Tn554: Isolation and characterization of plasmid insertions

PLASMID

5,292-305

Tn554: ELLEN

(1981)

Isolation

MURPHY,

and Characterization

SANDRA PHILLIPS,'

of Plasmid

IRIT EDELMAN,

Insertions

AND RICHARD

P. NOVICK

The Public Health Research Institute of the City of New York, Inc., New York, New York 10016 Received

December

31, 1980

Tn554, a transposon in Staphylococcus aureus that carries determinants of spectinomycin resistance and inducible macrolide-lincosamide resistance, is characterized by a highly efficient transposition, exceptional site specificity for insertion, and inhibition oftransposition by a copy of the transposon inserted at its preferred chromosomal site. In this communication we describe the characteristics of a number of rare, secondary-site insertions of Tn554 into several related penicillinase plasmids. These plasmid insertions display considerable variation in the frequencies with which they can act as transposon donors, as well as in the frequencies at which they undergo apparently precise excision. Transposition from the plasmid transposon donors is ordinarily a duplicative process and these subsequent transposition events always return Tn554 to its preferred site in the S. aureus chromosome; such derivatives are indistinguishable from the primary chromosomal insertion from which they were originally derived. We also report an unusual relationship between Tn554 and the transducing phage, 411, in which Tn554 is frequently transferred independently of its plasmid carrier. We suggest that the bacteriophage may play an important role in the mobility of Tn554, in addition to the usual transduction mechanism, in a process that we have referred to as “hitchhiking.”

Tn554 is a transposon in Staphylococcus au-em that carries spectinomycin resistance and the inducible macrolide-lincosamide-streptogramin B (MLS)2 resistance (Weisblum, 1975). Closely related or identical elements have been found to occupy a constant chromosomal site in a number of wild-type S. aureus strains (Krolewski in preparation) and et al., manuscript previous studies have revealed that Tn.554 has a very high preference for the “primary” site following transfer by transformation or transduction to a recA recipient as well as to a ret+ (Phillips and Novick, 1979; Phillips, 1978). Moreover, the frequency of transfer (via transduction) of Tn554 is, if anything, higher than that of most plasmids. In a recA 1 Present address: The Long Island College Hospital, Brooklyn, N. Y. 11201. y Abbreviations used: kb, kilobase pairs; bp, base pairs; Tsr, temperature-sensitive for replication; EmP, Sp’, Cd’, resistances to erythromycin, spectinomycin and cadmium ions, respectively; MLS, macrolidelincosamide-streptogramin B; uv, ultraviolet; PFU, plaque-forming units.

0147-619X/81/030292-14$02.00/0 Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

292

recipient, in which this transfer must occur almost exclusively by transposition, transfer is as efficient as in the ret+ host, where it can also occur by conventional meansi.e., as part of a larger chromosomal fragment. If the primary site for Tn554 insertion is occupied by a copy of the transposon, the frequency of transfer is dramatically reduced. This effect is most pronounced in the recA recipient where recombinational displacement cannot occur; in fact, we have been unable ever to detect transposition to a secondary chromosomal site. Secondary insertions into plasmids can be obtained, however, albeit with considerable difficulty, and these have permitted a more detailed functional analysis. With the first of these secondary insertions, pRN4166, the transposon was found to undergo excision from its carrier plasmid both in the donor strain during growth of the transducing phage +ll, and concomitant with transposition following transduction, and the latter two processes were apparently prevented by the

293

PLASMID INSERTIONS OF Tt-1554 TABLE 1 STRAINSAND PLASMIDS~

Plasmid

(M%l)

Host strain

pRNl053 pRNllO7

21.1 26.6

RN492 RN3212

~I524 blall repA ~I524 repA A 137 [ bla -+ asi]

pRN2040 pRN3218 pRN4166 pRN4169

17.4 18.7 30.7 55.0

RN3608 RN1902 RN3192 RN3146

pRN4171 pRN4174 pRN4175 pRN4176 pRN4177 pRN4007 ~I6187

22.8 21.5 21.5 25.2 25.2 24.5 15.3

RN3240 RN3665 RN3666 RN3801 RN3802 RN47 RN647

pII147 bIa1300 repA38Al40 [mer] ~1258bIa1443 asa ermB20 repA pRNll07 0171 [EcoA::Tn554 ermll spc-Z] 4llfIl77 [EcoF::Tn554 ermll spc-1] 0172 [EcoB::pRNl053] pRN3218 fll74 [EcoA::Tn554 ermll spc-l] pRN2040 al75 [EcoB::Tn554 ermll spc-I ] pRN2040 al76 [EcoD::Tn554ermll spc-I ] pRNl053 al78 [EcoA::Tn554ermll spc-l] pRNl053 0179 [EcoA::Tn554ermll spc-1] ~I524 blall al08 [EcoA::Tn551 Em]

Strain RN27 RN450 RN45 1 RN1030 RN2864 RN2876 RN2867 RN2868 RN2897 RN33 17 RN368 1 RN3683 RN3689 RN3735

Lysotype

bla cad asa asi mer

ermll

Wyman and Novick, 1974 Spontaneous rearrangement of pRNl053 Spontaneous deletion of ~11147 Novick et al., 1979b Phillips and Novick, 1979 This paper This paper This paper This paper This paper This paper Murphy and Novick, 1979 Shalita et al., 1980

