JOURNAL OF BACTKIUOWGY, Mar. 1976, p. 800-810Copyright 0 1976 American Society for Microbiology

Vol. 125, No. 3Printed in U.S.A.

Transposition of a Deoxyribonucleic Acid Sequence EncodingTrimethoprim and Streptomycin Resistances from R483 to

Other RepliconsP. T. BARTH, NAOMI DATTA,* R. W. HEDGES, AND N. J. GRINTER

Department of Bacteriology, Royal Postgraduate Medical School, London W12 OHS, England

Received for publication 22 September 1975

R483, a plasmid of the Ia incompatibility group, contained a deoxyribonucleicacid (DNA) sequence encoding resistance to trimethoprim (TpR) and streptomy-cin (SmR) that could be transposed to other replicons, i.e., to the Escherichia colichromosome and to related and unrelated plasmids. Each transposition resultedin the acquisition by the recipient replicon of a segment ofDNA of about 9 x 106daltons, both resistance genes, but never the colicin Ia or pilus genes of R483.Transposition took place at a single chromosomal site between dnaA and ilv anddid not suppress the DnaA phenotype, in contrast to integration of the wholeR483 plasmid. The chromosome, having received the transposition, could sec-ondarily act as a transposition donor to another plasmid. Such a plasmid wasindistinguishable from one having received a direct transposition from R483.TpR SmR transposition was very site specific and did not require a functionalrecA + gene. We postulate that the TpR SmR segment of R483 is a transposon(TnC) with specific boundary sequences.

In an accompanying paper (6) we noted that astrain carrying two plasmids of Ia incompati-bility group, J62(R483) (R144) (where R483 hadbeen first resident and R144 was introducedsubsequently), retained the resistance markersof both plasmids during growth, but the trans-missibility of the trimethoprim (TpR) and strep-tomycin (SmR) resistances of R483 was lost orgreatly reduced. We also observed clones inwhich these R483 markers had become linkedin their transmissibility to those of R144. Theseobservations seemed analogous to the transpo-sition of the gene(s) specifying ampicillin resist-ance (ApR) from RP4 to R64 (9), to other plas-mids (14), or to the chromosome of Escherichiacoli K-12 (27). Transposition, it has been sug-gested, may involve the integration of a specificdeoxyribonucleic acid (DNA) sequence (a"transposon") by a mechanism not involvingconventional recombination (14). We reporthere our studies on the transposition ofTpR andSmR from R483 to other replicons, includingevidence for the size of the transposed DNAsegment, the specificity of its site of insertion,and the influence of the bacterial recA gene.

MATERIALS AND METHODS

Media and materials. Media and materials wereas described previously (2).

Bacterial strains and plasmids. Strains and plas-mids used are listed in Table 1.

Resistance transfer, demonstration of colicino-geny, and I pilus production. The procedures wereas described (7).

Isolation of a recA derivative of C600. StrainMA1079 (Hfr recA ser thi, obtained from W. Maas)has a counterclockwise mode of chromosome trans-fer, with lysA + (55 min) being an early marker. Aculture of MA1079 was mixed with C600Thy- (de-rived by trimethoprim selection by V. Salisbury) for30 min and plated out, selecting for Thy+ C600clones. These were tested for ultraviolet light sensi-tivity. One such sensitive clone was designatedHH26 (Table 1).

Plasmid curing. A washed broth culture ofJ62(JR66a), in which the TpR SMR markers of R483were present but nontransmissible, was treatedwith graded doses of ultraviolet light (potency notmeasured). Irradiated samples were introduced intobroth containing graded doses of acriflavine (2 to 50Ag/ml) and incubated overnight. Cultures showingslight turbidity in the highest acriflavine concentra-tion for each ultraviolet dose were diluted, spread onnutrient agar plates, and incubated. The resultingcolonies were replicated to nutrient agar plates con-taining 25 ,ug of kanamycin (Km) per ml, and Km-sensitive clones were looked for.

Isolation of plasmid DNA. The DNA of bacterialcultures was labeled and plasmid DNA was isolatedby ethidium bromide-CsCl equilibrium centrifuga-tion as previously described (2).

Sucrose gradient sedimentation analysis. La-beled plasmid DNA was analyzed by sedimentationthrough freeze-thaw-generated neutral 5 to 20% su-crose gradients; molecular weights were calculatedas previously described (2).

