TRANSITORY DEREPRESSION AND THE MAINTENANCE OF CONJUGATIVE PLASMIDS

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1 Copyright by the Genetics Society of America TRANSITORY DEREPRESSION AND THE MAINTENANCE OF CONJUGATIVE PLASMIDS PETER D. LUNDQUIST AND BRUCE R. LEVIN Department of Zoology, University of Massachusetts, Amherst, Massachusetts Manuscript received August 2 1, 1985 Revised copy accepted March 8, 1986 ABSTRACT 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 extrachromsomal 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. ACTERIA isolated from natural populations abound with plasmids of B known and unknown function (JAMIESON et al. 1979; TAYLOR et al. 1982). In the E. coli isolated from the feces a single human host, for example, CAU- CANT, LEVIN and SELANDER (1981) found that 50 of 53 distinct enzyme electrophoretic types (ETs) carried at least one plasmid, and 42 appeared to carry more than one. For plasmids (or any other autonomous genetic elements) to become established and maintained in populations, their rate of replication has to be as great or greater than their rate of loss due to vegetative segregation and negative selection. In the case of plasmids, this overreplication (CAMP- BELL 1981) can be achieved by either positive selection for plasmid-determined phenotypes or by infectious transmission. Based on the analysis of a mathematical model of the population dynamics of conjugative plasmids, STEWART and LEVIN (1977) concluded that there are broad conditions under which these self-transmissible replicons could be maintained by infectious transfer alone, even when their carriage imposes a signif- To whom correspondence should be addressed. Genetics 113: July, 1986.

2 484 P. S. LUNDQUIST AND B. R. LEVIN icant burden on their host cell. Nevertheless, LEVIN (1980) and LEVIN and LENSKI (1983) argued that these conditions are unlikely to be met in natural populations of E. coli. Their assertions are based on estimates of the parameters of the models used by STEWART and LEVIN (LEVIN, RICE and STEWART 1979; LEVIN 1980). In populations of realistic densities, the anticipated rate of transfer for wild-type plasmids (those with repressible conjugative pili synthesis; WATANABE 1963) were considered to be too low to overcome loss by segregation and negative selection. For nonconjugative plasmids, the a priori conditions for establishment and maintenance in the absence of positive selection are extremely limited, even if these replicons are readily mobilized by conjugative plasmids (LEVIN and STEWART 1980). While the hypothesis that plasmids could not be maintained by infectious transfer alone may be compelling (it certainly was to one of us, B.R.L.), its theoretical foundation is limited. The mathematical models on which it is based (STEWART and LEVIN 1977; LEVIN and STEWART 1980) do not consider some potentially important features of the biology of wild-type, conjugative plasmids. One of these features is transitory derepression of conjugative pili synthesis, where for a time after the receipt of a wild-type plasmid, conjugative pili synthesis is derepressed in the transconjugant and its immediate descendants. The transitory derepression period can be quite long (WILLETTS 1974), and during this transient phase, the rate of transfer of repressed plasmids may equal that of high fertility, permanently derepressed mutants (CULLUM, COL- LINS and BRODA 1978). The importance of retransfer to the kinetics of plasmid spread is very clearly illustrated by the results of FRETER, FRETER and BRICK- NER (1983). The empirical base of this no neutral plasmid hypothesis is also limited. The studies that provided the necessary estimates of the fertility of conjugative plasmids and costs of their carriage (CULLUM, COLLINS and BRODA 1978; LEVIN 1980; LEVIN, RICE and STEWART 1979; FRETER, FRETER and BRICKNER 1983) have considered only three species of conjugative plasmids; RI, RlOO and F. All of these elements had been maintained in the laboratory for at least 10 years before the study of their transfer kinetics. To more extensively evaluate the importance of retransfer and transitory derepression of conjugative pili synthesis to the population biology and existence conditions of plasmids, we have examined the population dynamics of conjugative plasmids isolated from natural populations of E. coli. We give particular consideration to the conditions under which these replicons will increase in frequency when they are initially rare. The results of this study suggest that some naturally occurring conjugative plasmids may be maintained by infectious transfer alone. MATERIALS AND METHODS Plasmids and bacterial strains: Seven plasmids were examined. Two were the laboratory plasmids, R1 and its permanently derepressed derivative R1-drdl 9. The remaining five plasmids were from E. coli isolated from fecal samples of two humans under antibiotic treatment (B. R. LEVIN, D. A. CAUCANT and R. K. SELANDER, unpublished results). These wild plasmids were transferred from their original hosts to E.

