By using genetic complementation tests with various in vitro-constructed mutants with mutations in the cap region (which is essential for encapsulation in Bacillus anthracis), we identified three cistrons, capB, capC, and capA, in this order of arrangement. Minicell analysis revealed that these cistrons produce proteins of 44, 16, and 46 kilodaltons, respectively. The complete nucleotide sequence of 3,244 base pairs covering the whole cap region was determined and revealed the existence of the three open reading frames of capB (397 amino acid residues; molecular weight, 44,872), capC (149 amino acid residues; molecular weight, 16,522), and capA (411 amino acid residues; molecular weight, 46,420) arranged in the order predicted by complementation tests. These three cistrons were all transcribed in the same direction from promoters unique to each cistron. Judging from the predicted amino acid sequence of the three proteins and from their localization and their sensitivity to various physicochemical treatments, they appeared to be membrane-associated enzymes mediating the polymerization of D-glutamic acid via the membrane. Capsular peptides immunologically identical to that of B. anthracis were found in B. subtilis, B. megaterium, and B. licheniformis, but no sequence homologous to the cap region was found in any of these bacilli other than B. anthracis. Using strains of B. anthracis with or without insertional inactivation of the cap region, we found that the capsule of B. anthracis conferred strong resistance to phagocytosis upon the bacterial host.

In genetic analysis of bovine Staphylococcus aureus isolates that are recognized as an important pathogenic bacterium in bovine mastitis, multilocus sequence typing (MLST) showed strong correlation to the results of pulsed-field gel electrophoresis, coa PCR-restriction fragment length polymorphism (RFLP), spa typing, and the coagulase serotyping method. According to MLST results, strains derived from sequence type 97 (ST97) and ST705 were suggested as not only dominant bovine S. aureus lineages in Japan but also pandemic bovine S. aureus lineages. Although both lineages seem to be distantly related to each other by phylogenetic analysis, both had common characteristics, i.e., lukM/lukF-PV and coagulase serotype VI. These characteristics were very rare among minor bovine strains and human strains and may contribute to the host specificity of these lineages. Four methicillin-resistant S. aureus (MRSA) isolates were first confirmed from bovine milk in Japan; these isolates showed geno-and serotypes that were identical or similar to those of human MRSA isolates in Japan (ST5, staphylococcal cassette chromosome mec type II [SCCmec II], Spa type t002 or t375, and coagulase serotype II, and ST89, SCCmec IIIa, Spa type t5266, and coagulase serotype I). ST5 and ST89 are uncommon among bovine isolates in the world, whereas these STs are common among human MRSA isolates in Japan.

The 184-kb Bacilus anthracis plasmid pXO0, which is required for virulence, contains three genes encoding the protein components of anthrax toxin, cya (edema factor gene), kef (lethal factor gene), and pag (protective antigen gene). Expression of the three proteins is induced by bicarbonate or serum. Using a pagklacZ transcriptional construct to measure pag promoter activity, we cloned in BaciUus subtilis a gene (alxA) whose product acts in trans to stimulate anthrax toxin expression. Deletion analysis located aIxA on a 2.0-kb fragment between cya and pag. DNA sequencing identified one open reading frame encoding 476 amino acids with a predicted Mr of 55,673, in good agreement with the value of 53 kDa obtained by in vitro transcriptiontranslation analysis. The cloned airA gene complemented previously characterized Tn917 insertion mutants UM23 tp29 and UM23 tp32 (J. M. Hornung and C. B. Thorne, Abstr. 91st Gen. Meet. Am. Soc. Microbiol. 1991, abstr. D-121, p. 98), which are deficient in synthesis of all three toxin proteins. These results demonstrate that the atxA product activates not only transcription of pag but also that of cya and lef. 3-Galactosidase synthesis from the pag-lacZ transcriptional fusion construct introduced into an insertion mutant (UM23 tp62) which does not require bicarbonate for toxin synthesis indicated that additional regulatory genes other than atxA play a role in the induction of anthrax toxin gene expression by bicarbonate.

Virulent typical strains (Shikan, Morioka, Shizuoka) and Pasteur vaccine strains (no. 1, no. 2-H, no. 2-17JB) of Bacillus anthracis harboured two plasmid species with molecular masses of 110 MDal and 60 MDal. All of the 110 MDal plasmids isolated from the various strains showed indistinguishable patterns of digestion with restriction endonucleases. All the 60 MDal plasmids were also indistinguishable. Strain Davis, which is encapsulated but is asporogenous and avirulent, harboured only the 60 MDal plasmid while three non-encapsulated vaccine strains (34F2, Smith, Mukteswer) harboured only the 110 MDal plasmid. Four non-encapsulated variant strains obtained from the encapsulated strains Shikan, Pasteur no. 1, Pasteur no. 2-17JB and Davis had lost the 60 MDal plasmid, suggesting that encapsulation of B. anthracis may be associated with the 60 MDal plasmid.

SummaryThe two major virulence factors of Bacillus anfhracis are the tripartite toxin and the polyglutamate capsule, which are encoded by genes on the large plasmids, pXOl and pX02, respectively. The genes atxA, located on pXO1, and acpA, located on pX02, encode positive trans-acting proteins that are involved in bicarbonatemediated regulation of toxin and capsule production, respectively. A derivative strain cured of pXOl produced less capsular substance than the parent strain harbouring both pXOl and pX02, and electroporation of the strain cured of pXOl with a plasmid containing the cloned afxA gene resulted in an increased level of capsule production. An acpA-null mutant was complemented by not only acpA but also the afxA gene. The cap region, which is essential for encapsulation, contains three genes capB, cape, and capA, arranged in that order. The afxA gene stimulated capsule synthesis from the cloned cap region. Transcriptional analysis of cap by RNA slot-blot hybridization and primer-extension analysis revealed that afxA activated expression of cap in trans at the transcriptional level. These results indicate that cross-talk occurs, in which the pXOl -located gene, atxA, activates transcription of the cap region genes located on pX02. We identified two major apparent transcriptional start sites, designated P1 and P2, located at positions 731 bp and 625 bp, respectively, upstream of the translationinitiation codon of caps. Transcription initiated from P1 and P2 was activated by both atxA and acpA, and activation appeared to be stimulated by bicarbonate. Deletion analysis of the upstream region of the cap