Structure of a Conjugative Element in Streptococcus pneumoniae

Transcription

1 JOURNAL OF BACTERIOLOGY, June 1986, p /86/0609t8-07$02.00/0 Copyright 1986, American Society for Microbiology Vol. 166, No. 3 Structure of a Conjugative Element in Streptococcus pneumoniae MOSES N. VIJAYAKUMAR, SCOTT D. PRIEBE,t AND WALTER R. GUILD* Department of Biochemistry, Duke University, Durham, North Carolina Received 3 January 1986/Accepted 22 March 1986 We have cloned and mapped a 69-kilobase (kb) region of the chromosome of Streptococcus pneumoniae DP1322, which carries the conjugative Qk(cat-tet) insertion from S. pneumoniae BM6001. This element proved to be 65.5 kb in size. Location of the junctions was facilitated by cloning a preferred target region from the wild-type strain Rxl recipient genome. This target site was preferred by both the BM6001 element and the cat-erm-tet element from Streptococcus agalactiae B109. Within the BM6001 element cat and tet Were separated by 30 kb, and cat was flanked by two copies of a sequence that was also present in the recipient strain Rxl DNA. Another sequence at least 2. kb in size was found inside the BM6001 element and at two places in the Rxl genome. Its role is unknown. The ends of the BM6001 element appear to be the same as those of the B109 element, both as seen after transfer to S. pneumoniae and as mapped by others in pfp5 after transposition in Streptococcus faecalis. We see no homology between the ends of the BM6001 element and find no evidence suggesting that it ever circularizes. Plasmid-free isolates of Streptococcus pneumoniae and other streptococci can carry antibiotic resistance genes as parts of large chromosomal insertions that can be transferred in whole or in part to laboratory pneumococcal strains by transformation, provided the genes in the donor are flanked by DNA that shows some homology to the recipient S. pneumoniae genome (5, 7, 19, 22). Some of these also can transfer both within and between species by a DNaseresistant filter mating process that fits the operational definition of conjugation (2,, 7, 9, 19). At least two such insertions, Tn9O6 from Streptococcus faecalis () and fl(caterm-tet) from Streptococcus agalactiae B109 (21), now designated Tn3951, can transpose between replicons within a cell, but the mechanism of transfer and its relation to transposition remain obscure (7). Further work would be facilitated by availability of restriction maps and cloned segments of one or more such insertions and their target regions in the recipient chromosomes. We chose to concentrate first on the cat-tet insertion found in S. pneumoniae BM6001. Transformation evidence had suggested this element was at least 30 kilobases (kb) in size (18), and its general similarity in behavior to Tn3951 suggested it could easily be 60 kb or more (21). We needed a strategy and a handle for cloning in a large region, much of it devoid of selectable markers, and for repeated checking to detect possible rearrangements or other problems. As described in the preceding paper (2), we took advantage of the transformability of S. pneumoniae to introduce an Escherichia coli replicon, pva891, carrying a resistance marker and a segment of S. pneumoniae DNA that could direct insertion of the vector into the chromosome. DNA preparations from pneumococcal clones selected for the plasmid resistance marker were then used as sources of plasmids that replicate in E. coli and carry segments of the pneumococcal chromosome adjacent to the site of insertion, available for mapping and recycling. The strategy worked, yielding pneurhococcal clones with pva891 inserted into various sites in the conjugative element. Here we describe the use of these strains to generate a detailed restriction map of k(cat-tet) * Corresponding author. t Present address: Biology Department, Brookhaven National Laboratory, Upton, NY BM6001 and its preferred target segment in the recipient chromosome. MATERIALS AND METHODS Bacteria, plasmids, and procedures. All strains were as described in the preceding paper (2) or as presented below. S. pneumoniae Rxl is our laboratory wild type, and DP1322 is Rxl carrying fl(cat-tet) BM6001. Derivatives of DP1322 were created by directed insertion of E. coli plasmid pva891 into fl(cat-tet) BM6001 as described (2). pva891 confers erythromycin resistance to streptococci and chloramphenicol resistance to E. coli. Recombinant plasmids were generated in recombination-deficient E. coli strains HB101 or DH1 by transformation according to Hanahan (8). Growth of pneumococcal cultures, conjugation, competence regimen, and plating techniques have been described (6, 17-19). Pneumococcal strains with pva891 inserts were maintained under erythromycin selection (1,ug/ml), and E. coli strains with recombinant plasmids were grown in chloramphenicol (15,ug/ml). DNA isolation and manipulation. High-molecular-weight DNA from pneumococcal insertion mutants was prepared for cloning after sedimentation through 5 to 20% sucrose gradients, as described (2). Chromosomal DNA for other purposes was prepared by the method of Marmur (13). E. coli transformants were screened for recombinant plasmids by agarose gel electrophoresis of rapid alkaline lysates (11). For restriction nuclease mapping, plasmid DNA was isolated from E. coli by using standard methods of cell lysis (11) followed by sedimentation on cesium chloride-ethidium bromide density gradients. Highly purified EcoRI endonuclease was a gift from P. Modrich. Other restriction endonucleases and T DNA ligase were purchased from Bethesda Research Laboratories and International Biotechnologies, Inc. Reactions were carried out as suggested by the suppliers. Agarose gel electrophoresis. Electrophoresis of DNA on horizontal agarose gels was done at room temperature in TBE buffer (89 mm Tris hydrochloride, ph 8.3, 89 mm boric acid, 2.5 mm disodium EDTA). Defined DNA restriction fragments for subcloning were isolated by electroelution after electrophoresis on Bio-Rad Ultrapure agarose gels. For all other purposes, Seakem ME agarose was used. After 978

2 VOL. 166, 1986 TABLE 1. CONJUGATIVE ELEMENTS IN S. PNEUMONIAE 979 S. pneumoniae strains with pva891 insertions and some E. coli plasmids derived from their chromosomal DNA Straina Directing DNA fragmentb sites (coordinates) E. coli plasmid Passenger DNA (source)c coordinatesd (kb) SPQ7 HindIII-BglII ( ) pdp5 (BamHI) pdp52 (BgIII) pdp53 (KpnI) pdp58 (SstI) GP2e Unknown Sau3A fragment from S. faecalis (see text) pdp30 (BamHI) pdp36 (KpnI) e pdp59 (BgIII) e VG51 Sau3A-HindIII ( ) pvj162 (Bglll) VG31 BamHI-BamHI ( ) pvj91 (KpnI) pvj92 (spon.)f pvj93 (SphI) VG32 BglII-HindIII ( ) pvj97 (spon.f pvj16 (BglII) a Derivatives of DP1322 with all or part of pva891 inserted by transformation. b Fragment ligated to pva891 and used to direct its insertion (see text). c Treatment of chromosomal DNA to produce plasmid (see text). d In kilobases from an arbitrary zero, as shown in Fig. 1 and Table 2. e GP2 carries a 1.1-kb deletion adjacent to the site of the vector insertion, near 3.6 kb on the overall map. f Spontaneous excision of the vector plus one copy of the directing fragment. completion of electrophoresis, gels were stained for 15 min in ethidium bromide (1,ug/ml), destained in de-ionized water, and photographed under UV light. Molecular weight markers for agarose gels to be photographed as described above were HindIII digests of lambda phage ci ts857 Sam7 (16) and HaeIII digests of X17 (15) obtained from Bethesda Research Laboratories. Sizes of fragments smaller than 2 kb were estimated from 1.2 or 2.0% gels, and the sizes of