Amelioration of the Cost of Conjugative Plasmid Carriage in Eschericha coli K12

Corresponding author: Cecilia Dahlberg, Solna, S 171 82, Sweden., cecilia.dahlberg{at}smi.ki.se (E-mail)

Communicating editor: H. OCHMAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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.

OUR capacity to decrease antibiotic resistance among clinically important bacteria depends in part on the fitness costs associated with the resistances. If a cost is associated with a resistance phenotype, sensitive strains should be able to invade and outcompete resistant bacterial populations when the use of antibiotics is reduced. However, several studies examining the effects of reducing antibiotic use have shown that for chromosomally encoded resistances the cost is often ameliorated by compensatory mutations without loss of resistance (SHRAG and PERROT 1996 ; SHRAG et al. 1997 ; BJORKMAN et al. 1998 , BJORKMAN et al. 2000 ; REYNOLDS 2000 ; NAGAEV et al. 2001 ).

The potential cost to a bacterial host of plasmid-borne antibiotic resistance may be associated with the resistance function itself or with plasmid regulation. For instance, plasmid-borne antibiotic resistances often act through alteration or efflux of the antibiotic and these mechanisms may disturb bacterial growth. In addition, plasmid replication and gene expression may interfere with bacterial growth in ways independent of any resistance functions carried by that plasmid. These costs can be expected to be specific for each plasmid/bacteria combination.

In the absence of the selecting antibiotics, a cost associated with plasmid carriage has been observed for nonconjugative plasmids bearing antibiotic-resistance genes (ZUND and LEBEK 1980 ; HELLING et al. 1981 ; NOACK et al. 1981 ; CAULCOTT et al. 1983 ; DYKHUIZEN and HARTL 1983 ; COOPER et al. 1987 ; LENSKI and BOUMA 1987 ; BOUMA and LENSKI 1988 ; MODI and ADAMS 1991 ). Since these elements are also transmitted vertically during cell division one would anticipate that natural selection operating on the host, on the element, or both would either ameliorate the fitness costs or favor the ascent of bacteria that have lost these elements, i.e., segregation (LEVIN and LENSKI 1983 ). Amelioration of the cost of carriage for simpler, nonconjugative plasmids has been obtained by host evolution after evolution with selecting antibiotics (BOUMA and LENSKI 1988 ) as well as plasmid-host coevolution when evolution occurred without antibiotic selection (MODI and ADAMS 1991 ). Although the data are scarce, naturally occurring conjugative resistance plasmids are also generally considered costly when there is no selection for the resistance phenotype (GODWIN and SLATER 1979 ; ZUND and LEBEK 1980 ). A major difference between the high-copy-number cloning vector type of plasmids used in the experiments mentioned above and many of the conjugative low-copy-number plasmids is, in addition to their potential for horizontal dissemination within and between bacterial species, the presence of stability systems in the latter. These systems are found in most conjugative plasmids and ensure faithful inheritance to the daughter cells at cell division.

Since plasmids contribute greatly to antibiotic-resistance development in bacteria, it is fundamentally important to understand how plasmid-borne resistances may respond to a decreased use of antibiotics. This report focuses on two well-characterized plasmids with different replication systems, R1 (100 kb) and RP4 (60 kb). We examine how the costs of these plasmids on an Escherichia coli host can be ameliorated by natural selection in the absence of antibiotics.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains and designations:
Table 1 lists the E. coli K12 strains and plasmids used in this study. The plasmids R1 and RP4 were selected for this study on the basis of their differences in replication control and host range. Plasmid R1 has an inhibitor-target mechanism of replication control and has a narrow host range whereas RP4 has an iteron-based control of replication and a very broad host range. A notation where b refers to the bacterial host and p to the plasmid it carries was used for this study. The clones used to found long-term serial cultures were hence designated b₀p₀ or b₀ and clones isolated at the end of the experiment were designated b₁p₁ or b₁* (b₁* was used for clones that had evolved in the absence of plasmids to distinguish them from constructions where b₁p₁ were turned into b₁ by curing). Further strain constructions made from these isolates were designated as shown in Fig 1 and Table 1. Strain J53-1 was used as the host in all long-term selection experiments. Strains CD104 and CD105 are derivatives of J53-1 where plasmid R1 or plasmid RP4 was, respectively, introduced by conjugation. Strain CD100 is a spontaneous mutant of J53-1 resistant to bacteriophage T5 and was used as a common competitor in all the pairwise competition experiments (see below).

