The nucleotide sequence of comC, the gene encoding the 17-residue competence-stimulating peptide (CSP) of Streptococcus pneumoniae (L. S. Håvarstein, G. Coomaraswamy, and D. A. Morrison, Proc. Natl. Acad. Sci. USA 92:11140-11144, 1995) was determined with 42 encapsulated strains of different serotypes. A new allele, comC2, was found in 13 strains, including the type 3 Avery strain, A66, while all others carried a gene (now termed comC1) identical to that originally described for strain Rx1. The predicted mature product of comC2 is also a heptadecapeptide but differs from that of comC1 at eight residues. Both CSP-1 and CSP-2 synthetic peptides were used to induce competence in the 42 strains; 48% of the strains became competent after the addition of the synthetic peptide, whereas none were transformable without the added peptides.Genetic transformation not only is a valuable tool for molecular genetic analysis of Streptococcus pneumoniae (pneumococcus) but also appears to play a significant role in the evolution of this species, as in the assembly of mosaic antibiotic resistance genes containing blocks of information from other bacteria (6, 17) and in mediating a rapid mixing of alleles among natural populations (4). Yet, although transformation has long been described as a characteristic of the pneumococci, as a practical matter most studies involving this process have focused on a few laboratory strains, mainly descended from a single unencapsulated subclone (R36A) (2), for which optimal media and protocols were developed. Indeed, it has never been established just what proportion of pneumococcal isolates is transformable. This fraction has been difficult to assess, both because the pneumococcal capsule reduces or abolishes competence for genetic transformation (3,5,7,14,20,25) and because the optimal conditions for competence vary between strains.Competence for transformation in pneumococcus is not constitutive but is regulated by a quorum-sensing pheromone signal (23, 24). It was previously shown that culture supernatants from one strain, Rx1 (16), activate some other strains to competence and can induce competence in some encapsulated strains but not in others, including the type 3 strain A66 (25), even though an unencapsulated derivative of that strain is transformable (22). Following the recent identification of a small peptide from strain Rx1 with competence-stimulating activity (a competence-stimulating peptide [CSP]) (12), we reinvestigated the paradoxical behavior of A66, as a pure pheromone might be more effective than the crude culture supernatants used previously, but the results were still negative. We now trace the unresponsive character of strain A66 to the competence pheromone regulatory circuit itself and show that it reflects allelic variation of the pheromone gene in different isolates of this species.Failure of A66 to respond to the CSP of Rx1. Strain Rx1 is descended from R36A, an unencapsulated transformable derivative of D39S, a type 2 clinical isolate that was employed in early studies o...

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Streptococcus pneumoniae is the major cause of bacterial pneumonia, and it is also responsible for otitis media and meningitis in children. Apart from the capsule, the virulence factors of this pathogen are not completely understood. Recent technical advances in the field of bacterial pathogenesis (in vivo expression technology and signature-tagged mutagenesis [STM]) have allowed a large-scale identification of virulence genes. We have adapted to S. pneumoniae the STM technique, originally used for the discovery of Salmonella genes involved in pathogenicity. A library of pneumococcal chromosomal fragments (400 to 600 bp) was constructed in a suicide plasmid vector carrying unique DNA sequence tags and a chloramphenicol resistance marker. The recent clinical isolate G54 was transformed with this library. Chloramphenicol-resistant mutants were obtained by homologous recombination, resulting in genes inactivated by insertion of the suicide vector carrying a unique tag. In a mouse pneumonia model, 1.250 candidate clones were screened; 200 of these were not recovered from the lungs were therefore considered virulence-attenuated mutants. The regions flanking the chloramphenicol gene of the attenuated mutants were amplified by inverse PCR and sequenced. The sequence analysis showed that the 200 mutants had insertions in 126 different genes that could be grouped in six classes: (i) known pneumococcal virulence genes; (ii) genes involved in metabolic pathways; (iii) genes encoding proteases; (iv) genes coding for ATP binding cassette transporters; (v) genes encoding proteins involved in DNA recombination/repair; and (vi) DNA sequences that showed similarity to hypothetical genes with unknown function. To evaluate the virulence attenuation for each mutant, all 126 clones were individually analyzed in a mouse septicemia model. Not all mutants selected in the pneumonia model were confirmed in septicemia, thus indicating the existence of virulence factors specific for pneumonia.

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Electrotransformation was used to introduce both plasmid and linear DNA into Streptococcus pyogenes. The method was optimized using strain NZ131, for which transformation frequencies up to 10(7) per micrograms of plasmid DNA were obtained. A linear fragment of DNA, containing the streptokinase gene (ska) in which an internal fragment had been replaced with an erythromycin resistance gene (erm), was transformed into strain NZ131 with a frequency of 10(3) per micrograms DNA. The introduction of linear DNA into S. pyogenes by electrotransformation should be useful for future genetic analyses as well as targeted gene replacement.