Homologous recombination at the border: Insertion-deletions and the trapping of foreign DNA in Streptococcus pneumoniae

Abstract

Integration of foreign DNA was observed in the Gram-positive human pathogen Streptococcus pneumoniae (pneumococcus) after transformation with DNA from a recombinant Escherichia coli bacteriophage carrying a pneumococcal insert. Segments of DNA replaced chromosomal sequences adjacent to the region homologous with the pneumococcal insert, whence the name insertion-deletion. Here we report that a pneumococcal insert was absolutely required for insertion-deletion formation, but could be as short as 153 bp; that the sizes of foreign DNA insertions (289 -2,474 bp) and concomitant chromosomal deletions (45-1,485 bp) were not obviously correlated; that novel joints clustered preferentially within segments of high GC content; and that the crossovers in 29 independent novel joints were located 1 bp from the border or within short (3-10 nt long) stretches of identity (microhomology) between resident and foreign DNA. The data are consistent with a model in which the insert serving as a homologous recombination anchor favors interaction and subsequent illegitimate recombination events at microhomologies between foreign and resident sequences. The potential of homologydirected illegitimate recombination for genome evolution was illustrated by the trapping of functional heterologous genes. G enetic transformation, which was discovered in the Grampositive Streptococcus pneumoniae (pneumococcus) (1), is believed to play a central role in the biology of this human pathogen through its contribution to genetic plasticity (see ref.2 for a review). Transformation with naked DNA allows intraspecies and interspecies gene transfer. As such exchanges involve homologous recombination, they are generally ''conservative,'' i.e., they do not result in the creation of novel sequences but simply in a redistribution of previously existing genes. However, a pre-existing gene also can be modified when the two interacting sequences are partly divergent. Homologous recombination then leads to the production of mosaic genes, as exemplified in the case of the pbp genes of S. pneumoniae that encode altered penicillin-binding proteins with decreased affinity for ␤-lactam antibiotics (see ref. 3 for a review).Besides these conservative facets of transformation, is there any potential for the creation of novel sequence combinations or genes? The observation that transformation with chimeric donor DNA is mutagenic (4) suggested that shuffling and reassembly of previously unrelated sequences could readily occur in S. pneumoniae. The chimeric DNA extracted from a recombinant Escherichia coli bacteriophage carrying a pneumococcal insert produced illegitimate recombinants at a frequency of about 0.5% that of homologous recombinants (4). Illegitimate recombinants resulted from simultaneous insertion of heterologous vector (i.e., ) sequences and deletion of chromosomal sequences adjacent to the region homologous to the insert. These illegitimate events were therefore termed insertion-deletions (InsDels). As the pneumococcal insert in the chimeric donor seemed r...

Genetic Basis for the New Pneumococcal Serotype, 6C

“…Horizontal gene transfers between S. pneumoniae and another bacterial species have been demonstrated with antibiotic resistance genes (5,18) and have been suggested to occur for capsule gene loci because they have low GϩC content (2). The "flanking regions" are known to be critical for homologous recombination in pneumococci (21). Indeed, an examination of wciN 6C shows evidence for the two flanking regions that may have participated in the homologous recombination.…”

Relative Fitness of Fluoroquinolone-resistantStreptococcus pneumoniae

“…The QRDR mutations in gyrA and parC of the clinical QRSP (donor strains CT01147 and UAB169), the mutants (Phe/Phe and Phe/Tyr), and parent strain (EF3030) were sequenced to confirm the presence of QRDR mutations and because genetic transformation has been associated with increased mutation frequency (21,22). The transformation fragment for gyrA consisted of 1,325 bp, of which 660 inclusive of the QRDR were sequenced.…”

The RecBCD and SbcCD DNases suppress homology-facilitated illegitimate recombination during natural transformation of Acinetobacter baylyi

“…Foreign DNA having only a single small homologous segment can also be integrated through homology-facilitated illegitimate recombination (HFIR; de Vries & Wackernagel, 2002), which proceeds by homologous recombination in the homologous segment (anchor) accompanied by illegitimate recombination of the adjacent foreign DNA. HFIR was first observed in Streptococcus pneumoniae (Claverys et al, 1980), and was subsequently studied in detail in Gram-positive and Gramnegative bacteria, including S. pneumoniae (Prudhomme et al, 2002), Acinetobacter baylyi (de Vries & Wackernagel, 2002;de Vries et al, 2004;Harms et al, 2007;Hülter & Wackernagel, 2008) and Pseudomonas stutzeri (Meier & Wackernagel, 2003. In A. baylyi transformation, HFIR is about 10 000 times less frequent than fully homologous recombination, but at least 100 000-fold more IP: 52.183.12.225…”