View larger version (16K): In this window In a new window Download PPT slide   Figure 1. Experimental evolution and strain construction. b refers to the bacterial host and p to the plasmid it carries. (A) Three independent populations of the E. coli host J53-1 carrying either plasmid R1 or plasmid RP4 (b0p0) were propagated by daily serial transfer. Clones were isolated from each population after 166 days (b1p1). One random clone from each population was subjected to curing, leading to a plasmid-free evolved host (b1). Curing was accomplished through infection by an incompatible replicon: pKG339 for R1-carrying clones and pFF1ts97 for RP4-carrying clones (see main text for details). The respective ancestral plasmid (p0) was further transferred into the cured clones (b1p0). Finally constructs were made where the evolved plasmids were transferred into the ancestral plasmid-free host (b0p1). (B) One population of the plasmid-free E. coli host J53-1 (b0) was propagated by daily serial transfer. A random clone from this population was isolated after 166 days (b1*). In addition, constructions were made where either of the ancestral plasmids R1 or RP4 was transferred into this evolved host (b1*p0).

Growth media:
The bacterial populations were propagated in Davis minimal (DM) medium (CARLTON and BROWN 1981 ) supplemented with 230 µg/ml proline, 45 µg/ml methionine, and with a glucose concentration of 100 µg/ml for liquid cultures. This glucose concentration gave a stationary-phase bacterial concentration of 5 x 10⁸ cells/ml. DM agar with a glucose concentration of 1000 µg/ml was used for plating. Final concentrations of antibiotics in Luria agar were 100 µg/ml nalidixic acid, 25 µg/ml kanamycin sulfate, 10 µg/ml tetracycline, 25 µg/ml chloramphenicol, 20 µg/ml streptomycin, and 100 µg/ml ampicillin. For phage resistance selection, a lysate of phage T5 corresponding to 10⁹ phages was spread onto DM plates prior to spreading of the bacteria.

Long-term serial cultures:
A schematic presentation of the experimental evolution and the construction of new plasmid-host combinations are shown in Fig 1. Three replicate populations 1, 2, and 3 of CD104 and three replicate populations 4, 5, and 6 of CD105 were initiated from stationary-phase cultures grown in LB with selective antibiotics for the respective plasmid. Following the notation developed for this study (see Strains and designations), the bacteria and plasmid used to start either of these triplicate populations were all designated b₀p₀. Population 7 was started with the plasmid-free J53-1 and the starting clone was given the designation b₀. The populations were serially propagated every 24 hr with 40 µl of culture into 4 ml of fresh DM without selecting antibiotics in 16-mm tubes. The cultures were incubated at 37° in a slanted rack, shaking at 250 rpm. Aerosol-resistant filter tips were used in all work associated with transfers of the evolving populations. The transfers continued for 166 days corresponding to 1100 generations. About every 50 generations the presence of the plasmids was determined on the appropriate antibiotic plates for 100 colonies from each population. Plasmid isolation was used to verify the presence of plasmids if resistance markers were absent. One random clone from each end-population was isolated for further analysis [CD111–116 (b₁p₁) and CD117 (b₁*) (Table 1)]. Clones with new resistance phenotypes that had evolved during the experiment were also isolated (CD139 and CD142–143) and further analyzed when they represented >5% of the total population. All clones isolated at the end of the experiment are referred to as evolved in the following text.

Curing and construction of new plasmid-host combinations from evolved clones:
The plasmids from the evolved clones were transferred into the ancestral host J53-1. Transfer was done either by conjugation in two steps, where E. coli LE392-1 served as an intermediate host, or by transformation using the CaCl₂ procedure (MANIATIS et al. 1989 ), and resulted in clones CD130–135 (b₀p₁). The plasmids R1 and RP4 both carry multiple stability systems, including postsegregational killing and partitioning, which makes natural segregant formation very rare. Clones cured of their plasmids (b₁) were instead obtained by using two slightly different protocols. Plasmid pKG339 (JENSEN et al. 1995 ), which contains the R1 copA gene under control of a LacI-regulated promoter, was transformed into CD111–113. Transformants were isolated and selected clones were plated on isopropyl thiogalactoside (IPTG)-containing plates. The addition of IPTG results in a large production of CopA RNA that will inhibit R1 replication and make the isolation of evolved R1 segregants possible. After confirmation that the evolved R1 plasmid had been lost the clones were grown in the absence of selecting antibiotics and natural segregants of pKG339 were isolated [CD120–122 (b₁)]. For clones CD114–116 the mini-RP4 replicon pFF1ts97 (FANG and HELINSKI 1991 ; VALLA et al. 1991 ) was used for curing. This temperature-sensitive replicon was transformed into the cells and transformants were grown at 30°. The pFF1ts97 replicon is incompatible with the wild-type RP4 plasmid and selection for pFF1ts97 made it possible to select for segregants of the evolved RP4 plasmids. After one additional plating and confirmation that the evolved RP4 plasmid was lost, the clones were allowed to grow without selective antibiotics at 37°, which would lead to segregation of pFF1ts97, and clones CD123–124 (b₁) were then isolated. Each step in the curing procedures was monitored by electrophoresis of plasmid isolations. Controls were made where the plasmids that had been removed by either of the curing procedures above were reintroduced into their host. The ancestral plasmids were finally introduced into the evolved, cured clones and CD117, resulting in CD125–129 (b₁p₀) and CD148–149 (b₁*p₀).

