Copyright 0 1983 by the Genetics Society of America

EVOLUTION OF TRANSPOSONS: NATURAL SELECTION FOR Tn5 IN ESCHERICHIA COLI K l 2

SUSAN WURSTER BIEL' and DANIEL L. HARTL'

Deportment of Microbiology and Immunology' and Department of Genetics: Woshington University SchooI of Medicine, St. Louis, Missouri 63120

Manuscript received August 30, 1982 Revised copy accepted October 10, 1982

ABSTRACT

A novel in vivo effect of the transposable element Tn5 has been observed in chemostats when certain isogenic Tn5 and non-Tn5 strains of Escherichia coli compete for a limiting carbon source in the absence of kanamycin. The Tn5- bearing strain has a more rapid growth rate and increases in frequency from 50% to 90% within the first 15 to 20 generations. The effect occurs when Tn5 is inserted at a variety of chromosomal locations or when the element is carried by an episome, but it is strain specific, having been observed in two out of three strains examined. (For reasons unknown, the effect has not been observed with derivatives of strain CSH12.) Although the growth-rate advantage of Tn5 is independent of nutrient concentration and generation time, it can be reduced by prior adaptation of the strains to limiting conditions, and the amount of reduction is proportional to the length of prior adaptation. The growth-rate effect is evidently not caused by beneficial mutations induced by Tn5 transpo- sition, as Tn5-bearing strains selected in chemostats retain their initial Tn5 position and copy number. However, the effect does not occur in Tn5-112, a transpositionless deletion mutation missing the transposase-coding region of the right-hand IS sequence flanking the element. Since Tn5-112 retains a functional kanamycin-phosphotransferase gene, this gene is not responsible for the growth-rate effect. Thus, the effect evidently requires transposase function, but it does not involve actual transposition of the intact element. Altogether, these data provide a mechanism for the maintenance of Tn5 in bacterial populations in the absence of kanamycin, and they suggest a model for the proliferation and the maintenance of IS sequences and transposable elements in the absence of other identifiable selection pressures.

RANSPOSABLE elements are discrete segments of DNA capable of moving T within and between replicons in the absence of DNA sequence homology (reviews in CAMPBELL et al. 1979; CALOS and MILLER 1980; KLECKNER 1981). They are ubiquitous in both eukaryotes and prokaryotes, and the class of transposable elements in prokaryotes called transposons has a characteristic physical struc- ture consisting of direct or inverted terminally repeated squences that flank a central region carrying genetic determinants for functions such as antibiotic resistance, pathogenicity, and a wide range of other phenotypes (review in KOPECKO 1980). In the composite transposons, such as Tn5, Tn9 and TnlO, the terminally repeated sequences are IS elements, themselves small transposable elements. These IS elements usually encode proteins necessary for their own

Genetics 103 581-592 April, 1983.

582 SELECTION FOR Tn5

transposition and catalyze the transposition of sequences they flank; this is their sole currently known function.

Transposons are responsible for much of the naturally occurring resistance to antimicrobial agents. Clinical studies of ANDERSON (1968) and RUBENS et al. (1981) have shown that antibiotic resistance provides a decisive growth advan- tage to cells in an antibiotic-rich environment, and this results in the prolifera- tion of resistance transposons in such environments. Transposons provide a means by which resistance determinants can be incorporated into different replicons, and they can change the resistance spectrum of plasmids and increase the genetic variability of bacterial populations. Similar logic can be invoked to explain the occurrence of transposons containing genes for any potentially advantageous phenotype.

Although the selection pressures maintaining antibiotic-resistance transpo- sons in antibiotic-rich environments are clear, the situation regarding IS ele- ments is very uncertain. Since IS elements have no known effects beyond transposition, and can occasionally give rise to lethal mutations by inserting into essential genes (KLECKNER et al. 1975; STARLINGER 1977) or by causing deletions due to imperfect excision (KLECKNER and Ross 1979; SAEDLER et al. 1980), it seems paradoxical from an evolutionary point of view that IS elements should be maintained in bacterial genomes in such great variety and abundance (NYMAN et al. 1981). Several authors, notably DOOLITTLE and SAPIENZA (1980), SAPIENZA and DOOLITTLE (1980), and ORGEL and CRICK (1980), have proposed on theoretical grounds that transposition alone will lead to a proliferation of transposable elements in populations, irrespective of any phenotypic effects of the elements. This hypothesis does not exclude other possibilities, one being that IS elements and transposons might be maintained in populations partly because they may occasionally promote advantageous mutations. Here we propose and provide evidence for a third possibility, which is that IS sequences and other transposable elements are maintained in populations because they have as yet unrecognized beneficial effects upon their bacterial hosts. Transpo- sase proteins are believed to bind and interact with DNA, and KLECKNER (1981) has speculated that they are evolutionarily related to topoisomerase-like pro- teins. Genes that encode DNA-binding proteins that are frequently present in many copies per genome could have manifold subtle phenotypic effects owing to their interactions with other proteins involved in nucleic acid processing.