Genotype

Derivation or reference

Chr::Tn554 ermll spc-1 recA1, Chr::Tn554 ermA5 Chr::TnSS4 ermA4 Chr::Tn554 ermA4 Chr::Tn554 ermA4 Chr::Tn554 ermll spc-I Chr::Tn554 ermll spc-1 Chr::Tn554 ermll spc-1 Chr: :Tn554 ermll spc-1 Chr::Tn554 ermA4

NCTC8325 cured of 411, 412; 8001lysogen NCTC8325 cured of 411, 412, 413 RN450, $11 lysogen Wyman and Novick, 1974 Tn554 ermll spc-I + RN451; Phillips, 1978 Tn554 ermA5 + RN1030; Phillips, 1978 Tn554 ermA4 -+ RN450; Phillips, 1978 Tn554 ermA4 + RN451; Phillips, 1978 Tn554 ermA4 + RN27 RN3 192 + RN27 RN3240 + RN27 RN3665 -+ RN27 RN3666 + RN27 RN3 146 -+ RN2868

413, 8Oa! $11 $11 $11 411 411 411 413, 80a 413, 8Oar 413, 80a $13, 80a 413, 80a #~ll::nl77 Tn554

Derivation or reference

Genotype

spc-1

n All strains are derivatives of Staphylococcus aureus NCTC8325. +, Transfer; A, deletion; Q, insertion. spc, spectinomycin resistance; ermA, erythromycin (Tn554) erml, erythromycin inducibility locus.

presence of a copy at the primary site,in the recipient (Phillips and Novick, 1979). On the basis of these results, it was suggested that the transposon was a phage-like element that was controlled by a transposonencoded diffusible repressor and that its transposition occurred by a process akin to zygotic induction upon entering a recipient lacking a copy. Isolation and study of several additional plasmid insertions, reported in this com-

munication, have revealed that the situation is more complex; the secondary plasmid insertions vary in their ability to act as transposon donors and precise excision is the exception rather than the rule: when a plasmid carrying an insertion of Tn554 is transferred by transformation, Tn554 most often transposes by a duplicative process, similarly to other transposons (Shapiro, 1979), rather than by excision-insertion; and the reduced transposition frequency

294

MURPHY

ET AL.

into Tn554-containing recipients is probably manufacturers’ specifications. Digests of an effect of site interference as well as plasmid DNA were analyzed by electropossibly involving a diffusible repressor. phoresis on 0.7% horizontal agarose gels in We have also observed that the transducing Tris- borate buffer. Electron microscopic phage, $11, often transfers Tn554 inde- heteroduplex analysis, with plasmid DNA pendently of its plasmid carrier; because linearized with BarnHI, was according to Davis et al. (1971). this transfer might well involve insertion into the phage genome, followed by subsequent transposition into the chromosome, RESULTS we have come to refer to it as “hitchhiking.” A preliminary report of some of these Isolation and Characterization of Plasmid results has appeared elsewhere (Novick Insertions of Tn554 et al., 1980). Insertions of Tn554 into plasmids were obtained by preparing a 4 11lysate of a strain MATERIALS AND METHODS containing a chromosomal Tn554 ermll Strains of Staphylococcus aweus used in spc-I and a temperature-sensitive (Tsr) this study are listed in Table 1. Transduction penicillinase plasmid, and selecting for with 411, a generalized transducing phage, transductants with both the Tn554 marker, was as described (Novick, 1963). Lysates of erythromycin resistance (Em’), and the defective lysogens were prepared by uv plasmid marker, cadmium resistance (Cd’). induction and superinfection with wild-type The recipient used was a strain containing $11 at a multiplicity of 2.5-3 PFU/cell. an erythromycin-sensitive (Em”) mutant of Media and buffers were as in Novick and Tn554. Approximately 1 in IO6 of the Cd’ Brodsky (1972). Transformation of S. aureus transductants was also Em’; these preprotoplasts was according to Chang and sumptive insertions were challenged at the Cohen (1979), modified for S. aUreUS as nonpermissive temperature for the plasmid follows: 3.5 x lOlo cells in early log phase (43°C) to identify those in which Em resistwere washed and incubated with 40 &ml ance was cured along with the plasmid lysostaphin in 5 ml 2x Penassay medium markers Cd’ and penicillinase. Trans(Difco) containing 0.5 M sodium succinate, ductants in which Cd and Em resistance pH 6.5, 0.02 M maleic acid, and 0.02 M appeared by this test to be linked were MgClz until protoplasted, as judged by test- selected for further study; of these, 10% ing an aliquot for lysis with 2% sarkosyl. (or about one in 1012 phage particles, Then 0.5 ml of the washed protoplasts were overall) proved to have insertions of Tn554 mixed with 0.8-2.0 pg plasmid DNA and in the target plasmid. In a slightly different 1.5 ml 40% polyethylene glycol (PEG-6000, protocol, Tn554 ermll spc-I was transUnion Carbide Corp.) for 2 min; after wash- duced to a recipient containing Tn554 ing, the protoplasts were incubated in the ermA.5 and the target plasmid, with selection above medium at 30°C for 4 h and then for Em’ at 37”C, a temperature that is plated on DM3 regeneration medium con- intermediate for plasmid replication. Transtaining 3 &ml erythromycin. ductants in which the plasmid was stabilized Plasmid DNA was prepared by CsCl- by the Em selection were identified by stainethidium bromide density gradient centrifu- ing for penicillinase activity or by replica gation of cleared lysates as modified for plating. Both pRN4166 and pRN4169 were S. aureus (Novick et al., 1979a,b). Restric- obtained by this method; pRN4166 was tion endonucleases were from New England originally obtained in a recA background Biolabs and Bethesda Research Labora- and subsequently transferred by transtories and were used in accordance with the duction to the ret+ host.

