h Natural genetic transformation of Streptococcus pneumoniae, an important human pathogen, mediates horizontal gene transfer for the development of drug resistance, modulation of carriage and virulence traits, and evasion of host immunity. Transformation frequency differs greatly among pneumococcal clinical isolates, but the molecular basis and biological importance of this interstrain variability remain unclear. In this study, we characterized the transformation frequency and other associated phenotypes of 208 S. pneumoniae clinical isolates representing at least 30 serotypes. While the vast majority of these isolates (94.7%) were transformable, the transformation frequency differed by up to 5 orders of magnitude between the least and most transformable isolates. The strain-to-strain differences in transformation frequency were observed among many isolates producing the same capsule types, indicating no general association between transformation frequency and serotype. However, a statistically significant association was observed between the levels of transformation and colonization fitness/virulence in the hypertransformable isolates. Although nontransformable mutants of all the selected hypertransformable isolates were significantly attenuated in colonization fitness and virulence in mouse infection models, such mutants of the strains with relatively low transformability had no or marginal fitness phenotypes under the same experimental settings. This finding strongly suggests that the pneumococci with high transformation capability are "addicted" to a "hypertransformable" state for optimal fitness in the human host. This work has thus provided an intriguing hint for further investigation into how the competence system impacts the fitness, virulence, and other transformation-associated traits of this important human pathogen. Streptococcus pneumoniae (the pneumococcus) is a commensal in the upper airway of humans and an opportunistic pathogen for numerous infections, including pneumonia, otitis media, and meningitis (1). Genetic plasticity of S. pneumoniae mediated by horizontal gene transfer is a primary driving force behind the success of this pathogen in its adaptation to the increasingly hostile environment in humans, the only known natural host (2). S. pneumoniae is capable of horizontal gene transfer via transduction, transformation, conjugation, and specialized transduction (or pseudotransduction) (3). Natural genetic transformation, referred to as natural transformation herein, alters the bacterial genome via uptake of foreign DNA and subsequent integration of new sequences into recipient genomes by homologous recombination (4). Although natural transformation appears to be the sole mechanism of transferring chromosomal DNA in S. pneumoniae, conjugation mobilizes the transfer of integrative and conjugative elements (ICEs) between pneumococcal cells or between S. pneumoniae and other Gram-positive bacteria (3).Natural transformation has been attributed to the acquisition of exogenous genes for immune ev...