Anne-Marie Gasc

Organization around the dnaA gene of Streptococcus pneumoniae

The dnaA gene region of Streptococcus pneumoniae was cloned and sequenced. A tRNA gene, seven ORFs and three DnaA box clusters were identified. The order of the genes and intergene regions found was tRNA(Arg)-orf1-DnaA box cluster 3-htrA-spoOJ-DnaA box cluster 2-dnaA-DnaA box cluster 1-dnaN-orfX-orfY. Five ORFs are homologous to known bacterial genes. The tRNA(Arg) gene and orf1, also called orfL, have already been described in pneumococci and have been reported to be preceded by the competence regulation locus comCDE. In Escherichia coli, htrA encodes a serine protease. In Bacillus subtilis, spoOJ plays a role in sporulation and partition. dnaA encodes an initiator replication protein, very well conserved in several bacteria and dnaN encodes the beta subunit of DNA polymerase III in E. coli. The function of orfX is unknown. The N-terminal part of another reading frame, orfY, revealed high homology with a GTP-binding protein, DnaA box clusters were found upstream and downstream from dnaA. The presence of two such clusters suggests that the chromosomal origin of S. pneumoniae is located within this region. The position of dnaA, and therefore the putative origin of replication, were localized on the physical map of S. pneumoniae.

Mismatch repair during pneumococcal transformation of small deletions produced by site-directed mutagenesis.

SummaryThe genetic behaviour of short non-homologous regions has been studied during transformation of Streptococcus pneumoniae. Amethopterin-resistant mutants belonging to the amiA locus were used for these investigations. Five mutants deleted for 1–5 bp were obtained by oligonucleotide-direcrted mutagenesis. Their efficiency of transformation was measured using recipient strains either able to excise and repair mismatched bases (Hex+) or Hex- derivatives. Deletions or insertions of 1 and 2 bp are fully recognized by the Hex system, and are efficiently repaired whereas 3-bp deletions or insertions are only partially excised and repaired. The efficiency of repair is inversely related to the size of the non-homology. Markers with 5-bp deletions or insertions are poorly repaired and thus transform at very high frequency: similar results are obtained in reciprocal crosses. It is proposed that 1-or 2-bp deletions or insertions are included in the heteroduplex structure as transition mutations. The Hex system would detect only small deviations from the normal DNA structure.

DNA sequences required to induce localized conversion in Streptococcus pneumoniae transformation

SummaryIn pneumococcal transformation a particular point mutation belonging to the amiA locus is able markedly to enhance recombination frequency when crossed with any other markers of this gene. This results from a polarized conversion of the mutation towards the wild-type sequence. In this report, by site-directed oligonucleotide mutagenesis, we have generated a series of mutants showing various degrees of conversion. We have found that the substitution 5′-ATTCAT→5′-ATTAAT is a sufficient signal for localized conversion. Changing individual bases within this sequence results in decreased conversion frequencies to levels that depend on the mutation, suggesting that there is a family to related sequences which may act as a substrate for a conversion system. Moreover, the length over which this conversion occurs has been estimated to be 12 base pairs on the average.

Inhibition of DNA repair by neighbouring mismatched bases in Streptococcus pneumoniae.

A set of pneumococcal strains containing immediately adjacent or nearby double mutations at the amiA locus, conferring resistance to amethopterin, has been isolated by oligonucleotide site-specific mutagenesis. Repair of these double mutations has been measured by transformation of wild-type strains with DNA extracted from these strains. In several transformations we have observed an inhibition of repair by neighbouring mismatches. This inhibition ranges from mild to severe depending upon the interfering mismatch. Unrepaired mismatches can strongly inhibit repair of an adjacent repairable mutation. This suggests that the repair-complex proteins attach not only to repairable mismatches but also to some mismatches known to escape the repair system.