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TRANSPOSITION OF LINKED Tp AND Sm RESISTANCE 801

TABLE 1. Bacterial strains and plasmids useda

Strain or plas- Relevant characters5 Reference or sourcemid

StrainJ53 proA metF (X) (4)J62 proC his trp lac (X) (4)AB1157 thr leu thi proA his argE str (1)AB1450 argH his metB ilvD str B. J. BachmannCRT46 thr leu thi ilv thy dnaA (ts) (18)W3110T- thy dra (1)C600 thr leu thi lacY (1)HH26 thr leu thi lacY recA MA1079 x C600Thy- (see text)

PlasmidR483 Tp Sm/Sp ColIa I pili Incla (6)R144 Tc Km ColIb I pili IncIa (13)JR66a Sm Km I pili IncIa (13)Flac lac+ IncFI (23)R391 Km Hg IncJ (15)R7K Ap IncW (11)RP4 Ap Tc Km IncP (11)a Chromosomal marker symbols are according to Taylor and Trotter (30).b Plasmid marker symbols are resistances to trimethoprim (Tp), streptomycin-spectinomycin (Sm/Sp),

tetracycline (Tc), kanamycin (Km), mercury (Hg), and ampicillin (Ap). Production of colicins Ia and Ib,ColIa and ColIb, respectively; incompatibility group, Inc.

P1 transduction. Plkc transducing lysates weremade basically as described by Walker and Ander-son (31). Temperature-resistant donor strains weregrown in broth containing 10 mM CaCi2 at 37 C withaeration to a titer of about 5 x 108 cells/ml. Temper-ature-sensitive (dnaA) strains were similarlygrown, but in Hershey medium (17) with 10 mMCaCl2 at 30C; subsequent steps were also at thistemperature. A 0.25-ml sample of such a culture wasmixed with a 0.1-ml suspension of Plkc (about 5 x107 plaque-forming units per ml), and phage adsorp-tion was allowed for 25 min at 37 C. A 2.5-ml sampleof soft minimal agar, containing 20 mM CaCl2, wasadded, and the mixture was poured onto a pre-warmed, undried plate of 50 ml of nutrient agarcontaining 10 mM CaCl2. Plates were incubated at37 C overnight. The soft top layers were collected,mixed with 0.25 ml ofchloroform, and centrifuged at12,000 x g for 10 min at 4 C. The supernatant liquidwas diluted 10-fold in P1 buffer (31). Phage titerswere assayed after adsorption and plating (on ordi-nary dried nutrient agar plates) and were typically 5x 108 to 10 x 108 plaque-forming units/ml after thedilution. Cultures of the recipient strain were grownas above. The cells were centrifuged and resus-pended in 0.1 volume of donor phage suspension (inP1 buffer) to give a multiplicity of infection of 0.01 to0.05. A 25-min period adsorption was allowed (at 37or 30 C). Sodium citrate solution was added to afinal concentration of 10 mg/ml, and the cells werecentrifuged again. The cells were resuspended in 2.5ml of broth containing sodium citrate (10 mg/ml),incubated for 1 h at 37 or 30 C, and then plated onsuitable selective media.DNA-DNA hybridization. DNA sequence homolo-

gies were assayed at 75 C by using the single-strand-specific S1 endonuclease as previously de-scribed (3).

Restriction of DNA by EcoRl. Plasmid DNA was

restricted in a buffer of 100 mM tris(hydroxy-methyl)aminomethane, pH 7.5, containing 10 mMMgCl2 with 0.05 volume ofEcoRl (a gift of S. Cohen)at 37 C for 30 min.

RESULTS

Derivation of cultures carrying no plasmidcharacters other than TpR SmR. Introduction ofR144 or JR66a into a culture of E. coli K-12carrying R483 led to loss of the colicin Iamarker ofR483 and the normal transmissibilityof its TpR and SmR markers (6, 13). Cultures ofE. coli K-12 with nontransmissible TpR SmR,without plasmids R144 or JR66a, were obtainedby two methods.

(i) Curing. After treatment with ultravioletlight and acriflavine, a single clone lackingJR66a was isolated out of about 5,000 tested.The culture was no longer Km resistant, butretained nontransmissible TpR SmR. It wasdesignated J62-TnC1.

(ii) Mobilization. On several occasions avery low frequency of transfer (<10-6 per donorcell) of TpR was observed from R144+ or JR66a+cultures that had lost the ColIa and normaltransmissibility of R483. The majority of theTpR transconjugants were also KmR, but a mi-nority were resistant only to TpSm, their resist-ance was nontransmissible, and they producedno detectable I pili. Independently isolatedclones were designated J62-TnC2, J53-TnC3,J53-TnC4, and J62-TnC5. In all these, R144 wasthe conjugative plasmid that had acted as mobi-lizing agent.