larger fragments were obtained from 0.6 or 0.8% agarose gels. For blot hybridization experiments, a set of internal size standards was constructed from appropriate digests of pva891 or derivative plasmids, each of which reacted with the probes used. Blot hybridization. Membranes used for Southern blot transfers were either Trans-Blot (Bio-Rad Laboratories) or GeneScreen Plus (New England Nuclear Corp.). For the latter the manufacturer's suggested methods were used both for transfer and for hybridization with 32P-labeled probe DNA. For the Trans-Blot membrane, standard methods were used for both one-way and two-way transfer (11) and for hybridization (23). Radioactive probe DNA was prepared by nick translation (11) of the appropriate plasmid with [32P]dCTP (New England Nuclear Corp.). Unincorporated nucleotides were removed by gravity flow on Sephadex G-50 (10 ml) or by three ethanol precipitations with an excess of salmon sperm DNA. Each hybridization used about 5 x 106 cpm of 32P-DNA. RESULTS Cloning of segments of *l(cat-tet) BM6001. Strain DP1322 carries a conjugative fl(cat-tet) in an Rxl S. pneumoniae background, having acquired it by successive transformations from strains BM6001 to DP1302 to DP1322 (18). Cloning was initiated by generating Emr derivatives in which pva891 had been inserted into strain DP1322 by transformation, directed initially by Sau3A fragments from S. faecalis carrying the BM6001 insertion, as described (2). DNA preparations from the initial Emr transformants were digested with one of several restriction nucleases, selfligated, and used to transform recombination-deficient E. coli. Plasmids from the resulting Cmr transformants were restriction nuclease mapped, and the passenger and vector DNA segments were identified. Defined fragments well away from the vector were recovered and ligated into pva891 for further rounds of insertion into DP1322, cloning, and mapping. Table 1 lists four derivative strains and one of the original set, GP2, which has a deletion and is useful for special purposes. All strains listed here cotransferred the vector Emr marker with cat and tet in filter matings, suggesting that the vector had inserted within the element as expected. Chromosomal DNA from these strains was then digested with restriction nucleases that recognized sites within or flanking the vector. These digests were self-ligated and used to transform E. coli to generate new plasmids, of which 13 are listed (Table 1). These and others were mapped. Mapping the conjugative element. Analysis of the passenger DNA from a given strain started with the smallest plasmid obtained from it and extended to progressively larger plasmids, generating self-consistent maps of the DNA surrounding the pva891 insert in this strain. Overlapping maps of plasmids derived from different strains allowed orientation of the passenger segments and deletion of the vector portions, to yield a linear map of over 68 kb of the DP1322 chromosome, most of which proved to be within the conjugative element (see below). Table 2 lists 97 sites, all those detectable for 11 nucleases, and Fig. 1 presents the data for four of these in map form. Figure 1 also shows the passenger segments carried on the plasmids used to generate the final map. Use of these and other plasmids as probes in blot hybridizations to digests of DP1322 DNA confirmed directly the entire map (data not shown). Along with the fact that most regions were mapped more than once, as parts of several plasmids, these results give us a high degree of confidence in the map as shown. In terms of precision, most entries reflect summations of measurements reproducible to about 10 base pairs on double-digest fragments that usually were smaller than 1.0 kb. The major uncertainty in absolute values is due to a 9-kb region with no sites in the 35- to -kb interval, introducing an additive uncertainty of perhaps 0.3 kb to coordinates above and to distances spanning this region. Locating cat and tet. cat was localized by a series of experiments leading ultimately to its cloning on a 1.3-kb ClaI fragment (coordinates ) inserted in pneumococcal plasmid pdp1 (20). This fragment is within a 2.3-kb HindlIl