The subject of this report is Tn5, a 5.7 kilobase kanamycin-neomycin resist- ance transposon originally isolated from an R factor transferred from Klebsiella to E. coli K12 (BERG et al. 1975). Tn5 carries terminal inverted repeats of the insertion sequence IS50 (BERG et al. 1982), which bracket the segment encoding the aminoglycoside phosphotransferase responsible for resistance (DAVIES and SMITH 1978). IS50 is itself transposable and encodes the transposase protein(s) responsible for transposition (BERG et al. 1980a; ROTHSTEIN et al. 1980). Owing to an ochre mutation in the left-hand IS50 element (IS50L), only the right-hand IS50 element (IS50R) encodes functional transposase. However, both IS50L and IS50R contain transposase recognition sites and thereby allow Tn5 to transpose as an intact unit. This element was chosen for study because of its single

S . W. BIEL AND D. L. HARTL 583

TABLE 1

Strain list

Strain Genotype Source or reference

DB1506 DB1506-204 DB1633-3 c u 4 CU585 CSHl2 SWB23 SWB147 SWB148 SWB179 SWB188

SWB203

SWB205

SWBllOO

F'lac proB/Alac-pro AtrpE5 A- F'IacP204::Tn5 proB/Afac-pro AtrpE5 A- F'lacP204::Tn5-112 proB/Alac-pro AtrpE5 A- F galTl2 A- F- ilvD502 leu-455 galTl2 h- F lacZ thi rpsL F A- F Tn5 h- F- tonA A- F lacZ tonA thi rpsL F- lacZ tonA A-

F ilvD:Tn5 leu-455 galTl2 A-

F lac::Tn5 thi rpsL

F- lacZ:Tn5 A-

BERG et al. (1980b) BERG et al. (1980b) BERG (1980) PLEDGER and UMBARGER (1973) SMITH, SMOLIN and UMBARGER (1976) MILLER (1972) Gal+ revertant of CU4 Tn5 insertion in SWB23 Spontaneous TSR mutant of SWB23 Spontaneous T5R mutant of CSHlZ Spontaneous lacZ mutation in

SWB148 Tn5 insertion in ilvD, transduced into

CU505 (WATSON, WILD and UM-

Unmapped insertion of Tn5 in lac using Lac+ revertant of CSH12

Tn5 insertion in SWB23

BARGER 1979)

functional transposase-coding region, the availability of various mutant Tn5 derivatives (ROTHSTEIN et al. 1980), and the absence of sequences homologous to Tn5 in normal E. coli K12 (BERG and DRUMMOND 1978).

Here we show that Tn5 has an in vivo effect on E. coli K12 when strains are grown in carbon-source limited chemostats in the absence of the specific selective agents kanamycin or neomycin. Some strains containing Tn5 exhibit a faster initial rate of growth than do their isogenic non-Tn5 counterparts. This effect occurs in the absence of transposition of intact Tn5 but requires a functional IS50R. A preliminary report of this work has previously appeared (BIEL and HARTL 1981). Our results suggest that prokaryotic transposable ele- ments may have beneficial effects upon their hosts that are mediated by their element-specific transposases.