Analysis of DNA from strains with non-

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transmissible R483 resistance markers. J62-TnC1 and J62-TnC2 were grown in the presenceof L3H]thymine and washed with cold TESbuffer, and each was mixed with an equal vol-ume of a TES-washed, ['4C]thymine-labeledculture of W3110T-(R1). The mixtures werethen lysed and centrifuged to equilibrium withethidium bromide and CsCl. The admixture ofa known plasmid-containing strain acts as acontrol of successful isolation of supercoiledDNA by this technique.The radioactivity in each fraction after cen-

trifugation is shown in Fig. 1. Whereas the 14Clabel appeared in two bands corresponding tosupercoiled (R1) and linear (chromosomal)DNA as expected, the 3H-labeled materiallacked any significant supercoiled DNA bandin either J62-TnC1 or J62-TnC2. We concludethat the TpR and SmR markers of R483 couldexist in these strains on a nonconjugative plas-mid that does not appear as supercoiled DNA,but, since such an event has no precedent to ourknowledge, it is more likely that these markershave become inserted into the bacterial chro-mosome, as postulated previously (13).Mapping TpR and SmR from R483 in the E.

coli chromosome. We sought an approximatemap position for TpR in J62-TnC1 and J62-TnC2

lO5-

10 -

H3JH62-TnCIcl4C RI

30 400.1 ml fracions

by testing for its co-mobilization with chromo-somal markers mediated by Flac. After 90 miinof uninterrupted conjugations of J62-TnCl(Flac) and J62-TnC2 (Flac) with AB1157, TpRclones of AB1157 were selected and scored forthe acquisition of several markers carried onthe donor chromosome. The results (Table 2)suggest that TpR may have a map position be-tween argE and mtl. The SmR marker was al-ways co-mobilized with TpR.

TABLE 2. Flac mobilization ofTPR and chromosomalmarkers from J62-TnCl and J62-TnC2 into

AB1157a

Unselected marker Cotransfer with Tpm from:

Gene Map posi- J62-TnCl J62-TnC2tion (min) (Flac) (Flac)

leu+ 2 24 (34)b 20 (28)argE+ 79 39 (55) 37 (52)mtl+ 71 23 (32) 32 (45)galK+ 17 0 (0) 0 (0)

a After 90 min of uninterrupted conjugations ofJ62-TnC1(Flac) and J62-TnC2(Flac) with AB1157 at37 C in broth, 71 TpR clones from each cross werepurified and tested for the cotransfer of chromo-somal markers to the recipient.

b Numbers in parentheses are percentages.

. 3H Jg2-TnC2o0 C RI

30 400.1 ml fractions

50

FIG. 1. Ethidium bromide-CsCl equilibrium centrifugations of 3H-labeled DNA from J62-TnCl and J62-TnC2 [strains ofJ62(R483) that had lost their normal TpR Sm" transmissibility; see text]. Each was mixedwith a 14C-labeled culture ofan Rl + strain before lysis as a control ofplasmid isolation. After centrifugationand fractionation, a 1O0-p. sample from each fraction was assessed for radioactivity (expressed logarithmicallyon the ordinate). The CsCl density decreases from left to right. The '4C-labeled supercoiled peak is on the left ineach graph.

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TABLE 3. Pl cotransduction of markers from J62, J62-TnCl, and J62-TnC2 into CRT46 (ilv dnaA)a

P1 donor Selected marker Unselectedmarker Cotransduction frequency Mapping dis-

tance (min)

J62-TnC1 TpR Ilv+ 30/112 (26.8)b 0.82J62-TnC2 TpR Ilv+ 26/115 (22.6) 0.90J62-TnC1 Ilv+ TpR 46/112 (41.1) 0.59J62-TnC2 Ilv+ TpR 50/110 (45.5) 0.53J62-TnC1 TpR DnaA+ 78/114 (68.4) 0.27J62-TnC2 TpR DnaA+ 75/115 (65.2) 0.31J62-TnCl DnaA+ TpR 76/111 (68.5) 0.27J62-TnC2 DnaA+ TpR 44/71 (62.0) 0.34J62 Ilv+ DnaA+ 78/408 (19.1) 0.98J62-TnC1 Ilv+ DnaA+ 22/407 (5.4) 1.43J62-TnC2 Ilv+ DnaA+ 32/609 (5.3) 1.44J62 DnaA+ liv 55/409 (13.4) 1.12J62-TnC1 DnaA+ liv 6/245 (2.4) 1.63J62-TnC2 DnaA+ Ilv 8/323 (2.5) 1.63

a P1 transductants of CRT46 were selected for trimethoprim resistance (TpR), ability to grow in theabsence of isoleucine and valine (Ilv+) or for growth at 42 C (DnaA+). Purified clones were then scored for theunselected markers shown. Mapping distances were calculated according to Wu (32), using 2.3 min for thesize of P1 (unpublished data).

b Numbers in parentheses are percentages.