295

PLASMID INSERTIONS OF Tn554

penicillinase plasmids, at a minimum of five locations that are clustered within several kb of the regions encoding plasmid replication functions (Novick et al., 1979a,b; Fig. 2). Although pRN4166 and pRN4177 appeared at first sight to have Tn554 insertions at different sites, closer examination revealed that they always had in common at least one of the junction fragments. This results from the fact that pRN1107 was FIG. 1. Restriction endonuclease digests of plasmids derived from pRN1053 (the respective containing insertions of Tn554. Lanes a-k, EcoRI target plasmids) by a duplication of 15 kb; digests; a, pRN1053; b, pRN4176; c, pRN4177; d, pRN4166; e, pRN1107; f, pRN2040; g, pRN4174; h, the Tn554 insertion in pRN4166 is located pRN4175, i, 411; j, pRN4169, k, pRNlO53. Lanes 1 very near the end of the duplicated region and m, HpaI digests; 1, pRN4171; m, pRN3218. at a site that appears to be indistinguishable (at the level of resolution provided by agarose gel electrophoresis) from that of To date we have isolated seven insertions pRN4177. The remaining insertion, pRN4169, has a of Tn554 into secondary sites on plasmids (see Table 1). Analysis of these has revealed size of about 80 kb and contains the a 6.5kb insertion; representative restriction genomes of the Tsr plasmid pRN1053, the endonuclease digests are shown in Fig. 1. transducing phage $11, and Tn554. pRN4169 These restriction patterns have permitted arose spontaneously during the selection for the assignment of the locations of the Tn554 plasmid-linked insertions. The plasmid and insertions into plasmids whose maps have ~$11were found to be joined at a site within been previously determined. Six of the $11 EcoRI-B, overlapping 411 HueIII-E, insertions are into four closely related that corresponds to 41 latt (Lofdhal et al.,

ill77 Tn554

FIG. 2. EcoRI restriction (inner) and genetic (outer) maps of plasmids containing insertions of Tn554. 411 restriction and genetic maps from Lofdhal et al. (1981a,b); plasmid maps from Novick et al. (1979b) and Murphy and Novick (1979). The inverted repeats of pRN1053 are indicated by the bold arrows. pRN1107 is derived from pRN1053 and contains an extensive inverted duplication. The locations of Cl176 and a175 with respect to cadB and rep38 of pRN2040 are not known for certain.

296

MURPHY ET AL.

FIG. 3. Heteroduplex of pRN4171 and ~I6187 (Shalita et al., 1980);the Tn551 insertion on pRN4171 was used as an internal standard; it is 5.2 kb in length and has terminal inverted repeats of 35 bp (Khan and Novick, 1980).

1981a); this insertion results in the inactivation of the arsenate-arsenite resistance locus of the plasmid, similar to previously described plasmid-phage cointegrates (Schwesinger and Novick, 1975). pRN4169 expresses 411 immunity and is Tsr; therefore, it must replicate under the control of the penicillinase plasmid. Its 411 moiety is defective, most likely as a result of the

Tn554 insertion into the $11 EcoRI-F fragment, a region encoding late structural genes (Lofdahl et al., 1981b). The locations of the plasmid and Tn554 insertion sites in 411 were confirmed by digestion with other enzymes (not shown). For each enzyme, two $11 fragments and one pRN1053 fragment were missing from the digest of pRN4169; where the locations of the restric-

TABLE 2 TRANSFORMATION WITH PLASMIDS CARRYING Tn554 INSERTIONS~

Donor

Recipient strain

pRN4166 pRN4166 pRN4175 pRN4175

RN450 RN2867 (Tn554 ermA4) RN450 RN2867 (Tn554 ermA4)

Percentage of Em’ transformants resistant to Cd (No. tested) 100 (50) 100 (loo)

100 (50) loo (50)

Percentage of nonlinkage of Cd and Em (No. tested)b

loo(SO) 2 ww 67 (18) 0 (13)

’ Transformants were selected on DM3 plates containing 3 &ml erythromycin (see Materials and Methods). b Linkage of Cd and Em tested by cocuring of temperature-sensitive plasmids.