MATERIALS AND METHODS

Strains and strain construction: E. coli K12 strains are described in Table 1. Chromosomal Tn5 insertions were obtained using h::Tn5 b221 cI857 as in BERG (1977) and D. BOTSTEIN (personal communication). Unmapped prototrophic insertions were selected on minimal glucose agar plus 30 pg/ml kanamycin. Insertions in lac were selected on Macconkey lactose (Difco) containing 30 p g / ml kanamycin (Sigma). /3-Galactosidase activity was determined using ONPG (0-nitrophenyl-b-D- galactopyranoside) as described in MILLER (1972). Plcm clrlOO transductions were performed as in ROSNER (1972). Spontaneous Lac- mutants were selected on TONPG (0-nitrophenyl-B-D-thiogalac- topyranoside) as in MILLER (1972). Spontaneous tonA mutations were used to mark certain non-Tn5 strains and were obtained by selecting cells resistant to bacteriophage T5 (CURTISS 1965). Our data, and those of others (KUBITSCHEK and BENDICKEIT 1964; DYKHUIZEN and HARTL 1980), indicate that tonA mutants are selectively neutral in carbon-source limited chemostats, and tonA can therefore be used as a neutral marker in competition experiments.

Chemostat culture conditions: The assay of selection is based on the relative ability of two strains

584 SELECTION FOR Tn5

to compete for limiting nutrient in a chemostat. Chemostat apparatus, operating conditions, and media were as described in DYKHUIZEN and DAVIES (1980) with the following exceptions. Batch culture minimal media was supplemented with glucose, lactose, glycerol or proline; final concentra- tions were 0.2%. Chemostat media contained 0.04% or 0.01% carbon source as specified (0.1% in the case of proline). Amino acids and vitamins were added as required to a concentration of 20 pg/ml and 1 pg/ml, respectively. Cells were prepared for chemostat inoculation as in DYKHUIZEN and HARTL (1980). Tn5-bearing and non-Tn5 strains were inoculated at frequencies of 0.5 in all experiments. Generation times of approximately 2 hr were used with exceptions to be noted.

Estimation of strain frequency: Resistance to phage T5 was used to monitor the frequency of tonA as described in DYKHUIZEN and HARTL (1980), and the frequency of the Tn5-bearing strain was assayed directly on L-agar plates (MILLER 1972) with and without kanamycin. The tonA mutation was used to mark the non-Tn5 strain, so a decrease in the frequency of the tonA strain indicates a corresponding increase in the Tn5-bearing strain. Doubly marking strains in this manner allows the frequencies of both competing strains to be estimated directly and thereby enhances precision.

Data analysis: If A(t) and B(t) represent the number of Tn5-bearing and non-Tn5 cells, respec- tively, in a chemostat at time t hours after inoculation, then, theoretically, ln[A(t)/B(t)] = ln[A(O) /B(O)] + st, where s is the selection coefficient and measures the competitive ability of the Tn5- bearing strain relative to non-Tn5 (DYKHUIZEN and HARTL 1980). The Tn5-bearing strain is favored, neutral, or disfavored according to whether s > 0, s = 0, or s c 0. The selection coefficient is estimated as the slope of the linear regression of ln[A(t)/B(t)] against time, and the statistical significance of s against the null hypothesis s = 0 is tested by means of analysis of variance (SNEDECOR and COCHRAN 1967). Because carbon source limitation in chemostats requires about 12 hr to be achieved under our conditions, data obtained prior to this time were not used in the regressions. In some cases ln[A(t)/B(t)] does not change linearly throughout the entire course of an experiment, implying that s can change during growth in chemostat culture.

RESULTS

Growth-rate advantage conferred by Tn5: The fundamental observation per- taining to the selective effect of Tn5 is illustrated in Figure 1. When a Tn5- containing strain and its isogenic non-Tn5 counterpart are grown together in chemostats, the strain containing Tn5 increases in frequency. Thus, under conditions of glucose limitation, the Tn5-bearing strain grows faster than its competitor. During the period required for the chemostat population to reach its equilibrium density, very little change in frequency occurs. After approxi- mately 12 hr limiting conditions are reached, and at this point a change in frequency begins and continues for the next 30-40 hr (15-20 generations). This selective advantage abates after 60-70 hr, so that both strains are maintained in the population during a 100- to 120-hr experiment. [Experiments were termi- nated at 100-120 hr because the phenomenon of interest occurs by about 70 hr, and complications resulting from periodic selection can increase significantly after 100 hr (NOVICK and SZILARD 1950).] This leveling off is not an artifact of cells growing on the chemostat wall because, when such mixed cultures are transferred to clean chemostats after 70 hr of growth, no immediate change in frequency is observed (data not shown). Were wall growth the cause of the diminished advantage, this constancy of frequency upon transfer would not occur.

