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At least 19 recordsLinked to original sources
PubMed1991

Comparative analysis of Bacillus anthracis, Bacillus cereus, and related species on the basis of reverse transcriptase sequencing of 16S rRNA.

The primary structures of the 16S rRNAs of Bacillus anthracis, Bacillus cereus, Bacillus mycoides, and Bacillus thuringiensis were determined by using the reverse transcription-dideoxy sequencing method. All of the strains exhibited very high levels of sequence similarity (greater than 99%) that were consistent with the close relationships shown by previous DNA hybridization studies. The sequences of B. anthracis Sterne and B. cereus emetic strain NCTC 11143 were found to be identical for a continuous stretch of 1,446 bases and differed from the sequence of B. cereus NCDO 1771T (T = type strain) by only a single nucleotide. The 16S rRNA sequences of B. mycoides and B. thuringiensis differed from each other and from the sequences of B. anthracis and B. cereus by four to nine nucleotides.

Bacillus anthracis↗
PubMed1985

Mating system for transfer of plasmids among Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis.

To facilitate the analysis of genetic determinants carried by large resident plasmids of Bacillus anthracis, a mating system was developed which promotes plasmid transfer among strains of B. anthracis, B. cereus, and B. thuringiensis. Transfer of the selectable tetracycline resistance plasmid pBC16 and other plasmids from B. thuringiensis to B. anthracis and B. cereus recipients occurred during mixed incubation in broth. Two plasmids, pXO11 and pXO12, found in B. thuringiensis were responsible for plasmid mobilization. B. anthracis and B. cereus transcipients inheriting either pXO11 or pXO12 were, in turn, effective donors. Transcipients harboring pXO12 were more efficient donors than those harboring pXO11; transfer frequencies ranged from 10(-4) to 10(-1) and from 10(-8) to 10(-5), respectively. Cell-to-cell contact was necessary for plasmid transfer, and the addition of DNase had no effect. The high frequencies of transfer, along with the fact that cell-free filtrates of donor cultures were ineffective, suggested that transfer was not phage mediated. B. anthracis and B. cereus transcipients which inherited pXO12 also acquired the ability to produce parasporal crystals (Cry+) resembling those produced by B. thuringiensis, indicating that pXO12 carries a gene(s) involved in crystal formation. Transcipients which inherited pXO11 were Cry-. This mating system provides an efficient method for interspecies transfer of a large range of Bacillus plasmids by a conjugation-like process.

Bacillus↗
PubMed1997

Pyrolysis mass spectrometry studies on Bacillus anthracis, Bacillus cereus and their close relatives.

Pyrolysis mass spectrometry was used to examine strains of B. anthracis, of B. cereus, of B.cereus either proven to cause emetic illness or connected with outbreaks of emetic food poisoning and of B.thuringiensis. Analysis of the data-set for all strains allowed differentiation between B.anthracis, the emetic B.cereus and B.thuringiensis but B.cereus strains could not be clearly discriminated. Removal of data for the B.thuringiensis and the emetic B.cereus strains, followed by re-analysis, allowed clear separation of the B. anthracis and B. cereus groups. Furthermore, PyMS was found to be capable of discriminating between some strains of B.anthracis, and demonstrating sub-groupings of others. This work provides further evidence of the ability of PyMS to distinguish rapidly between very closely related organisms and indicates its potential in epidemiology.

Bacillus anthracis↗
PubMed1984

Glycosidase activities of Bacillus anthracis.

Bacillus anthracis could be distinguished from the taxonomically related species B. cereus, B. mycoides, and B. thuringiensis by a comparison of glycosidase activities. All the bacilli tested possessed alpha-glucosidase activity, as evidenced by the hydrolysis of p-nitrophenyl-alpha-D-glucoside. In B. anthracis, the glucosidase activity could be enhanced by the addition of agents which damage cellular surface structures. Treatment of B. anthracis strains with toluene. Triton X-100, or mutanolysin or cellular disruption by sonication resulted in higher rates of alpha-glucoside hydrolysis than were accomplished by cells suspended in buffer. It is suggested that intact B. anthracis cells have a limited permeability to the glucosidase substrate. In contrast to the results obtained for B. anthracis, Triton X-100 markedly diminished the enzymatic hydrolysis of p-nitrophenyl-alpha-D-glucoside by strains of B. cereus, B. mycoides, and B. thuringiensis. Triton X-100 also enhanced the alpha-maltosidase activity of B. anthracis but not that of the other bacilli. B. mycoides possessed an apparently inducible N-acetylglucosaminidase although the enzyme was absent in B. anthracis. The glucosaminidase was inducible in the presence of p-nitrophenyl-N-acetylglucosamine in the absence of conventional nitrogen sources. Chloramphenicol prevented the induction of the glucosaminidase in B. mycoides. In several B. cereus and all B. thuringiensis strains, the glucosaminidase was constitutive. The results suggest a means for the rapid laboratory differentiation of B. anthracis from other closely related bacilli. Assays for alpha-glucosidase and alpha-maltosidase, in the presence and absence of Triton X-100, can be used to distinguish B. anthracis from B. cereus, B. mycoides, and B. thuringiensis. Similarly, the hydrolysis of p-nitrophenyl-beta-N-acetylglucosamine induced by B. mycoides but not by B. anthracis provides an additional means for differentiating these similar bacilli.