3 PLASMID MAINTENANCE TABLE 1 Strains and genetic markers 485 Strain" SUP' Amp Kan Tet Crb Chl CSH7 L+d CSH7 L- Rl/CSH7L- R1 drd-l9/csh7lpm1 l/csh7l- (L+) pb 1 5/CSH 7 L- (L') pb23/csh 7 L- (L') pb39/csh 7L- (L') pb40/csh 7 L- (L') - + +/- +/- + +/- + a A11 hosts were nal str prototrophs derived from CSH7 (MILLER 1972), an E. coli K-12. * + = > resistant; - = > sensitive; +/- = > low-level resistance. ' Antibiotics tested were as follows: Amp, ampicillin; Crb, carbenicillin; Tet, tetracycline; SUI, sulfonamide and trimethoprim; Ghl, chloramphenicol; Kan, kanamycin. In addition to those mentioned, these strains were also tested for resistance to cefoxitin, tobramycin and gentamycin. All were sensitive to these antibiotics. L- = >Lac-; L+ = >Lac+; L- (L+) = >Lac- and Lac+ variants were prepared. coli K-12 by classical procedures (selection for transconjugants in mixtures of donors and recipients growing exponentially in broth). Two variants of a single strain of E. coli K-12 were used in all these experiments, CSH7 (lacy rps nal) and a spontaneous lac+ revertant of it (MILLER 1972). The antibiotic resistances of the various plasmids and bacterial hosts used in this study are listed in Table 1. Plasmid electrophoresis and restriction: Plasmid DNA was extracted according to the method of BIRNBOIM and DOLY (1979). The relative molecular weights of native and restriction endonuclease digested plasmid DNA were examined by electrophoresis on 0.7% agarose gels using the buffers and procedures described in MEYERS et al. (1976). Two restriction endonucleases were used, EcoRI and BamHI (New England Biolabs). The procedure for preparing the restriction digests was identical to that described by MANIATIS, FRITSH and SAMBROOK (1982, pp ). Experimental cultures and medium: All experimental cultures were maintained in chemostats of a design described in the appendix to CHAO, LEVIN and STEWART (1977). The minimal salt solution used for these experimental cultures contained per liter of water 7 g K2HP04, 2 g KH2P04, 1 g (NH4)2S04, g MgS04.7H20, 0.5 g Na3C6H50,.2H20, and 1 ml of a % solution of thiamine. Glucose at 50 rg/ml was the sole and limiting carbon source. Unless specifically noted, the dilution rates in the chemostats used were maintained at approximately 0 hr-'. Under these conditions, total cell densities averaged about 1 X 10' in established chemostats. Sampling procedures and medium: Samples were taken from the chemostat vessels, serially diluted and plated on selective and nonselective solid media. Tetrazolium lactose and minimal lactose agar were used (LEVIN, STEWART and CHAO 1977). Tetracycline hydrochloride (Sigma) was added at 25 kg/ml for selection of cells carrying the plasmid pb23. Kanamycin sulfate (Sigma) was added at 40 pg/ml to select for cells carrying the other six plasmids. In most of the experiments, the donors were lac- and the recipients lac+. The density of donors was estimated from the numbers of lac- (red) colonies on tetrazolium lactose plates containing the selecting antibiotic (kanamycin or tetracycline). Transconjugant densities were estimated from the number of lac+ (white) colonies on these antibioticcontaining tetrazolium lactose plates or from colony counts on minimal lactose agar supplemented with kanamycin or tetracycline. The recipient densities were calculated from the difference in the density estimates for lac+ cells on antibiotic-free tetrazolium - + +/

