Lawrence K. Nakamura

Transfer of Bacillus alginolyticus, Bacillus chondroitinus, Bacillus curdlanolyticus, Bacillus glucanolyticus, Bacillus kobensis, and Bacillus thiaminolyticus to the genus Paenibacillus and emended description of the genus Paenibacillus

We determined the taxonomic status of six Bacillus species (Bacillus alginolyticus, Bacillus chondroitinus, Bacillus curdlanolyticus, Bacillus glucanolyticus, Bacillus kobensis, and Bacillus thiaminolyticus) 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-C15:0 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 Bacillaceae 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.

Relationship of Bacillus subtilis clades associated with strains 168 and W23: a proposal for Bacillus subtilis subsp. subtilis subsp. nov. and Bacillus subtilis subsp. spizizenii subsp. nov.

Earlier phylogenetic studies based on the inferred DNA sequences of the polC, rpoB and gyrA genes suggested that strains of the species Bacillus subtilis formed two clusters, indicating the presence two closely related taxa; one contained the laboratory strain 168 and the other the laboratory strain W23. Significant sexual isolation was found between strain 168 and members of the group containing W23, but no sexual isolation was observed between strain 168 and other members of the 168 group. DNA reassociation between the two groups ranged from 58 to 69% and intragroup DNA relatedness ranged from 82 to 100%. Because group 168 strains were highly related to the B. subtilis type strain, they were considered to be bona fide members of the species. About 99.5% sequence identity was observed between the 16S rRNA genes of the 168 and W23 groups. Ribitol and anhydroribitol were principal cell wall constituents of the W23 but not of the 168 group. These observations revealed two closely related but genetically and phenotypically distinct groups within B. subtilis that correspond to two historically important strains. Subspecies distinction is proposed for the 168 and W23 groups, with the names Bacillus subtilis subsp. subtilis subsp. nov. and Bacillus subtilis subsp. spizizenii subsp. nov., respectively. The type strain of the former is NRRL NRS-744T and the latter NRRL B-23049T.

Bacillus mojavensis sp. nov., distinguishable from Bacillus subtilis by sexual isolation, divergence in DNA sequence, and differences in fatty acid composition.

A number of Bacillus strains isolated from desert soil samples were shown to belong to a previously unidentified species, for which we propose the name Bacillus mojavensis. The type strain is RO-H-1 (= NRRL B-14698). On the basis of restriction digest data, B. mojavensis is most closely related to Bacillus amyloliquefaciens, Bacillus atrophaeus, and Bacillus subtilis. So far, B. mojavensis can be distinguished from B. subtilis only by differences in whole-cell fatty acid composition, divergence in DNA sequence, and resistance to genetic transformation between taxa (in addition to reduced genome relatedness values). Sequence divergence and sexual isolation may prove to be more useful than metabolic characteristics for delimiting cryptic Bacillus species.

Bacillus sonorensis sp. nov., a close relative of Bacillus licheniformis, isolated from soil in the Sonoran Desert, Arizona.

Eight Bacillus strains isolated from Sonoran Desert soil were shown to belong to a previously unidentified species, for which the name Bacillus sonorensis sp. nov. is proposed. The type strain is strain L87-10T (= NRRL B-23154T). On the basis of phenotypic and genetic data, B. sonorensis is most closely related to Bacillus licheniformis. B. sonorensis can be distinguished from B. licheniformis by salt tolerance, pigmentation, multilocus enzyme electrophoresis, reassociation of genomic DNA and sequence differences in protein-coding genes and 16S rRNA.

Emended description of Paenibacillus amylolyticus and description of Paenibacillus illinoisensis sp. nov. and Paenibacillus chibensis sp. nov.

The taxonomic position of unidentified group 6 of Bacillus circulans as described by Nakamura and Swezey (L.K. Nakamura and J. Swezey, Int. J. Syst. Bacteriol. 33:46-52, 1983) was determined, and the taxonomy of Paenibacillus amylolyticus was reexamined. The results of PCR amplification of a 16S rRNA gene fragment with a specific primer and comparative analysis of 16S rRNA gene sequences warranted placing the two taxa in the genus Paenibacillus. The levels of DNA reassociation among the strains revealed four groups (designated groups I, II, III, and 6), each with a high level of intragroup relatedness (> 72%). Clustering based on phenotypic characteristics correlated well with DNA relatedness grouping. P. amylolyticus strains were scattered in groups I, II, and III. Strains labeled the type strain of P. amylolyticus from different culture collections appeared in groups I and III. Strains found in group I were identified as P. amylolyticus sensu stricto, and the one strain found in group III was identified as Paenibacillus lautus. Group 6 encompassed strains formerly assigned to B. circulans group 6, and group II contained other strains identified as P. amylolyticus. Groups 6 and II were phenotypically and genetically distinct taxa that were distinguishable from the previously described species. These findings showed that groups 6 and II were new species, for which we propose the names Paenibacillus illinoisensis and Paenibacillus chibensis, respectively.

Bacillus vallismortis sp. nov., a close relative of Bacillus subtilis, isolated from soil in Death Valley, California

Five Bacillus strains isolated from Death Valley soil were shown to belong to a previously unidentified species, for which we propose the name Bacillus vallismortis. The type strain is strain DV1-F-3 (= NRRL B-14890). On the basis of previously published restriction digestion data, B. vallismortis is most closely related to Bacillus subtilis. At this time B. vallismortis can be distinguished from B. subtilis only by differences in whole-cell fatty acid compositions, DNA sequences, and levels of reassociation of genomic DNA.