Estimates of fitness:
Pairwise competition experiments were performed against a common competitor, CD100. The competing clones were separately preconditioned in DM for one 24-hr cycle. For each competition experiment cultures were inoculated with the preconditioned cultures of the competitors at a 1:1 ratio and were then propagated for one 24-hr cycle as described above. The initial and final ratios of the two competitors were assayed on DM plates with and without phage T5. The relative fitness (see method of fitness estimation below) of the T5-resistant common competitor was 0.992 ± 0.014 relative to the J53-1 ancestor. At least 10 independent competitions were conducted for each strain. An increased sensitivity can also be obtained by measuring fitness over several cycles but this method was avoided to minimize potential effects of plasmid transfer between the competitors.

Calculation of Malthusian parameter:
Relative fitness (W) was determined as described by LENSKI 1988 and is the ratio of the number of doublings for the tested clone and the common competitor

where N_i(0) and N_j(0) are the initial densities of the tested clone and the common competitor, respectively, and N_i(1) and N_j(1) are their corresponding final densities.

Determination of relative copy number changes:
Minimal inhibitory concentrations of ampicillin were determined for the ancestral and evolved strains as an indirect measure of plasmid copy number (UHLIN and NORDSTROM 1977 ). Drops (10 µl) corresponding to 10² cells from overnight cultures were spotted onto plates containing different concentrations of ampicillin. The lowest concentration of ampicillin where inhibition of growth was detected was determined after overnight incubation at 37°. In a second approach to determine copy number change, DNA band intensities on agarose gels from plasmid isolations were measured and expressed as a ratio to an internal standard in the plasmid isolation. Equal volumes from overnight cultures of CD104 (b₀p₀) and CD111–113 (b₁p₁) were each mixed with an equal volume from one single overnight culture of a tester plasmid containing strain (the relevant characteristic of the tester plasmid is that it differs in size from the plasmids under study). Plasmid DNA was isolated from the mixtures and run on an agarose gel. The tester plasmid was included to make the assay independent of the DNA extraction efficiency and functioned as a standard for quantification. A similar procedure was done for CD105 (b₀p₀) and CD114–116 (b₁p₁). The gel was then stained for 2 hr in ethidium bromide. The intensity of the ancestral or evolved R1 or RP4 plasmid band was determined and normalized against the intensity of the coextracted tester plasmid band using Scion Image Version 3b (Scion Corporation, Frederick, MD). The relative copy number of each evolved plasmid is expressed as the ratio of the evolved plasmid and the tester plasmid band intensities from one coextraction, divided by the ratio of the ancestral plasmid and its corresponding tester plasmid band intensity. Any differences in yield between the ancestral and evolved plasmids in the overnight cultures were first corrected for.

Phenotypic and molecular analysis of evolved plasmids:
The resistance phenotype of 100 isolates from each end population was determined by replica plating on antibiotic-containing media. Restriction enzyme analysis was performed on plasmid DNA that was isolated by alkaline lysis (BIRNBOM and DOLY 1979 ) and digested with either EcoRI (R1) or EcoRV and PstI (RP4). Digestions were performed according to the manufacturer's recommendations (New England Biolabs, Beverly, MA) and separated on 0.9% agarose TAE gels. The rate of conjugal transfer (SIMONSEN et al. 1990 ) was determined for evolved and ancestral plasmids in evolved and ancestral hosts in experiments equal to one 24-hr cycle of serial transfer. The number of transconjugant CD100 cells was determined on DM plates supplemented with kanamycin and phage T5.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plasmid stability and general fitness changes:
Plasmid-free cells were never detected in the populations founded by R1- or RP4-bearing clones over the 1100 generations of selection in an antibiotic-free environment. The plasmids initially decreased the fitness of E. coli J53-1, with 6% (t-test, P < 0.002) for R1 (CD104, b₀p₀) and 21% (P < 0.001) for RP4 (CD105, b₀p₀; Table 2). The random clones isolated at the end of the experiment (b₁p₁) had increased their fitness, with up to 70% as compared to their plasmid-bearing ancestors (b₀p₀; Table 2).