4 486 P. S. LUNDQUIST AND B. R. LEVIN lactose agar and the estimated transconjugant density. In a few experiments, the original donors were lac+ and the recipients lac-. Under these conditions, the frequency transconjugants could only be estimated when they were relatively common (in excess of 1 OT2 of the total) from the number of lac- colonies on tetrazolium lactose agar containing the selecting antibiotic. The donor densities were estimated from colony counts on antibiotic-containing minimal lactose agar, or as lac+ on selecting or nonselecting tetrazolium lactose agar. The recipient densities were calculated from the difference in the total numnber of lac- cells on tetrazolium lactose agar containing and not containing the selecting antibiotics. RESULTS The wild plasmids on E. coli K-12 hosts: Four of five naturally occurring E. coli strains used as sources of these plasmids exhibited multiple plasmid bands on agarose gels; however, the E. coli K-I2 transconjugants used in this investigation had only one distinct plasmid band. This band corresponded to one of the high molecular weight plasmid bands present in the native plasmid gels of the donor strains. Retransfer from the K-12 transconjugants to other K-12 strains yielded second order transconjugants with the same array of plasmid-determined phenotypes and the same single band plasmid profile as their respective donors. For these reasons, we assume that each of the five wild plasmid-bearing strains examined carried only a single plasmid that is transmissible by conjugation. Three lines of evidence indicated that the wild plasmids considered here were different replicons: (1) they showed different antibiotic resistance patterns (see Table 1); (2) their bands in undigested extracts had somewhat different mobilities on the agarose gels; and (3) they showed different fragment patterns after EcoRI and BamHI digestion. Primarily as a display of frugality, we have elected not to include the photographs of the gels illustrating these points. These photographs were made available to the reviewers, and on request, copies will be sent to interested readers. Invasion-when-rare experiments: To ascertain whether these plasmids can invade and be maintained by transfer alone, we introduced plasmid-bearing cells, donors, into chemostats with established populations of recipients and then monitored the frequencies of donors, recipients and transconjugants. The donors were taken from overnight, stationary phase cultures or from established chemostats. To minimize the likelihood of the plasmid-bearing bacteria becoming established due to intrinsic fitness advantages, we introduced the donors at relatively low frequencies, lo-* or less. Neither R 1 nor R1 -drd 19 increased in frequency in chemostat populations of E. coli K-12 (Figure la and b). For the repressed transfer plasmid, R1, few transconjugants were observed. For R1-drdl9, a substantial number of transconjugants were detected, but the intrinsic disadvantage of cells carrying this plasmid appeared to be too great to be overcome by infectious transfer. This can be seen from the continuous decline in the frequencies of both donors and transconjugants (Figure 1 b). Three additional invasion-when-rare experiments were performed with the RI plasmid. In one, the RI-bearing cells had an initial frequency of 2 x

5 PLASMID MAINTENANCE a I * +...,...,... c *...,... t b 487 i 0 n. m m TIME in HOURS FIGURE 1.-Invasion-when-rare experiments with laboratory plasmids. Abbreviations: n = recipients; n+ = donors; n* = transconjugants (total). a, R1; b, R1-drdl9. no transconjugants were observed and, after 9 days, the plasmid was no longer detected. In the second replicate, the R1-bearing cells were introduced at a relative frequency of 7 X lo-. In the course of this 6-day experiment, the original donors were maintained at approximately the same frequency as that to which they were introduced, and the transconjugants achieved a relative frequency of IO-. the last R1 invasion-when-rare experiment was performed with both donors and recipients on the same lac- cell line. The R1-bearing cells were introduced at a relative frequency of 3 X and after the 6th day this plasmid was no longer detectable. Two additional invasion-when-rare experiments were performed with R 1 - drdl9. In one of these, the donors were introduced at a frequency of 6 X 10-. Although more transconjugants were produced in that experiment than in the one presented in Figure lb, at the final sample of this 4-day experiment, the relative frequency of R1-drdl9 was nearly an order of magnitude lower than its initial frequency (7 X In the third R1-drdl9 invasion-when-rare experiment, the frequency of the plasmid went from 4 X to less than lo- in the course of the 4 days that the chemostat was run. In Figure 2a, b and c we present the results of an analogous set of invasionwhen-rare experiments with pm 11, pb23 and pb40 plasmids. When introduced at low frequencies, none of these plasmids showed substantial increases in frequency, and few or no transconjugants were observed. The pb23 invasion-when-rare experiment was repeated with an initial frequency of 4 x No transconjugants were observed, and after the seventh day, bacteria carrying this tet plasmid were no longer detectable. It should be noted, however, that pb23 transconjugants were observed when this plasmid was introduced at higher frequencies. The pm11 and pb40 invasion-when-rare experiments were performed two and three additional times. In one pm11 replicate, the plasmid was introduced at a relative frequency of 6 X and remained at approximately that level for the 7 days that chemostat was run.

