The generation of genetic variation (somatic hypermutation) is an essential process for the adaptive immune system in vertebrates. We demonstrate the targeted single-nucleotide substitution of DNA using hybrid vertebrate and bacterial immune systems components. Nuclease-deficient type II CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated) and the activation-induced cytidine deaminase (AID) ortholog PmCDA1 were engineered to form a synthetic complex (Target-AID) that performs highly efficient target-specific mutagenesis. Specific point mutation was induced primarily at cytidines within the target range of five bases. The toxicity associated with the nuclease-based CRISPR/Cas9 system was greatly reduced. Although combination of nickase Cas9(D10A) and the deaminase was highly effective in yeasts, it also induced insertion and deletion (indel) in mammalian cells. Use of uracil DNA glycosylase inhibitor suppressed the indel formation and improved the efficiency.

In eukaryotes, the CRISPR-Cas9 system has now been widely used as a revolutionary genome engineering tool. However, in prokaryotes, the use of nuclease-mediated genome editing tools has been limited to negative selection for the already modified cells because of its lethality. Here, we report on deaminase-mediated targeted nucleotide editing (Target-AID) adopted in Escherichia coli. Cytidine deaminase PmCDA1 fused to the nuclease-deficient CRISPR-Cas9 system achieved specific point mutagenesis at the target sites in E. coli by introducing cytosine mutations without compromising cell growth. The cytosine-to-thymine substitutions were induced mainly within an approximately five-base window of target sequences on the protospacer adjacent motif-distal side, which can be shifted depending on the length of the single guide RNA sequence. Use of a uracil DNA glycosylase inhibitor in combination with a degradation tag (LVA tag) resulted in a robustly high mutation efficiency, which allowed simultaneous multiplex editing of six different genes. The major multi-copy transposase genes that consist of at least 41 loci were also simultaneously edited by using four target sequences. As this system does not rely on any additional or host-dependent factors, it may be readily applicable to a wide range of bacteria.

The KOTO (K 0 at Tokai) experiment aims to observe the CP-violating rare decay K L → π 0 ν ν by using a long-lived neutral-kaon beam produced by the 30 GeV proton beam at the Japan Proton Accelerator Research Complex. The K L flux is an essential parameter for the measurement of the branching fraction. Three K L neutral decay modes, K L → 3π 0 , K L → 2π 0 , and K L → 2γ were used to measure the K L flux in the beam line in the 2013 KOTO engineering run. A Monte Carlo simulation was used to estimate the detector acceptance for these decays. Agreement was found between the simulation model and the experimental data, and the remaining systematic uncertainty was estimated at the 1.4% level. The K L flux was measured as (4.183 ± 0.017 stat. ± 0.059 sys. ) × 10 7 K L per 2 × 10 14 protons on a 66-mm-long Au target.