Compensatory evolution:
The respective contributions of host- and plasmid-encoded mutations to the general fitness increase observed in the evolved clones (b₁p₁) were measured on combinations of ancestral and evolved hosts and plasmids.

The costs of the evolved plasmids were tested in both the evolved and ancestral host backgrounds. With one exception (see below, clone CD122) there were no significant differences in fitness between the evolved clones (b₁p₁) and the evolved clones cured of their plasmids (b₁). The cost of the evolved plasmids on the ancestral host (b₀p₁) was further tested and showed that five of the six evolved plasmids had a lower cost than the corresponding ancestral plasmid (b₀p₀). In contrast, one of the evolved plasmids, the R1 derivative in clone CD112, showed an increased cost on the ancestral host (CD131, 17%, P < 0.001).

The effect of host evolution was assessed by measuring the cost of the ancestral plasmids on the evolved hosts. Where the ancestral plasmids were transferred to the evolved hosts that were cured of their evolved plasmids (b₁p₀) we found no significant costs of the plasmids. For the host that had evolved in the absence of plasmids reduced costs of the ancestral plasmids (b₁*p₀) were demonstrated, as compared to the costs on the ancestral host (b₀p₀). For the ancestral plasmid R1 on this host (CD148, b₁*p₀), we found no significant cost and the ancestral plasmid RP4 (CD148, b₁*p₀) had reduced its cost to 9% (P < 0.002; Table 2).

Control experiments where the evolved plasmid was reintroduced into the cured host (i.e., reconstruction of b₁p₁ from b₁) showed that the curing procedure did not affect fitness (data not shown) with one exception. This cured clone CD122 showed a higher fitness when its plasmid had been reintroduced compared to the fitness of the plasmid-bearing clone before curing.

A second problem we encountered when curing the evolved clones was observed in the evolved RP4-bearing clone CD116 (b₁p₁). During curing when this clone was transformed with the plasmid pFF1ts97 that carries the same basic replicon as the ancestral RP4, the expected loss of the resident evolved RP4 plasmid was not seen. Both plasmids were still present, as determined by plasmid isolation, after several successive platings on media selective for pFF1ts97.

Phenotypic evolution:
Clones that had lost one or more antibiotic resistances emerged in all R1 populations but represented a minority of the cells (Table 3). The plasmids from representatives of these minority phenotypes were further analyzed by restriction endonuclease digestion and showed loss of restriction fragments of the size corresponding to the respective antibiotic-resistance genes (Fig 2A; CLERGET et al. 1981 ). Consequently all resistance gene fragments were absent in the digestions of the clones CD139 and CD142 that had lost all the plasmid resistance phenotypes. Clone CD143 had retained only those resistance gene fragments containing the kanamycin resistance and its flanking insertion sequence (IS1) elements.

View larger version (126K): In this window In a new window Download PPT slide   Figure 2. Restriction fragments of ancestral and evolved (A) R1 derivative plasmids digested with EcoRI and (B) RP4 derivative plasmids digested with EcoRV and PstI. The molecular weight marker (Mr) is a 1-kb ladder.

Several changes in the evolved plasmids from the randomly drawn clones were also revealed. The RP4 isolates CD114 and CD115 (b₁p₁) differed from the ancestral plasmid in their restriction endonuclease digestion patterns (Fig 2B) and showed several novel restriction sites in the transfer and replication regions (data not shown). These changes were probably associated with transposition of chromosomally located IS elements into the plasmids. For the plasmids in both CD114 and CD115 possible insertions into the trbE region that is involved in pilus production were found (PANSEGRAU et al. 1994 ). An additional insertion in the oriV region of the plasmid in CD115 was also found. One structural change was detected in the random R1 clones as CD113 showed a reduction in size of the restriction fragment that in the ancestral R1 plasmid harbors one IS1 copy and a kanamycin resistance gene (Fig 2A). The clone CD113 (b₁p₁) was kanamycin resistant and the size reduction was probably due to excision of the IS1 element.