Acetylglucosaminidase↗
PubMedDate not supplied

Molecular tools for the study of transcriptional regulation in Bacillus anthracis.

Bacillus anthracis produces two toxins composed of three proteins. Genetic tools were constructed to study the regulation of toxin synthesis. They included transcriptional fusions with various reporter genes, in replicative and integrative vectors. The reporter gene xylE, encoding catechol 2,3-dioxygenase, may be valuable for screening of strong promoters, as expression of the gene can be visualized directly and the studies of regulation in B. anthracis. Therefore, transcriptional fusions between a lacZ reporter gene and the toxin genes were constructed. Experiments with a multicopy plasmid in trans suggested that the transcriptional activator(s) of the toxin genes were not titrated. B. anthracis strains, which contain pXO1 carrying multiple copies of fusions, were analysed. Expression of the reporter gene was proportional to the fusion copy number. Indeed, single integration of a suicide plasmid can be distinguished from multiple integration according to the level of resistance to an appropriate antibiotic. Finally, recombination in B. anthracis was found to be very efficient (approximately 10(-2) recombinants per transconjugant cell.

Bacillus anthracis↗
PubMed1999

Genetic diversity in the protective antigen gene of Bacillus anthracis.

Bacillus anthracis is a gram-positive spore-forming bacterium that causes the disease anthrax. The anthrax toxin contains three components, including the protective antigen (PA), which binds to eucaryotic cell surface receptors and mediates the transport of toxins into the cell. In this study, the entire 2,294-nucleotide protective antigen gene (pag) was sequenced from 26 of the most diverse B. anthracis strains to identify potential variation in the toxin and to further our understanding of B. anthracis evolution. Five point mutations, three synonymous and two missense, were identified. These differences correspond to six different haploid types, which translate into three different amino acid sequences. The two amino acid changes were shown to be located in an area near a highly antigenic region critical to lethal factor binding. Nested primers were used to amplify and sequence this same region of pag from necropsy samples taken from victims of the 1979 Sverdlovsk incident. This investigation uncovered five different alleles among the strains present in the tissues, including two not seen in the 26-sample survey. One of these two alleles included a novel missense mutation, again located just adjacent to the highly antigenic region. Phylogenetic (cladistic) analysis of the pag corresponded with previous strain grouping based on chromosomal variation, suggesting that plasmid evolution in B. anthracis has occurred with little or no horizontal transfer between the different strains.

Anthrax↗
PubMed1982

Evaluation of a microfluorometer in immunofluorescence assays of individual spores of Bacillus anthracis and Bacillus cereus.

A microfluorometer was constructed by modifying a standard fluorescent microscope with a fibre optic eyepiece and a simple photometric system. It was evaluated in direct immunofluorescence assays of Bacillus anthracis and Bacillus cereus spores immobilised on multispot microscope slides. From measurements of stable fluorescent crystals comparable in size to the spores, it was inferred that the fluorescence intensity of a stained bacterium could be measured with good precision. Fluctuation of a exciting light from a mercury vapour lamp did not contribute significantly to the distribution of fluorescence measurements obtained when samples of 20 spores were assessed. Attempts to correlate spore size with fluorescence intensity suggest that spore fluorescence does not increase in a 1 : 1 ratio with surface area; it is therefore possible that the density of antigenic sites on the surface decreases with increasing spore size. It is concluded that differences in the observed fluorescence of individual spores truly reflect differences in fluorescent antibody binding, but the relative contribution of antigenic variability and of artefacts of the staining procedure remain unknown.

Animals↗
PubMed1998

The capsule and S-layer: two independent and yet compatible macromolecular structures in Bacillus anthracis.

