Molecular recognition specificity of Bacillus anthracis spore antibodies

The sensitivity and specificity of polyclonal and monoclonal antibodies raised against anthrax spore preparations has been assessed by Western blotting. None of the antibodies studied were completely specific in recognizing the anthrax spore surface. A polyclonal serum recognized a wide range of spore surface epitopes and demonstrated limited cross-reaction with the near-neighbour species Bacillus cereus spore surface. Two monoclonal antibodies studied demonstrated more extensive cross-reaction with distant-neighbour species B. globigii and B. subtilis. These monoclonal antibodies did not react with spore surface epitopes but did bind strongly to vegetative cell epitopes in all four Bacillus species studied.     

Monoclonal antibodies against spore antigens of Bacillus anthracis

A murine monoclonal antibody produced against heat inactivated spores of Bacillus anthracis Ames, reacted with live or inactivated spores of several anthrax strains in indirect immunofluorescence (IF) tests. The reactive anthrax strain gave only a moderate degree of reaction. No staining of anthrax vegetative cells was observed. The monoclonal did not react with spores of non-anthrax Bacillus strains that gave cross reactions with mouse hyperimmune antiserum raised against Ames spores. The staining of individual spores in B. anthracis preparations was more heterogeneous with the monoclonal antibody than with the hyperimmune serum. Evidence is produced that the epitope for this monoclonal is not stable during long-term storage of inactivated spore preparations, and is not fully available for reaction with antibody until late in spore maturation. The monoclonal did not react by immunoblotting (Western blotting) of spore extracts. A monoclonal antibody produced against Ames spore extracts reacted with about 1% of Ames spores in IF tests, but not reproducible reactions with other anthrax strains were recorded. This monoclonal interacted with three bands in Western blots of anthrax spore extracts.     

Monoclonal antibodies for Bacillus anthracis spore detection and functional analyses of spore germination and outgrowth

All members of the Bacillus genus produce endospores as part of their life cycle; however, it is not possible to determine the identity of spores by casual or morphological examination. The 2001 anthrax attacks demonstrated a need for fast, dependable methods for detecting Bacillus anthracis spores in vitro and in vivo. We have developed a variety of isotypes and specificities of mAbs that were able to distinguish B. anthracis spores from other Bacillus spores. The majority of Abs were directed toward BclA, a major component of the exosporium, although other components were also distinguished. These Abs did not react with vegetative forms. Some Abs distinguished B. anthracis spores from spores of distantly related species in a highly specific manner, whereas others discriminated among strains that are the closest relatives of B. anthracis. These Abs provide a rapid and reliable means of identifying B. anthracis spores, for probing the structure and function of the exosporium, and in the analysis of the life cycle of B. anthracis.     

Molecular cloning of a Bacillus subtilis gene involved in spore outgrowth

A lambda Charon 4A derivative carrying the outB gene of Bacillus subtilis has been identified by transformation of a B. subtilis mutant temperature-sensitive in spore outgrowth. The cloned region is a single EcoRI fragment 14 kb in length. In addition to outB, the cloned DNA includes at least part of the amyE and aroI loci.     

The Bacillus anthracis spore

In response to starvation, Bacillus anthracis can form a specialized cell type called the spore, which is the infectious particle for the disease anthrax. The spore is largely metabolically inactive and can resist a wide range of stresses found in nature. In spite of its dormancy, the spore can sense the presence of nutrient and rapidly return to vegetative growth. These properties help the spore to persist for long periods of time in the environment, survive host defenses after entering the body, and cause disease when the correct location in the host is reached. The anatomy of the spore is unique among bacteria, being comprised of a series of specialized concentric shells, each of which provides specific critical functions. Surrounding the spore core (which houses the chromosome) is a peptidoglycan layer important for spore dormancy, a protein shell that resists a variety of toxic molecules, and finally an exterior protein and glycoprotein layer that, among other functions, mediates interactions with surfaces, including those encountered by the spore within the host. Detailed molecular analysis of these shells has shed considerable light on how each layer determines specific spore properties. Future work, especially on the outermost spore layer, is likely to advance therapeutics, methods for spore decontamination and other critical biodefense technologies.     

Recognition sequence for the restriction endonuclease BglI from Bacillus globigii

Restriction endonuclease BglI recognizes the DNA sequence (Formula: see text) and cleaves each strand at the site indicated, thus generating 3' protruding ends. The recognition sequence was deduced by correlating mapping data with nucleotide sequence information and the position of cleavage was unambiguously determined by 32P labeling of 5' termini produced by BglI digestion.     

Biochemical and genetic properties of site-specific restriction endonucleases in Bacillus globigii

Bacillus globigii contains two site-specific endonucleases, BPGLI AND BglI. A rapid technique for selection of mutants deficient in each of these enzymes was developed using sensitivity to infection by bacteriophage SP50 as an indication of the levels of enzyme. Mutants defective in BglI, BglII, and both BglI and BglII retained the wild-type modification phenotype. Genetic and biochemical studies have established that these enzymes are involved in restriction in vivo. Simplified purification procedures for BglI and BglII using these mutants are described.     

Purification and properties of the restriction endonuclease BglII from Bacillus globigii

The restriction endonuclease BglII from Bacillus globigii has been purified to homogeneity. The enzyme is a dimer of two subunits of Mr = 27000. The reaction mechanism does not involve the accumulation of a DNA intermediate nicked in one strand and the enzyme is not affected by superhelical twists in the substrate DNA, indicating that DNA binding does not involve either winding or unwinding of the double helix. Antibodies were prepared against BglII. These antibodies did not cross react with any other restriction endonucleases tested, including other enzymes from B. globigii or from closely related strains. It is thus unlikely that type II restriction enzymes represent a closely related group of proteins.     