The evolved RP4 plasmids in clones CD114 and CD115 were transfer deficient in both the evolved and the ancestral host. The evolved RP4 plasmid in clone CD116 had retained its transferability but the transfer rates of this plasmid and of the ancestral RP4 could not be determined for the liquid culture conditions used in these experiments, as they did not exceed the transfer that occurred after plating on the selective media. The evolved R1 clones (b₁p₁) showed lower transfer rates compared to the ancestral R1 clone (b₀p₀). The reduction in transfer rate of the evolved plasmids was not seen in the ancestral host (b₀p₁; Table 4). Transfer of the ancestral plasmid R1 was also measured from the host that had evolved in the absence of plasmids, CD148 (b₁*p₀). This transfer rate was of the same magnitude as transfer from the ancestral host (b₀p₀; Table 4).

The values obtained by the two different methods used for copy number measurements did not fully correlate. Only when both methods showed a similar deviation in copy number was an average of these values used to assign changes in copy number. The RP4 derivative in clone CD116 (b₁p₁) showed an increase in copy number of 70% and the R1 clone CD111 (b₁p₁) showed a 20% reduction in copy number (Table 5).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Many antibiotic-resistance genes are carried by conjugative plasmids but studies to date on compensatory evolution of antibiotic resistance have focused on genes encoded either on chromosomes or on nonconjugative plasmids (BOUMA and LENSKI 1988 ; MODI and ADAMS 1991 ; SHRAG and PERROT 1996 ; BJORKMAN et al. 1998 , BJORKMAN et al. 2000 ; REYNOLDS 2000 ; NAGAEV et al. 2001 ). We examined how the cost of carrying conjugative antibiotic-resistance plasmids is affected by evolution in an antibiotic-free environment. Parallel populations of an E. coli host carrying the natural conjugative plasmids R1 or RP4 were propagated for 1100 generations by serial transfer. The cost associated with plasmid carriage before and after experimental evolution was determined in competition experiments using a plasmid-free common competitor. Both plasmids R1 and RP4 imposed fitness costs on E. coli J53-1 at the start of the experiment (Table 2). At the end of the experiment all tested clones showed an increased fitness compared to the respective ancestral plasmid-host combination (Table 2). Genetic adaptation to the general growth conditions was expected to account for part of this fitness increase but changes in response to the cost of plasmid carriage were also anticipated.

Reduction of the cost of plasmid carriage can be accomplished through two main processes: segregation and compensatory mutation. For plasmid-borne antibiotic resistance segregation will lead to the loss of resistance by the formation of plasmid-free cells whereas compensatory mutations may or may not abolish resistance. Both segregation and compensatory mutation are likely to occur in a large population, resulting in a race between the two. Under conditions of antibiotic-resistance selection, compensatory mutation is primarily available, but under conditions where antibiotic selection is released it is unclear which of these processes will dominate.

Like most natural conjugative plasmids, the plasmids used in this study have stability systems such as postsegregational killing and partitioning that make their segregation rates very low (NORDSTROM et al. 1980 ; SCHMIDHAUSER and HELINSKI 1985 ). The fact that plasmid-free cells were never detected in the evolved populations was most likely affected by the action of these stability systems. Earlier studies have also shown that plasmid RP4 can be stably maintained in continuous culture (MELLING et al. 1977 ; JONES et al. 1980 ). There is a possibility that segregants were reinfected if they appeared, but the very low transfer rates we found under these conditions (see below) makes this explanation unlikely.

Since segregation was not detected in these experiments we directed our efforts to examine the effect of compensatory evolution. The first indication of compensatory mutations was seen when the fitness of plasmid-free clones (b₁) constructed from evolved plasmid-bearing clones (b₁p₁) was measured. These measurements showed no difference in fitness between the pairs of plasmid-carrying (b₁p₁) and plasmid-free (b₁) clones with one exception (see below, clone CD122; Table 2). Compensatory mutations in either the chromosome or the plasmid could explain these results. The costs of five of the six tested evolved plasmids on the ancestral host (b₀p₁) were lower than the costs of the corresponding ancestral plasmid (b₀p₀; Table 2), suggesting that these evolved plasmids had acquired compensatory mutations. The fact that four of six evolved plasmids had no cost at all suggests that compensatory mutations on the plasmid may prevent selection for segregants and hence plasmid loss from the population.

If compensatory mutations on the evolved plasmids were alone responsible for the decreased cost one would expect that the ancestral plasmids would still impose a similar cost on the evolved hosts as on the ancestral host. This was, however, not seen; instead we found that the ancestral plasmids had no significant cost on the evolved hosts that were cured of their evolved plasmids (b₁p₀; Table 2). This result indicates that additional compensatory mutations had occurred in the chromosome. The surprising aspect of these results lies in the apparent redundancy of the compensatory mutations shown by the compensatory mutations occurring in both the plasmid and the host. When a mutation compensatory for the cost of plasmid carriage occurs on either the host or the plasmid replicon, selection for a second compensatory mutation on the second replicon should be abolished.

