The nonconjugative streptococcal plasmid pMV158 can be mobilized by the conjugative streptococcal plasmid pIP501. We determined the sequence of the 1.1-kilobase EcoRI fragment of pMV158 to complete the DNA sequence of the plasmid. We showed that an open reading frame, mob (able to encode a polypeptide of 58,020 daltons), is required for mobilization of pMV158. An intergenic region present in the EcoRI fragment contains four lengthy palindromes that are found also in one or more of the staphylococcal plasmids pT181, pE194, and pUBIIO. One palindromic sequence, palD, which is common to all four plasmids, also appeared to be necessary for mobilization. Circumstantial evidence indicates that this sequence contains both an oriT site and the mob promoter. The Mob protein is homologous in its amino-terminal half to Pre proteins encoded by pT181 and pE194 that were shown by others to be essential for site-specific cointegrative plasmid recombination; their main biological function may be plasmid mobilization.A variety of conjugative and nonconjugative plasmids are found in streptococcal species (for a review, see reference 6). The conjugative plasmid pIP501 confers erythromycin resistance (Emr) and chloramphenicol resistance (Cm'-). This 30-kilobase (kb) plasmid was found by Horodniceanu and co-workers in a strain of Streptococcus ag(llactiae (15) and shown to pass conjugatively into various streptococcal species (13). The 5.5-kb nonconjugative plasmid pMV158 confers tetracycline resistance (Tcr); it was found by Burdett in another strain of S. agalactiae (5). Guild and co-workers transferred the two plasmids into Str eptococcils pneitnoniae by transformation (32). They showed that when both plasmids were present in the same cell, pIP501 was able to mobilize pMV158 for conjugative transfer. In this work, we inquired into the molecular basis of such mobilization.A derivative of pMV158 called pLS1, which was constructed by removal of a 1.1-kb EcoRI fragment, replicates normally in S. pneirmoniae (33). Both pMV158 and pLS1 also replicate in other gram-positive bacteria, such as Bacillus sibtilis (11), and in gram-negative bacteria, such as Escherichia coli (20 Novick and co-workers found that recombination between two staphylococcal plasmids, pT181 and pE194, to give a cointegrate product occurred within closely related 70-basepair (bp) sequences (called RSA) in these plasmids (12,25). This site-specific recombination was independent of a )ecA mutation, but it required a protein called Pre (for plasmid recombination), encoded by a plasmid gene, pi-e (12). The * Corresponding author. pre gene product of either pT181 or pE194 could promote recombination, and the pT181 protein was shown to act in tr'atis, that is, when the active pre gene was present on a plasmid not participating in the cointegrate formation (12). The Pre proteins predicted from the DNA sequence of pT181 and pE194 appeared to be homologous, and it was pointed out that another staphylococcal plasmid, pUB110, contains a sequence identifical to part of RSA and ...
The Hex system of heteroduplex DNA base mismatch repair operates in Streptococcus pneumoniae after transformation and replication to correct donor and nascent DNA strands, respectively. A functionally similar system, called Mut, operates in Escherichia coli and Salmonella typhimurium. The nucleotide sequence of a 3.8-kilobase segment from the S. pneumoniae chromosome that includes the 2.7-kilobase hexA gene was determined. An open reading frame that could encode a 17-kilodalton polypeptide (OrfC) was located just upstream of the gene encoding a polypeptide of 95 kilodaltons corresponding to HexA. Shine-Dalgarno sequences and putative promoters were identified upstream of each protein start site. Insertion mutations showed that only HexA functioned in mismatch repair and that the promoter for hexA transcription was located within the OrfC-coding region. The HexA polypeptide contains a consensus sequence for ATP- or GTP-binding sites in proteins. Comparison of the entire HexA protein sequence to that of MutS of S. typhimurium, which was determined by Haber et al. in the accompanying paper (L. T. Haber, P. P. Pang, D. I. Sobell, J. A. Mankovitch, and G. C. Walker, J. Bacteriol. 170:197-202, 1988), showed the proteins to be homologous, inasmuch as 36% of their amino acid residues were identical. This homology indicates that the Hex and Mut systems of mismatch repair evolved from an ancestor common to the gram-positive streptococci and the gram-negative enterobacteria. It is the first direct evidence linking the two systems.
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...
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...
Sequence homology is expected to influence recombination. To further understand mechanisms of recombination and the impact of reduced homology, we examined recombination during transformation between plasmid-borne DNA flanking a double-strand break (DSB) or gap and its chromosomal homolog. Previous reports have concentrated on spontaneous recombination or initiation by undefined lesions. Sequence divergence of approximately 16% reduced transformation frequencies by at least 10-fold. Gene conversion patterns associated with double-strand gap repair of episomal plasmids or with plasmid integration were analyzed by restriction endonuclease mapping and DNA sequencing. For episomal plasmids carrying homeologous DNA, at least one input end was always preserved beyond 10 bp, whereas for plasmids carrying homologous DNA, both input ends were converted beyond 80 bp in 60% of the transformants. The system allowed the recovery of transformants carrying mixtures of recombinant molecules that might arise if heteroduplex DNA--a presumed recombination intermediate--escapes mismatch repair. Gene conversion involving homologous DNAs frequently involved DNA mismatch repair, directed to a broken strand. A mutation in the PMS1 mismatch repair gene significantly increased the fraction of transformants carrying a mixture of plasmids for homologous DNAs, indicating that PMS1 can participate in DSB-initiated recombination. Since nearly all transformants involving homeologous DNAs carried a single recombinant plasmid in both Pms+ and Pms- strains, stable heteroduplex DNA appears less likely than for homologous DNAs. Regardless of homology, gene conversion does not appear to occur by nucleolytic expansion of a DSB to a gap prior to recombination. The results with homeologous DNAs are consistent with a recombinational repair model that we propose does not require the formation of stable heteroduplex DNA but instead involves other homology-dependent interactions that allow recombination-dependent DNA synthesis.
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