Finer and unequivocal mapping wasachieved by P1 transduction. TpH was cotrans-ducible with ilv and dnaA, closer to dnaA (Ta-ble 3, lines 1-8). Since the distance between ilvand dnaA was about 1 min (lines 9 and 12), TpRmust be between them (see also next section),mapping close to 74 min on the E. coli K-12 map(30).

Specificity of the chromosomal integrationsite. The transposition of TpR SmR from R483into the chromosome took place in J62-TnC1and J62-TnC2 at apparently the same site.Three more strains carrying transposed TpRSmR (J53-TnC3, J53-TnC4, and J62-TnC5) weretested for P1 cotransduction between Ilv andTpR. The results showed that the E. coli chro-mosome probably has a unique or single pre-ferred locus for TpR integration (Table 4). Theseresults also show that the TpR SmR genes hadbeen mobilized from one chromosome to anotherby R144. We have not done experiments to dis-tinguish between the various conceivablemechanisms by which this may have comeabout, but we consider the most likely to bechromosomal mobilization without establish-ment of the mobilizing plasmid.Length of transposed DNA measured by

transduction. We estimated the length ofDNAtransposed from R483 into the chromosome bymeans of P1 transduction. We scored the co-transduction frequency of dnaA + and ilv+ fromJ62 and its derivatives, J62-TnC1 and J62-TnC2, into strain CRT46 (dnaA, ilv). By anal-ogy with the integration of A between gal andbio (28), we expected the insertion of DNA be-tween dnaA + and ilu + to reduce their cotrans-

TABLE 4. Pl cotransduction of ilv and TPe inchromosomally transposed strainsa

Selected Unselected CotransductionP1 donor marker marker frequency

J53-TnC3 FjpR Ilv+ 32/76 (42)bJ53-TnC3 Ilv+ TpR 15/49 (31)J53-TnC4 Tp Ilv 22/65 (34)J53-TnC4 Ilv Tp 4/13 (31)J62-TnC5 Tp liv 38/78 (49)J62-TnC5 Ilv Tp 21/52 (40)

a TpR colonies were selected from P1 transuctionsinto AB1450 and scored for their ability to grow inthe absence of isoleucine and valine.

b Numbers in parentheses are percentages.

duction frequency. The last six lines in Table 3show that this expectation was fulfilled: afterselection for Ilv+, the dnaA ilv cotransductionfrequency was reduced from 19.1 to 5.4% by thepresence of TpR SmR in the donor strain. Afterselection for growth at 42 C, TpR SmR in thedonor strain reduced the cotransduction fre-quency from 13.4 to 2.5%. J62-TnC1 and J62-TnC2 behaved indistinguishably. Using Wu'sequation (32) and the molecular weight of vege-tative P1 phage DNA as 64 x 106 (unpublisheddata), we calculated from these results a molec-ular weight for the interposed DNA of 12.8 x106 and 14.2 x 106, respectively.Length of transposed DNA measured by

DNA-DNA hybridization. We also measuredthe proportion of the R483 molecule that hadbecome transposed into the chromosome byDNA-DNA hybridization between labeled R483plasmid DNA and unlabeled chromosomalDNA from J62, J62-TnCl, and J62-TnC2. The

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latter strains had gained DNA sequences ho-mologous to about 15% of R483, i.e., had gainedan average of 9.6 Mdal from the 62-Mdal R483plasmid (Table 5).Lack of suppression of the DnaA phenotype

by TpR SmR integration. P1 transduction ofTrpRfrom J62-TnC1 or J62-TnC2 into the dnaAstrain CRT46 did not lead to temperature rever-sion of all the TpR recipients (Table 3, lines 5-8).Temperature reversion of all the clones wouldbe expected if integration into the chromosomeof the DNA segment from R483, carrying TpRSmR, gave suppression of the DnaA phenotype,as does the whole R483 molecule itself (7).Transposition of TpR and SmR into Ia group

plasmids. Clones from J62(R483)(JR66a) orJ62(R483) (R144), i.e., strains containing two Iaplasmids with R483 being the first resident (6),were occasionally observed to have become sta-ble, but in contrast to the above, Tp and Smresistance determinants remained transmissi-ble, linked to the markers of the second plas-mid. The colicin Ia marker was never retained.We considered that this might be due to thetransposition of TpR and SmR from R483 ontothe second plasmid, analogously to their trans-position into the chromosome.