297

PLASMID INSERTIONS OF Tn554 TABLE 3 TRANSDUCTION WITH PLASMIDS CARRYING Tn554 INSERTIONS

Transductants per PFU (x 106)”

Singly resistant transductants (Fraction) Em

Cd

Transposition*

Donor

Recipient genotype

Em

Cd

pRN4171

RN451 RN2868 Tn554 RN 1030 recA1 RN2876 Tn554 recA I

917 425 45.8 2.2

246 210 0.4 0.4

0.65 0.46 0.96 0.02

l/14 o/20 5112 o/12

pRN4174

RN45 1 RN2868 Tn554 RN1030 recAl RN2876 Tn554 recAl

160 92 18.1 2.3

72 44 0.3 0.5

0.35 0.24 0.88 0.01

12124 O/23 N.D.’ N.D.c

pRN4175

RN45 1 RN2868 Tn554 RN1030 recA1 RN2876 Tn554 recA1

360 220 192 7.0

150 100 2.7 3.6

0.60 0.27 0.99 0.03

7127 O/26 N.D.C N.D.’

pRN4176

RN45 1 RN2868 Tn 554 RN 1030 recA2 RN2876 Tn554 recA1

386 213 9.2 3.6

196 69 0.6 0.7

0.54 0.63 0.85 0.01

4144 O/18 2112 o/11

pRN4177

RN45 1 RN2868 Tn554 RN1030 recA1 RN2876 Tn554 recA1

1830 117 50 1.1

196 64
0.88 0.50 0.97 0.02

27137 O/16 -

pRN4166

RN45 1 RN2868 Tn554

N.D.’ N.D.’

N.D.’ N.D.’

0.60 0.43

~RN4007~

RN45 1

48

32

<1o-a

0.19 0.11 < 10-4

18125 O/25

-

a Transduction frequencies include both singly and doubly resistant transductants, for each marker. These frequencies are expressed as transductants/PFU, not per viable recipient cell. Because of the reduced viability of the recA strain, the frequencies appear lower than for the ret+. * Transposition measured by the cocuring test on doubly resistant transductants (No. transposed/No. tested). c N.D., not determined. d pRN4007 is a derivative of ~1524carrying an insertion of Tn551 (Novick et al., 1979a), which carries an erythromycin resistance determinant unrelated to that of Tn554.

tion sites in pRN1053 and in Tn554 are known, the relative sizes of the junction fragments allowed the unequivocal determination of the insertion sites. For example, Tn554 lacks a Hue111 site; thus the junction fragment containing Tn554 is easily identified as the junction fragment that is 6.5 kb larger than a fragment present in the Hue111 digests of either 411 or pRN1053, but missing from pRN4169; in this case, a 2.9-kb fragment from $I11 is replaced in pRN4169 by a 9.1-kb fragment, placing the Tn554 insertion in the doll moietv.

Analysis of heteroduplexes of one of the Tn554 insertions, pRN4171, with ~16187 (a plasmid closely related to the target plasmid) (Shalita er al., 1980), revealed a simple 6.5-kb insertion loop without visible terminal inverted repeats (Fig. 3). Genetic Analysis of Tn554 Insertions: Transformation and Transduction When pRN4166 or pRN4175 plasmid DNAs were used to transform RN450 and RN2867 (without and with Tn554. resoec-

298

MURPHY

tively), all transformants selected for Em resistance were also resistant to Cd (selection for Cd resistance was not possible for technical reasons) (Table 2). Transposition from these plasmids was tested by scoring the apparent linkage of Em and Cd resistances among these doubly resistant transformant colonies. Each transformant was streaked on nonselective medium and incubated at 43°C. From each streak, three colonies were scored for Cd and Em resistances. Usually, all three colonies had the same phenotype; mixed colonies containing both Em’ and Em” were scored according to the majority class, and those in which the plasmid (Cd) was not eliminated by the temperature challenge were discarded. Transposition to the chromosomal site was assumed to have occurred in each transformant in which Em resistance was not eliminated at 43°C. Note that this test does not distinguish between cells containing two similarly marked copies of Tn554 (chromosomal and plasmid) and those containing only a chromosomal copy; in neither case will there appear to be linkage with the plasmid. Using this test, we found that transposition of Tn554 to the chromosome following transformation was very strongly dependent upon the genotype of the recipient strain with regard to Tn554; transposition did not occur if the recipient contained Tn554 (Table 2). In 11 transformants into RN450, with both pRN4166 and pRN4175, in which the genetic test had indicated that transposition had occurred, the plasmid DNAs were examined and were found to be unchanged (not shown); as with other transposons, it would appear that excision does not typically accompany transposition. In contrast, when transfer was via transduction, a large fraction of the Em’ transductant colonies contained no plasmid markers, and there was a corresponding excess of transductants resistant only to Em over those resistant to both Em and Cd (Table 3). This effect was not reciprocal; most of the transductants selected for resistance to cadmium were also resistant to

ET AL.

erythromycin. Since the results obtained in the transformation experiments indicated that intact plasmid DNA molecules, once taken up by the recipient cell, were not physically disrupted or lost from the cells following transposition of Tn554 to the chromosome, the high frequency of singly Em’ transductants would appear to be an effect of bacteriophage growth in the donor. For comparison, Table 3 also shows the results of transduction with the plasmid pRN4007, a derivative of ~1524 carrying an insertion of an unrelated, low frequency erythromycin-resistance transposon, Tn55 1 (Novick et al., 1979a) showing that there is little or no marker effect on transduction frequency and no disruption of the Cd-Em linkage. Note that lysates of Tn554-containing plasmids tend to transduce both Cd’ and Em’ markers with higher frequencies than do lysates of other plasmid-containing strains. The significance of this finding is unclear. The frequency of the Em’ Cd” class of transductants (both the absolute frequency and as a fraction of the total Em’ transductants) is, for most of the plasmid insertions, only slightly reduced by Tn554 in the chromosome of a WC+ recipient (Tables 3 and 4). However, in the recA recipients the effect of a copy of Tn554 is the same for the plasmid Tn554 carriers as for donors with Tn554 in the chromosome. Although transposition (estimated from the fraction of transductants resistant only to erythromytin) into recA strains lacking Tn554 is lOOfold more common than is transduction of the entire plasmid, the latter appears to be essentially the only way in which EmP transductants can be obtained in the recA, Tn554-containing lysogenic recipient. In the ret+ host there are two possible recombinational routes by which Tn554 can be transferred to a Tn554+ recipient: recombination between the incoming and resident copies of the transposon and recombination between an incoming $111genome carrying the transposon, and the resident prophage. These events evidently occur at sufficiently high frequency to mask the inhibition of