SWB147 and SWB148 are isogenic except for the Tn5 insertion in SWB147 (site unknown) and tonA in SWB148. To rule out the spread of Tn5 by means of some unknown type of genetic exchange in the chemostat, SWB147 and SWB148 were grown together in a chemostat until the frequency of KanR cells

S . W. BlEL AND D. L. HARTL 585

I 6.0 } luc: T n 5 ton A+ (SWBllOO) /vs lac n o n - T n 5 tonA(SWB188)

’- 0.9%

-0.99 9 C

I-

5.0 -

4.0 - - - 0.95 .-. 6

5 3.0 2; C

U & 2.01 \ h D . T n 5 (SWB203)

VI //vD n o n - T n 5 (CU585) - V.Y

- 0.7 - 0.5

I I I I I I 1 I I 40.3 20 40 60 80 100

TIME (hrs.) FIGURE 1.-Growth-rate advantage conferred by Tn5. Triangles: SWBllOO (lac: :Tn5) vs. SWB188

(lac tonA); s = 0.047 f 0.014. Squares: SWB203 (ilvD:Tn5) vs. CU585 (ilvD); s = 0.046 f 0.014. Circles: SWB148 (tonA) vs. SWBZI (tonA+) control: s = 0.004 +- 0.004.

exceeded 95%. At this point 1050 T5R colonies were replica plated to L-agar plus kanamycin, and all colonies exhibited the expected KanS phenotype.

Strain SWB147 is unsuitable for characterizing the effect of Tn5 in detail because the location of Tn5 in SWB147 is unknown. We therefore studied insertions at known locations in order to address the following issues: 1) Is the selective advantage limited to particular sites of insertion of Tn5? 2) Does it depend on the host genotype? 3) Is it altered or eliminated by different culture conditions? 4) Is it “physiological” in the sense that it can be altered by adapting strains to limiting growth conditions before competition? 5) Is it caused by transposition of Tn5? 6) Is it a pleiotropic effect of the kanamycin phosphotrans- ferase gene?

The selective advantage is independent of insertion site: Figure 1 illustrates that the selective effect of Tn5 occurs irrespective of the site of insertion. Strains SWBllOO and SWB188 are isogenic except for tonA and lac (SWB1100 has a lac: :Tn5 insertion, SWB188 has a spontaneous lac mutation: both lack P-galac- tosidase activity); the selection coefficients for the Tn5-bearing strain are s = 0.047 0.014 (triangles) in one replicate and s = 0.050 & 0.024 in another. Corresponding slopes for an independently isolated lac: :Tn5 insertion that retains P-galactosidase activity (SWB1200) are 0.051 f 0.016 and 0.058 f 0.014. Virtually identical selection occurs for ilvD insertions (squares). Strain SWB203 has an ilvD::Tn5 insertion whereas cu585 has an ilvD point mutation. The selection coefficient favoring the Tn5-bearing strain is still around 5% per hour, namely s = 0.046 f 0.014. The circles in Figure 1 pertain to strains SWB23 and an isogenic tonA derivative (SWB148); this is a control showing the selective

586 SELECTION FOR Tn5

TABLE 2

Selection involving E. coli K12 strains

Strains In competition S F Significance*

DB1506-204 vs. DB1506' 0.018 k 0.0004 2307.75 <0.001 DB1506-204 vs. DB1506".d 0.034 -C 0.002 307.56 <0.001 SWB205 vs. SWB179 0.002 k 0.004 0.18 n.s.= SWB205 vs. SWB179 0.009 f 0.005 2.80 n s . SWBllOO vs. SWB188' 0.032 f 0.008 16.89 0.010 SWBll00 vs. SWB188g 0.028 k 0,011 6.74 0.05 SWBll00 vs. SWB188h 0.042 k 0.009 21.58 0.001

a F test of significance of s = 0. * DB1506-204 contains one copy of Tn5. e Selection coefficient pooled over four separate experiments.

e Not significant. Subclone of DB1506-204 containing at least two copies of Tn5 per genome.

Glucose concentration 0.01%; generation time 2 hr. Glucose concentration 0.04%; generation time 4 hr.

* Glucose concentration 0.0470, generation time 2 hr.

neutrality of tonA under these conditions. In some experiments the non-Tn5 strain was not marked with tonA, yet the selective advantage of Tn5 was still observed, indicating that tonA itself is not involved in the phenomenon.