3H-labeled plasmid DNA was isolated from afew such clones and analyzed by sedimentationthrough 5 to 20% sucrose gradients togetherwith "4C-labeled plasmid DNA from JR66a+ orR144+ strains, according to which was "paren-tal." Figure 2 shows the cosedimentation ofR144 with R144-ThC1 [i.e., the transpositionplasmid derived from J62(R483)(R144)I. R144-

TABLE 5. Measurement by DNA-DNA hybridizationof the R483 segment transposed into J62-TnC1 and

-TnC2

Source of Hybridiza- N I. . Mol wt ofunla- tion with oruazn transposedbeled ['H1R483 homologous segmentrDNA (9b reaction (M)' (Mdal)

J62(R483) 74.8 100J62 1.9J62-TnC1 12.3 14.3 8.9J62-TnC2 14.1 16.7 10.4

a Samples of sheared [3H]R483 DNA (isolated byethidium bromide-CsCl equilibrium centrifugation)were mixed with sheared unlabeled DNA isolated(25) from the strains shown to give final concentra-tions of 10 and 150 gg/ml, respectively. After dena-turation, they were incubated at 75 C for 22 h andassayed for annealed DNA by using the single-strand-specific S1 endonuclease (3).

b The nonspecific (J62) reaction was first sub-tracted.

' Taking the molecular weight of R483 as 62 Mdal(Table 6).

TnC1 had a higher molecular weight than itsparent, R144. Similarly, analysis of two inde-pendently isolated JR66a-derived plasmids(JR66a-TnC1 and JR66a-TnC2) revealed thateach had increased in molecular weight com-pared with its parent (Table 6). The molecularweight gains of the transposition plasmids of7.4 to 9.8 Mdal are, within experimental error,not significantly different from one another orfrom the gain by the chromosome upon transpo-sition of TpR SmR.

Transposition of TpR and SmR into plasmidsof other compatibility groups. Transpositionof markers from R483 into other Ia group plas-mids, which are all related, sharing about 50 to70% DNA homology (3, 11), does not suggestthat a mechanism other than normal recombi-nation need be occurring. Hedges and Jacob(14), however, have shown that ampicillin re-sistance is transposed freely from RP4 to plas-mids unrelated by compatibility group or DNAhomology (11), suggesting a mechanism dis-tinct from conventional recombination. Wetherefore looked for TpR transposition into plas-mids R391(IncJ), R7K(IncW), and RP4(IncP) byselecting for TpR transfer from J62-TnC1 carry-ing each of these plasmids into J53 or anothersuitable recipient. Transconjugants were testedfor their resistance markers and transmissibil-ity.

(i) Transposition of TpR SmR into R391.R391 gave two classes of transconjugants, viz.,transmissible and nontransmissible (all had re-tained the Km, Hg resistance markers of R391and had acquired TpR SmR from J62-TnC1). Weattempted to isolate radiolabeled plasmid DNAfrom a tra+ clone [J53(R391-TnC1)], a tra-clone [J53(R391-TnCO1)], and the parent[W3110T-(R391)]. None of them had a visiblesupercoiled DNA band after ethidium bromideCsCl centrifugation, nor were there any signifi-cant counts detectable below the chromosomalDNA after fractionation. Molecular weightanalysis of the plasmids was therefore impossi-ble. It has been confirmed in our laboratorythat R391, and other J plasmids, cannot beisolated as supercoiled DNA by our normal pro-cedure (15). This seems unlikely to be due totheir having too large a molecular weight to beisolated intact; our procedure permits the isola-tion of intact molecules of at least 170 Mdal(group S plasmids; P. T. Barth, manuscript inpreparation).

(ii) Transposition of TpV SmR into R7K. Onetransconjugant from J62-TnCl(R7K), J53(R7K-TnC1), showed linked transfer of ApR (the R7Kmarker), TpR, and SmR. Plasmid DNA was iso-lated and cosedimented with R7K DNA. The

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TRANSPOSITION OF LINKED Tp AND Sm RESISTANCE

3. H R144-TnCl14C R144

0 40

1600

1400

120C

500

400

-200

0.1 ml fractions

FIG. 2. Sucrose gradient analysis ofR144 plasmid DNA with the TpR SmR transposition (R144-TnC1, 3Hlabeled) and without (14C labeled). Sedimentation, from right to left, was for 100 min at 100,000 x g at 20 C.

