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Thirty-five Bacillus brevis strains obtained from culture collections, including protein-producing isolates, were taxonomically studied by using numerical analysis, DNA base composition, and DNA-DNA hybridization. Six DNA relatedness groups were represented, and these groups correlated well with clusters based on the numerical analysis. The B. brevis strains were separated into B. brevis sensu stricto, four new species, and an unidentified species of the genus Bacillus. Bacillus migulanus sp. nov., Bacillus choshinensis sp. nov., Bacillus parabrevis sp. nov., and Bacillus galactophilus sp. nov, are proposed.The heterogeneity of Bacillus brevis has been indicated by the wide range of maximum growth temperatures (10) and the results of numerical classification (13,26,27) and classification based on API test data (17). The protein producers identified as B. brevis (45) had guanine-plus-cytosine (G+C) contents ranging from 47 to 55 mol%. These observations suggest that this taxon is genetically heterogeneous. Recently, Nakamura (23) reported that the phenotypic heterogeneity of B. brevis was due to variability introduced by the presence of genetically unrelated strains. Previously, Udaka (44) and Takagi et al. (37) reported the bacterial production of large amounts of proteins in culture fluids; many of the bacteria involved appeared phenotypically to be B. brevis (37,44). These strains showed either weak or no detectable proteolytic activity in the culture supernatant (37). B. brevis 47 and HPD31 have been employed successfully as host bacteria for extracellular production of foreign proteins, such as bacterial thermophilic a-amylase (38) and human epidermal growth factor (49). Additional unique characteristics of B. brevis that have been reported are gramicidin production (9) and molluscicidal activity (31).In order to clarify the taxonomic positions of 14 B. brevis strains from culture collections and of 21 protein-producing isolates, we examined their phenotypic characteristics and processed the data obtained by using standard numerical analysis. In addition, the relationships between clusters resulting from the numerical analysis were evaluated by DNA-DNA hybridization.As a result, the above-mentioned 35 strains assigned to B. brevis were divided into six independent species, four of which are proposed below as the new species Bacillus migulanus, Bacillus choshinensis, Bacillus parabrevis, and Bacillus gala c toph ilus. MATERIALS AND METHODSBacterial strains. B. brevis strains and reference strains obtained from culture collections and other sources and the protein-producing strains described previously (37, 44) were * Corresponding author. used in this study. The sources and histories of these strains are listed in Table 1. Working stocks were cultivated on T2 agar (44) for 24 h at 30 or 37°C and were stored at a room temperature.Characterization, Unless indicated otherwise, the methods described by Gordon et al. (10) were used for characterization of organisms. Acid production from carbohydrates was determin...

Bacillus cereus secretes phospholipases C , which hydrolyze phosphatidylcholine, sphingomyelin and phosphatidylinositol. A 7.5-kb HindIII fragment of B. cereus DNA cloned into Escherichia coli, with pUC18 as a vector, directed the synthesis of the sphingomyelin-hydrolyzing phospholipase C, sphingomyelinase. Nucleotide sequence analysis of the subfragment revealed that it contained two open reading frames in tandem. The upstream truncated open reading frame corresponds to the carboxy-terminal portion of the phosphatidylcholine-hydrolyzing phospholipase C, and the downstream open reading frame to the entire translational portion of the sphingomyelinase. The two phospholipase C genes form a gene cluster.As inferred from the DNA sequence, the B. cereus sphingomyelinase has a signal peptide of 27 amino acid residues and the mature enzyme comprises 306 amino acid residues, with a molecular mass of 34233 Da. The signal peptide of the enzyme was found to be functional in protein transport across the membrane of E. coli.The enzymatic properties of the sphingomyelinase synthesized in E. coli resemble those of the donor strain sphingomyelinase. The enzymatic activity toward sphingomyelin was enhanced 20 -30-fold in the presence of MgCI2, and the adsorption of the enzyme onto erythrocyte membranes was accelerated in the presence of CaCI2.Phospholipases hydrolyze several phospholipids present in biomembranes, liposomes and micelles, and play important roles in lipid metabolism. Among them only phospholipases A2 from various snake venoms and mammalian pancreas have been extensively characterized as to their primary, secondary and tertiary structures [l -51. The gene for phospholipase B of Escherichia coli (so-called 'detergent-resistant phospholipase A', which splits two fatty acids residues from phosphatides in a random order) was cloned and sequenced [6]. Phospholipases C, which cleave the bond between the hydrophobic moiety and the polar head of phospholipids, have been demonstrated in microorganisms as well as mammalian tissues, and studied for their enzymatic properties [7 -121. Phospholipases C specific for various phospholipids have been isolated from several microorganisms, such as Pseudomonas aeruginosa [13], Staphylococcus aureus [I41 and Bacillus cereus [15]. Bacillus cereus produces extracellularly three enzymes : phospholipases C, which hydrolyze phosphatidylcholine, sphingomyelin and phosphatidylinositol. Sphingomyelinase, the sphingomyelin-hydrolyzing phospholipase C produced in B. cereus, has been shown to be different from that produced in Staphylococcus aureus in several respects, such as amino acid composition and isoelectric point.Correspondence to N. Tsukagoshi,