6 488 P. S. LUNDQUIST AND B. R. LEVIN *,...I ". * * a -*-...."...".*. "..."..._.._..,*...!?. C.,._"..." \- \-.- Y -*/.----'\.-, n. e-- t TIME (HOURS) FIGURE 2.-Invasion-when-rare experiments with wild plasmids. Abbreviations: n = recipients; n+ = donors; 12% = transconjugants (total). a, pm11; b, pb23; C, PB40. J '0 50 loo 150 In the second of these pm11 replicates, the plasmid was introduced at an initial frequency of and was no longer detectable after the eighth day. In one of the pb40 replicates, the relative frequency of this plasmid went from lo-' to 8 x in the course of the 7-day experiment, with occasional transconjugants being detected. In the second pb40 experiment, in the course of the 4-day experiment, the relative frequency of cells carrying this plasmid declined from an initial value of 4 X to 1 X lov5, and no transconjugants were detected. In the final pb40 invasion-when-rare experiment, this plasmid was introduced at a relative frequency of and although a few transconjugants were observed during the course of this experiment, bacteria carrying this plasmid were no longer detectable after the sixth day. The population dynamics of pb15 and pb39 are very different from that of the laboratory plasmids presented in Figure 1 and the three wild plasmids considered in Figure 2. The frequency of bacteria carrying pb15 and pb39 rose more than four orders of magnitude in the course of a single day (roughly five generations). For both of these replicons, this ascent is through an increase in the frequency of transconjugants, rather than that of donors (Figure 3a and b). The pb 15 and pb39 invasion-when-rare experiments were repeated three and two times, respectively, with the same donors and recipients. While the rate of ascent varied among replicates, the same basic result was obtained. In the pb 15 invasion-when-rare experiments, the initial frequencies of cells carrying these elements were 2 X 4 x lo-' and 5 x lo-'. After two days in each of these chemostats, the frequency of cells carrying pb15 exceeded 0.5, and the rise of that plasmid could be attributed primarily to the increase in the frequency of transconjugants. In one pb39 replica, the plasmid went from an initial frequency of 2 X to 1 X lo-* by the third day, and by the fifth day, the frequency of cells carrying this replicon exceeded In other replicates, bacteria carrying the pb39 plasmid went from an initial frequency of 8 x to 0.97 by the third day. In all these cases, the ascent of the pb39 can be attributed almost exclusively to increases in the frequency of transconjugants.?

7 PLASMID MAINTENANCE 489 Hypotheses and models: We interpret the observations that the plasmids R1, R1-drdl9, pm11, pb23 and pb40 were unable to increase in frequency when rare as results consistent with the hypothesis that plasmids cannot become established by infectious transfer alone. On the other hand, the rapid ascent of pb15 and pb39 in these invasion-when-rare experiments appears to be inconsistent with this hypothesis. Both of these replicons spread through the population in the absence of direct selection for known genes. There are two distinct classes of hypotheses that could account for the pb15 and pb39 result: (1) the direct selection for some, as yet undetermined, phenotype coded for by these elements, or (2) the rates of conjugational transfer of these plasmids being sufficiently high for them to become established and be maintained by infectious transfer alone. Our analysis supports a hypothesis of the latter type. We postulate that the capacity for the pb15 and pb39 plasmids to invade when rare is a consequence of a high rate of retransfer during the period of transitory derepression of conjugative pili synthesis. The arguments in support of this hypothesis require a brief consideration of a model of the population biology of conjugative plasmids. The model developed here is an extension of that considered by STEWART and LEVIN (1977) and LEVIN, RICE and STEWART (1979). It is similar in general form to that employed by FRETER, FRETER and BRICKNER (1983), but differs from it in the treatment of vegetative segregation and transitory derepression. Our model differs from that employed by CULLUM, COLLINS and BRODA (1978) in that we assume a continuous flow (chemostat) habitat, rather than a discrete (batch) culture, and separately treat the population dynamics of the various component populations, their reproduction and contribution to plasmid transfer. We consider a vessel of unit volume into which a limiting resource flows at a constant rate, p hr-', from a reservoir where it is maintained at a concentration C pg/ml. The rate of flow into the vessel is equal to the rate at which unused resources, wastes and bacteria flow out. At any given time, the internal concentration of the limiting resource is r pg/ml. There are four distinct populations of bacteria: (1) original plasmid-free recipients (n), (2) original repressed transfer plasmid-bearing donors (n+), (3) repressed transconjugants (n* and (4) transitorially derepressed transconjugants (n*2). The variables n, n+, n*1 and n*2 are the densities (bacteria per milliliter) of the component bacterial populations. These bacterial populations grow at rates that are monotonically increasing functions of the resource concentration, #(r) (1 - a,), where a, is the selection coefficient for the xth population. Resources are taken up by the bacteria at rates proportional to their densities, growth rates and a conversion efficiency constant, e pg. All plasmid-bearing cells lose these elements by vegetative segregation at the same rate T hr-', and upon plasmid loss these bacteria become members of the recipient population. Plasmid transfer occurs at random at a rate proportional to the product of the densities of the plasmid-bearing and plasmid-free cells and a conjugational transfer rate constant. The latter parameter takes two values, y1 and 72, for the repressed transfer (n+ and n* 1) populations and transitorially derepressed (n*2) popula-