TABLE 6. Molecular weights ofparental and transposition plasmids

Plasmid . No. of plas-Incompati- Mol wta Mol wt difference mid copies

Paetlnbility (Mdal) (Mdal) per chromo-Parental Transposition group someb

R483 Ia 62.0 1.0R144 Ia 65.0 1.1

R144-TnCl Ia 72.4 7.4 0.88JR66a Ia 62.0 0.86

JR66a-TnCl Ia 69.6 7.6 1.0JR66a-TnC2 Ia 71.8 9.8 0.49r

R7K W 24.4 1.3R7K-TnC1 W 33.5 9.1 1.7

RP4 P 36.3 1.8RP4-TnCl P 43.8 7.5 1.8RP4-TnC2 P 44.7 8.4 2.1

Avg = 8.3 ± 1.0a Parental plasmid molecular weights were determined by sucrose gradient cosedimentation with Ri (60

Mdal) or other suitable molecular weight marker. Transposition plasmids were analyzed by cosedimenta-tion with their parental plasmids.

b Calculated from the ratio of supercoiled (plasmid) DNA to chromosomal DNA found in the ethidiumbromide-CsCl centrifugation analyses, using the plasmid molecular weights given and 2.5 x 109 for the E.coli .chromosome molecular weight (5).

r This particular preparation was centrifuged twice in ethidium bromide-CsCl because of a collectionfailure after the first centrifugation; hence a lower yield of supercoiled DNA is expected.

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cpm

0

. 3H R7K-TnCl4C R7K

00.1 ml fractions

FiGos3. Sucrose gradient analysis of R7K ("4C-labeled) and its TpR Sm" transposition derivative, R7K-TnCl (3H labeled). Sedimentation at 100,000 x g, 20 C, was for 120 min.

transposition plasmid had a higher molecularweight than its parent (Fig. 3). The gain inmolecular weight of 9.1 Mdal (Table 6) wassimilar to the estimate above of the length ofDNA transposed from R483 into the J62-TnClchromosome. It was also similar to the gains inweight of the Ia plasmids that had acquiredDNA directly from R483. It therefore seemedlikely that R7K-TnC1 had gained the same se-quence ofDNA from the J62-TnC1 chromosomethat the latter, in turn, received from R483.Clearly, however, transposition of TpR SmRcould take place from the chromosome into aplasmid.

(iii) Transposition of Tpe SmR into RP4. Weused RP4 derivatives to test whether plasmidsgenerated by direct and indirect transpositionwere distinguishable. We selected TpR trans-conjugants from J62-TnC1(RP4). All six testedfor transfer of KMR or TpR gave cotransfer ofboth resistances; they carried plasmids thathad acquired TpR SmR indirectly. One clone wasnamed J53(RP4-TnC2). Direct transpositionwas obtained by crossing J62(R483)(RP4) withProteus mirabilis 13 and selecting for TpR Pro-teus transconjugants. Plasmids ofthe I complexcannot successfully enter or replicate within

this species (8). All 48 TpR transconjugants werealso ApR and KMR". Four tested for transmissi-bility gave cotransfer of KMR and TpR. One ofthese transconjugants, J53(RP4-TnCl), wasused for the isolation of plasmid DNA, whichwas then cosedimented with lP4 DNA througha sucrose gradient (Fig 4). Plasmid DNA fromthe indirectly transposed RP4-TnC2 gave avery similar sedimentation pattern. Both plas-mids had gained about 8 Mdal and were similarto the other transposition plasmids analyzed(Table 6). The two (direct and indirect) transpo-sition plasmids were also sedimented togetherto permit a finer comparison of their molecularweights. The two plasmids were identicalwithin the limits of the technique (Fig. 5).

(iv) Failure of some plasmids to accept thetransposition of TpR SmR. We looked for trans-position of TpR SmR onto R386 (TcR, IncFI, con-stitutive F pilus production; 10) by selectingTpR transconjugants from J62-TnC1(R386). Notransposition plasmid was obtained. Transposi-tion of TpR SmR onto R300B (SmR SuR) (2) wassought in two ways. First, transconjugantsfrom J62-TnC1(R300A)(R300B) crossed with J53were selected. R300A is a conjugative plasmid(2). The level ofSmR conferred by R300B (50 ,ug/

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. 3H RP4-TnClo14C RP4

O 10 20 30 40 50

0.1 ml fractionsFIG. 4. Sucrose gradient analysis of RP4 (14C labeled) and its TPe SmR transposition derivative, RP4-

TnCl (3H labeled). Sedimentation at 100,000 x g, 20 C, was for 120 min.

