PspA is a cell surface protein of Streptococcus pneumoniae that is present on a number of clinical isolates as well as the nonencapsulated laboratory strain Rx1. In a previous report we have shown that mAbs directed against PspA can protect mice from at least some of the pneumococcal strains bearing this protein. In our present report we have produced insertional inactivation mutants that lack PspA and have used these mutants to demonstrate that PspA can play a role in pneumococcal virulence and that anti-PspA immunity can lead to protection against pneumococcal infection. PspA- mutants were obtained using derivatives of plasmid pVA891 carrying chromosomal fragments from Rx1. From one of the mutants, we cloned a 550 bp fragment of the pneumococcal gene into pVA891 and transferred this chimeric plasmid, designated pKSD300, into Escherichia coli. After transformation of pKSD300 into Rx1, PspA production is not detected. In colony hybridization experiments, the 550 bp fragment hybridizes specifically to pneumococcal isolates in a pattern consistent with the hypothesis that the fragment is a portion of the pspA structural gene that is different from the portions coding for the antigenic determinants detected by mAbs Xi64 or Xi126. When X-linked immunodeficient (xid) CBA/N mice were immunized with wild-type Rx1, they were resistant to challenge with type 3 strain WU2. However, when these mice were immunized with a PspA- mutant of Rx1, they failed to survive the subsequent challenge, indicating that immunity to PspA can contribute to the resistance to pneumococcal infection. Using pKSD300 we insertionally inactivated pspA in D39, a virulent strain of S. pneumoniae. When injected intravenously there was a 10-fold greater reduction of the mutant pneumococci in the blood, as compared to the wild-type D39.

that the conjugative transfer of all these elements shares a common mechanism, as the elements are derived from an ancestral element with transfer properties.Tn5253, formerly called the fl(cat tet) element (36), was originally detected as a heterologous insertion in the chromosome of the plasmid-free clinical isolate S. pneumoniae BM6001 (3, 9). By inserting the Escherichia coli vector plasmid pVA891 (22) (which is incapable of autonomous replication in streptococci) at many sites specifically within Tn5253, we were able to clone and recover parts of the element in E. coli (37). Physical analysis of the passenger DNAs from these plasmids made it possible to construct a detailed restriction map of this 65.5-kb element, to localize the drug resistance determinants, and to identify its junction and target regions in the pneumococcal chromosome (36), (Fig. 1)

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 2.4 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 (2,4,7,9,19). At least two such insertions, Tn9O6 from Streptococcus faecalis (4) 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 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 (24) 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 (24). pVA891 confers erythromycin resistance to streptococci and chlo...

The 47-kb, broad-host-range, streptococcal conjugative transposon Tn5252 is capable of site-specific integration into the pneumococcal chromosome. We present the nucleotide sequence of the terminal regions of the transposon and its target site in the pneumococcal genome. No inverted repeats were found at the termini of the transposon. A 72-bp region of the target was present on either side following the insertion of Tn5252 and appeared to serve as a signal for its integration and excision. The data suggest that the left copy of the 72-bp segment was a part of the conjugative element, the crossover point of integration was nonrandom within this region, and the mechanism of insertion could resemble that of the site-specific temperate phages.Sudden emergence of multiple-antibiotic resistance in clinical streptococci in the 1970s (6,8,15,16,21) has been chiefly due to a highly promiscuous class of mobile elements termed conjugative transposons (4,12,17,22,29,31). These mediate self-transfer by a DNase-resistant process requiring cell-to-cell contact (8,16,30 BM6001 (8,26), is a 65.5-kb self-transmissible element that encodes resistances to chloramphenicol and tetracycline (29).We had previously cloned the entire element in fragments in Escherichia coli and generated its restriction endonuclease map (34, 35). The transposition behavior of a DNA segment carrying Tetracycline resistance when separated from the context of surrounding DNA led to the identification of TnS253 as a composite structure of two independent conjugative transposons with an 18-kb element, TnS251 (Tcr), inserted in the central region of another, TnS252 (47 kb, Cmr) (2). Similarly, Bouguenec et al., found that Tn3701 in Streptococcus pyogenes was also a composite structure containing a transposon, Tn3703 (Tcr), in its central region (5). TnS251, which is homologous to Tn3703, has been shown to be structurally and functionally very similar to Tn916 and TnlS45 (2, 5). The structure and mechanism of transposition of Tn916 (9,17,18,27,28)

