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We determined the taxonomic status of six Bacillus species (Bacillus alginolyticus, Bacillus chondroitinus, Bacillus curdlanolyticus, Bacillus glucanolyticus, Bacillus kobensis, and Bacillus thiuminolyticus) by using the results of 16s rRNA gene sequence and cellular fatty acid composition analyses. Phylogenetic analysis clustered these species closely with the Paenibacillus species. Like the Paenibacillus species, the six Bacillus species contained anteiso-C,,,, fatty acid as a major cellular fatty acid. The use of a specific PCR primer designed for differentiating the genus Paenibacillus from other members of the BaciUaceae showed that the six Bacillus species had the same amplified 16s rRNA gene fragment as members of the genus Paenibacillus. Based on these observations and other taxonomic characteristics, the six Bacillus species were transferred to the genus Paenibacillus. In addition, we propose emendation of the genus Paenibacillus.Rod-shaped, aerobic, endospore-forming bacteria have generally been assigned to the genus Bacillus, a systematically diverse taxon (5). 16s rRNA gene sequence analyses have identified at least 10 phylogenetic groups in the genus Bacillus (2, 3, 7 , 23, 26, 31, 33, 34, 36). (12), that P. pulvifaciens was a subspecies of P. larvae (ll), and that Bacillus lautus and Bacillus peoriae should be transferred to the genus Paenibacillus (13). In addition, Nakamura proposed the new species Paenibacillus apianus (22). Consequently, the genus Paenibacillus consists of 13 species and one subspecies.Members of the genus Paenibacillus are facultatively anaerobic organisms that produce spores in definitely swollen sporangia and have G+C contents ranging from 45 to 54 mol%, and some of these organisms excrete diverse assortments of extracellular polysaccharide-hydrolyzing enzymes (5, 25). Interestingly, many recently described Bacillus species possess the general characteristics of the genus Paenibacillus. The noteworthy ability of these species to hydrolyze complex carbohydrates, including alginate (19), chondroitin (19), chitin (25), curdlan (14), and other polysaccharides (25), suggests that some of them may be related to the genus Paenibacillus. To understand the taxonomic position of these Bacillus species * Corresponding author. Mailing address: Research Laboratory, Higeta Shoyu Co., Ltd., 2-8 Chuo-cho, Choshi, Chiba 288, Japan. Phone: 81-479-22-1180. Fax: 81-479-24-3422. E-mail: LDX05744 @niftyserve.or.jp. 289among the Bacillaceae, we determined the sequences of their 16s rRNA genes and compared these sequences with homologous sequences available for other members of the Bacillaceae. In addition, a highly specific PCR amplification primer was designed on the basis of the 16s rRNA gene sequence alignments for differentiating the genus Paenibacillus from other aerobic, endospore-forming rods. MATERIALS AND METHODSBacterial strains. The bacterial strains used in this study are listed in Table 1. Working stocks were cultured on tryptic soy agar plates (Difco Laboratories, Detroit, Mich.) fo...

DNA base composition was determined by reversed‐phase high‐performance liquid chromatography (HPLC). DNA was hydrolysed into nucleosides with nuclease P1 and bacterial alkaline phosphatase. The mixture of nucleosides was applied to HPLC without any further purification. One determination by chromatography needed 2 μg of hydrolysed nucleosides and took only 8 min. The relative standard error of nucleoside analysis was less than 1%. The system described here gives a direct and precise method for determining DNA base composition.

