Genome engineering in Bacillus anthracis using tyrosine site-specific recombinases.

Tyrosine site-specific recombinases (T-SSR) are polynucleotidyltransferases that catalyze cutting and joining reactions between short specific DNA sequences. We developed three systems for performing genetic modifications in Bacillus anthracis that use T-SSR and their cognate target sequences, namely Escherichia coli bacteriophage P1 Cre-loxP, Saccharomyces cerevisiae Flp-FRT, and a newly discovered IntXO-PSL system from B. anthracis plasmid pXO1. All three tyrosine recombinase systems were used for creation of a B. anthracis sporulation-deficient, plasmid-free strain deleted for ten proteases which had been identified by proteomic analysis as being present in the B. anthracis secretome. This strain was used successfully for production of various recombinant proteins, including several that are candidates for inclusion in improved anthrax vaccines. These genetic tools developed for DNA manipulation in B. anthracis were also used for construction of strains having chromosomal insertions of 1, 2, or 3 adjacent atxA genes. AtxA is a B. anthracis global transcriptional regulator required for the response of B. anthracis virulence factor genes to bicarbonate. We found a positive correlation between the atxA copy number and the expression level of the pagA gene encoding B. anthracis protective antigen, when strains were grown in a carbon dioxide atmosphere. These results demonstrate that the three T-SSR systems described here provide effective tools for B. anthracis genome editing. These T-SSR systems may also be applicable to other prokaryotes and to eukaryotes.