Photoreactivation of ultraviolet-irradiated, plasmid-bearing, and plasmid-free strains of Bacillus anthracis

The effects of toxin- and capsule-encoding plasmids on the kinetics of UV inactivation of various strains of Bacillus anthracis were investigated. Plasmids pXO1 and pXO2 had no effect on bacterial UV sensitivity or photoreactivation. Vegetative cells were capable of photoreactivation, but photo-induced repair of UV damage was absent in B. anthracis Sterne spores.     

Photoreactivation, photoproduct formation, and deoxyribonucleic acid state in ultraviolet-irradiated sporulating cultures of Bacillus cereus

Photoreactivation of ultraviolet-irradiated Bacillus cereus T declined markedly during the development of stage IV forespores. During ultraviolet irradiation of a culture containing early and late stage IV forespores, both vegetative- and spore-type photoproducts were formed. The formation of vegetative-type photoproducts (mainly thymine dimers) decreased to nearly half during late stage IV, remaining constant until lysis of the mother cells began, when it fell to zero. Spore-type photoproducts were first observed during late stage IV and increased with the increase in numbers of late stage IV forespores. The occurrence of spore-type photoproducts preceded the development of refractile forespores by about 1 h. At stage III the nuclear material occupied a central position, and the ribosomes were at the periphery of the forespore protoplast. During stage IV the deoxyribonucleic acid (DNA) occurred in a peripheral position, and bundles of fibers ("transition" DNA) could be seen. By stage V, all of the DNA appeared to be of the spore type and was peripheral, and the forespore protoplast center was packed with ribosomes. Forespore stages II, III, and IV were classified by light and electron microscopy. The curve for electron microscope classifications preceded that for light microscope classifications by approximately one stage. The formation of spore-type photoproducts preceded differentiation of DNA by about 1 h, the latter coinciding with the development of refractility. Spore-type photoproducts have been associated with DNA in the A state, and the progressive change of the forespore DNA into this state is discussed in relation to the spore differentiation process.     

Global analysis of a model of plasmid-bearing,plasmid-free competition in a chemostat

A model of competition between plasmid-bearing and plasmid-free organisms in a chemostat was proposed in a paper of Stephanopoulis and Lapidus. The model was in the form of a system of nonlinear ordinary differential equations. Such models are relevant to commercial production by genetically altered organisms in continuous culture. The analysis there was local (using index arguments). This paper provides a mathematically rigorous analysis of the global asymptotic behavior of the governing equations in the case of uninhibited specific growth rate.Research supported by the National Council of Science, Republic of ChinaResearch supported by National Science Foundation Grant, DMS-9204490Research supported by the Natural Science and Engineering Council of Canada. This author's contribution was made while on research leave visiting the Department of Ecology and Evolutionary Biology at Princeton University. She would especially like to thank Simon Levin for his guidance as well as for providing an exceptional working environment     

Identification of Bacillus anthracis strains in China

As part of a project to establish a reference strain collection, phenotypic, biochemical and genetic analyses were carried out on 84 strains of Bacillus anthracis, 81 of them of Chinese origin from various sources. Particular differences from reports on isolates of other origins were the possession of fimbriae and single polar flagella with consequent motility in 77, self-agglutination by 64 and failure to ferment maltose in 60 of the Chinese strains. The findings were considered to be of significance to identification, classification and evolution of B. anthracis.     

Comparison of PCR-RFLP, ribotyping and ERIC-PCR for typing Bacillus anthracis and Bacillus cereus strains

PCR-RFLP analysis of the vrrA gene and cerAB gene was used to investigate the genomic diversity in 21 strains of Bacillus anthracis and 28 strains of Bacillus cereus, and was compared with results obtained by ribotyping and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) analysis. VrrA-typing divided the B. anthracis into four groups. Except for one Pasteur vaccine strain, the vrrA PCR-RFLP profiles of the B. anthracis were separated into three groups, which were different from those of the B. cereus strains. Ribotyping separated the B. anthracis isolates into seven ribotypes, and a common fragment of an approximately 850 bp band from the ERIC-PCR fingerprints separated most B. anthracis strains into two groups. VrrA/cerAB PCR-RFLP, ribotyping and ERIC-PCR generated 18, 22 and 23 types, respectively, from B. cereus strains. The results suggest that a combination of all three methods provides a high resolution typing method for B. anthracis and B. cereus. Compared with ribotyping and ERIC-PCR, PCR-RFLP is simple to perform and has potential as a rapid method for typing and discriminating B. anthracis strains from other B. cereus group bacteria.     

Characterization of Bacillus anthracis strains used for vaccination

Three Bacillus anthracis strains, formerly used as anti-anthrax vaccine strains in Argentina, were characterized from genetic and pathogenic perspectives. Southern blotting and PCR with pXO1 and pXO2 probes and primers, as well as pathogenicity and protection tests in guinea pigs and mice, were performed. Two of the B. anthracis strains contained both pXO1 and pXO2 plasmids, as did the fully virulent strains, while the third was a Sterne-type strain (pXO1+, pXO2-). The three strains were, however, markedly less pathogenic than a wild-type virulent strain. The methodology applied here may be used to characterize other B. anthracis strains.     

