Genome editing is an effective tool for the functional examination of bacterial genes and for live attenuated vaccine construction. Here, we report a method to edit the genomic DNA of Bacillus anthracis and Bacillus cereus using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)9 system. Using two prophages in B. anthracis as targets, large-fragment deletion mutants were achieved with rates of 100 or 20%. In B. cereus, we successfully introduced precise point mutations into plcR, with phenotypic assays showing that the resulting mutants lost hemolytic and phospholipase enzyme activities similar to B. anthracis, which is a natural plcR mutant. Our study indicates that CRISPR/Cas9 is a powerful genetic tool for genome editing in the Bacillus cereus group, and can efficiently modify target genes without the need for residual foreign DNA such as antibiotic selection markers. This system could be developed for use in the generation of marker-free live anthrax vaccines or for safer construction of microbiological candidate-based recombinant B. cereus.

Plasmid-cured derivative strains of Bacillus anthracis are frequently used in laboratory studies. Plasmid incompatibility, which does not increase the risk of chromosomal mutation, is a useful method for plasmid curing. However, in bacteria containing multiple plasmids, it often requires the sequential introduction of multiple, specific incompatibility plasmids. This lengthy process renders the traditional plasmid incompatibility method inefficient and mutation-prone. In this study, we successfully cured plasmids pXO1 and pXO2 from B. anthracis A16 simultaneously using only one recombinant incompatible plasmid, pKORT, to obtain a plasmid-free strain, designated A16DD. This method may also be useful for the simultaneous, one-step curing of multiple plasmids from other bacteria, including Bacillus thuringiensis and Yersinia pestis.

Three worldwide historical plague pandemics resulted in millions of deaths. Yersinia pestis, the etiologic agent of plague, is also a potential bioterrorist weapon. Simple, rapid, and specific detection of Y. pestis is important to prevent and control plague. However, the high similarity between Y. pestis and its sister species within the same genus makes detection work problematic. Here, the genome sequence from the Y. pestis CO92 strain was electronically separated into millions of fragments. These fragments were analyzed and compared with the genome sequences of 539 Y. pestis strains and 572 strains of 20 species within the Yersinia genus. Altogether, 97 Y. pestis-specific tags containing two or more single nucleotide polymorphism sites were screened out. These 97 tags efficiently distinguished Y. pestis from all other closely related species. We chose four of these tags to design a Cas12a-based detection system. PCR–fluorescence methodology was used to test the specificity of these tags, and the results showed that the fluorescence intensity produced by Y. pestis was significantly higher than that of non-Y. pestis (p < 0.0001). We then employed recombinase polymerase amplification and lateral flow dipsticks to visualize the results. Our newly developed plasmid-independent, species-specific library of tags completely and effectively screened chromosomal sequences. The detection limit of our four-tag Cas12a system reached picogram levels.

The Bacillus anthracis spore constitutes the infectious form of the bacterium, and sporulation is an important process in the organism’s life cycle. Herein, we show that disruption of SpoVG resulted in defective B. anthracis sporulation. Confocal microscopy demonstrated that a ΔspoVG mutant could not form an asymmetric septum, the first morphological change observed during sporulation. Moreover, levels of spoIIE mRNA were reduced in the spoVG mutant, as demonstrated using β-galactosidase activity assays. The effects on sporulation of the ΔspoVG mutation differed in B. anthracis from those in B. subtilis because of the redundant functions of SpoVG and SpoIIB in B. subtilis. SpoVG is highly conserved between B. anthracis and B. subtilis. Conversely, BA4688 (the protein tentatively assigned as SpoIIB in B. anthracis) and B. subtilis SpoIIB (SpoIIB_Bs) share only 27.9% sequence identity. On complementation of the B. anthracis ΔspoVG strain with spoIIB_Bs, the resulting strain pBspoIIB_Bs/ΔspoVG could not form resistant spores, but partially completed the prespore engulfment stage. In agreement with this finding, mRNA levels of the prespore engulfment gene spoIIM were significantly increased in strain pBspoIIB_Bs/ΔspoVG compared with the ΔspoVG strain. Transcription of the coat development gene cotE was similar in the pBspoIIB_Bs/ΔspoVG and ΔspoVG strains. Thus, unlike in B. subtilis, SpoVG appears to be required for sporulation in B. anthracis, which provides further insight into the sporulation mechanisms of this pathogen.

To investigate gene function in Bacillus anthracis, a high-efficiency cloning system is required with an increased rate of allelic exchange. Golden Gate cloning is a molecular cloning strategy allowing researchers to simultaneously and directionally assemble multiple DNA fragments to construct target plasmids using type IIs restriction enzymes and T4 DNA ligase in the same reaction system. Here, a B. anthracis S-layer protein EA1 allelic exchange vector was successfully constructed using the Golden Gate method. No new restriction sites were introduced into this knockout vector, and seamless assembly of the DNA fragments was achieved. To elevate the efficiency of homologous recombination between the allelic exchange vector and chromosomal DNA, we introduced an I-SceI site into the allelic exchange vector. The eag gene was successfully knocked out in B. anthracis using this vector. Simultaneously, the allelic exchange vector construction method was developed into a system for generating B. anthracis allelic exchange vectors. To verify the effectiveness of this system, some other allelic exchange vectors were constructed and gene replacements were performed in B. anthracis. It is speculated that this gene knockout vector construction system and high-efficiency targeted gene replacement using I-SceI endonuclease can be applied to other Bacillus spp.