299

PLASMID INSERTIONS OF Tn554 TABLE 4 INHIBITION OF TRANSPOSITIONBY AN OCCUPIED PRIMARYSITE Ratio of Em’ Cd” transductants into strains without/ with Tn554 Tn554 donor pRN4166 pRN4171 pRN4174 pRN4175 pRN4176 pRN4177 pRN4169 RN2864 (chromosomal)

ret+”

recilm

12.1 3.1 2.8 3.6 1.6 27.8 15.3’ 29.0c

N.D.* 1350 790 1030 150 150 N.D.b SOOC

(I ret+ recipients RN451 and RN2868; recA/ recipients RN1030 and RN2876. b N.D., not determined. c Ratios for pRN4169 and RN2864 are of total Em’ transductants.

transposition exerted by the recipient copy. Sometimes integration of the entire carrier plasmid occurs as well (unpublished observations). The frequency of transposition following transduction of plasmid-Tn554 complexes was estimated in two ways: from the fraction of singly-Em’ transductants, and from the Cd’-Em’ coelimination test with the doubly-resistant transductants (Table 3, last column). The latter estimate generally tends to be lower. This might reflect real differences in the transposition that occurs in the recipient and that which seems to involve the bacteriophage in the donor (see Discussion); other factors, such as the establishment of repression, could also contribute. Chromosomal Insertions Derived from Plasmid Insertions Strains derived by transduction of Em resistance from several of the plasmid insertions of Tn554 to RN27 were compared to RN2864 for their characteristics as donors of Tn554; all proved to be similar in the frequency of transduction of Tn554 and in

the inhibition exerted by an occupied primary site in the recipient (Table 5). These derivatives were shown to contain Tn554 reinserted at a site indistinguishable from that of the original chromosomal donor, RN2864 (Krolewski et al., manuscript in preparation). pRN4169 In any strain carrying pRN4169 (the 41 l::Tn554::pRN1053 complex), lytic phage growth virtually always resulted in the dissociation of the pRN1053 genome from 411 and Tn554; that is, Cd’ transductants were almost invariably found to contain intact pRN 1053plasmid DNA (not shown). Cd and Em were rarely cotransduced (
Derivation

RN2864 RN3317 RN3681 RN3683 RN3689

chromosome pRN4166 pRN4171 pRN4174 pRN4175

a Recipient strain.

RN27” 1170 900 660 950 200

RN2897” 48 45 46 36 15

MURPHY ET AL.

300

TABLE 6 LINKAGE OF Tn554 AND 411”

Transuctants per PFU (x 106) Donor

Recipient genotype

Em

RN3 146 (pRN4169)

RN450 RN2867 Tn554 RN451 (411) RN2868 (411) Tn554

166 4.7 5,260 269

RN3735 (chr:$ll R177 Tn554)

RN450 RN2867 Tn554 RN451 (411) RN2868 (411) Tn554

30,000 3,000 490,000 23,000

Em’ transductants (Fraction)

Cd’ transductants (Fraction)

Cd

Immune

Productive

Immune

Productive

82.5 99.5 152 325

0 0.18 1.0 1.0

0 0 1.0 0.08

0.02 0.05 1.0 1.0

0 0.05 1.0 1.0

N.A.b N.A.* N.A.* N.A.b

0.20 1.0 1.0 1.0

0.06 0.05 0.89 0.18

-

-

’ Transductions were performed at a constant multiplicity of 0.01 PFU/cell; unadsorbed phage were removed by washing and the mixtures were diluted and plated on erythromycin, and where applicable, on cadmium, plates. Immunity to 411 and phage production were tested by toothpicking a minimum of 25 purified transductant colonies to lawns of 411 or RN450, respectively. b N.A., not applicable.

conversion of the recipient to a defective lysogen provided a test equivalent to the Cd-Em linkage of the plasmid insertions. In a lysogenic recipient, Tn554 transposed efficiently to the chromosome if its primary site was vacant (Table 6); most such transductants retained wild-type prophage. However, if the chromosomal site was occupied, Em’ transductants, which arose at a frequency at least IO-fold lower, generally contained defective prophages, implying that recombination between the two phage genomes had occurred. Nonlysogenic recipients were sometimes converted to defective lysogens, with Tn554 in the prophage; defective lysogenic conversion was much more frequent if the recipient contained Tn554. Similar results were obtained with a plasmid-negative, defective lysogen, RN3735, as donor. RN3735 was derived from the cross RN3 146 -+ RN2868; it contains Tn554 ermA4 at the primary transposon site, as well as the RI77 Tn554 ermll spc-1 insertion into the prophage. The presence of two copies of Tn554 at these distinct sites was confirmed by blotting experiments (E. Murphy, unpublished data). The only difference between RN3146 and

