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Sequential markerless genetic manipulations of species from the Neisseria genus.

The development of simple and highly efficient strategies for genetic modifications are essential for post-genetic 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 counter selection gene rpsL. These cassettes can be transformed with high efficiency across the Neisseria genus while significantly reducing the number of false positives compared to similar approaches. We exemplify 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.