Construction of new unencapsulated (rough) strains of Streptococcus pneumoniae.

To construct rough strains of Streptococcus pneumoniae in which the capsule locus was completely deleted, a genetic cassette to be used as a donor DNA in transformation was developed. The cassette contained an aphIII gene, conferring kanamycin resistance, flanked by segments of dexB and aliA. Since, in all strains of S. pneumoniae the capsule locus is between dexB and aliA, the DNA segments of these two genes allow insertion of a 1354-bp DNA fragment containing aphIII into the pneumococcal chromosome, determining the deletion of the whole capsule locus. The capsule locus was deleted from the classic type 2 and type 3 Avery's strains, from R6 (whose complete genome sequence is released) and Rx1 (the two most commonly used transformation recipients), from a type 3 clinical strain and type 19F clinical isolate G54 (whose draft genome sequence is annotated). The effect of capsule removal was tested in 4 isogenic pairs. In unencapsulated strains, growth rate increased up to 56% and transformation frequency increased up to 1075-fold. A correlation was observed between the increase in growth rate and an increase in transformation frequency.