700 is * 3H RP4-TnC2 (indirect transposition)

Sa 14CC RP4-TnC1 (direct transposition)600

i.

500

400I

~~~~~~0

cpm ,300

~~~~~~~~~~~~~0200 d |aoOv aV0

0 10 20

0.1 ml fractionsFIG. 5. Cosedimentation through a sucrose gradient of TPR Sm5 transposition derivatives of RP4: RP4-

TnCl by direct transposition from R483 and RP4-TnC2 by indirect transposition from the chromosome ofJ62-TnCl. Sedimentation, as in Fig. 2 through 4, was for 110 min.

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808 BARTH ET AL.

ml) is much higher and therefore distinguisha-ble from the 15 ,ug/ml conferred by R483 (or itsderivatives). We obtained high-level SmR butno TpR transconjugants. Second, we made P1lysates from J62-TnC1(R300A)(R300B) andJ62(R483)(R300B). High-level SmR transduc-tants from these lysates were tested for TpR;none were found. We conclude that neitherR300A nor R300B readily accepts transpositionof the TpR SmR sequence.

Incompatibility properties of transpositionplasmids. The incompatibility properties of thetransposition J-, W-, and P-group plasmidswere tested. R391-TnC1 belongs to group IncJ(15) (R391-3b-1 is a derivative of R391-TnCl,lacking KmR). R7K-TnC1 was incompatiblewith plasmid S-a, IncW (12). RP4-TnC1 andRP4-TnC2 were incompatible with R906, IncP(16). The transposition plasmids thus retainedtheir parental incompatibility properties. Also,the replication properties of the transpositionplasmids, as reflected in the number of super-coiled plasmid copies isolated per chromosome,were not significantly different from their pa-rental plasmids (Table 6).

Site of TpR SmR insertion into RP4. RP4 has

0

500I

01400p X

cpm 0

300

200(

00 10 20

been shown to have a single site susceptible tothe restriction endonuclease EcoRl (20). Aga-rose gel electrophoreses of RP4-TnC1 and RP4-TnC2 DNA each produced two bands afterEcoRl restriction compared with only one forRP4, suggesting that the transposed DNA car-ries a restriction site. Consistent with this, wehave found that although R7K has no EcoRlrestriction sites, R7K-TnCl has one. Thus wecan use the EcoRl site on the transposed DNAto mark its position relative to the RP4 restric-tion site. Figure 6 shows the cosedimentation ofRP4-TnC1 and RP4-TnC2 after restriction byEcoRl. The sedimentation pattern confirmedthat each plasmid was cut into linear frag-ments. These two fragments, which had molec-ular weights of about 30 and 15 Mdal, werethe same for both plasmids. Two more indirecttransposition derivatives of RP4, RP4-TnC6and RP4-TnC10, were similarly cosedimentedwith RP4-TnC1 after EcoRl restriction. Thesedimentation patterns (not shown) again indi-cated that each plasmid was cut into two frag-ments, indistinguishable from the two pro-duced from RP4-TnC1. Since all four plasmidsgave the same sized fragments, we conclude

* 3H RP4-TnC2

14o C RP4-TnCl

30 40 50

0. 1 ml fractions

FIG. 6. Sucrose gradient cosedimentation of the direct (-TnC1) and indirect (-TnC2) transposition deriva-tives of RP4 after cutting by the restriction enzyme EcoRI. EcoRI-cut RP4 was used as a molecular weightmarker (36 Mdal) in a separate tube (not shown) in the same centrifugation (210 min).

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TRANSPOSITION OF LINKED Tp AND Sm RESISTANCE

that the TpR SmR DNA segment had insertedinto the same site on the RP4 molecule eachtime, or into sites symmetrically disposed oneither side of the EcoRl restriction site of RP4.

Transposition in a recA strain. The influ-ence of the bacterial recombination system ontransposition was tested in C600 and its recAderivative, HH26. Both were made first R483+and then JR66a+. Maintaining selection forJR66a with Km, evidence for transposition ofTpR to the chromosome was scored as clonesretaining TpR but losing colicinogeny and TpRtransmissibility. Both strains behaved identi-cally, evidence for transposition of TpR SmRbeing found in 4% of KMR transconjugants,whether rec+ or recA. Thus we conclude thatthe recA+ gene product is not needed for thistransposition.

