Michel Sicard

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.

Exclusion of long heterologous insertions and deletions from the pairing synapsis in pneumococcal transformation.

We have studied the mode of recombination of six insertions during genetic transformation of Streptococcus pneumoniae. The six heterologous insertions are located at the same site in the ami locus of the pneumococcal chromosome; insertion sizes range from 4 to 1374 bp. With respect to single-point markers we found that the number of transformants in one-point crosses is reduced, while the number of wild-type transformants in two-point crosses is drastically increased, what we call hyper-recombination. The magnitude of the shift is correlated with the size of the insert. This effect could result either from a special repair pathway of multibase heteroduplexes or from the exclusion of multibase heterologous insertions out of the pairing synapsis. To test these hypotheses we have used insertions in two kinds of three-point crosses. The repair model predicts that the excess of wild-type transformants remains in one set of crosses but is suppressed in the second set. The results we obtained are reversed, ruling out the hypothesis of a repair process, but in agreement with predictions based on the exclusion model. Moreover, we have re-examined the situation of deletions, our previous results suggesting that deletions were likely to be converted at the heteroduplex step. Genetic evidence we obtained in this work no longer supports this hypothesis. Thus, long heterologous insertions are partly excluded at the pairing step.

Genetic studies of cefotaxime resistance in Streptococcus pneumoniae: relationship to transformation deficiency

A laboratory pneumococcal strain resistant to cefotaxime was studied by DNA-induced transformation in order to characterize its genetic structure. At least three independent genes were required to confer the highest level of resistance to this beta-lactam antibiotic. The accumulation of mutations in these three genes accounted for three levels of resistance. Mutation of the gene encoding penicillin-binding protein 2x was very likely responsible for the first step of resistance, which was a prerequisite for sequential increase in resistance. Additionally, strains highly resistant to cefotaxime were defective for natural transformation. Revertants of these strains were frequently observed. Such strains had recovered full transformability, suggesting a correlation between the inability to be transformed and a high level of resistance to cefotaxime. The possibility of electrotransforming these highly resistant strains suggests that natural transformation is probably blocked at the DNA-uptake level.

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.