The selective advantage depends on host genotype: Experiments like those in Figure 1 were carried out with strains of E. coli K12 not closely related to SWB23. Strain DB1506-204, obtained from DOUGLAS BERG, carries an F'lacP: :Tn5 proB episome; in competition with its isogenic parent (DB1506), the Tn5-bearing strain was favored at the rate of about 0.018 per hour (Table 2, line 1). One isolate of DB1506-204 proved to have at least two copies of Tn5, as curing of the episome did not result in loss of KanR (Table 2, line 2). It is of interest that selection favoring this strain is somewhat greater than selection involving the single copy DB1506-204.

Table 2 also shows that strains derived from CSHl2 do not exhibit the Tn5- associated selection. SWB205 (lac::Tn5) and SWB179 (lac tonA) are both derived from CSH12, and in this genetic background Tn5 appears to be selectively neutral. We interpret this to mean that the advantage involving Tn5 observed in other strains is caused by an interaction between Tn5 and an unknown gene or genes in the bacterial genome.

Effect of culture conditions on the selective advantage: We have examined a limited range of generation times, equilibrium densities and limiting carbon sources, and none of these factors substantially alters the advantage conferred by Tn5.

Table 2 (line 5) shows the result of an experiment in which the limiting glucose concentration was reduced from 0.04% to 0.01%, which reduces equilib- rium cell density from 4-5 X 10' cells/ml to 1-2 X 10' cells/ml. Nevertheless, the selective effect of Tn5 is within the range observed in experiments at the higher density.

Most of our experiments have been carried out at a generation time of 2 hr. Table 2 (line 6) shows the result of an experiment carried out at a 4-hr generation

S. W. BIEL AND D. L. HARTL 587

TABLE 3

Preadaptation of J'WB1100 and SWB188

Time in monoculture S F Significance

12 hr" 0.060 f 0.01 24 hr" 0.040 f 0.007 72 hr" 0.021 f 0.013 Comparison of 12 and 24 hr 2.75 ns.' Pooled 12 and 24 hr 0.058 f 0.008 Comparison of pooled vs. 72 hr 29.75 0.001

Values of s for 12, 24 and 72 hr pooled over two experiments. Not significant. (F test for difference of two slopes.)

time. Again, the Tn5-associated effect is comparable to that observed in other experiments. The selective advantage of Tn5 also occurs in chemostats limited for glycerol, proline or lactose, and selection coefficients ranging from 0.04 to 0.08 were observed. The effect of Tn5 is thus not a peculiarity of glucose limitation but occurs with any carbon source limitation and under a variety of densities and doubling times.

Preadaptation to chemostat culture eliminates the selective advantage: The diminution of the selective advantage with time (Figure 1) suggests that the advantage may be a temporary physiological effect occurring immediately upon carbon and/or energy limitation and becoming less important as the strains become adapted to their stringent growth conditions. To examine this possibil- ity, strains were grown in monoculture chemostats for various lengths of time before inoculation into mixed culture chemostats. Results are shown in Table 3. By 72 hr of preadaptation the selective differential disappears. The pooled selection coefficient for 12 and 24 hr of preadaptation is significantly greater than the one observed at 72 hr, and the selection coefficient at 72 hr does not differ significantly from 0. This finding coincides with the earlier observation that after 65 to 70 hr changes in the frequencies of Tn5 and non-Tn5 strains no longer occur. Either the growth rate of the Tn5-bearing strain decreases or that of the non-Tn5 strain increases. Such an increase could be attributed to physi- ological adaptation of the non-Tn5 strain. Alternatively, the non-Tn5 strain could evolve genetically, but 72 hr (36 generations) seems too brief a time for a newly arisen mutation to become nearly fixed.

The time required for the increase in frequency of the Tn5-bearing strain also implies that the selection is not caused by advantageous Tn5-induced mutations. NESTMAN and HILL (1973) and Cox and GIBSON (1974) investigated the kinetics of selection for strains carrying various mutator alleles. In their experiments, selection for the mutator strain did not begin for aproximately 50 generations and continued at a relatively constant rate for 500 generations. The kinetics of the Tn5 effect are quite different and would seem to rule out a mutational origin.