Bacillus anthracis, the etiological agent of anthrax, is a gram-positive spore-forming bacterium. Fully virulent bacilli are toxinogenic and capsulated. Two abundant surface proteins, including the major antigen, are components of the B. anthracis surface layer (S-layer). The B. anthracis paracrystalline S-layer has previously only been found in noncapsulated vegetative cells. Here we report that the S-layer proteins are also synthesized under conditions where the poly-gamma-D-glutamic acid capsule is present. Structural and immunological analyses show that the capsule is exterior to and completely covers the S-layer proteins. Nevertheless, analysis of single and double S-layer protein mutants shows that the presence of these proteins is not required for normal capsulation of the bacilli. Similarly, the S-layer proteins assemble as a two-dimensional crystal, even in the presence of the capsule. Thus, both structures are compatible, and yet neither is required for the correct formation of the other.

Animals↗
PubMed1998

Fermentation, purification, and characterization of protective antigen from a recombinant, avirulent strain of Bacillus anthracis.

Bacillus anthracis, the etiologic agent for anthrax, produces two bipartite, AB-type exotoxins, edema toxin and lethal toxin. The B subunit of both exotoxins is an M(r) 83,000 protein termed protective antigen (PA). The human anthrax vaccine currently licensed for use in the United States consists primarily of this protein adsorbed onto aluminum oxyhydroxide. This report describes the production of PA from a recombinant, asporogenic, nontoxigenic, and nonencapsulated host strain of B. anthracis and the subsequent purification and characterization of the protein product. Fermentation in a high-tryptone, high-yeast-extract medium under nonlimiting aeration produced 20 to 30 mg of secreted PA per liter. Secreted protease activity under these fermentation conditions was low and was inhibited more than 95% by the addition of EDTA. A purity of 88 to 93% was achieved for PA by diafiltration and anion-exchange chromatography, while greater than 95% final purity was achieved with an additional hydrophobic interaction chromatography step. The purity of the PA product was characterized by reversed-phase high-pressure liquid chromatography, sodium dodecyl sulfate (SDS)-capillary electrophoresis, capillary isoelectric focusing, native gel electrophoresis, and SDS-polyacrylamide gel electrophoresis. The biological activity of the PA, when combined with excess lethal factor in the macrophage cell lysis assay, was comparable to previously reported values.

Antigens, Bacterial↗
PubMed1993

Identification of a novel gene, dep, associated with depolymerization of the capsular polymer in Bacillus anthracis.

Bacillus anthracis produces a gamma-linked poly-D-glutamic acid capsule that is essential for virulence. A 6.2 kb fragment of B. anthracis DNA (cap), when present in Escherichia coli, produces a capsular polymer that is immunologically identical to that produced by B. anthracis. By immunodiffusion analysis of E. coli strains carrying varying portions of the cap region, we identified a novel gene (dep) responsible for degradation of the capsular polymer of B. anthracis. The simultaneous presence of the cap region and the dep gene caused production of low-molecular-weight, degraded capsular polymer both in E. coli and in B. anthracis, whereas the cap region alone caused production of a high-molecular-weight capsule. The dep gene mapped immediately downstream of the cap region within a 1.8 kb fragment and was transcribed in the same direction. This fragment was sequenced and a 1401 bp open reading frame (ORF) was found that is predicted to encode a peptide with molecular weight of 51,460. By in vitro transcription-translation analysis, this ORF was shown to be the dep gene product. The deduced amino acid sequence of the dep product has sequence similarity to E. coli and mammalian gamma-glutamyltranspeptidase (GGT). However, the Dep protein did not have GGT activity. The Dep protein appears to be an enzyme that catalyses the hydrolysis of the poly-D-glutamic acid capsule. Although the biological functions of the dep gene are unknown, it is possible that low-molecular-weight, diffusible polyglutamates produced through the action of the dep gene may act to inhibit host defence mechanisms.

Amino Acid Sequence↗
PubMed1983

Differentiation between spores of Bacillus anthracis and Bacillus cereus by a quantitative immunofluorescence technique.

A quantitative immunofluorescence assay based on fiber optic microscopy was used to measure the reaction of formalized spores of Bacillus anthracis and Bacillus cereus isolates with fluorescein conjugates prepared by hyperimmunization with B. anthracis Vollum spores. The spores of 11 of the 20 B. cereus strains reacted with the anti-anthrax conjugate to such an extent that they were indistinguishable from the spores of the several B. anthracis isolates tested. However, absorption of the conjugate with spores of B. cereus NCTC 8035 and B. cereus NCTC 10320 greatly reduced the cross-reaction with the B. cereus preparations so that the mean specific fluorescence of samples of B. cereus spores was in no case higher than 14% of the fluorescence of a reference B. anthracis Vollum preparation.

Absorption↗
PubMed1993

Determination of carbohydrate profiles of Bacillus anthracis and Bacillus cereus including identification of O-methyl methylpentoses by using gas chromatography-mass spectrometry.