The host that evolved in the absence of plasmids (b₁*) expectedly demonstrated an increase in fitness in response to the growth conditions. However, a second surprising result showed that this well-adapted host naive to plasmid carriage was more tolerant to the cost of a plasmid. For the ancestral plasmid R1 on this host we found no significant cost and the ancestral plasmid RP4 had greatly reduced its cost (Table 2).

As an explanation for the results on compensatory evolution we suggest that during the experiment both the host and the plasmids acquire mutations that are selected but for different purposes. The plasmids evolve compensatory mutations that are selected because they enhance fitness of the host whereas the host evolves toward the general growth conditions with a side effect of decreasing the cost of plasmid carriage. If the plasmid evolves compensatory mutations more rapidly than the host evolves mutations with compensatory side effects this would result in the apparent redundancy of compensatory mutations. It would also explain why a host that has evolved in the absence of plasmids suffers a lower cost of an ancestral plasmid than an ancestral host does. This explanation is also consistent with the results that an evolved plasmid does better than its ancestor in the ancestral host.

The cured clone CD122 showed a higher fitness when its plasmid had been reintroduced compared to the fitness of the plasmid-bearing clone before curing in the control experiment. This suggests that chromosomal genetic changes occurred during the curing procedure and the fitness values measured for the cured state of this clone are uncertain. A reasonable explanation for this result is that a mutualistic association resulting in strong plasmid dependency had evolved in this clone. We believe that the strong selection applied when the resident plasmid was forced out by an incompatible replicon during the curing procedure selected for a mutation compensatory for the loss of the plasmid. BOUMA and LENSKI 1988 and MODI and ADAMS 1991 previously showed that evolution leading to plasmid dependency occurred during experimental evolution of small nonconjugative plasmids. They were able to demonstrate, by the isolation of plasmid-free cells by natural segregation that had a significantly lower fitness compared to the plasmid-bearing cells, that plasmid dependency had evolved.

What was the basis for the decreased costs of the evolved plasmids from five out of six independent clones? First one may consider conjugation. Plasmid RP4 has a constitutively expressed transfer function whereas R1 is repressed for transfer. One part of the cost may be associated with pilus production. We found that two of the evolved RP4 isolates were transfer deficient and that this deficiency correlated with changes in their restriction endonuclease digestion patterns in genes involved in pilus production. The evolved R1 clones (b₁p₁) showed lower transfer rates than the ancestral R1 clone (b₀p₀). This reduction in transfer rate was likely due to host evolution resulting in a host-directed suppression of transfer since the reduced transfer of these plasmids was observed only in the evolved host (b₁p₁) and not in the ancestral host (b₀p₁; Table 4). The transfer rate of the ancestral R1 from the host that had evolved in the absence of plasmids (b₁*p₀) was of the same magnitude as transfer from the ancestral host (b₀p₀). This result supports the idea that the mechanism responsible for the reduced transfer rates in the former case evolved in direct response to plasmid carriage (Table 4). We did not attempt to localize the chromosomal mutations responsible for this effect but several host-encoded factors that influence plasmid transfer have been identified in previous studies. For example, the ArcA protein, involved in cellular redox sensing, affects R1 transfer (STROHMAIER et al. 1998 ).

Copy number reduction is another possible route for cost reduction (MODI and ADAMS 1991 ). The copy number has been shown to be 4–5/cell for plasmid R1 and 4–6 for plasmid RP4 (FIGURSKI et al. 1979 ; NORDSTROM et al. 1980 ). For one evolved R1 clone (CD111) we could observe a 20% reduction in copy number (Table 5). In contrast, an increased copy number corresponding to 7–10 copies per cell was observed for the evolved RP4 clone CD116 (Table 5). The mutation that made this plasmid increase its copy number could account for our failure to cure this clone by making it compatible with the original replicon.

Antibiotic-resistance expression is a third type of plasmid function that can be altered to reduce cost. Most of the clones from the evolved populations had retained all their antibiotic resistances and had obviously reduced their costs by other means. However, R1-carrying populations did contain minor fractions of clones that had lost resistances. The antibiotic sensitivities of these evolved clones correlated with deletions of the resistance genes and included loss of all the plasmid-borne resistances. Similar results have previously been presented for the conjugative plasmid TP120, where loss of resistances was observed after selection in chemostats (GODWIN and SLATER 1979 ).

