Rapid targeted gene disruption in Bacillus anthracis
1 UES Inc, 4401 Dayton-Xenia Road, Dayton, OH 45432, USA
2 Henry Jackson Foundation, 6720-A Rockledge Drive, Suite 100, Bethesda, MD 20817, USA
3 Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712-0159, USA
4 Air Force Research Laboratory, Air Force Research Laboratory, 711th HPW/RHXBC, Molecular Signatures Section, 2510 Fifth Street, Area B, Bldg 840, Room W220, Wright-Patterson AFB, OH 45433-7913, USA
5 Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0524, USA
BMC Biotechnology 2013, 13:72 doi:10.1186/1472-6750-13-72Published: 18 September 2013
Anthrax is a zoonotic disease recognized to affect herbivores since Biblical times and has the widest range of susceptible host species of any known pathogen. The ease with which the bacterium can be weaponized and its recent deliberate use as an agent of terror, have highlighted the importance of gaining a deeper understanding and effective countermeasures for this important pathogen. High quality sequence data has opened the possibility of systematic dissection of how genes distributed on both the bacterial chromosome and associated plasmids have made it such a successful pathogen. However, low transformation efficiency and relatively few genetic tools for chromosomal manipulation have hampered full interrogation of its genome.
Group II introns have been developed into an efficient tool for site-specific gene inactivation in several organisms. We have adapted group II intron targeting technology for application in Bacillus anthracis and generated vectors that permit gene inactivation through group II intron insertion. The vectors developed permit screening for the desired insertion through PCR or direct selection of intron insertions using a selection scheme that activates a kanamycin resistance marker upon successful intron insertion.
The design and vector construction described here provides a useful tool for high throughput experimental interrogation of the Bacillus anthracis genome and will benefit efforts to develop improved vaccines and therapeutics.