Bacillus anthracis and Bacillus cereus are closely related pathogenic organisms that are difficult to differentiate phenotypically or genotypically. It is well known that vegetative and spore forms of bacilli are quite distinct both morphologically and chemically, but spore-specific chemical markers allowing these species to be distinguished have not been previously described. By using gas chromatography-mass spectrometry, vegetative cells and spores of the two species were shown to exhibit distinct carbohydrate profiles. Profiles of vegetative B. anthracis typically contained high levels of galactose but did not contain galactosamine, whereas B. cereus contained galactosamine and generally low levels of galactose. Spore cultures exhibited unique carbohydrate profiles compared with those of vegetative cultures. B. anthracis spore profiles contained rhamnose alone, whereas B. cereus spore profiles contained rhamnose and fucose. Additionally, two spore-specific O-methylated methylpentoses were discovered. Both B. anthracis and B. cereus spores contained 3-O-methyl rhamnose, whereas B. cereus spores also contained 2-O-methyl rhamnose. Carbohydrate profiling is demonstrated to be a powerful tool for differentiating the two closely related species. Differentiation does not depend on whether organisms are in the vegetative or spore stage of growth.

Bacillus anthracis↗
PubMed1991

Differentiation of Bacillus anthracis from Bacillus cereus by gas chromatographic whole-cell fatty acid analysis.

Three strains of Bacillus anthracis and seven strains of Bacillus cereus were grown on complex medium and on synthetic medium. Gas chromatographic analysis of whole-cell fatty acids of strains grown on complex medium gave nearly identical fatty acid patterns. Fatty acid patterns of strains grown on synthetic medium showed a high content of branched-chain fatty acids. Significant differences between the fatty acid patterns of the two species were found. Odd iso/anteiso fatty acid ratios were about equal in B. anthracis strains, whereas in B. cereus strains the fractions of iso acids were at least twice as high as the fractions of anteiso acids. The method described herein is used in our diagnostic laboratory to help differentiate between these two species.

Bacillus anthracis↗
PubMed1998

The expression of the protective antigen of Bacillus anthracis in Bacillus subtilis.

The expression of Bacillus anthracis protective antigen (PA) in B. subtilis from the pag gene in pPA101-1 was explored in different genetic backgrounds in an attempt to identify opportunities to maximize expression. Introduction of AtxA, which positively regulates PA expression in B. anthracis did not improve expression levels in the protease-deficient strain WB600. Plasmid pPA101-1 was found to carry a deletion which created a new fusion point between vector and insert sequence, and which removed part of the AtxA binding site. The deletion may have occurred as a consequence of recombination between TCTAT sequences within both the vector and insert. Host mutations could influence expression; PA levels from pPA101-1 are threefold higher in a ccpA mutant than in an otherwise isogenic parent, and eightfold higher in an abrB mutant. These data demonstrate that the introduction of mutations affecting catabolite repression and growth phase regulation results in an increase in the yield of PA in this host-vector system. Combining these mutations with a multiply protease-negative background could potentially allow further improvements in PA yield.

Antigens, Bacterial↗
PubMed1968

Fatty acids in the genus Bacillus. II. Similarity in the fatty acid compositions of Bacillus thuringiensis, Bacillus anthracis, and Bacillus cereus.

The nature and relative abundance of fatty acids produced by two strains each of Bacillus thuringiensis and of B. anthracis were studied by gas-liquid chromatography on a 12,000 theoretical plate polyester column capable of partially resolving iso- and anteiso-fatty acids with the same number of carbon atoms. Unsaturated fatty acids as the bromo derivatives were separated from the saturated acids and resolved in a short SE-30 column by use of programmed-temperature gas chromatography. All four strains produced 16 major fatty acids: 9 branched (i-C(12), i-C(13), i-C(14), i-C(15), i-C(16), i-C(17), a-C(13), a-C(15), and a-C(17)), 3 normal (n-C(14), n-C(15), and n-C(16)), and 4 monounsaturated (i-C(16) (1=), i-C(17) (1=), a-C(17) (1=), and n-C(16) (1=)), in addition to some minor fatty acids. In all cases, 12 branched acids, including saturated and monounsaturated, made up over 70% of the total fatty acids, and iso-C(15) acid was most abundant. These fatty acid distribution patterns were very similar to those of B. cereus and B. cereus var. mycoides. There were, however, minor but clear differences between the fatty acid distribution patterns of B. thuringiensis and B. anthracis. B. thuringiensis, like B. cereus, produced higher proportions of i-C(13), a-C(13), and i-C(14) fatty acids than did B. anthracis. This difference between these two species could be useful as a supplemental criterion in their differentiation. Indications are that the enzyme systems for monounsaturated fatty acid synthesis in B. thuringiensis and B. anthracis prefer normal fatty acids as substrates rather than branched-chain fatty acids.

Bacillus↗