Sequential markerless genetic manipulations of species from the Neisseria genus

The development of simple and highly efficient strategies for genetic modifications is essential for postgenetic studies aimed at characterizing gene functions for various applications. We sought to develop a reliable system for Neisseria species that allows for both unmarked and accumulation of multiple genetic modifications in a single strain. In this work, we developed and validated three-gene cassettes named RPLK and RPCC, comprising of an antibiotic resistance marker for positive selection, the phenotypic selection marker lacZ or mCherry, and the counterselection gene rpsL. These cassettes can be transformed with high efficiency across the Neisseria genus while significantly reducing the number of false positives compared with similar approaches. We exemplified the versatility and application of these systems by obtaining unmarked luminescent strains (knock-in) or mutants (knock-out) in different pathogenic and commensal species across the Neisseria genus in addition to the cumulative deletion of six loci in a single strain of Neisseria elongata.