Interspecies genetic exchange is an important evolutionary mechanism in bacteria. It allows rapid acquisition of novel functions by transmission of adaptive genes between related species. However, the frequency of homologous recombination between bacterial species decreases sharply with the extent of DNA sequence divergence between the donor and the recipient. In Bacillus and Escherichia, this sexual isolation has been shown to be an exponential function of sequence divergence. Here we demonstrate that sexual isolation in transformation between Streptococcus pneumoniae recipient strains and donor DNA from related strains and species follows the described exponential relationship. We show that the Hex mismatch repair system poses a significant barrier to recombination over the entire range of sequence divergence (0.6 to 27%) investigated. Although mismatch repair becomes partially saturated, it is responsible for 34% of the observed sexual isolation. This is greater than the role of mismatch repair in Bacillus but less than that in Escherichia. The remaining non-Hex-mediated barrier to recombination can be provided by a variety of mechanisms. We discuss the possible additional mechanisms of sexual isolation, in view of earlier findings from Bacillus, Escherichia, and Streptococcus.Bacteria from all major taxa are able to exchange genes across species by homologous recombination (26). While the various bacteria take up donor DNA by a diversity of mechanisms, all studied systems of homologous recombination share at least one homologous feature: recombination depends ultimately on the activity of the RecA protein and its homologues (26). Similarly, there is one system that hinders recombination across species in both the Proteobacteria and the gram-positive bacteria (3). This is the mismatch repair system encoded by mutS, mutL, and their homologues. Because some of the molecular basis for interspecies recombination is shared across disparate taxa, we might expect that recombination between species is constrained in similar ways throughout the bacterial world.In addition, previous studies have shown that the frequency of homologous recombination decreases with the sequence divergence between donor and recipient, in a manner that is similar across a wide range of organisms. In Bacillus transformation as well as in Escherichia conjugation, the frequency of recombination decreases exponentially with the degree of DNA sequence divergence between donor and recipient (23,28,31). A similar exponential relationship has been observed for the frequency of intrachromosomal crossovers in Saccharomyces cerevisiae (5). Nevertheless, the major mechanisms producing recombinational barriers have been shown to differ in each of the above cases. In Escherichia coli, the predominant barrier to recombination is presented by the methylation-directed mismatch repair system (21). In Bacillus, mismatch repair is only marginally effective in preventing recombination between divergent sequences; the most significant barrier is that donor stran...

The aim of the present study was to examine the stability and evolution of tet(M)-mediated resistance to tetracyclines among members of different clonal lineages of Streptococcus pneumoniae. Thirty-two tetracyclineresistant isolates representing three national (Spanish serotype 14, Spanish serotype 15, and Polish serotype 23F) and one international (Spanish serotype 23F) multidrug-resistant epidemic clones were all found to be tet(M) positive and tet(O), tet(K), and tet(L) negative. These isolates all carried the integrase gene, int, which is associated with the Tn1545-Tn916 family of conjugative transposons. High-resolution restriction analysis of tet(M) products identified six alleles, tet(M)1 to tet(M)6: tet(M)1 to tet(M)3 and tet(M)5 in isolates of the Spanish serotype 14 clone, tet(M)4 in both the Spanish serotype 15 and 23F clones, and tet(M)6, the most divergent allele, in the Polish 23F clone. This indicates that tet(M) variation can occur at the inter-and intraclone levels in pneumococci. Two alleles of int were identified, with int1 being found in all isolates apart from members of the international Spanish 23F clone, which carried int2. Susceptibility to tetracycline, doxycycline, and minocycline was evaluated for all isolates with or without preincubation in the presence of subinhibitory concentrations of tetracyclines. Resistance to tetracyclines was found to be inducible in isolates of all clones; however, the strongest induction was observed in the Spanish serotype 15 and 23F clones carrying tet(M)4. Tetracycline was found to be the strongest inducer of resistance, and minocycline was found to be the weakest inducer of resistance.The gram-positive pathogen Streptococcus pneumoniae (the pneumococcus) is a major cause of pneumonia, otitis media, and meningitis (12). The evolution and broad global distribution of multiple antibiotic resistance determinants in bacteria have resulted in a situation in which pneumococci are commonly resistant to penicillin, the broad-spectrum cephalosporins, macrolides, lincosamides, co-trimoxazole, chloramphenicol, and tetracyclines, as well as rifampin (11), sulphonamides (42), and fluoroquinolones (13,24,30), making the treatment of serious pneumococcal disease increasingly difficult (17,22). The transformable nature of S. pneumoniae (which has played an important role, along with point mutations) in the evolution of resistance [1,4,11,13,24,30] has in no small part also led to a population structure characterized by free genetic exchange, punctuated by clonal expansion of successful variants. The best studied of these are the Spanish 23F, Spanish 6B, and French/ Spanish 9V14 multidrug-resistant clones that have now spread intercontinentally (see reference 10 for a recent review).One class of antimicrobial agents found most often in clinical use is the tetracyclines, broad-spectrum bacteriostatic drugs shown to be active against pneumococci (33). In some European (9, 16, 23), Asian (35,36,41,47), and African (31, 52), countries lack of susceptibility to tetracyclines...

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Rifampicin resistance has arisen in several different species of bacteria because of alterations to one or more regions in the target of the antibiotic, the beta-subunit of RNA polymerase encoded by rpoB. Nucleotide sequence analysis of a 270 bp fragment of rpoB from 16 clinical rifampicin-susceptible isolates of Streptococcus pneumoniae, 8 clinical rifampicin-resistant isolates, and 3 spontaneous rifampicin-resistant mutants, has revealed that, as with previously examined species, point mutations within the cluster I region of rpoB, at sites encoding Asp516 and HiS526, also confer resistance to rifampicin in this important human pathogen. Moreover, the residues within cluster I, that were altered within the rifampicin-resistant mutants of S. pneumoniae, were in the same position as those previously found to alter in resistant isolates of Escherichia coli and Mycobacterium tuberculosis. Sequence analysis of rpoB, both from these isolates of S. pneumoniae and from two strains of S. mitis, reveals that, among a number of clinical isolates, resistance to rifampicin in S. pneumoniae has arisen by point mutation. However, the nucleotide sequence of rpoB from one isolate examined suggests that interspecies gene transfer may also have played a role in the evolution of rifampicin-resistance in S. pneumoniae.

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