In the chemotaxis of Escherichia coli, polar clustering of the chemoreceptors, the histidine kinase CheA, and the adaptor protein CheW is thought to be involved in signal amplification and adaptation. However, the mechanism that leads to the polar localization of the receptor is still largely unknown. In this study, we examined the effect of receptor covalent modification on the polar localization of the aspartate chemoreceptor Tar fused to green fluorescent protein (GFP). Amidation (and presumably methylation) of Tar-GFP enhanced its own polar localization, although the effect was small. The slight but significant effect of amidation on receptor localization was reinforced by the fact that localization of a noncatalytic mutant version of GFP-CheR that targets to the C-terminal pentapeptide sequence of Tar was similarly facilitated by receptor amidation. Polar localization of the demethylated version of Tar-GFP was also enhanced by increasing levels of the serine chemoreceptor Tsr. The effect of covalent modification on receptor localization by itself may be too small to account for chemotactic adaptation, but receptor modification is suggested to contribute to the molecular assembly of the chemoreceptor/histidine kinase array at a cell pole, presumably by stabilizing the receptor dimer-to-dimer interaction.Spatial regulation of the subcellular localization of proteins is important for various cellular events in both prokaryotic and eukaryotic cells. In prokaryotes, for example, polar protein localization has been implicated in cell division, virulence, and chemotaxis (24, 31). In the chemotaxis of Escherichia coli, a set of transmembrane receptors named chemoreceptors or methyl-accepting chemotaxis proteins (MCPs), together with the histidine kinase CheA and the adaptor CheW, cluster at a cell pole (26,34). This polar clustering of the chemotactic machinery is thought to be required for normal signal amplification and adaptation (1,4,8,32,36).E. coli has four chemoreceptors (Tsr for serine, Tar for aspartate and maltose, Tap for dipeptides, and Trg for ribose and galactose) and one MCP-related protein involved in redox taxis (Aer). A chemoreceptor forms a homodimer regardless of its ligand occupancy state (27). Other chemotaxis signaling proteins (i.e., CheY, which controls the rotational sense of the flagellar motor, and CheZ, which facilitates dephosphorylation of CheY, the methyltransferase CheR, and the methylesterase CheB) also target to the receptor-kinase cluster (2, 5, 33, 34). However, despite growing knowledge of the three-dimensional structures of and interactions between the signaling components, the mechanism that leads to the polar localization of the receptor is still largely unknown.Receptor methylation, a key process of adaptation, might be a good candidate for a factor affecting the localization of chemoreceptors. First, the formation of an in vitro complex consisting of a cytoplasmic fragment of Tar, CheA, and CheW is facilitated by receptor amidation, which is equivalent to methylation (18, ...

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As an issue of biosecurity, species-specific genetic markers have been well characterized. However, Bacillus anthracis strain-specific information is currently not sufficient for traceability to identify the origin of the strain. By using genome-wide screening using short read mapping, we identified strainspecific single nucleotide polymorphisms (SNPs) among B. anthracis strains including Japanese isolates, and we further developed a simplified 80-tag SNP typing method for the primary investigation of traceability. These 80-tag SNPs were selected from 2,965 SNPs on the chromosome and the pXO1 and pXO2 plasmids from a total of 19 B. anthracis strains, including the available genome sequences of 17 strains in the GenBank database and 2 Japanese isolates that were sequenced in this study. Phylogenetic analysis based on 80-tag SNP typing showed a higher resolution power to discriminate 12 Japanese isolates rather than the 25 loci identified by multiple-locus variable-number tandem-repeat analysis (MLVA). In addition, the 80-tag PCR testing enabled the discrimination of B. anthracis from other B. cereus group species, helping to identify whether a suspected sample originates from the intentional release of a bioterrorism agent or environmental contamination with a virulent agent. In conclusion, 80-tag SNP typing can be a rapid and sufficient test for the primary investigation of strain origin. Subsequent whole-genome sequencing will reveal apparent strain-specific genetic markers for traceability of strains following an anthrax outbreak.Many potential bioterrorism agents, including anthrax, present as pulmonary disease. Anthrax is caused by the spore-forming bacterium Bacillus anthracis, which is among the most severe zoonoses posing a serious threat to both public and animal health (7,14). B. anthracis belongs to the Bacillus cereus group of bacteria, which is composed of closely related Gram-positive organisms with highly divergent virulent properties (14,18). Infection with this bacterium can occur through the skin, gastrointestinal tract, or respiratory apparatus following contact, ingestion, or inhalation of spores, respectively (7, 14).As an issue of biosecurity, a comprehensive molecular diagnosis system is considered for detecting potential infectious agents. For most potential bioterrorism agents, species-specific genetic markers have been well characterized (9), but strain-specific information is not sufficient for traceability to identify the origin of the strain.A liquid suspension of B. anthracis was dispersed by the Aum Shinrikyo religious cult in Japan in 1993. The genotype of the B. anthracis isolate released was identical to that of the Sterne 34F2 strain, which is a member of the A3b diversity cluster (10). Fortunately, there were no victims of this attack because the strain was pXO2 plasmid defective and a low-virulent derivative used commercially in Japan to vaccinate animals against anthrax. The recent "postal anthrax attacks" in the United States aimed at the intentional release of B. anthracis ...

Fluoroquinolone (FQ) resistance of