3 980 VIJAYAKUMAR ET AL. J. BACTERIOL. TABLE 2. Restriction nuclease sites in the Ql(cat-tet) region of the chromosome of S. pneumoniae DP1322' BamHI KpnI BgII EcoRI HindIII ClaI XbaI AvaI SstI SphI Sall b c d Distances in kb from a BamHI site designated 0.0 (see text). Four pairs of close sites have been ordered as follows: HindIII sites are to the left of a BgllI site near 5.5, a ClaI site near 8.0, and ari SphI site near 59.; a BgII site is to the left of the AvaI site near 27.. b Another ClaI site in this interval is 0.2 kb from the nearest site. c Another ClaI site in this interval is 0.1 kb from the nearest site. fragment that is interhal to a 6.7-kb EcoRI fragment, and each of these.expresses chloramphenicol resistance when cloned on pneumococcal vectors (J. Hageman, S. Priebe, G. Pozzi, and W. Guild, manuscript in preparation). Strain DP1333 carries the point mutation tet-3 (22) and was transformed to Tcr at high efficiency by pdp36, pdp59, and pvj162, but not by pdp30 or pvj91. GP2, the parent strain of three of these plasmids, has pva891 inserted near the HindIII site at coordinate.7. However, comparison of plasmids pdp36 and pdp59, each derived from GP2, with pvj162 revealed that GP2 is missing 1.1 kb of DNA that extends to the right of the vector and includes this HindIII site but not the ClaI site at 5.6. Strain GP2 is Tcs. When its DNA was used as donor to DP1322, every Emr transformant was also Tcs. However, strain GP2 DNA gave Tcr Ems transformants of DP1333. Thus the deletion does not cover the tet-3 point mutation, which therefore has to be between the HindIII site at.7 and the KpnI site at 6.5. Locating boundaries. Identification of boundaries of the conjugative element proved complex because some internal segments showed homology to the wild-type genome, as indicated first by the fact that plasmids carrying them could transform both Rxl cells and DP1322 to Emr and then by DNA-DNA blot hybridization experiments. Starting from plasmids carrying large passenger DNA segments, smaller fragments were subcloned into the pva891 vector and examined for whether they induced insertion of the vector into Rxl. Two plasmids, pdp5 and pvj16 (Table 1), appeared to contain continuous segments of Rxl DNA extending to the right and left, joined to other segments clearly not present in Rxl by transformation or blot hybridization criteria. As will be shown below, these contained the true junctions. However, pdp52, pdp53, pdp58, pvj91, and pvj97 also transformed Rxl, and the subcloning revealed three internal regions that induced insertion of pva891 into Rxl in vivo and hybridized to blots of Rxl digests in vitro. Two of these include 0.58-kb EcoRI fragments, near coordinates 11 and 18 kb, which flank cat and appear to be parts of larger direct repeats. Analysis of these and their relation to spontaneous curing of cat from this and cat 1I111 I III I 11 I- i I - Eco RI I I I I Kpn I II 11 Bom HI 11 1 I II I l I SBg/I I 0 10 a I J. tet i * t L kb I FIG. 1. Recognition sites in map form for four restriction nucleases in the Ql(cat-tet) BM6001 region in, DP1332. See Table 2 for further data. Also indicated are the passenger regions carried on the plasmids used to generate the map. The arrows indicate the positions of the vector plasmid insertions (see Table 1). The left and right junctions are near positions 1.9 and 67., within 3.6- and 3.3-kb EcoRI fragments, respectively (see text and Fig. 3).

