We have generated mutator strains of Bacillus anthracis Sterne by using directed gene knockouts to investigate the effect of deleting genes involved in mismatch repair, oxidative repair, and maintaining triphosphate pools. The single-knockout strains are deleted for mutS, mutY, mutM, or ndk. We also made double-knockout strains that are mutS ndk or mutY mutM. We have measured the levels of mutations in the rpoB gene that lead to the Rif r phenotype and have examined the mutational specificity. In addition, we examined the mutational specificity of two mutagens, 5-azacytidine and N-methyl-N-nitro-N-nitroso-guanidine. The mutY and mutM single knockouts are weak mutators by themselves, but the combination of mutY mutM results in very high mutation rates, all due to G:C 3 T:A transversions. The situation parallels that seen in Escherichia coli. Also, mutS knockouts are strong mutators and even stronger in the presence of a deletion of ndk. The number of sites in rpoB that can result in the Rif r phenotype by single-base substitution is more limited than in certain other bacteria, such as E. coli and Deinococcus radiodurans, although the average mutation rate per mutational site is roughly comparable. Hotspots at sites with virtually identical surrounding sequences are organism specific.Mutational pathways, mutagenesis, and DNA repair systems are an integral part of biology. On the one hand, unchecked mutagenesis can lead to aberrant cellular physiology, cell death, and myriad human diseases, including cancer (for a review, see reference 8). On the other hand, mutational pathways can be a driving force in adaptive evolution, can create sufficient antigenic variation to allow pathogens to overcome host immune responses, and are instrumental in generating antibody diversity (for a review, see reference 19). Surprisingly, mutagenesis and repair have been studied in detail in only a very small number of organisms, such as Escherichia coli and Salmonella, yeast, and humans. As the field of genomics opens up possibilities for examining mutational pathways and repair strategies in a wide range of microorganisms, it is important to study these processes in microorganisms of interest. We will no doubt uncover many new mechanisms and will also learn more about the organism in question. In this study we focus on Bacillus anthracis, a bacterium of great interest for biodefense.Bacillus anthracis is a gram-positive spore-forming bacillus that can infect humans, causing anthrax (for a review, see reference 15). Two plasmids are responsible for the virulence of B. anthracis. These are pXO1, which encodes the toxin protein and the lethal and edema factors, among other proteins (11, 24), and pXO2, encoding the capsule biosynthesis and depolymerization proteins, as well as additional factors (4, 10, 15, 30). The durable endospore and the lethality of inhalation anthrax make B. anthracis a powerful bioweapon. The complete 5.23-Mb sequence of the B. anthracis Ames genome has been determined, and it encodes 5,508 predicted proteincoding ...