RN3735 as donors of 4 11R177Tn554 would appear to be one of relative frequencies; the packaging of this (bll::Tn554 complex is evidently much more efficient (ca. 2 logs) when it originates in the bacterial chromosome at the prophage site than when it is inserted in a plasmid. These events are illustrated schematically in Fig. 4. Note that none of the Cd’ transductants of RN3146 were converted to defective lysogens, indicating that Em but not Cd transduction was linked to $11. In addition, there was significant killing of the nonlysogenic recipients that received the #I l::Tn554 complex: whereas the frequency of Cd’ transduction was at most 3-fold higher with an immune recipient than a sensitive one, Em’ transductants were 50-fold more frequent when the recipient was immune. Thus it appears likely that the transducing particles containing Tn554 have the ability to kill potential transductants, even though the $11 genome carrying Tn554 is defective, while potential Cd’ transductants have received transducing particles that have no lethal capabilities. Note that a defective phage such as $llde (Novick, 1967) that carries the ~1258 replicon linked

301

PLASMID INSERTIONS OF Tn554 DONORS

411:: Tn554 EdSp-

pRN4169

0 pRNl053 RN3146

RN3735

Juv\ Em’

tronrducing par ticks ($ll::Tn554En’Sp~

Cdr

tronsducinq PWtiCkbS

+

Cd’ transductanb

(pRNl053)

1

RECIPIENTS

EITlr

TRANSDUCTANTS

FIG. 4. Schematic drawing of the fate of transducing particles containing ~$110177[EcoF::Tn554 ermll spc-I ] into lysogenic and nonlysogenic recipients with and without Tn554 ermA4. Large circles represent chromosomes; small black boxes represent vacant primary sites for Tn554.

to a 411 segment with only late bacteriophage functions does not cause killing of the recipient strain; rather, lysates prepared from strains containing it are presumably Hft because the phage segment must confer a selective advantage (relative to ordinary plasmids) for the phage packaging system. Excision

of Tn554

With pRN4166, unlike the other plasmid insertions of Tn554, a large proportion (up to 20%) of the Cd’ transductants were Em”.

One such transductant was obtained from pRN4171. Six transductants, five from pRN4166 and the one from pRN4171, were examined by agarose gel electrophoresis and found to contain plasmids with apparently precise excisions of Tn554 (not shown). We have not succeeded in isolating any Cdr Ems transductants derived from the remaining plasmid insertions (including pRN4177, which is in other respects very similar to pRN4166); therefore for these insertions the excision frequency cannot be greater than 10e2to 10P3.Precise excision

302

MURPHY

of Tn554 from pRN4169 also appears to be very infrequent, about 3 x IO-’ per viable cell, based on the frequency of infective centers following uv irradiation of cells carrying pRN4169. DISCUSSION

On the basis of the results presented here and previously (Phillips and Novick, 1979), it appears that Tn554 is mechanistically similar to other transposons. It is a discrete DNA segment that is conserved during transposition, it can transpose to multiple nonhomologous sites, shows sequential transpositions, and ordinarily transposes by a duplicative process. Nucleotide sequence data on termini, insertion sites, and flanking sequences are not yet available. In a number of respects, however, Tn554 is strikingly different from most other transposons and it appears to represent a different strategy for genetic mobility. Site Speci$city Tn554 shows an extreme preference for a single, specific chromosomal site; quantitatively, this preference amounts to at least a factor of lo4 over known plasmid or possible secondary chromosomal insertion sites. Transposition from the rare secondary site insertions (in plasmids or phage) always returns Tn554 to its preferred chromosomal location. Furthermore, attempts to isolate secondary chromosomal insertions have thus far failed; the frequency of transduction to a recA, Tn554-containing recipient is about 10e4in comparison with that of a recipient lacking Tn554, and the few transductants thus far scored were found by genetic tests or by DNA hybridization to contain only a single copy of Tn554 (Phillips, 1978; Krolewski et al., manuscript in preparation). Inasmuch as the recA strain employed has a recombination frequency of about 1O-4in comparison with the wild type (Wyman et al., 1974), it appears that these represent recombinants, rather than secondary site insertions of Tn554. It seems

ET AL.

very probable that the high target site specificity of Tn554 involves recognition of a nucleotide sequence of considerable length: based on its frequency of once per chromosome, and considering no other factors, one would predict that the target is at least 11 nucleotides in length. Role of Flanking Sequences We have described here the characteristics of six secondary site insertions of Tn554 into several penicillinase plasmids, and one insertion into the transducing phage 411. These insertions display considerable variation in the frequency with which they can act as transposon donors (from about 5 to lOO%), suggesting that not only are the ends of the transposon and the target site recognized, but also the flanking donor sequences. A simple possibility is that the more closely the secondary site flanking sequences match the primary target site, the higher the transposition frequency. Repression of Transposition Transposition of Tn554, from any of the secondary site insertions as well as from the naturally occurring chromosomal donor, is very sensitive to inhibition by a copy of Tn554 occupying its primary site. This might simply be due to an interruption of the primary target sequence, in which case an examination of the relevant DNA sequences should prove informative. On the other hand, some of our observations support the possibility of regulation via a diffusible repressor, namely that there seems to be a burst of transposition immediately following entry of a plasmid::Tn554 donor, followed by a decline in the transposition frequency. This is supported by findings such as that with pRN4174 (Table 3) where 50% of the primary Cd’- Em’ transductants are composed largely of cells in which a transposition has occurred and 50% are composed largely of cells in which there has been no transposition. The occurrence of these two