The selective advantage is not associated with transposition of Tn5: The kinetic argument is rather weak, but the possibility that Tn5 transposes to other genomic sites and creates favorable mutations can be ruled out by two direct

588 SELECTION FOR Tn5

experiments. In one experiment, strains DB1506-204 and its isogenic counterpart, DB1506, were grown together in a chemostat until DB1506-204 had reached a frequency of 90-95%. At this time individual KanR colonies were obtained and cured of their episome by growth to saturation in L-broth plus 250 pg/ml acridine orange (MILLER 1972). Cells that have lost their episome give rise to white colonies on Macconkey lactose agar in contrast to the pink coloration caused by leakiness of the lacP::Tn5 allele. Loss of the episome was confirmed by the inability of the cells to grow without added proline. From the DB1506- 204 cells recovered from the chemostat, 35 were cured of their episome and all were Kans, indicating simultaneous loss of Tn5. In no case had transposition of Tn5 from the episome to the chromosome occurred.

In a second experiment, SWlloO and SW188 were competed until the fre- quency of SWllOO exceeded 90%. Individual KanR colonies were streaked on Macconkey lactose agar and the red Lac+ revertants tested for their resistance to kanamycin. Ninety-one out of 96 cells from the original chemostat became KanS simultaneously with reversion to Lac+. Again there is no evidence for transposition as the cause of the selective effect, as 5% multiple copy cells is insufficient to account for the increase in frequency of the Tn5-bearing strain from 50 to 90%. Five percent of cells with multiple Tn5s is also in good agreement with values reported in non-chemostat experiments (BERG 1977).

IS50R is required for 7315's selective effect: To determine whether the bene- ficial effect of Tn5 is a pleiotropic effect of an aminoglycoside phosphotrans- ferase gene or is associated with IS50, experiments were carried out using Tn5- 112 (JORGENSEN, ROTHSTEIN and REZNIKOFF 1979). Tn5-112 was created from Tn5 by deletion of an internal 3.0-kilobase fragment. Tn5-112 retains the outermost 186 base pairs of IS50R, an intact kanamycin resistance gene, and all of IS50L. The deletion removes the transposase coding region of IS50R and approximately half of the central region of Tn5, so the element is defective in transposition. However, Tn5-112 retains transposase recognition sites at both termini and renders cells kanamycin resistant.

As shown in Figure 2, DB1506-204, which contains wild-type Tn5, has a selective advantage of s = 0.018 k 0.0004. Strain DB1633-3, which contains Tn5- 112 at the same site, shows no significant selection (s = -0.0008 k 0.001). This result indicates that the selective advantage that Tn5 confers upon E. coli K12 requires the presence of an IS50R coding for functional transposase.

DISCUSSION

Our results reveal a selective advantage of Tn5 in certain strains of E. coli K12. This favorable effect appears as a growth-rate advantage associated with an abrupt shift from carbon source excess to the carbon source limitation that occurs in chemostats. The advantage is substantial, ranging from 2 to 8%/hr in various experiments, and is sufficient to increase the frequency of the Tn5- bearing strain from an initial 50% to 95% or more in 40 hr of growth.

The effect of Tn5 is not dependent upon its actual site of insertion as it occurs with lac: :Tn5, ilvD: :Tn5, F'lacP: :Tn5, and insertions at unknown locations. Tn5 can insert in essential genes, and in a natural environment these detrimental

S. W. BIEL A N D D. L. HARTL

1.5

1 .o T

5 C

c; .5 C \ 9 I.

589

1

-0.5

FIGURE 2.-1S50R required for selective effect of Tn5. Circles: DB1506-204 (F'lacP: :Tn5) VS. DB1506 (F'hc); s = 0.018 1- 0.0004. Triangles: DB1633-3 (F'IacP:Tn5-112) vs. DB1506; s = -0.0008 f 0.001. DB1506-204 and DB1633-3 are phenotypically Lac- but leaky, whereas DB1506 is Lac+.

F'/acP204::Tn5-112 (DB1633-3) - vs F' lac* (DB1506)

I I I I I I I I l l 1

mutations could override the beneficial effect of Tn5. Insertions resulting in auxotrophy have been reported to occur in only 2% of all cells into which Tn5 has been introduced (BERG 1977), and in our experiments involving prototrophic strains carrying unmapped Tn5 insertions, none had an altered maximal growth rate.

The beneficial effect of Tn5 involves an interaction between the element and the genome of the host, as evidenced by the finding that the effect is absent in strains of the CSH12 lineage. We do not know how widespread the phenomenon may be in lineages other than E. coli K12. Natural isolates of E. coli exhibit an extraordinary amount of genetic variation (SELANDER and LEVIN 1980), and it is tempting to speculate that the interaction responsible for the phenomenon would occur in some proportion of natural isolates of E. coli and other bacterial species.