Allelopathy of plasmid-bearing and plasmid-free organisms competing for two complementary resources in a chemostat

We consider a model of competition between plasmid-bearing and plasmid-free organisms for two complementary nutrients in a chemostat. We assume that the plasmid-bearing organism produces an allelopathic agent at the cost of its reproductive abilities which is lethal to plasmid-free organism. Our analysis leads to different thresholds in terms of the model parameters acting as conditions under which the organisms associated with the system cannot thrive even in the absence of competition. Local stability of the system is obtained in the absence of one or both the organisms. Also, global stability of the system is obtained in the presence of both the organisms. Computer simulations have been carried out to illustrate various analytical results.     

Virulotypes of Bacillus anthracis strains isolated in Poland

Bacillus anthracis--the causative agent of anthrax--possesses several virulence genes located in the chromosome as well as in two B. anthracis virulence plasmids: pXO1 and pXO2. In the presented study, we determined occurrence of six virulence markers located in the virulence plasmids (capA, capB, capC, pagA, lef and cya) for capsule and toxin production together with virulence-associated gene gerXA and chromosomal gene sap, which are responsible for germination and S-layer biosynthesis respectively. Fourteen strains of B. anthracis isolated in Poland and belonging to five different MLVA genotypes were analyzed by PCR for presence of the aforementioned genes. Two virulotypes were found in tested strains. The only variation was absence of capA, capB and capC due to a lack of pXO2.     

Competition between plasmid-bearing and plasmid-free organisms in a chemostat with an inhibitor

A model of competition in the chemostat with an inhibitor is combined with a model of competition in the chemostat between plasmid-bearing and plasmid-free organism to produce a model that more closely approximates the way chemostat-like devices are used in biotechnology. The asymptotic behavior of the solutions of the resulting system of nonlinear differential equations is analyzed as a function of the relevant parameters. The techniques are those of dynamical systems although perturbation techniques are used when the parameter reflecting plasmid-loss is small.Research Supported by National Council of Science, Republic of ChinaResearch Supported by National Science Foundation Grant MCS 9204490     

Global analysis of a model of plasmid-bearing,plasmid-free competition in a chemostat with inhibitions

A model of competition between plasmid-bearing and plasmid-free organisms in a chemostat was proposed in a paper of Stephanopoulis and Lapidus. The model was in the form of a system of nonlinear ordinary differential equations. Such models were relevant to commercial production by genetically altered organisms in continuous culture. The analysis there was local. The rigorous global analysis was done in a paper of Hsu, Waltman and Wolkowicz in the case of the uninhibited specific growth rates. This paper provides a mathematically rigorous analysis of the global asymptotic behavior of the governing equations in the cases of combinations of inhibited and uninhibited specific growth rates.Research Supported by the National Council of Science, Republic of China     

Use of long-range repetitive element polymorphism-PCR to differentiate Bacillus anthracis strains.

The genome of Bacillus anthracis is extremely monomorphic, and thus individual strains have often proven to be recalcitrant to differentiation at the molecular level. Long-range repetitive element polymorphism-PCR (LR REP-PCR) was used to differentiate various B. anthracis strains. A single PCR primer derived from a repetitive DNA element was able to amplify variable segments of a bacterial genome as large as 10 kb. We were able to characterize five genetically distinct groups by examining 105 B. anthracis strains of diverse geographical origins. All B. anthracis strains produced fingerprints comprising seven to eight bands, referred to as "skeleton" bands, while one to three "diagnostic" bands differentiated between B. anthracis strains. LR REP-PCR fingerprints of B. anthracis strains showed very little in common with those of other closely related species such as B. cereus, B. thuringiensis, and B. mycoides, suggesting relative heterogeneity among the non-B. anthracis strains. Fingerprints from transitional non-B. anthracis strains, which possessed the B. anthracis chromosomal marker Ba813, scarcely resembled those observed for any of the five distinct B. anthracis groups that we have identified. The LR REP-PCR method described in this report provides a simple means of differentiating B. anthracis strains.     

Strategy for identification of Bacillus cereus and Bacillus thuringiensis strains closely related to Bacillus anthracis

