Natural Genetic Transformation: A Direct Route to Easy Insertion of Chimeric Genes into the Pneumococcal Chromosome
The ability of Streptococcus pneumoniae (the pneumococcus) to transform is particularly convenient for genome engineering. Several protocols relying on sequential positive and negative selection strategies have been described to create directed markerless modifications, including deletions, insertions, or point mutations. Transformation with DNA fragments carrying long flanking homology sequences is also used to generate mutations without selection but it requires high transformability. Here, we present an optimized version of this method. As an example, we construct a strain harboring a translational fusion ftsZ-mTurquoise at the ftsZ locus. We provide instructions to produce a linear DNA fragment containing the chimeric construction and give details of the conditions to obtain optimal pneumococcal transformation efficiencies.
Key wordsNatural genetic transformation Gene transfer Markerless integration/mutagenesis Overlapping PCR
We warmly thank Jean-Pierre Claverys and Bernard Martin for their prominent contribution to development of pneumococcal genetics. We thank Dave Lane and Calum Johnston for critical reading of the manuscript. We also thank all past members of the Claverys lab, past and present members of the Polard lab who participated in development of the method. This work was funded by the Centre National de la Recherche Scientifique, Université Paul Sabatier and Agence Nationale de la Recherche (Grant ANR-13-BSV8-0022 and ANR-17-CE13-0031).