Markerless Genome Editing in Competent Streptococci
Selective markers employed in classical mutagenesis methods using natural genetic transformation can affect gene expression, risk phenotypic effects, and accumulate as unwanted genes during successive mutagenesis cycles. In this chapter, we present a protocol for markerless genome editing in Streptococcus mutans and Streptococcus pneumoniae achieved with an efficient method for natural transformation. High yields of transformants are obtained by combining the unimodal state of competence developed after treatment of S. mutans with sigX-inducing peptide pheromone (XIP) in a chemically defined medium (CDM) or of S. pneumoniae with the competence-stimulating peptide (CSP) together with use of a donor amplicon carrying extensive flanking homology. This combination ensures efficient and precise integration of a new allele by the recombination machinery present in competent cells.
Key wordsPheromone Competence Natural transformation Markerless mutagenesis Streptococcus mutans Streptococcus pneumoniae Streptococcus XIP CSP
This work was partially supported by the National Science Foundation, grant no. MCB-1020863, by the Faculty of Dentistry, University of Oslo, and by the Norwegian surveillance system for antibiotic resistance in microbes (Norsk overvåkingssystem for antibiotikaresistens hos mikrober—NORM). We thank Kunal Desai for assistance with exploratory experiments.