Multiplex gene transfer by genetic transformation between isolated S. pneumoniae cells confined in microfluidic droplets.

Abstract

Gene exchange via genetic transformation makes major contributions to antibiotic resistance of the human pathogen, Streptococcus pneumoniae (pneumococcus). The transfers begin when a pneumococcal cell, in a transient specialized physiological state called competence, attacks and lyses another cell, takes up fragments of the liberated DNA, and integrates divergent genes into its genome. Recently, it has been demonstrated that the pneumococcal cells can be enclosed in femtoliter-scale droplets for study of the transformation mechanism, offering the ability to characterize individual cell-cell interactions and overcome the limitations of current methods involving bulk mixed cultures. To determine the relevance and reliability of this new method for study of bacterial genetic transformation, we compared recombination events occurring in 44 recombinants recovered after competence-mediated gene exchange between pairs of cells confined in femtoliter-scale droplets vs. those occurring in exchanges in parallel bulk culture mixtures. The pattern of recombination events in both contexts exhibited the hallmarks of the macro-recombination exchanges previously observed within the more complex natural contexts of biofilms and long-term evolution in the human host.