8 490 P. S. LUNDQUIST AND B. R. LEVIN a r I -..% /- 0...'... n*..a... /-.--I.- f....*...,.. n. n :?G/-+ n+ b '5p Id0 151 TIME in HOURS FIGURE 3.-Invasion-when-rare experiments with wild plasmids. Abbreviations: n = recipients; n+ = donors; n* = transconjugants (total). a, pb15; b, pb39. tions, respectively. All newly formed transconjugants enter the n*2 population, but members of this population become repressed and enter the n*1 transconjugant population at a rate m hr-'. With these definitions and assumptions, rates of change in the concentration of resources and the component bacterial populations are given by li+ 1: = p(c - Y) - $(r)[n + n+ f n*l + w 2]e n = n$(r) - nyl(n+ + ml) - ny~n*~ - pn = n++(r)( 1 - a+) - pn+ = n*l+(r)(l - a*') + mn*2 - pn*l 1 n*2 = n**+(r)(l - a**) + nyl(n+ + n*') + ny2n*2 - mn*2 - pn*2 (5) where a dot (') denotes differentiation with respect to time. Transitory derepression and the population dynamics of pb15 and pb39: It is clear from Figure 3a and b that the rapid ascent of the pb15 and pb39 plasmids cannot be attributed to an intrinsically higher fitness of the original donor (n+) population, since the frequencies of the original donors for both of these plasmids either declined or remained constant. Furthermore, the rise in the frequency of cells carrying these plasmids cannot be readily attributed to a constant higher fitness for transconjugants produced in these cultures. In two experiments, pb15 lac+ transconjugants isolated from pb15 lac- X lac+ invasion-when-rare cultures were used as donors in reciprocal invasion-whenrare experiments using a lac- recipient. In one of these experiments, the donors (nee transconjugants) did increase in frequency, but the newly formed lactransconjugants dominated. In the second of these reciprocal invasion-whenrare experiments, the donors (nee transconjugants) declined in frequency, whereas the newly formed transconjugants swept through the population in a manner similar to that depicted in Figure 3a. The rapid increase in the frequency of the pb15 and pb39 transconjugants (1) (2) (3) (4)

9 PLASMID MAINTENANCE 49 1 FIGURE 4.--Simulated invasion-when-rare experiments [numerical solutions to equations (1)-(5)]. In all runs: C = 50 rg; p = 0; e = 5 X lo-'; T = lo-'; and +(r) = 0.7/(4 + r). Abbreviations: n = recipients; n+ = donors; n: = transconjugants (total). a, No derepression: yi = y2 = 9 X IO-"; m = 0; (YI = 012 = b, Transitory derepression of transfer: y1 = 9 x lo-"; y2 = IO-'; m = 0; (YI = 012 = c, Transitory increase in fitness; y1 = y2 = 9 X lo-"; m = 0; (YI = 0.05; 012 = might be explained by the permanent derepression of conjugative pili synthesis in the transconjugants produced in these populations, but this too can be ruled out. Using the procedures described in LEVIN, RICE and STEWART (1979), we estimated the first order rate constant of plasmid transfer, yl, for pb15 and pb39 in their original K-12 donors and the transconjugant clones isolated from these experiments. These rates (on the order of 9 X lo-") are too low to account for the results depicted in Figure 3a and b. This is illustrated by numerical solutions to equations (1)-(5) depicted in Figure 4a. For these and the following numerical simulations presented in Figures 4 and 6, we assume a 0.05 growth rate disadvantage for plasmid-bearing cells (a+ = a1 = a2 = 0.05), a vegetative segregation rate of 7 = 10-3/cell/hr and population growth parameters in a range similar to that estimated for E. coli in similar culture conditions (LEVIN, STEWART and CHAO 1977; LEVIN, RICE and STEWART 1979). The resource concentration (C = 50 pg/ml), dilution rates and initial population densities employed in these simulations are similar to those for the pb 15 and pb39 invasion-when-rare and invasion-when-common experiments. As suggested by our hypothesis, the rapid rise in the frequency of transconjugants for the pb 15 and pb39 plasmids in the invasion-when-rare experiments can be accounted for by transitory derepression of conjugative pili synthesis. This is illustrated in Figure 4b, where we assume plasmid transfer rate constants 9 x lo-" and lo-' for y1 and 72, respectively, and a derepression halftime, T = hr (e-mt = 0.5). There is a caveat to the above interpretation. Reasonably good fit to the pb15 and pb39 invasion-when-rare experiments also obtain if, instead of assuming transitory derepression of conjugative pili syntheses, we assume transitory high fitness in transconjugants (Figure 4c). However, while transitory increase in fitness is a potential explanation for these observations, we consider it to be a highly unlikely one. First, we are unaware of demonstrated mecha-