DISCUSSIONWe have demonstrated that a segment of

DNA from R483, containing its TpR SmRmarkers, can be transposed into a variety ofreplicons: the bacterial chromosome, otherplasmids of the Ia incompatibility group, andalso plasmids unrelated to R483. The chromo-some containing this transposed segment canalso act as a donor, transposing TpR SmR to aplasmid. The transposition appears to be rea-sonably efficient; on screening, clones contain-ing a transposition plasmid or chromosome ap-pear frequently. The observations that a signif-icant extent of homology between R483 and atransposon acceptor plasmid is not required,that transposition is nevertheless a frequentoccurrence, and that the process is independentof a functional recA+ gene suggest that we areobserving an "illegitimate" recombination sys-tem.Within the limitations of the three tech-

niques used, the size of the DNA sequencetransposed from R483 is probably identical eachtime. We have always observed both TpR andSmR and never the ColIa or I pilus genes of R483after transposition to another replicon. Theseobservations and the indistinguishability of theplasmids produced from RP4 by direct or indi-rect transposition lead us to consider that thesetranspositions involve a precise segment of theR483 DNA, i.e., a transposon (14). We desig-nate the TpR SmR sequence from R483 as trans-poson C (TnC).

Transposon A has been shown to have atleast 12 distinct insertion sites in a region cor-responding to one-third of a 5.5-Mdal plasmid(16a). The authors concluded that the siteswere not random although they were clearlymultiple. In contrast, transposon C seems to be

very site specific. Transposition into RP4 hasprobably taken place at the same site eachtime. Some plasmids (R300A, R300B, and R386)seem to have no available site, but R391 musthave at least two sites (giving tral and tra-plasmids). The E. coli chromosome has only onepreferred site; all five independently isolatedclones had TpR mapping close to ilv (Tables 3and 4). It should be noted that this site is re-mote from the site where the whole R483plasmid can integrate to give suppression of theDnaA phenotype (7).

Since the original recognition of a transposa-ble region (transposon A) from RP4 conferringampicillin resistance (14), several other trans-posable sequences coding for antibiotic resist-ances recently have been described. TransposonA has been shown by Heffron et al. (16a) to bebounded by inverted repeated sequences about140 base pairs long. Berg et al. (3a) have de-scribed the transposition of a 4-Mdal DNAsequence conferring kanamycin resistance froman R factor to a lambdoid phage. This trans-poson is bordered by inverted repetitions 1,400base pairs long. Transposable sequences con-ferring tetracycline resistance have been de-scribed by Foster et al. (lla) and also byKleckner et al. (21a). The latter authors havedemonstrated that their 5-Mdal transposon isalso flanked by 1,400 base pair inverted re-peats. Ptashne and Cohen (26) have shown thatthe inverted repetition (1,300 base pairs long)bordering the tetracycline transposon in R6-5 ishomologous with the IS3 insertion sequence ofMalamy et al. (24). Direct repetitions of inser-tion sequences (21, 29) in plasmids have alsobeen reported (19, 26).Transposon C is the largest (9 Mdal) transpo-

son described so far. We do not yet knowwhether it is also bounded by inverted repeti-tions or, if so, whether these and other invertedrepetitions will turn out to be insertion se-quences. It is difficult to imagine that an en-zyme could "measure off' a 9-Mdal segment ofDNA for transposition; we consider that specificenzyme-recognizable boundary sequences mustbe involved.

It seems clear that the transposition phenom-enon we have described here could be related tothe transposition of insertion sequences and tothe recA-independent insertion of a segment ofone plasmid into one of (several) separate siteson another, as described by Kopecko and Cohen(22); personal communication. Inverted repeti-tions are located at the junctions of these plas-mid insertions. It seems likely that these phe-nomena are related by the same fundamentalinsertion mechanism.

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810 BARTH ET AL.

ACKNOWLEDGMENTS

We are grateful to the Medical Research Council of theU.K. for supporting this work.

ADDENDUMWe have now compared the sedimentation pat-

tern of plasmid DNA from 20 independently isolatedTnC derivatives of RP4 with RP4-TnCl after EcoRltreatment of each DNA. Each plasmid is cut intotwo fragments, but we have now distinguished atleast seven different sedimentation patterns andthus conclude that there are at least seven sites onRP4 at which TnC can be inserted. These sites ap-parently include the TcR and KmR genes of RP4since a few derivatives have lost TcR or KmR with-out any loss of molecular weight. We have alsorecently obtained TnC derivatives of R300A. It thusseems that with respect to plasmids at least, TnCis not as site specific as we concluded above.

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