4 VOL. 166, 1986 CONJUGATIVE ELEMENTS IN S. PNEUMONIAE 981 ABC DE F G HI J K L 8 *5- a b c d e f 9 h i j k I m i:l.: ir t.?. 0 0 _ 0 a kb _ *75-1 A _ X 9 FIG.. Site specificity for insertion of fil(cat-tet) BM6001. Autoradiogram showing DNA-DNA blot hybridization of 31P-labeled _ 113 FIG. 2. DNA sequence homology between pvj97 and the Rxl pvj183 to EcoRI digests of DNA from (a through g) seven transconjugants, (i) Rxl, (j) DP1302, (k) DP1322, (I) 8R1, and (in) DP3117 chromosome. Digests of Rxl chromosomal DNA were resolved on a 0.8% agarose gel, transferred, and hybridized to [32P]dCTP-labeled [8Rl::(cat-tet) BM6001]. Lane h shows standards. Fragment sizes pvj97. Chromosomal digests with (A) BglII, (B) EcoRI, (C) are given in kb. The transconjugants were isolated after mating Rxl Hindlll, (D) BglII plus EcoRI, (E) BglII plus Hindlll, (F) EcoRI str-j nov-1 cells with DP1302 donors on filters for only 1 h, instead plus HindIII, (G) standards described below, (H) BamHI, (I) BamHI of the usual h, to reduce the possibility of isolating sister clones. plus EcoRI, (J) BamHI plus HindIII, (K) BamHI plus BglII, and (L) BamHI plus KpnI. The sizes indicated are for the standards in lane U, co] nsisung o0 a set or calirated tragments trom pva891 or results suggested that the passenger DNAs in pdp5 and derivaltive plasmids, all of which react with the probe. pvj16 extended from within the element into contiguous Rxl DNA without complexities like those above. The target other insertions will be described elsewhere (S. D. Priebe region from the Rxl chromosome was cloned as follows. A and VV. R. Guild, manuscript in preparation). DNA fragment from pdp5, from the BamHI site at 0.0 to A t:hird region contained a 2.-kb EcoRI fragment (coor- the BglII site at 0.8 on the map, was ligated to BamHIdinate.s 62.0 to 6.), which we thought might contain the cleaved pva891, and the ligation mixture was used to juncti[on. However, pvj97, used as a probe, blotted to two transform Rxl cells. DNA from Emr transformant VG72 was regioris in Rxl cells (Fig. 2) and to these plus a third one in digested with PstI, self-ligated, and used to transform E. DP13 22 (not shown), as though the presence of Q1(cat-tet) coli. A Cmr clone gave pvj183, whose passenger DNA (Fig. added......~~ ~ ~~~~~~... ~ ~~~~~ I another copy to the two already there. Other results 3a) was expected to include the target region for Ql(cat-tet) impli d that DNA unique to the element was to the right of BM6001 as carried in DP1322. Alignment of this map with the 2..-kb EcoRI segment (data not shown). Cloning and the left and right ends of the DP1322 map (Fig. 3b and c) analy: sis of the target region from Rxl showed that the right shows excellent agreement up to the Sall site at the left and junctiion was past coordinate 67 (see below) and not within to an EcoRI site on the right. The one exception is that the 2. -kb EcoRI segment. DP1322 has a BglII site at 0.8 kb (Table 2) that is outside the Junction and target regions. Transformation and blotting insertion but not in Rxl. Analysis of the target. To ask about target preference, we used pvj183 as a probe for hybridization with EcoRI digests of seven transconjugants and other strains (Fig. ). In each t - transconjugant tested, and in DP1322 and DP1302 (the donor for the conjugations), the probe hybridized to four EcoRI / I -- (a) -fragments I of sizes 3.3, 3.6, 3.8, and 8.5.kb. However, it - -M hybridized to only three fragments of Rxl. The 3.3- and E3oHo 3.6-kb fragments disappeared and were replaced by a new 00 L" _ Q V) 2.1-kb fragment, with retention of the 3.8- and 8.5-kb (b) pieces _(Fig., lane i). This result implies that the 2.1-kb EcoRI fragment includes the target and that the 3.6- and 3.3-kb IdJȘ< k k, EcoRI fragments represent left and right junctions, tively. These three respec- (c) fragments are identifiable in Fig. 3, and the faintness of the 3.3-kb band reflects the shortness of its kb I and overlap of different with the probes probe. Further comparisons (compare Fig. 5 with of Fig. intensities FIG 3. Alignment of the target region in S. pneumoniae Rxl that the 3.8- and 8.5-kb bands extend to the right and ) left show of with tlhe ends of Qk(cat-tet) BM6001, as seen in strain DP1322. (a) Map oif the passenger DNA from Rxl in pvj183. (b and c) Left and the target, respectively. Similar hybridizations showed the right ends, respectively, of the DP1322 map as given in Fig. 1 and target to be contained within 3.2-kb BamHI, 1.75-kb BglII, Table 2.. Thin lines represent DNA that is not present in Rxl. Dotted and 5.0-kb PstI fragments in Rxl (data not shown). These region indicates uncertainty as to where the Ql(cat-tet) DNA starts results all fit the map of the target (Fig. 3), and the junction and en Ids (see text). fragments fit the DP1322 map and extend it slightly.