10 49 2 P. S. LUNDQUIST AND B. R. LEVIN a b TIME in HOURS FIGURE 5.-Invasion-when-common experiment with wild plasmids. Abbreviations: n = recipients; n+ = donors; n* = transconjugants (total). a, pb15; b, pb39. a n,...,... n.i. -$*;:ih, I%...,,...,,~, m +,..,.+***' n+ I I I r TIME FIGURE 6.--Simulated invasion-when-common experiments [numerical solutions to equations (1)-(5)]. In all runs: C = 50 fig; p = 0; e = 5 X IO-'; 7 = IO-'; and +(r) = 0.7/(4 + r). Abbreviations: n = recipients; n+ = donors; n* = transconjugants (total). a, Transitory increase in fitness: y1 = 72 = 9 X IO-"; m = 0; a, = 0.05; a2 = b, Transitory derepression of transfer: yi = 9 X IO-"; y2 = IO-'; m = 0; a, = a2 = nism for transitory increases in fitness, and it is difficult to imagine any mechanisms that increase fitness temporarily that would not become permanently established. Second, the results of high-frequency invasion experiments with pb15 and pb39 (Figure 5a and b) are not consistent with those anticipated from the model with transitory high growth rates for transconjugants. Under these conditions, the donor density should decline rapidly as the transconjugants become more frequent (Figure 6a). Although there was some decline in the density of the pb15 donors in Figure 5a, the rate was very much less than that anticipated in Figure 4a. Also, the pb39-bearing donors did not decline in frequency (Figure 5b). If, however, there is transitory derepression of plasmid transfer, the observed and anticipated trajectories in these high invasionwhen-common experiments would be much more similar (Figure 6b). Finally,

11 PLASMID MAINTENANCE 493 for transitory increases in fitness to account for the observed rise in the frequency of transconjugants, the fitness differential would have to be extremely large (a three-fold greater growth rate for transitorially derepressed transconjugants relative to other plasmid-free and plasmid-bearing cells). This is a particularly high fitness differential to account for, especially without a postulated mechanism to generate it in a transitory manner. CONCLUSIONS AND IMPLICATIONS The results of this investigation provide evidence for the existence of naturally occurring plasmids with the potential to be maintained by infectious transfer alone. When introduced at low frequencies, these replicons rapidly sweep through bacterial populations, and cells carrying them quickly dominate. These plasmids are repressed for conjugative pili synthesis, and their capacity to spread through populations is a consequence of a high rate of transfer during the period of transitory derepression in newly formed transconjugants. They can become established and, in bacterial populations of modest density, can do so without direct selection for plasmid-determined phenotypes. We interpret these observations as support for the hypothesis that, in natural populations of bacteria, there are plasmids that are maintained as pure parasites, i.e., by infectious transfer (horizontal transmission) alone. The justification for this extrapolation requires elaboration. The two plasmids exhibiting this capacity for maintenance by infectious transfer, pb15 and pb39, were obtained directly from natural sources. They had not been kept in the laboratory for an extended period, less than 2 yr, and most of that time they were kept at -70". Save for their transfer to an E. coli K-12, they had not been subject to experimental manipulation. Certainly, minimal medium and chemostats are not a realistic analogue of the natural habitat of E. coli; nevertheless, there is empirical justification for assuming that results in this experimental habitat have implications for natural habitats. Using strains of E. coli K-12 and the plasmids RI and R1-drdl9, FRETER, FRETER and BRICKNER (1983) examined the transfer kinetics and population dynamics of plasmids in complex multi-species communities maintained in anaerobic chemostats and in the intestines of mice. The estimates of the first-order plasmid transfer parameters obtained in these more realistic environments were nearly identical to those obtained by LEVIN, RICE and STEWART (1979) under conditions similar to those in the present experiment. Furthermore, and most critically, there was reasonably close agreement between the plasmid population dynamics anticipated from the mass-action models used by FRETER, FRETER and BRICKNER (1983) and those observed in their experiments. The absolute rate of transfer of a plasmid will depend on the physiological conditions of its host, which in turn depends on environmental conditions (LEVIN, RICE and STEWART 1979). However, for any given plasmid and environment, whether that replicon can be maintained by transfer alone depends primarily on density of potential recipients (STEWART and LEVIN 1977; FRE- TER, FRETER and BRICKNER 1983). In the experiments presented here, pb15

