Unbiased homeologous recombination during pneumococcal transformation allows for multiple chromosomal integration events

Abstract

The rapid spread of antimicrobial resistance and vaccine escape in the opportunistic human pathogen Streptococcus pneumoniae can be largely attributed to competence-induced transformation. To better understand why competence-induced transformation is so effective, we studied the dynamics of this process at the single-cell level. We show that within isogenic populations, all cells become naturally competent and bind exogenous DNA. In addition, we find that transformation is highly efficient and that the chromosomal location of the integration site or whether the transformed gene is encoded on the leading or lagging strand has limited influence on recombination efficiency. Indeed, we have observed multiple recombination events in single recipients in real-time. However, because of saturation of the DNA uptake and integration machinery and because a single stranded donor DNA replaces the original allele, we find that transformation efficiency has an upper threshold of approximately 50% of the population. Counterintuitively, in the presence of multiple transforming DNAs, the fraction of untransformed cells increases to more than 50%. This results in a fail-safe strategy for the population as half of the population generally keeps an intact copy of the original genome. Together, this work advances our understanding of pneumococcal genome plasticity.

Direct Visualization of Horizontal Gene Transfer by Transformation in Live Pneumococcal Cells Using Microfluidics

“…We also detected 41 % of the cells expressing FtsZ-GFP only after division of the transformed cell ( Figure 5 , yellow arrowhead). Altogether, and in line with earlier reports [ 13 , 14 , 15 ], these findings indicate that either strand of the recipient chromosome can serve for transformation and that their transformability is statistically comparable.…”

“…In that case, expression of the newly acquired gene allows the accumulation of the FtsZ-GFP fusion in the transformed cell and its transmission to the progeny by cell division. Yet, as resolution of the transformation heteroduplex by replication generates one transformed and one parental chromosome, only one daughter cell has the potential to give rise to a truly transformed population, as previously observed [ 15 , 34 , 35 ]. The first transformants exhibiting the FtsZ-GFP signal were detected before division of the transformed cell (17 cells, 59% of the transformed cells, Figure 5 , white arrowhead).…”

“…We confirm that transformed cells can express newly acquired genes before or after the first cell division event, probably depending on which strand of the recipient chromosome was transformed. We also directly visualized the phenomenon of non-genetic inheritance recently demonstrated in Vibrio cholerae and S. pneumoniae [ 15 , 36 ]. This mechanism, for which genetic evidence was presented in the 60s [ 34 , 35 ], involves the transient transmission of the transformed phenotype from the transformed cell to the part of its progeny that is not genetically transformed.…”

“…The kinetics of recombinant formation indicate that integration of internalized DNA into the recipient chromosome occurs in less than 15 min [ 11 , 12 ]. Integration can occur into either strand of the recipient DNA with equal probability [ 13 , 14 , 15 ]. The resulting heteroduplex is subsequently resolved by replication and segregation, producing one daughter chromosome identical to the recipient genome and one containing the donor sequence.…”

“…These methods are powerful to analyze the average behavior of competent cells, especially given that all pneumococcal cells are able to develop competence in a coordinated manner. More recently, single cell analyses using fluorescence microscopy have been used to detect competent cells [ 16 , 17 , 18 , 19 , 20 ], to localize transformation proteins [ 12 , 15 , 21 ], to analyze the binding of transforming DNA at the surface of competent cells and to visualize transformants [ 12 , 15 ]. The development of the droplet microfluidic technology to study transformation also allowed the characterization of the genetic outcome of individual cell–cell interactions.…”