pheS* as a counter-selectable marker for marker-free genetic manipulations in Bacillus anthracis.

Several genetic tools have been developed for use in Bacillus anthracis, but there is still a need for a more marker-free gene inactivation protocols. Thus, we report a method to generate unmarked mutations in B. anthracis. This approach was based on the counter-selectable pheS* gene with assistance by the I-SceI homing endonuclease. Using this strategy, the NprR gene, a transcriptional activator of B. anthracis, was deleted at an extremely high efficiency. Our study indicates that mutated pheS is a useful counter-selective marker to design a valuable genetic tool for in-frame and unmarked gene deletions of B. anthracis.