We used a directed insertion method to introduce a nonreplicating vector plasmid into the large conjugative cat-tet element foubd in the chromosome of Streptococcus pneumoniae BM6001 and derivatives. To direct insertion preferentially to the conjugative element, we transferred it by conjugation to Streptococcus faecalis and then used DNA from this strain as a source of restriction nuclease fragments for ligation to digests of the vector pVA891, which can replicate in Escherichia coli but not in streptococci. This ligation mix was used to transform pneumococcal cells carrying the cat-tet element, with selection for the erythromycin resistance carried by pVA891. Eight such isolates were found, and transformation and conjugation tests showed that in each case the vector had inserted into the conjugative element, as expected. DNA from these pneumococcal strains generated a variety of E. coli plasmids which provide tools for obtaining a detailed restriction map and for defining other structural features of the streptococcal conjugative element.The recent appearance and horizontal spread of multiple antibiotic resistance among clinical strains of pneumococci (Streptococcus pneumoniae) and other gram-positive-organisms is of considerable medical importance and biological interest (2, 6). Among streptococci, most drug resistance determinants are parts of large nonhomologous insertions into the chromosome (8, 9, 18). Several of these elements exhibit a new form of conjugal transfer within and among species carrying no detectable plasmids (3, 8, 18, 18a). Two of these conjugative elements, Tn916 (4) and Ql(cat tet erm) of Streptococcus agalactiae B109 (19), are known to transpose to plasmids, and the term "conjugative transposon" has been applied to them. From sedimentation and transformation studies, it was found that fQ(cat-tet) of S. pneumoniae BM6001, fl(cat-erm-tet) of S. agalactiae B109, fl(cattet-erm-aphA) of S. pneumoniae BM4200, and fl(cat-tet) of S. pneurhoniae N77 were located in the chromosome (6). Using tet-3, a point mutation conferring drug sensitivity, it was shown that the tet determinants derived from various fQ elemenits were homologous to each other, whereas plasmidderived tet genes were not (20). The results thus far indicate that there is substantial homnology among the nl elements, which suggests that the various drug resistance determinants insert within or delete from a basic QI tet unit (6). If so, a detailed analysis of one such element should provide a framework to gain understanding of other related elements. Hence, we chose to study the well-characterized (6,18,20) fl(cat-tet) strain BM6001 element in detail.The initial scope of the project involved cloning fragments of the fl element in Escherichia coli and generating a restriction map. One could then focus on the location of the drug resistahce and transfer genes, the target sites for the insertion of the fl element in the pneumococcal chromo-* Corresponding author. some, and other features which may be pertinent to the mode of its transfer...

To obtain a functional map of TnS252, a 47.5-kb streptococcal conjugative transposon, a series of defined deletion and insertion mutations were introduced within the transposon. Interruptions at several regions were found to affect the conjugal transposition functions of the element in filter-mating experiments. The nucleotide sequence of the left terminus of Tn5252 showed two open reading frames, ORFR and ORF2, adjoining the at site. The organization of this region and the structure of the predicted integrase encoded by ORFi were found to be similar to those of other site-specific recombination systems.Even though Streptococcus pneumoniae can receive and stably maintain a variety of plasmids from other streptococci, endogenous plasmids are rare in this species. However, since the 1970s multiple antibiotic resistance has begun to appear in this species and a novel class of mobile elements conveniently termed "conjugative transposons" has been shown to be the chief carriers of the multiple antibiotic resistance (5,9,10,14,17,24). The chromosomally integrated conjugative transposons are able to transfer themselves to other cells by a process requiring cell-to-cell contact (5,10,12,23). Two classes of streptococcal conjugative transposons have been recognized (2, 16). Even though both types are capable of conjugal transposition, no structural similarity has been observed (2, 15). Also, the class of elements, typified by the well-characterized Tn916 and Tn1545, integrate at several sites in the recipient's genome (2,7,8), whereas the larger conjugative transposons such as Tn5252 and Tn3701 preferred to integrate at a unique spot (2, 28). We have been studying the biology of Tn5253, a 65.5-kb conjugative transposon carrying resistance to tetracycline and chloramphenicol (2,(27)(28)(29). In a previous article (2), we showed that TnS253 is a composite element of two conjugative transposons (Fig. 1). The smaller element, now termed TnS251 (18 kb), was observed to be structurally and functionally similar to the Tn916 class of elements (2) and did not show homology to any other region in the parental element, TnS253 (2). The sequences beyond TnS251 within TnS253, now called Tn5252, were themselves capable of conjugative transposition (2). While considerable information regarding the Tn916 (TnS251) class of elements has been obtained (6), very little is known of the Tn5252 class of elements (6). To understand the molecular details of this novel class of elements, we had focused our efforts on studying TnS252. In this article, we report our studies relating to the genetic organization and nucleotide sequence of the left end of this transposon.Strategy for creating insertion and deletion mutations within Tn5252. The availability of Escherichia coli recombinant plasmids carrying DNA fragments derived from TnS252 facilitated generation of deletion and insertion mutations within the passenger segments. The basic strategy employed in this 744-6790. study, illustrated in Fig. 2, was as follows. DNA restriction fragments rangi...

Intracellular locations of 11 proteins associated with the development of competence in Streptococcus pneumoniae were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of subcellular fractions prepared from protoplasts. Controls showed that the competence-induced proteins were stable during the formation of protoplasts at 25C even though some had a half-life of only 8 min at 37°C. Five competence-induced proteins p38, p27, p19.5, p16, and p14.5, were found in the cytoplasm. Two, p52 and p41, were associated with the membrane, and one, plO, was extracellular. Three others, p50, p36, and p29, were recovered in both cytoplasmic anid membrane fractions. No competence-induced protein was detected in the periplasmic fraction except under conditions where leakage of all components was occurring, a phenomenon that was seen in many preparations. Similar fractionation of competent cells soon after uptake of [3HIDNA showed the "edipse complex" of single-stranded DNA and p19.5 was associated approximately one-third with membranes and two-thirds with cytoplasmic fractions, with almost none in the periplasm. This result suggests strongly that at the time the donor DNA entered the cytosol it was in single-stranded form and it had not yet paired with the recipient DNA.

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