Microfluidic Device for Studies of Bacterial Genetic Transformation in Streptococcus pneumoniae

Streptococcus pneumoniae (pneumococcus) is a Gram-positive bacterium that causes a million deaths of children, elderly, and immunocompromised annually due to pneumonia, septicemia, and meningitis. Two clinical strategies used to combat such infections are antibiotics to cure patients and vaccines to reduce the deadliest types of bacteria. However, the effectiveness of both strategies has recently been restrained due to the spread of antibiotic-resistant strains. The fundamental phenomenon behind the failure of both strategies is the bacterial genetic transformation by a pathway for the uptake of exogenous DNA from the bacterial surroundings and the incorporation of the acquired DNA into the genome. The study of pneumococcal transformation is currently challenged both biologically and methodologically: 1) while many steps of genetic transformation are known, the cell-cell attack and DNA uptake have not yet been directly observed in living cells, making the mechanism enigmatic, and 2) the conventional strategies to study genetic transformation, which usually employ bulk cultures, fail to identify specific donor cells or to capture all descendants from a single productive interaction. We hypothesize that the DNA uptake or cell attack by competent pneumococci is initiated by an external 1-2 μm long, 5-nm-diameter filament, called the com pilus. To obtain a more dynamic view of the pneumococcal competence pilus, we genetically introduced cysteine codons into major pilin ComGC with the objective that solvent-accessible cysteines could be labeled with fluorescently conjugated maleimide dyes. We demonstrated that this labeling strategy revealed that pneumococcal competence pili are highly dynamic, undergoing cycles of extension and retraction over timescales of seconds, and its retraction is required for DNA uptake. Then, to overcome the limitations of conventional bulk methods, we developed a droplet microfluidic system capable of isolating individual episodes of bacterial transformation between two pneumococci. To determine the relevance and reliability of this new method for the study of bacterial genetic transformation, we compared recombination events of recombinants recovered from the droplets vs. those in parallel bulk culture mixtures by whole genome sequencing. This study demonstrates that droplet microfluidics can provide a new method to study bacterial genetic transformation at the level of individual cells.