PLASMID

INSERTIONS

classes of colonies suggests that if transposition does not occur within a short time following entry, it subsequently occurs rarely, if at all. Mobilization Tn554 is transferred by transducing phages such as 4 11evidently independently of flanking DNA regions, accounting for considerably more than half of all transductants, including those derived from either the secondary site plasmid donors, or from the chromosome (Phillips and Novick, 1979). There are several possible mechanisms for this mobilization: (i) The transposon inserts into a secondary site on the phage genome and thence to its primary chromosomal site following phage infection. This possibility has given rise to the notion of “hitchhiking” (Novick et al., 1980). It is possible that pRN4169 represents an intermediate in this process, one which has been sidetracked from the normal pathway by the presence of Tn554 and 411 in the recipient plus an available secondary target. The apparent consequence has been the insertion of the $1 l::Tn554 intermediate into the plasmid target. (ii) Tn554 undergoes doublestranded excision and is packaged as a free molecule, not physically linked to the phage or plasmid DNA. This mechanism is probably not very common, since precise excision of Tn554 is, with one exception (pRN4166) very much rarer than is independent transduction of Tn554. In connection with these two mechanisms, at least two of the plasmid insertions have been found to undergo apparently precise excision during growth of bacteriophage $11. This may indicate a similarity in sequence between the transposon and the prophage termini; data on this point are not presently available, nor is the significance of this excision clear. (iii) The flanking DNA regions are packaged and transferred along with the transposon and transposition occurs after entry into the recipient organism. This very likely accounts for a substantial fraction of ret+ transductants with a chromosomal Tn554 donor;

OF Tn554

303

for the plasmid donors, in view of earlier evidence that the vast majority of transduced plasmids are viable (Novick, 1967), this mechanism would not appear to account satisfactorily for the large majority of transductants that receive only Tn554, unless one also postulates that transposition destroys the donor genome. This seems unlikely in view of the other evidence presented here. It appears that different transposons have evolved various means for ensuring their own survival without detriment to the host cell. Transposons which have relatively low target site specificity may be prevented from undergoing multiple, lethal transposition events within the bacterial chromosome via a low intrinsic transposition frequency. The prototype, Tn3, is associated with extremely low levels of a transposase whose synthesis is stringently limited by a transposon-encoded repressor; elevation of the transposase level is associated with conditional lethality (Heffron et al., 1979; Gill et al., 1978; Chou et al., 1979). Other means of regulating the activity of a transposon that displays little site specificity include, for Tn3, cis effects that prevent a second insertion into any single plasmid replicon (Robinson et al., 1977) or, as for Tn5, a trans-acting repressor (Biek and Roth, 1980). At the other extreme are transposons such as Tn554 and Tn7, which couple a high site preference with very efficient transposition. Thus for Tn554, transposition occurs with a frequency that may approach 100% under permissive conditions, but is completely inhibited when its site is already occupied. Similarly, Tn7, which has a highly preferred primary site in the chromosome but is able to utilize secondary sites in plasmids at significant frequencies (Barth et al., 1976), it typically found to be located at this chromosomal site in clinical isolates and in strains derived by conjugation with R plasmids carrying Tn7 (Datta et al., 1980). The Tn3 type of transposon appears well suited as a movable element for species that exchange genetic information primarily by plasmid-mediated conjugation, i.e.,

304

MURPHY ET AL.

insertion into a conjugative plasmid results in a stable and viable transferrable element, and can be responsible for wide dissemination. The Tn554 type seems better suited for species in which genetic transfer is primarily mediated by transduction. Here, insertion of the transposon into a transducing phage would in general inactivate the phage because of headful packaging requirements and could therefore be a dead end for the transposon. However, the ability to transpose with high frequency would enable it to exit from the inactivated carrier phage, thus ensuring its survival. In this case, the high degree of target site preference would follow of necessity from the high transposition frequency. The stability of the system could be improved by coupling high site preference with a diffusible repressor; in this case, there would be a zygotic induction-like burst of transpositions upon entry into a transposon-negative recipient. Establishment of an inhibitory concentration of repressor would then take place, preventing further movement. ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of Health (GM27253) to E.M. and from the National Science Foundation (PCM7725476) to R.N.