Phylogeny of Marine Bacillus Isolates from the Gulf of Mexico

The phylogeny of 11 pigmented, aerobic, spore-forming isolates from marine sources was studied. Forty-two biochemical characteristics were examined, and a 16S rDNA sequence was obtained for each isolate. In a phylogenetic tree based on 16S sequencing, four isolates (NRRL B-14850, NRRL B-14904, NRRL B-14907, and NRRL B-14908) clustered with B. subtilis and related organisms; NRRL B-14907 was closely related to B. amyloliquefaciens. NRRL B-14907 and NRRL B-14908 were phenotypically similar to B. amyloliquefaciens and B. pumilus, respectively. Three strains (NRRL B-14906, NRRL B-14910, and NRRL B-14911) clustered in a clade that included B. firmus, B. lentus, and B. megaterium. NRRL B-14910 was closely related phenotypically and phylogenetically to B. megaterium. NRRL B-14905 clustered with the mesophilic round spore-producing species, B. fusiformis and B. sphaericus; the isolate was more closely related to B. fusiformis. NRRL B-14905 displayed characteristics typical of the B. sphaericus-like organisms. NRRL B-14909 and NRRL B-14912 clustered with the Paenibacillus species and displayed characteristics typical of the genus. Only NRRL B-14851, an unusually thin rod that forms very small spores, may represent a new Bacillus species.

Protein-Coding Genes as Molecular Markers for Ecologically Distinct Populations: The Case of Two Bacillus Species

Bacillus globisporus and Bacillus psychrophilus are one among many pairs of ecologically distinct taxa that are distinguished by very few nucleotide differences in 16S rRNA gene sequence. This study has investigated whether the lack of divergence in 16S rRNA between such species stems from the unusually slow rate of evolution of this molecule, or whether other factors might be preventing neutral sequence divergence at 16S rRNA as well as every other gene. B. globisporus and B. psychrophilus were each surveyed for restriction-site variation in two protein-coding genes. These species were easily distinguished as separate DNA sequence clusters for each gene. The limited ability of 16S rRNA to distinguish these species is therefore a consequence of the extremely slow rate of 16S rRNA evolution. The present results, and previous results involving two Mycobacterium species, demonstrate that there exist closely related species which have diverged long enough to have formed clearly separate sequence clusters for protein-coding genes, but not for 16S rRNA. These results support an earlier argument that sequence clustering in protein-coding genes could be a primary criterion for discovering and identifying ecologically distinct groups, and classifying them as separate species.

Phylogeny of Bacillus sphaericus-like organisms.

The mesophilic round-spored bacteria embrace four species, namely Bacillus sphaericus, Bacillus fusiformis, Bacillus silvestris and Bacillus pasteurii. Although not displayed by all strains, mosquito pathogenicity is a noteworthy characteristic of B. sphaericus sensu lato. Phylogenetic analysis based on 16S rDNA sequences from 58 strains identified as B. sphaericus was used to examine the genetic heterogeneity of the taxon. Results from sequence analysis were compared with whole-cell fatty acid profiles and other phenotypic determinations. The B. sphaericus-like strains segregated into seven distinct clusters in a phylogenetic tree generated from 16S sequences. One cluster represented B. sphaericus and another B. fusiformis. A third cluster containing all of the pathogenic strains was closely related to, or was possibly part of, the B. fusiformis group. The remaining four groups were distinct and represented unnamed taxa that were more closely related to B. sphaericus and B. fusiformis than to the psychrophilic round-spored species, Bacillus globisporus and Bacillus psychrophilus. Groups based on phenotypic analysis corresponded to the 16S rDNA phylogenetic clusters. Data showed that B. sphaericus was genetically and phenotypically a highly heterogeneous taxon including at least seven genetically distinct taxa. The pathogenic strains were members of a distinct group and not of the species B. sphaericus sensu stricto. This heterogeneity partially accounts for the apparent variability of mosquito pathogenicity among B. sphaericus strains.

Proposal of Bacillus reuszeri sp. nov., Bacillus formosus sp. nov., nom. rev., and Bacillus borstelensis sp. nov., nom. rev.

Taxonomic studies of 26 putative Bacillus brevis strains (12 strains formerly assigned to groups 2, 4 through 7, and B and 14 ungrouped strains) were carried out. These organisms were placed in five distinct groups on the basis of phenotypic characteristics, the results of DNA base composition and reassociation analyses, and the results of cellular fatty acid and isoprenoid quinone composition analyses. Group 2, which contained three strains, was identified as Bacillus choshinensis, and group 4, which contained one strain, was identified as Bacillus parabrevis. The three remaining unidentified groups were clearly distinguishable phenotypically and genetically from the type strains of B. brevis and the recently described species Bacillus agri, Bacillus centrosporus, Bacillus migulanus, Bacillus choshinensis, and Bacillus parabrevis. Therefore, the names Bacillus reuszeri sp. nov., Bacillus formosus sp. nov., nom. rev., and Bacillus borstelensis sp. nov., nom. rev. are proposed for groups 5 (3 strains), 6 (3 strains), and 7 (16 strains), respectively. The S-layer proteins found in the three new species were immunologically and genetically similar to the S-layer proteins found previously in B. brevis, B. agri, B. centrosporus, B. choshinensis, and B. parabrevis. The G+C content of NRRL NRS-1206, the type strain of B. reuszeri sp. nov., was 46.5 mol%; the G+C content of NRRL NRS-863, the type strain of B. formosus sp. nov., nom. rev., was 47.2 mol%; and the G+C content of NRRL NRS-818, the type strain of B. borstelensis sp. nov., nom. rev., was 51.3 mol%.