16s rRNA gene sequences of the type strains of 11 species belonging to the Bacillus brevis and Bacillus aneurinolyticus groups were determined. On the basis of the results of gene sequence analyses, these species were separated into two clusters. The B. brevis cluster included 10 species, namely, Bacillus brevis, Bacillus agri, Bacillus centrosporus, Bacillus choshinensis, Bacillus parabrevis, Bacillus reuszeri, Bacillus formosus, Bacillus borstelensis, Bacillus luterosporus, and Bacillus thermoruber. Bacillus aneurinolyticus and Bacillus migulunus belonged to the B. aneurinolyticus cluster. Moreover, the two clusters were phylogenetically distinct from other Bacillus, Amphibacillus, Sporoluctobacillus, Paenibacillus, and Alicyclobacillus species. On the basis of our data, we propose reclassification of the B. brevis cluster as Brevibacillus gen. nov. and reclassification of the B. aneurinolyticus cluster as Aneurinibacillus gen. nov. By using 16s rRNA gene sequence alignments, two specific PCR amplification primers were designed for differentiating the two new genera from each other and from other aerobic, endospore-forming organisms.The aerobic, rod-shaped, endospore-forming genus Bacillus is a systematically diverse taxon (5). The members of this genus exhibit a wide range of DNA base compositions, and the major amino acid compositions of the cell walls of these organisms vary (6, 22, 32). Analyses of 16s rRNA gene sequences have identified at least eight phylogenetic groups in the genus Bacillus (2,3,7,20,(22)(23)(24)33,38). Two of these groups have been reclassified as new genera. One genus, the genus Alicyclobacil-.lus (38), consists of thermoaciduric species that contain rarely encountered cellular o-cyclic fatty acids. The other new genus, the genus Paenibacillus (3), was distinguished on the basis of the results of slot blot hybridization in which a specific probe was used.Recent taxonomic studies have shown that strains previously assigned to Bacillus brevis should be separated into nine species, namely, Bacillus brevis, Bacillus agri, Bacillus centrosporus, Bacillus choshinensis, Bacillus parabrevis, Bacillus reuszeri, Bacillus fonnosus, Bacillus borstelensis, and Bacillus migulanus (18,19, 28,29,34). A closely related species, Bacillus aneurinolyticus, has also been revived by Shida et al. (30). On the basis of the results of comparisons of their phenotypic characteristics, chemosystematic profiles, and conserved specific Slayer proteins, the 10 species mentioned above were separated into two groups (28-30, 34). One group, designated the Bacillus brevis group, included all of the species derived from Bacillus brevis except Bacillus miplanus. The other group, called the Bacillus aneurinolyticus group, included Bacillus aneurinolyticus and Bacillus migulanus. These groups corresponded closely to the groups generated by a numerical analysis of electrophoretic whole-cell protein patterns (31). Moreover, the results of phylogenetic studies demonstrated that Bacillus laterosporus and Bacillus thermoruber f...

Eight Gram-negative, aerobic, rod-shaped and peritrichously flagellated strains were isolated from flowers of the orchid tree (Bauhinia purpurea) and of plumbago (Plumbago auriculata), and from fermented glutinous rice, all collected in Indonesia. The enrichment culture approach for acetic acid bacteria was employed, involving use of sorbitol medium at pH 35. All isolates grew well at pH 30 and 30 SC. They did not oxidize ethanol to acetic acid except for one strain that oxidized ethanol weakly, and 035 % acetic acid inhibited their growth completely. However, they oxidized acetate and lactate to carbon dioxide and water. The isolates grew well on mannitol agar and on glutamate agar, and assimilated ammonium sulfate for growth on vitamin-free glucose medium. The isolates produced acid from D-glucose, D-fructose, L-sorbose, dulcitol and glycerol. The quinone system was Q-10. DNA base composition ranged from 593 to 610 mol % GMC. Studies of DNA relatedness showed that the isolates constitute a single species. Phylogenetic analysis based on their 16S rRNA gene sequences indicated that the isolates are located in the acetic acid bacteria lineage, but distant from the genera Acetobacter, Gluconobacter, Acidomonas and Gluconacetobacter. On the basis of the above characteristics, the name Asaia bogorensis gen. nov., sp. nov. is proposed for these isolates. The type strain is isolate 71 T (l NRIC 0311 T l JCM 10569 T ).

Interrelation of bacteria has been shown by the indices of deoxyribonucleic acid (DNA)-deoxyribonucleic acid homology (DNA-DNA homology) and ribosomal ribonucleic acid (rRNA)-deoxyribonucleic acid homology (rRNA-DNA homology), and by the similarity of the sequence of ribosomal RNA. However, such parameters are difficult to apply in the classification and routine identification of bacteria. Instead, the chemical constituents of bacterial cells, such as DNA base compositions (G+ C contents), cellular fatty acids, quinone systems, and amino acids in the peptidoglycans, have been examined for use in classification and rapid identification.Previously, IKEMOTO et al. (I) reported the cellular fatty acid composition, and YAMADA et al. (2) investigated quinone systems in the strains of the genus Pseudomonas. Following these studies, the present authors further investigated the cellular fatty acid composition and quinone system in Pseudomonas species including phytopathogenic species, and found that the genus showed heterogeneity in 3-hydroxy fatty acid composition.

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