As to mechanism, the experiments with the mutant Tn5-112 indicate that the right-hand IS50 element is required for the Tn5 effect whereas the kanamycin resistance gene is not involved. It is unlikely that 1S50R induces its beneficial effect by acting as a mutagenic agent and creating new mutations that uniquely adapt the cells to growth in chemostats. The effect occurs while Tn5 remains in situ, so transposition of the intact element is not involved. Although IS50 alone can transpose and would not have been detected by our assay, BERG et al. (1981, 1982) have shown that IS50 transposes less frequently than the intact element

590 SELECTION FOR Tn5

2nd is therefore unlikely to promote the rapid frequency changes observed. It is also unlikely that IS50s transposase mobilizes other transposable elements endogenous to the E. coli K12 genome because complementation requires a high degree of sequence homology (CHOI et al. 1980; REED 1981) and no Tn5-homol- ogous sequences can be detected in E. coli K12 (BERG and DRUMMOND 1978). Transpositionless Tn5 mutants remain unable to transpose even when placed on an F’ episome in proximity to other IS elements. Although mediation of the effect by transposition of IS50 to new, advantageous positions remains a formal possibility, the genetic and kinetic data argue against it. We currently favor an explanation in which the transposase protein(s) encoded by IS50 are responsible for the selective advantage in the absence of transposition by virtue of its nonspecific interaction with DNA.

The selective advantage conferred by Tn5 is particularly interesting because it is not unique to Tn5. LIN, BITNER and EDLIN (1977) and EDLIN, LIN and BITNER (1977) have shown that lysogens carrying the highly specialized transposable elements, lambda, Mu, and Plcm (Plcm contains Tn9), are favored in chemostat competition over their nonlysogenic counterparts, and we have found that TnlO has effects in chemostats similar to those of Tn5 (s = 0.045 k 0.005, data not shown).

We have described a new growth-rate effect of Tn5, which seems to occur with other transposable elements as well. Although the effect is small relative to the effect of antibiotic resistance genes in the presence of their cognate antibiotics, it is likely to be important because natural populations of bacteria often fluctuate between nutrient excess and nutrient deficiency and between very different physiological states. Thus, the temporary, probably physiological, growth-rate advantage conferred by Tn5 is likely to be important in the adap- tation of natural populations to new conditions. Indeed, the effect is sufficiently strong to maintain the transposon, even during prolonged periods in which the antibiotic is absent from the environment. In a wider context, a slight growth- rate advantage of IS elements will lead to their proliferation and maintenance in natural populations and account for their great abundance and variety. At some later time in their evolution, some of these IS elements become integral parts of transposons carrying strongly beneficial genes, such as those associated with antibiotic resistance, and thus the selection pressures that initially main- tained the IS elements become overshadowed.

We wish to thank BARBARA MCCLINTOCK for helpful suggestions and criticisms of a preliminary draft of the manuscript. Thanks also to DOUGLAS BERG for providing strains and for many useful suggestions and discussion, and DANIEL DYKHUIZEN for technical advice in using chemostats. This work was supported by Public Health Service Grant GM30201.

LITERATURE CITED

ANDERSON, E. S., 1968 The ecology of transferable drug resistance in the Enterobacteria. Ann. Rev. Microbiol. 22: 131-180.

BERG, D. E., 1977 Insertion and excision of the transposable kanamycin resistance determinant Tn5, pp. 205-212. In: DNA Insertion Elements, Plasmids, and Episomes. Edited by A. BUKHARI, J. A. SHAPIRO and S. ADHYA. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

S . W. BIEL AND D. L. HARTL 591

BERG, D. E., 1980 Control of gene expression by a mobile recombinational switch. Proc. Natl. Acad. Sci. USA 77: 4880-4884.

BERG, D. E., J . DAVIES, B. ALLET and 1.-D. ROCHAIX, 1975 Transposition of R-factor genes to the bacteriophage lambda. Proc. Natl. Acad. Sci. USA 7 2 3628-3632.

BERG, D. E. and M. DRUMMOND, 1978 Absence of DNA sequences homologous to transposable element Tn5 (kan) in the chromosome of Escherichia coli K-12. J. Bacteriol. 136 419-422.

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Corresponding editor: G. MOSIG