5 982 VIJAYAKUMAR ET AL. There is, therefore, a strongly preferred target for insertion of Ql(cat-tet) BM6001 after conjugative transfer, and this is the same site at which it is located in the donor and to which it goes during transformation. In Rxl the target is within a 2.1-kb EcoRI fragment and has been narrowed further to a 0.-kb region between SaII and EcoRI sites, shown in dotted lines in Fig. 3. In strain 8R1, from a clinical isolate far removed from Rxl (1), the target for this element is contained in a 2.9-kb EcoRI fragment (Fig., lane m). Size of the element. We obtain the length of fl(cat-tet) BM6001 as the difference between the spacings of the Sall and the relevant EcoRI site in DP1322 and in Rxl. This number is 65.5 kb plus or minus the accumulated error between the SalI site at 1.7 and the EcoRI site at 67.7 (Table 2) and is independent of the precise location of the junctions within the 0.-kb dotted regions in Fig. 3. The junctions are, then, near 1.9 and 67. kb in Fig. 1 and Table 2. Target and junction regions of fk(cat-erm-tet) B109 after its transfer from S. agalactiae B109 to S. pneumoniae. S. pneumoniae DP150 is an Rxl transconjugant carrying a cat-ermtet element from S. agalactiae B109 (7, 9; M. D. Smith, Ph.D. thesis, Duke University, Durham, N.C., 1981). Whereas Smith obtained Rxl derivatives carrying copies of both the BM6001 element and one from S. pneumoniae BM200, he found that the B109 element seemed to replace that from BM6001, as if they preferred the same target. Cotransformation mapping data (Priebe and Guild, in preparation) also suggested that the targets for these elements were close together. We probed EcoRI digests of DP150 and DP1322 with pvj186, a subclone of the Rxl target that carries the 3.2-kb BamHI fragment from pvj183 ligated to BamHI-cleaved pbr322. The results (Fig.5) show that the two elements (lanes b and g) inserted into the same 2.1-kb EcoRI fragment of Rxl, and further, that the EcoRI junction fragments of the two elements were identical in size. DP1320. Strain DP1320 arose during transformation of Rxl recipients with DP1302 donor DNA and selection for Cms Tcr transformants (18). This strain carries tet as part of a large insertion but is transfer deficient, in contrast to strains that have lost cat by spontaneous excision (22). The DNA blot in Fig. 5 (lane c) shows that strain DP1320 contains an intact 2.1-kb EcoRI target fragment and also the right junction fragment, but lacks the left junction fragment. Therefore the crossover that allowed integration of tet without cat did not involve the DNA immediately surrounding the target. 3.8 k b 2 1 a b c d e f g FIG. 5. Comparison of target and junction regions in some S. pneumoniae strains carrying elements derived from BM6001 or from S. agalactiae B109. EcoRI digests of DNA from strains (a) Rxl, (b) DP1322, (c) DP1320, (d) DP1302, (e) 8R1, (f) DP3117, and (g) DP150 were probed with [32P]dCTP-labeled pvj186. ~~~~~ A B C _ _ A!- 8 0 kb - - O09 FIG. 6. Absence of detectable fusion fragments. [32P]dCTPlabeled pvj91 was hybridized to fragments of DP1322 chromosomal DNA digested with (A) BglIH, (B) EcoRI, or (C) HindlIl. Numbers indicate fragment sizes in kb. Failure to detect a circular intermediate in DP1322. A model often suggested for transfer of these elements invokes excision, circularization, transfer as a plasmid, and reinsertion (3, 10, 12). This model conflicts with evidence from in vivo restriction experiments in S. pneumoniae (7), but Inamine and Burdett (10) reported that probes to both ends of the B109 element hybridized to a 5.5-kb EcoRI fragment in digests of an S. faecalis transconjugant. This band was fainter than others on the blot. They suggested it might be a fusion fragment representing about 20% of the copies of the insertion in circular form (10). We used pvj91, carrying about 19 kb of passenger DNA that terminates 2 kb inside the right junction (Table 1), to probe digests of DP1322 DNA. If the BM6001 element circularizes at a detectable frequency, this probe should hybridize to 10.3-kb BgllI, 6.5-kb HindIII, and.8-kb EcoRI fusion fragments. The DNA blot analysis showed no trace of fragments of these sizes (Fig. 6), and all bands can be accounted for as expected from the element in linear form. To do so, however, we have to include the bands seen in Fig. 2 for Rxl digests (because the probe carries the internal 2.