmed the hypothesis that retromobilization is a one-step conjugation process.     

Conjugal transfer of cloning vectors derived from ColE1.

The transfer properties of five cloning vectors derived from ColE1 were studied. Two of the vectors (pSF2124 and pGM706) behaved like wild type ColE1 in that they could be transferred efficiently in the presence of the conjugative plasmid F. The mobilization of the remaining three vectors (pMB9, PBR313 and pBR322) by F was barely detectable. The transfer defect in pBR313 and pBR322 could be complemented by ColK when R64drd11, but not F, was used as the conjugative plasmid. The transferred plasmids could be recovered unchanged from recipients. Conjugal transfer is a potentially useful technique for screening hybrid plasmids in low-risk cloning experiments involving poorly transformable strains.