12 ~~ ~~~ 494 P. S. LUNDQUIST AND B. R. LEVIN TABLE 2 Minimum density for plasmid invasion - Simulation results Rate constants _~ Selection Derepressed Dilution Minimum recipient coefficients half time rate density 10-1 O-R lo-'* IS lo O-s 1 o O-s 1 O-s 1 IO-@ 1 O+ lo-@ o -~ x lo6u 3.0 x 105" 2.1 x 107" 2.1 x lo1ou 2.2 x X 3.0 x io7 1.8 x x x x x x x IOH 2.6 X 10' Unless otherwise noted, invasion is considered to be a net increase in the frequency of plasmidbearing cells at 100 hr. In all runs, the initial ratio of plasmid-bearing to plasmid-free cells is a Calculated from the invasion condition specified in the model of STEWART and LEVIN (1977), Up + T N=- Y where y = y1 = y2; a+ = al = ap = a; e = 5 X IO-'; and T = The density presented is that for an indefinite period. For 100 hr, the minimum invasion density in these runs is no more than 6% higher than the tabulated value for an indefinite period. The remaining minimum invasion densities were calculated by numerical solutions to equations (1)-(5) with #(r) = 0.7/(4 + r); a+ = a1 = a*; C = 50; and T = lo-'. The conversion efficiency parameter, e, was varied to determine the minimum density needed for a net increase in the frequency of plasmid-bearing cells. The other parameters used in these solutions are specified in the table. 200-hr invasion period. and pb39 became established in populations of 10' cells/ml. The results of simulation experiments suggest that plasmids with conjugative transfer rates in a range similar to those of pb15 and pb39 could be maintained in populations of densities less than lo', even when their carriage substantially reduces the fitness of their host. The model used for these simulation experiments is that specified by equations (1)-(5). By manipulating the conversion efficiency parameter, e, we adjusted the equilibrium population density. For different sets of parameter values, we calculated the minimum density necessary for a net increase in the frequency of plasmid-bearing cells. Unless otherwise noted, these simulated populations were initiated with repressed-transfer donors and recipients in a ratio of 1, and successful invasion is considered to be a net increase in the frequency of plasmid-bearing cells by the 100th hr. The results of these simulation experiments are presented in Table 2. With