-kb EcoRI region that shows homology to Rxl) and a 5.7-kb HindIII right junction fragment extending to 69.9 kb, beyond the end of Fig. 1. This site is inferred from Fig. 3a, the map for the Rxl target region. Experiments using pvj16, carrying the right junction region, also failed to detect signs of a circular intermediate in DP1322. Thus, circularization is at most a rare event in DP1322, and we have no evidence that it occurs at all. Also implicit in these results is lack of homology between the ends of the element. DISCUSSION J. BACTERIOL. The objective was to obtain cloned DNA segments and a restriction map covering the cat-tet conjugative insertion from S. pneumoniae BM6001, as represented in a laboratory

6 VOL. 166, 1986 Rxl derivative, DP1322. Using directed insertion of an E. coli vector into the chromosome, an approach to cloning first described for S. pneumoniae by Mejean et al. (1), we have cloned nearly 69 contiguous kb of the DP1322 chromosome and have mapped this region for 11 restriction nuclease enzymes. An important point is that the method required cycling into and out of the chromosome and allowed repeated checks that independent clones generated the same restriction site map for the segments of interest. Thus, although some clones not shown here contained major rearrangements, these could be bypassed to generate the final map as given in Table 2 and Fig. 1. Significant results so far include: (i) Ql(cat-tet) BM6001 is 65.5 kb in size between junctions with normal chromosome DNA (transformation data had suggested greater than 30 kb [18]); (ii) cat and tet are about 1 and kb, respectively, in from the "left" end of Ql(cat-tet) BM6001; (iii) cat is flanked by sequences that also appear in the wild-type Rxl genome and are probably related to its frequent spontaneous curing (22) and to its ability to separate from tet during transformation (18); (iv) a third internal region at least 2. kb long shows homology to two places in the Rxl chromosome. A search for the presence of both types of sequence in other streptococci should be informative; they could be IS-like elements or their remains. Further, (v) there is a strongly preferred target to which fl(cat-tet) BM6001 transfers in Rxl, and (vi) this is the same target to which Tn3951 [fq(cat-erm-tet) B109] transfers when Rxl is mated with S. agalactiae B109. On the other hand, an element from S. pneumoniae BM200 goes to a different site in Rxl, such that strains carrying both it and the BM6001 element can be constructed (7). These results are consistent with cotransformation mapping results to be described elsewhere (Priebe and Guild, in preparation). In contrast to these elements, Tn916 goes to many sites in Rxl (Priebe and Guild, in preparation). Further findings include the following. (vii) Restriction sites are clustered near the ends of the BM6001 element, leaving a large central portion relatively barren of such sites. Of 97 sites determined for six-base recognition sequences (Table 2), only 5 fall inside the 18.8-kb segment from coordinates 32.2 to 51 kb, and of these are clustered near the tet gene that is common to most of these elements (22). The paucity of sites flanking tet might be related to the broad host range of such elements. (viii) We were unable to detect homology between the junction regions of the element, either within the element or neighboring it. The DNA blot results would not have detected five- to nine-base repeats if such were there, but larger terminal repeats would have been seen. Similar lack of homology was reported for clones thought to include the ends of the B109 element as carried on pdp5 in S. faecalis (10). We show below that our data for this element in S. pneumoniae DP150 imply that its ends are the same as those for the BM6001 element. Mode of transfer? Integrated plasmids in gram-negative species sometimes excise, transfer as plasmids, and reinsert into the chromosome. This possibility for S. pneumoniae was considered some time ago and found to be