Conclusions:
These results demonstrate that the fitness cost of conjugative resistance plasmid carriage can be reduced and is often completely ameliorated after selection in an antibiotic-free environment. Reversion to sensitivity by plasmid segregation was not observed in these experiments. Instead, compensatory evolution was the major pathway. As previously suggested a decreased usage of antibiotics may hence not be sufficient for dealing with the antibiotic-resistance problem as sensitive bacteria are prevented from invading resistant bacterial populations after compensatory evolution has occurred. However, the emergence of plasmids with deletion of all antibiotic-resistance markers from the basic replicon in some of our experiments shows that there is still a potential for selection of antibiotic-sensitive populations. A consequence of the plasmid-encoded compensatory mutations is that the resistance genes associated with these plasmids may have an enhanced potential to invade new populations by horizontal transfer as compared to the more limited chromosomal compensatory mutations that still are confined to one population. It remains to be elucidated if plasmid compensatory mutations are specific for the strain they were selected in or if they also reduce the cost of the plasmid carriage when it transfers to other strains or species.

	ACKNOWLEDGMENTS

We thank Dan Andersson, Diarmaid Hughes, Bruce Levin, Sophie Maisnier-Patin, and two anonymous reviewers for helpful comments and discussions; Kenn Gerdes, Donald Helinski, Daniel Dykhuizen, and Michael Travisano for the gifts of plasmids and phage; and Kamal Gandhi for excellent technical assistance. This work was funded by grants from the Swedish Research Council and The Royal Swedish Academy of Sciences to C.D and from the National Institutes of General Medical Sciences and National Institutes of Health to L.C.

Manuscript received May 23, 2003; Accepted for publication July 25, 2003.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

BIRNBOM, H. C. and J. DOLY, 1979  A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523.[Abstract/Free Full Text]

BJÖRKMAN, J., D. HUGHES, and D. I. ANDERSSON, 1998  Virulence of antibiotic-resistant Salmonella typhimurium.. Proc. Natl. Acad. Sci. USA 95:3949-3953.[Abstract/Free Full Text]

BJÖRKMAN, J., I. NAGAEV, O. G. BERG, D. HUGHES, and D. I. ANDERSSON, 2000  Effects of environment on compensatory mutations to ameliorate the cost of antibiotic resistance. Science 287:1479-1482.[Abstract/Free Full Text]

BOUMA, J. E. and R. E. LENSKI, 1988  Evolution of a bacteria/plasmid association. Nature 335:351-352.[Medline]

CARLTON, B. C., and B. J. BROWN, 1981 Gene mutation, pp. 222–242 in Manual of Methods for General Bacteriology, edited by P. GERHARDT. American Society for Microbiology, Washington, DC.

CAULCOTT, C. A., A. DUNN, H. A. ROBERTSON, N. S. COOPER, and M. E. BROWN et al., 1983  Investigation of the growth environment on the stability of low-copy-number plasmids in Escherichia coli.. J. Gen. Microbiol. 133:1881-1889.

CLERGET, M., M. CHANDLER, and L. CARO, 1981  The structure of R1drd19: a revised physical map of the plasmid. Mol. Gen. Genet. 181:183-191.[Medline]

COOPER, N. S., M. E. BROWN, and C. A. CAULCOTT, 1987  A mathematical method for analysing plasmid stability in micro-organisms. J. Gen. Microbiol. 181:1871-1880.

DYKHUIZEN, D. E. and D. L. HARTL, 1983  Selection in chemostats. Microbiol. Rev. 47:150-168.[Free Full Text]

FANG, F. C. and D. R. HELINSKI, 1991  Broad-host-range properties of plasmid RK2: importance of overlapping genes encoding the plasmid replication initiation protein TrfA. J. Bacteriol. 173:5861-5868.[Abstract/Free Full Text]

FIGURSKI, D., R. MEYER, and D. R. HELINSKI, 1979  Suppression of ColE1 replication properties by the IncP-1 plasmid RK2 in hybrid plasmids constructed in vitro.. J. Mol. Biol. 133:295-318.[Medline]

GODWIN, D. and J. H. SLATER, 1979  The influence of the growth environment on the stability of a drug resistance plasmid in Escherichia coli K12. J. Gen. Microbiol. 111:201-210.[Medline]

HELLING, R. B., T. KINNEY, and J. ADAMS, 1981  The maintenance of plasmid-containing organisms in populations of Escherichia coli. J. Gen. Microbiol. 123:129-141.[Medline]

JENSEN, R. B., E. GROHMANN, H. SCHWAB, R. DIAZ-OREJAS, and K. GERDES, 1995  Comparison of ccd of F, parDE of RP4, and parD of R1 using a novel conditional replication control system of plasmid R1. Mol. Microbiol. 17:211-220.[Medline]

JONES, I. M., S. B. PRIMROSE, A. ROBINSON, and C. C. ELLWOOD, 1980  Maintenance of some ColE1-type plasmids in continuous culture. Mol. Gen. Genet. 180:579-584.[Medline]

LENSKI, R. E., 1988  Experimental studies of pleiotropy and epistasis in Escherichia coli. I. Variation in competitive fitness among mutants resistant to virus T4. Evolution 42:425-432.