13 PLASMID MAINTENANCE 495 a 10% disadvantage for plasmid-bearing cells and a dilution rate of, for a plasmid with a unique transfer rate parameter, y1 = y2 = y, to invade a population with a density of 2 X lo, 7 would have to exceed lo-. Although first order transfer rate constants of this magnitude do obtain for permanently derepressed plasmids in exponentially growing cultures, much lower values of 71 (lo- or less) are anticipated for these mutant plasmids in slowly growing cultures, and for wild-type, repressed transfer plasmids under all conditions (LEVIN, RICE and STEWART 1979; FRETER, FRETER and BRICKNER 1983). With unique transfer rate constants of this magnitude and 10% selection against the plasmid-bearing cells, the minimum invasion density would be on the order of 2 x 10. However, even with these low values for 71, and only modest values of 72, these plasmids could become established in populations of densities on the order of 2 X lo7. The necessary condition is that the second order rate constant, 72, is 5 X lo- or higher. The estimated values of y2 for pb15 and pb39 are even greater than this. The period of transitory derepression, the relative fitnesses, time allowed for invasions, and initial density of donors all affect the minimum invasion density for the plasmids. However, the simulation results suggest that with these parameters in the, seemingly realistic, range considered, these invasion conditions appear to be robust. With y2 on the order of lo-, invasion densities of around 2 x lo7 obtain for a broad range of values for the other parameters (Table 2). Of interest is the relative insensitivity of invasion conditions to the dilution rate. In the single transfer rate model, the minimum invasion density is very sensitive to dilution rate. This is not the case for the transitory derepression model. We believe that this outcome in these simulations is largely because the time allowed for invasion is defined, rather than indefinite. We do not have estimates of the frequency of E. coli that can serve as donors and recipients of plasmids, and do so with efficiencies as great or greater than E. coli K-12. On the other hand, E. coli populations of 10 and lo per gram of feces have been reported (COOKE 1974; MASON and RICHARDSON 1981). Thus, it seems reasonable to assume that there are natural habitats in which the density of E. coli is as greater or greater than that needed for plasmids with transfer parameters similar to pb15 and pb39 to be maintained by infectious transfer alone. AN EVOLUTIONARY SPECULATION From an evolutionary perspective, repression of conjugative pili synthesis is somewhat of an enigma. It is a ubiquitous and relatively complex phenotype in conjugative plasmids. Consequently, it seems realistic to conclude that it is an adaptive and highly evolved character. However, as a consequence of repression of conjugative pili synthesis, the fertilities of conjugative plasmids are lower than they could be. If plasmids are, in fact, maintained by infectious transfer, then it seems reasonable to assume that, if all else were equal, selection should favor the maximum possible transfer rate. We believe that the reason for this apparent paradox is that in fact all else is not equal. Bacteria carrying mutant plasmids that are permanently derepressed for con-

14 496 P. S. LUNDQUIST AND B. R. LEVIN jugative pili synthesis appear to be at a competitive disadvantage relative to those carrying the wild-type repressed plasmids from which they were derived (LEVIN 1980). In the presence of donor (male)-specific bacteriophage, cells bearing conjugative pili are susceptible to infection, whereas those free of these organelles are not. In fact, the conditions for maintaining these types of bacterial viruses on cells carrying repressed transfer plasmids are considerably more restrictive than the conditions for maintaining them on permanently derepressed mutants (LERNER 1985). Thus, it is possible that repression of conjugative pili synthesis evolved, in part, as a defense against male-specific bacteriophage. The advantage of producing conjugative pili would decline with increases in the frequency of plasmid-bearing cells, whether they rise in frequency by infectious transfer or by direct selection. consequently, a mechanism for the horizontal transmission would be useful primarily when the plasmid is in a population dominated by bacteria that are potential recipients. If there were a cost associated with the synthesis of conjugative pili (as there appears to be; LEVIN 1979), selection would favor mechanisms to turn off the synthesis of these organelles. The intensity of this selection for mechanisms to repress conjugative pili synthesis would increase with the frequency of cells carrying the plasmid. Repression of conjugative pili synthesis can be considered a compromise between the advantages and costs of infectious transmission. It allows for the horizontal transmission of plasmids and, thus, for their spread by infectious transfer in naive populations and for the expansion of their range of hosts. At the same time, repression minimizes the overall cost of maintaining this mechanism for infectious spread. The present investigation suggests that, as a consequence of retransfer by transitorially derepressed cells, this compromise need not preclude the maintenance of these replicons by infectious transfer alone. We wish to thank ANNA HAJNAL FALUS for testing these strains for antibiotic resistance and for the electrophoretic assays. We are grateful to SANDRA COLLINS, RICHARD CONDIT, GORDON EDLIN, RALPH EVANS, RICHARD LENSKI and WILLIAM SHANNON for reading this manuscript and making useful suggestions. We also thank an anonymous reviewer for helpful comments. This research was supported by National Institutes of Health grants GM19848 and GM LITERATURE CITED BIRNBOIM, H. C. and J. DOLY, 1979 A rapid alkaline extraction procedure for screening plasmid DNA. Nucelic Acids. Res. 7: CAMPBELL, A., 1981 Evolutionary significance of accessory DNA elements in bacteria. Annu. Rev. Microhiol CAUCANT, D. A., B. R. LEVIN and R. K. SELANDER, 1981 Genetic diversity and temporal variation in the E. coli of a human host. Genetics 98: CHAO, L., B. R. LEVIN and F. M. STEWART, 1977 A complex community in a simple habitat: an experimental study with bacteria and phage. Ecology 58: COOKE, E. M., 1974 Escherichia coli and Man. Churchill Livingstone, Edinburgh. CULLUM, J., J. F. COLLINS and P. BRODA, 1978 Escherichia coli K12. Plasmid 1: The spread of plasmids in model populations of

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