unlikely in that the DpnII restriction system had no effect on transfer of the BM6001 or B109 elements in vivo, even in cells that simultaneously were restricting conjugative transfer of pip501 and pmv158 (7). Retaining the concept of a plasmid intermediate requires either making ad hoc assumptions or ignoring the above result. The only experimental data in streptococci that might support a circular intermediate are CONJUGATIVE ELEMENTS IN S. PNEUMONIAE 983 those of Inamine and Burdett (10), who interpreted a DNA blot fragment as representing a fusion of the ends of the B109 element. However, we found internal fragments of the BM6001 element that blotted to wild-type S. pneumoniae DNA (Fig. 2), and all fragments on the blot of Fig. 6 could be accounted for without invoking fusion fragments. It should still be noted that inasmuch as transfer is rare, failure to detect fusion fragments is not proof of their absence. Overall, the lack of restriction in vivo (7) is our primary reason for doubting that a plasmid intermediate is involved in transfer of these elements. It has been suggested that the integration step after transfer may resemble "ectopic integration" of hybrid plasmid molecules carrying DNA fragments homologous to the host (12). No source of the homologous DNA was cited, nor was it specified how the DNA transferred. However, ectopic integration leads to duplication of a segment of the insertion DNA, as in the cloning methodology used here, presumably with generation of enough homology to be detected by blot hybridization. As noted above, none was seen. Comparison to fl(cat-erm-tet) B109 (Tn3951). The B109 and BM6001 elements inserted into the same target in Rxl and generated junction fragments of the same sizes (Fig. 5), suggesting that the DNA sequences at the ends of these elements are closely similar. This suggestion is supported by comparison of Fig. 3 with corresponding maps reported by Inamine and Burdett (10) for the junction fragments of the B109 element in pdp5, which was created by its transposition from the chromosome of S. faecalis to pad1 (21). At the right ends the maps coincide closely for 2.8 kb from an EcoRI site to an XbaI site that appears to be just inside the true junction, and at the other end, an EcoRI site and two XbaI sites correspond. If we make one further assumption, that a PstI site arose by point mutation rather than by deletion in the pad1 target (10), then the junction in each case is 1.9 kb to the left of the EcoRI site and the left ends match for at least 1.9 kb. A further implication from this comparison is that the insertion site is just to the right of the Sall site in the Rxl target region, leaving an XbaI site just inside the end of the element (Fig. 3). The one clear difference so far is that the B109 element has an additional XbaI site not present in the BM6001 insertion, about 1.1 kb in from the right junction. For Tn3951 as a whole, Inamine and Burdett (10) found that pdp5 had a 67-kb insertion into pad1, and they reported a map of EcoRI sites for the insertion and for some cosmids constructed from S. agalactiae B109 itself. Their map shows similarities to our map for Qt(cat-tet) BM6001, but the arrangement of some of the larger fragments differs substantially. Since only EcoRI sites were reported, the differences cannot be analyzed further at this time. The data do not exclude the possibility that Tn3951 has simply added 1.5 kb including an erm gene to the BM6001 element. In any event, these elements show many similarities and appear nearly identical at the ends. Since the comparison has been between Tn3951 as seen on pdp5, after transfer from S. agalactiae to the chromosome of S. faecalis followed by transposition to pad1 (21), and the BM6001 element as seen in S. pneumoniae DP1322, a further suggestion is that all these transfer events involved precise or nearly precise endpoints. The present maps and cloned segments of Ql(cat-tet) BM6001 and of its target region, along with biological data on its behavior and that of other elements, provide a basis for further investigations both on epidemiology and on mechanisms of the transfer process.

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