Correlation between spore structure and spore properties in Bacillus megaterium

The spores of six strains of Bacillus megaterium were divided into two distinct groups on the basis of germination. Three of the strains germinated in a mixture of l-alanine and inosine (AL type spores), and three strains germinated in a mixture of glucose and potassium nitrate (GN type spores); recriprocal germination in the respective solutions did not occur. The AL spores and the GN spores were morphologically distinct. Other differences between the two spore groups included germination inhibition characteristics, dipicolinic acid content, hexosamine content, phosphorus and magnesium content, spore coat features, ion exchange properties, and heat resistance. A correlation appears to exist between spore morphology and certain other spore properties in strains of B. megaterium.     

Acid-soluble spore proteins of Bacillus subtilis

Acid-soluble spore proteins (ASSPs) comprise about 5% of the total protein of mature spores of different Bacillus subtilis strains. They consist of three abundant species, alpha, beta, and gamma, four less abundant species, and several minor species, alpha, beta, and gamma make up about 18, 18 and 36%, respectively, of the total ASSPs of strain 168, have molecular weights of 5,900, 5,9000, and 11,000, respectively, and resemble the major (A, C, and B) components of Bacillus megaterium ASSPs in several respects, including sensitivity to a specific B. megaterium spore endopeptidase. However, they have pI's of 6.58, 6.67, and 7.96, all lower than those of any of the B. megaterium ASSPs. Although strains varied in the proportions of different ASSPs, to overall patterns seen on gel electrophoresis are constant. ASSPs are located interior to the cortex, presumably in the spore cytoplasm, and are synthesized during sporulation and degraded during germination.     

Morphogenesis of the Bacillus anthracis spore

Bacillus spp. and Clostridium spp. form a specialized cell type, called a spore, during a multistep differentiation process that is initiated in response to starvation. Spores are protected by a morphologically complex protein coat. The Bacillus anthracis coat is of particular interest because the spore is the infective particle of anthrax. We determined the roles of several B. anthracis orthologues of Bacillus subtilis coat protein genes in spore assembly and virulence. One of these, cotE, has a striking function in B. anthracis: it guides the assembly of the exosporium, an outer structure encasing B. anthracis but not B. subtilis spores. However, CotE has only a modest role in coat protein assembly, in contrast to the B. subtilis orthologue. cotE mutant spores are fully virulent in animal models, indicating that the exosporium is dispensable for infection, at least in the context of a cotE mutation. This has implications for both the pathophysiology of the disease and next-generation therapeutics. CotH, which directs the assembly of an important subset of coat proteins in B. subtilis, also directs coat protein deposition in B. anthracis. Additionally, however, in B. anthracis, CotH effects germination; in its absence, more spores germinate than in the wild type. We also found that SpoIVA has a critical role in directing the assembly of the coat and exosporium to an area around the forespore. This function is very similar to that of the B. subtilis orthologue, which directs the assembly of the coat to the forespore. These results show that while B. anthracis and B. subtilis rely on a core of conserved morphogenetic proteins to guide coat formation, these proteins may also be important for species-specific differences in coat morphology. We further hypothesize that variations in conserved morphogenetic coat proteins may play roles in taxonomic variation among species.     

Optimizing Bacillus subtilis spore isolation and quantifying spore harvest purity

Investigating the biochemistry, resilience and environmental interactions of bacterial endospores often requires a pure endospore biomass free of vegetative cells. Numerous endospore isolation methods, however, neglect to quantify the purity of the final endospore biomass. To ensure low vegetative cell contamination we developed a quality control technique that enables rapid quantification of endospore harvest purity. This method quantifies spore purity using bright-field and fluorescence microscopy imaging in conjunction with automated cell counting software. We applied this method to Bacillus subtilis endospore harvests isolated using a two-phase separation method that utilizes mild chemicals. The average spore purity of twenty-two harvests was 88 ± 11% (error is 1σ) with a median value of 93%. A spearman coefficient of 0.97 correlating automated and manual bacterial counts confirms the accuracy of software generated data.     

Comparative antigenicity of spore coat proteins from Bacillus species using antibody to spore coat proteins of Bacillus megaterium

Spore coat proteins obtained by extraction with sodium dodecylsulfate/dithiothreitol from six Bacillus spores were compared by immunoblot analysis using antibodies to spore coat proteins from two strains of B. megaterium. Although the extract from spores of each strain had heterogenous proteins with various molecular weights, there were some bands which cross-reacted with specific antibodies from B. megaterium spores. Specific antibody to 48K protein from B. megaterium ATCC 12872 cross-reacted with 17K protein from B. megaterium ATCC 19213, 13K protein from B. cereus and 50K protein from B. subtilis 60015 and B. subtilis NRRL B558. Also, specific antibody to 22K protein from the same strain cross-reacted with 22K and 17K proteins from B. megaterium ATCC 19213 and 13K protein from B. cereus T. Specific antibody to 17K protein from B. megaterium ATCC 19213 reacted with 22K and 19K proteins in addition to 17K protein of own strain, and it was cross-reactive with 16K protein from B. megaterium ATCC 12872, 19K and 27K proteins from B. thiaminolyticus, 13K protein from B. cereus.