REFERENCES

DAVIS, R. W., SIMON, M. AND DAVIDSON, N. (1971). Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids. In Methods in Enzymology. (L. Grossman and K. Moldave, eds.), Vol. 21, pp. 413428. Academic Press, New York. GILL, R., HEFFRON, F., DOUGAN, G., AND FALKOW, S. (1978). Analysis of sequences transposed by complementation of two classes of transpositiondeficient mutants of Tn3. J. Bacterial. 136,742-756. HEFFRON, F., MCCARTHY, B. J., OHTSUBO,H., AND OHTSUBO, E. (1979). DNA sequence analysis of transposon Tn3: Three genes and three sites involved in transposition ofTn3. Cell 18,1153- 1163. KHAN, S., AND NOVICK, R. P. (1980). Terminal nucleotide sequences of Tn55 1, a transposon specifying erythromycin resistance in Staphylococcus aureus: Homology with Tn3. PIusmid 4, 148- 154. L~FDAHL, S., ZABIELSKI, J., AND PHILIPSON, L. (1981a). Structure and restriction enzyme maps of the circularly permuted DNA of the staphylococcal phage. 411. .I. Virol. 37, 784-794. L~FDAHL, S., SJOSTROM,J. E., AND PHILIPSON, L. (1981b). Cloning of restriction fragments of DNA from the staphyiococcal phage 411. J. Viral. 37, 795-801. MURPHY, E., AND NOVICK, R. P. (1979). Physical mapping of Staphylococcus aureus penicillinase plasmid ~1524: Characterization of an invertible region. Mol. Gen. Genet. 175, 19-30. NOVICK, R. P. (1963). Analysis by transduction of mutations affecting penicillinase formation in Staphylococcus

aureus.

J.

Gen.

Microbial.

33,

121-136. NOVICK, R. P. (1967). Properties of a cryptic highfrequency transducing phage in Staphylococcus aureus. .I. Virol. 33, 155-166. NOVICK, R. P., AND BRODSKY,R. (1972). Studies on plasmid replication I. Plasmid incompatibility and establishment in Staphylococcus aureus. J. Mol.

Biol. 68, 285-302. BARTH, P. T., DATTA, N., HEDGES, R. W., AND M., GRUSS, GRINTER, N. J. (1976). Transposition of a deoxy- NOVICK, R., EDELMAN, I., SCHWESINGER, A., SWANSON, E., AND PATTEE, P. A. (1979a). ribonucleic acid sequence encoding trimethoprim aureus. Genetic translocation in Staphylococcus and streptomycin resistances from R483 to other Proc. Nat. Acad. Sci. USA 76, 400-404. replicons. J. Bacferiol. 125, 800-810. BIEK, D., AND ROTH, J. R. (1980). Regulation of NOVICK, R. P., MURPHY, E., GRYCZAN,T. J., BARON, E., AND EDELMAN, I. (1979b). Penicillinase plasmids Tn5 transposition in Salmonella typhimurium. of Staphylococcus aureus: Restriction-deletion Proc. Nat. Acad. Sci. USA 77, 6047-6051. maps. Plasmid 2, 109- 129. CHOU, J., LEMAUX, P., CASADABAN,M., and COHEN, S. N. (1979). Identification and characterization of NOVICK,R. P., KHAN, S. A., MURPHY,E., IORDANESCU, S., EDELMAN, I., KROLEWSKI, J., AND RUSH, M. a self-regulated repressor of translocation of the Tn3 element. Proc. Nat. Acad. Sci. USA 76,4020-4024. (1980). Hitchhiking transposons and other mobile genetic elements and site-specific recombination CHANG, S., AND COHEN, S. N. (1979). High fresystems in Staphylococci. Cold Spring Harbor quency transformation of Bacillus subtifis protoplasts by plasmid DNA. Mol. Gen. Genet. 168, 111- 115. Symp. Quant. Biol. 45, 67-76. DATTA, N., NUGENT, M., AND RICHARDS,H. (1988). PHILLIPS, S. (1978). Ph.D. Thesis, New York UniTransposons in medically important bacteria. Cold versity, New York, N. Y. Spring Harbor Symp. Quant. Biol. 45,45-51. PHILLIPS, S., AND NOVICK, R. (1979). Tn554: A repres-

PLASMID INSERTIONS OF Tn554 sible site-specific transposon in Sfaphylococcus aureus. Nature (London)

278, 4X-478.

ROBINSON, M. K., BENNETT, P. M., AND RICHMOND,

M. H. (1977). Inhibition of TnA translocation by TnA. J. Bacterial. 129, 407-414. SCHWESINGER, M. D., AND NOVICK, R. P. (1975). Prophage-dependent plasmid integration in Staphylococcus aureus. J. Bacterial. 123, 724-738. SHALITA, Z., MURPHY, E., AND NOVICK, R. P. (1980). aureus: Penicillinase plasmids of Staphylococcus Structural and evolutionary relationships. Plasmid 3, 291-311. SHAPIRO, J. (1979). Molecular model for the transposition and replication of bacteriophage Mu and

305

other transposable elements. Proc. Nat. Acad. Sci. USA 76, 1933-1937. WEISBLUM, B. (1975). Altered methylation of ribosomal ribonucleic acid in erythromycin-resistant Staphylococcus aureus. In “Microbiology-1974” (D. Schlessinger, ed.), pp. 199-206, Amer. Sot. for Microbial., Washington, D. C. WYMAN, L., GOERING, R. V., AND NOVICK, R. P. (1974). Genetic control of chromosomal and plasmid recombination in Staphylococcus aureus. Genetics 76, 681-702. WYMAN, L., AND NOVICK, R. P. (1974). Studies on plasmid replication. IV. Complementation of replication-defective mutants by an incompatibilitydeficient plasmid. Mol. Gen. Genet. 135, 149- 161.