The size and position of heterologous insertions in a silent locus differentially affect pilin recombination in Neisseria gonorrhoeae

Gonococcal pilus antigenic and phase variation result from unidirectional, RecA-dependent recombination of DNA sequences from a silent pilin copy (pilS) into the expressed pilin gene (pilE). To develop a quantitative assay for pilin gene recombination that is independent of phase variation, a promoterless cat gene was inserted into pilS, and recombination of 'cat into pilE was detected by selection of chloramphenicol-resistant (Cm-R) variants expressing 'cat from the pilin promoter, Although RecA-dependent Cm-R variants occurred, none were generated by the simple transfer of 'cat into pilE, Instead, each Cm-R variant contained a new pilin locus that was a hybrid of sequences from the pilE and the pilS1::'cat loci in addition to the two starting loci, Therefore, this system could not be used to quantify antigenic variation, However, combined studies of these hybrid loci and of recombination products generated during additional pilS mutational analyses demonstrated that both the size and position of an insertion in pilS differentially affect pilin recombination. Also, the hybrid loci appear to be intermediates of antigenic variation. This enabled the creation of molecular models for the recombination reactions that result in pilin antigenic variation.