LENSKI, R. E. and J. E. BOUMA, 1987  Effects of segregation and selection on instability of plasmid pACYC184 in Escherichia coli B. J. Bacteriol. 169:5314-5316.[Abstract/Free Full Text]

LEVIN, B. R., and R. E. LENSKI, 1983 Coevolution of bacteria and their viruses and plasmids, pp. 99–127 in Coevolution, edited by D. J. FUTUYAMA and M. SLATKIN. Sinauer Associates, Sunderland, MA.

MANIATIS, T., E. F. FRITSCH and J. SAMBROOK, 1989 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

MELLING, J., D. C. ELLWOOD, and A. ROBINSON, 1977  Survival of R-factor carrying Escherichia coli in mixed cultures on the chemostat. FEMS Microbiol. Lett. 2:87-89.

MODI, R. I. and J. ADAMS, 1991  Coevolution in bacteria-plasmid populations. Evolution 45:656-667.

NAGAEV, I., J. BJÖRKMAN, D. I. ANDERSSON, and D. HUGHES, 2001  Biological cost and compensatory evolution in fusidic acid-resistant Staphylococcus aureus.. Mol. Microbiol. 40:1-8.[Medline]

NOACK, D., M. ROTH, R. GEUTHER, K. UNDISZ, and C. HOFFMEIER et al., 1981  Maintenance and genetic stability of vector plasmids pBR322 and pBR325 in Escherichia coli K12 strains grown in a chemostat. Mol. Gen. Genet. 184:121-124.[Medline]

NORDSTRÖM, K., S. MOLIN, and H. AAGAARD-HANSEN, 1980  Partitioning of plasmid R1 in Escherichia coli. I. Kinetics of loss of plasmid derivatives deleted of the par region. Plasmid 4:215-227.[Medline]

PANSEGRAU, W., E. LANKA, P. T. BARTH, D. H. FIGURSKI, and D. G. GUINEY et al., 1994  Complete nucleotide sequence of Birmingham IncP plasmids. J. Mol. Biol. 239:623-663.[Medline]

REYNOLDS, M. G., 2000  Compensatory evolution in rifampin-resistant Escherichia coli.. Genetics 156:1471-1481.[Abstract/Free Full Text]

SCHMIDHAUSER, T. J. and D. R. HELINSKI, 1985  Regions of broad-host-range plasmid RK2 involved in replication and stable maintenance in nine species of gram-negative bacteria. J. Bacteriol. 164:446-455.[Abstract/Free Full Text]

SHRAG, S. J. and V. PERROT, 1996  Reducing antibiotic resistance. Nature 381:120-121.[Medline]

SHRAG, S. J., V. PERROT, and B. R. LEVIN, 1997  Adaptation to the fitness costs of antibiotic resistance in Escherichia coli. Proc. R. Soc. Lond. Ser. B Biol. Sci. 264:1287-1291.[Medline]

SIMONSEN, L., D. M. GORDON, F. M. STEWART, and B. R. LEVIN, 1990  Estimating the rate of plasmid transfer: an end-point method. J. Gen. Microbiol. 136:2319-2325.[Medline]

STROHMAIER, H., R. NOIGES, S. KOTSCHAN, G. SAWERS, and G. HÖGENAUER et al., 1998  Signal transduction and bacterial conjugation: characterization of the role of ArcA in regulating conjugative transfer of the resistance plasmid R1. J. Mol. Biol. 277:309-316.[Medline]

UHLIN, B. E. and K. NORDSTRÖM, 1977  R plasmid gene dosage effects in Escherichia coli K-12: copy mutants of the R plasmid R1drd-19.. Plasmid 1:1-7.[Medline]

VALLA, S., K. HAUGAN, R. DURLAND, and D. R. HELINSKI, 1991  Isolation and properties of temperature-sensitive mutants of the trfA gene of the broad host range plasmid RK2. Plasmid 25:131-136.[Medline]

ZÜND, P. and G. LEBEK, 1980  Generation time-prolonging R plasmids: correlation between increases in the generation time of Esherichia coli caused by R plasmids and their molecular size. Plasmid 3:65-69.[Medline]

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