A GCDGC-specific DNA (cytosine-5) methyltransferase that methylates the GCWGC sequence on both strands and the GCSGC sequence on one strand.

5-Methylcytosine is one of the major epigenetic marks of DNA in living organisms. Some bacterial species possess DNA methyltransferases that modify cytosines on both strands to produce fully-methylated sites or on either strand to produce hemi-methylated sites. In this study, we characterized a DNA methyltransferase that produces two sequences with different methylation patterns: one methylated on both strands and another on one strand. M.BatI is the orphan DNA methyltransferase of Bacillus anthracis coded in one of the prophages on the chromosome. Analysis of M.BatI modified DNA by bisulfite sequencing revealed that the enzyme methylates the first cytosine in sequences of 5'-GCAGC-3', 5'-GCTGC-3', and 5'-GCGGC-3', but not of 5'-GCCGC-3'. This resulted in the production of fully-methylated 5'-GCWGC-3' and hemi-methylated 5'-GCSGC-3'. M.BatI also showed toxicity when expressed in E. coli, which was caused by a mechanism other than DNA modification activity. Homologs of M.BatI were found in other Bacillus species on different prophage like regions, suggesting the spread of the gene by several different phages. The discovery of the DNA methyltransferase with unique modification target specificity suggested unrevealed diversity of target sequences of bacterial cytosine DNA methyltransferase.

Complete Genome Sequences of 24 Strains of Bacillus cereus Isolated from Nosocomial Infection and Bacteremia Cases in Japan.

Bacillus cereus is mainly associated with foodborne illness but sometimes causes nosocomial infections. We previously reported that B. cereus strains of a specific sequence type, ST1420, were associated with nosocomial infection. Here, we determined the complete genome sequences of B. cereus strains isolated from nosocomial infection cases in Japanese hospitals.

Development of ELISA based on Bacillus anthracis capsule biosynthesis protein CapA for naturally acquired antibodies against anthrax.

Anthrax is a zoonotic disease caused by the gram-positive spore-forming bacterium Bacillus anthracis. Detecting naturally acquired antibodies against anthrax sublethal exposure in animals is essential for anthrax surveillance and effective control measures. Serological assays based on protective antigen (PA) of B. anthracis are mainly used for anthrax surveillance and vaccine evaluation. Although the assay is reliable, it is challenging to distinguish the naturally acquired antibodies from vaccine-induced immunity in animals because PA is cross-reactive to both antibodies. Although additional data on the vaccination history of animals could bypass this problem, such data are not readily accessible in many cases. In this study, we established a new enzyme-linked immunosorbent assay (ELISA) specific to antibodies against capsule biosynthesis protein CapA antigen of B. anthracis, which is non-cross-reactive to vaccine-induced antibodies in horses. Using in silico analyses, we screened coding sequences encoded on pXO2 plasmid, which is absent in the veterinary vaccine strain Sterne 34F2 but present in virulent strains of B. anthracis. Among the 8 selected antigen candidates, capsule biosynthesis protein CapA (GBAA_RS28240) and peptide ABC transporter substrate-binding protein (GBAA_RS28340) were detected by antibodies in infected horse sera. Of these, CapA has not yet been identified as immunoreactive in other studies to the best of our knowledge. Considering the protein solubility and specificity of B. anthracis, we prepared the C-terminus region of CapA, named CapA322, and developed CapA322-ELISA based on a horse model. Comparative analysis of the CapA322-ELISA and PAD1-ELISA (ELISA uses domain one of the PA) showed that CapA322-ELISA could detect anti-CapA antibodies in sera from infected horses but was non-reactive to sera from vaccinated horses. The CapA322-ELISA could contribute to the anthrax surveillance in endemic areas, and two immunoreactive proteins identified in this study could be additives to the improvement of current or future vaccine development.

Direct Regulons of AtxA, the Master Virulence Regulator of Bacillus anthracis.

AtxA, the master virulence regulator of Bacillus anthracis, regulates the expression of three toxins and genes for capsule formation that are required for the pathogenicity of B. anthracis. Recent transcriptome analyses showed that AtxA affects a large number of genes on the chromosome and plasmids, suggesting a role as a global regulator. However, information on genes directly regulated by AtxA is scarce. In this work, we conducted genome-wide analyses and cataloged the binding sites of AtxA in vivo and transcription start sites on the B. anthracis genome. By integrating these results, we detected eight genes as direct regulons of AtxA. These consisted of five protein-coding genes, including two of the three toxin genes, and three genes encoding the small RNAs XrrA and XrrB and a newly discovered 95-nucleotide small RNA, XrrC. Transcriptomes from single-knockout mutants of these small RNAs revealed changes in the transcription levels of genes related to the aerobic electron transport chain, heme biosynthesis, and amino acid metabolism, suggesting their function for the control of cell physiology. These results reveal the first layer of the gene regulatory network for the pathogenicity of B. anthracis and provide a data set for the further study of the genomics and genetics of B. anthracis. IMPORTANCE Bacillus anthracis is the Gram-positive bacterial species that causes anthrax. Anthrax is still prevalent in countries mainly in Asia and Africa, where it causes economic damage and remains a public health issue. The mechanism of pathogenicity is mainly explained by the three toxin proteins expressed from the pXO1 plasmid and by proteins involved in capsule formation expressed from the pXO2 plasmid. AtxA is a protein expressed from the pXO1 plasmid that is known to upregulate genes involved in toxin production and capsule formation and is thus considered the master virulence regulator of B. anthracis. Therefore, understanding the detailed mechanism of gene regulation is important for the control of anthrax. The significance of this work lies in the identification of genes that are directly regulated by AtxA via genome-wide analyses. The results reveal the first layer of the gene regulatory network for the pathogenicity of B. anthracis and provide useful resources for a further understanding of B. anthracis.