Bacillus cereus strains that are genetically closely related to B. anthracis can display anthrax-like virulence traits (A. R. Hoffmaster et al., Proc. Natl. Acad. Sci. USA 101:8449-8454, 2004). Hence, approaches that rapidly identify these "near neighbors" are of great interest for the study of B. anthracis virulence mechanisms, as well as to prevent the use of such strains for B. anthracis-based bioweapon development. Here, a strategy is proposed for the identification of near neighbors of B. anthracis based on single nucleotide polymorphisms (SNP) in the 16S-23S rRNA intergenic spacer (ITS) containing tRNA genes, characteristic of B. anthracis. By using restriction site insertion-PCR (RSI-PCR) the presence of two SNP typical of B. anthracis was screened in 126 B. cereus group strains of different origin. Two B. cereus strains and one B. thuringiensis strain showed RSI-PCR profiles identical to that of B. anthracis. The sequencing of the entire ITS containing tRNA genes revealed two of the strains to be identical to B. anthracis. The strict relationship with B. anthracis was confirmed by multilocus sequence typing (MLST) of four other independent loci: cerA, plcR, AC-390, and SG-749. The relationship to B. anthracis of the three strains described by MLST was comparable and even higher to that of four B. cereus strains associated with periodontitis in humans and previously reported as the closest known strains to B. anthracis. SNP in ITS containing tRNA genes combined with RSI-PCR provide a very efficient tool for the identification of strains closely related to B. anthracis.     

Allelopathy of plasmid-bearing and plasmid-free organisms competing for two complementary resources in a chemostat

We consider a model of competition between plasmid-bearing and plasmid-free organisms for two complementary nutrients in a chemostat. We assume that the plasmid-bearing organism produces an allelopathic agent at the cost of its reproductive abilities which is lethal to plasmid-free organism. Our analysis leads to different thresholds in terms of the model parameters acting as conditions under which the organisms associated with the system cannot thrive even in the absence of competition. Local stability of the system is obtained in the absence of one or both the organisms. Also, global stability of the system is obtained in the presence of both the organisms. Computer simulations have been carried out to illustrate various analytical results.     

Siderophores of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis

Three Bacillus anthracis Sterne strains (USAMRIID, 7702, and 34F2) and Bacillus cereus ATCC 14579 excrete two catecholate siderophores, petrobactin (which contains 3,4-dihydroxybenzoyl moieties) and bacillibactin (which contains 2,3-dihydroxybenzoyl moieties). However, the insecticidal organism Bacillus thuringiensis ATCC 33679 makes only bacillibactin. Analyses of siderophore production by previously isolated [Cendrowski et al., Mol. Microbiol. 52 (2004) 407-417] B. anthracis mutant strains revealed that the B. anthracis bacACEBF operon codes for bacillibactin production and the asbAB gene region is required for petrobactin assembly. The two catecholate moieties also were synthesized by separate routes. PCR amplification identified both asbA and asbB genes in the petrobactin producing strains whereas B. thuringiensis ATCC 33679 retained only asbA. Petrobactin synthesis is not limited to the cluster of B. anthracis strains within the B. cereus sensu lato group (in which B. cereus, B. anthracis, and B. thuringiensis are classified), although petrobactin might be prevalent in strains with pathogenic potential for vertebrates.     

Competition between plasmid-bearing and plasmid-free organisms in a chemostat with nutrient recycling and an inhibitor

The asymptotic behavior of solutions of a model for competition between plasmid-bearing and plasmid-free organisms in the chemostat with two distributed delays and an external inhibitor is considered. The model presents a refinement of the one considered by Lu and Hadeler [Z. Lu, K.P. Hadeler, Model of plasmid-bearing plasmid-free competition in the chemostat with nutrient recycling and an inhibitor, Math. Biosci. 167 (2000) p. 177]. The delays model the fact that the nutrient is partially recycled after the death of the biomass by bacterial decomposition. Furthermore, it is assumed that there is inter-specific competition between the plasmid-bearing and plasmid-free organisms as well as intra-specific competition within each population. Conditions for boundedness of solutions and existence of non-negative equilibrium are given. Analysis of the extinction of the organisms, including plasmid-bearing and plasmid-free organisms, and the uniform persistence of the system are also carried out. By constructing appropriate Liapunov-like functionals, some sufficient conditions of global attractivity to the extinction equilibria are obtained and the combined effects of the delays and the inhibitor are studied.     

Immuno-detection of anthrose containing tetrasaccharide in the exosporium of Bacillus anthracis and Bacillus cereus strains

Aims:  Bacillus anthracis strains of various origins were analysed with the view to describe intrinsic and persistent structural components of the Bacillus collagen-like protein of anthracis glycoprotein associated anthrose containing tetrasaccharide in the exosporium.
Methods and Results:  The tetrasaccharide consists of three rhamnose residues and an unique monosaccharide – anthrose. As anthrose was not found in spores of related strains of bacteria, we envisioned the detection of B. anthracis spores based on antibodies against anthrose-containing polysaccharides. Carbohydrate–protein conjugates containing the synthetic tetrasaccharide, an anthrose–rhamnose disaccharide or anthrose alone were employed to immunize mice. All three formulations were immunogenic and elicited IgG responses with different fine specificities. All sera and monoclonal antibodies derived from tetrasaccharide immunized mice cross-reacted not only with spore lysates of a panel of virulent B. anthracis strains, but also with some of the B. cereus strains tested.
Conclusions:  Our results demonstrate that antibodies to synthetic carbohydrates are useful tools for epitope analyses of complex carbohydrate antigens and for the detection of particular target structures in biological specimens.
Significance and Impact of the Study:  Although not strictly specific for B. anthracis spores, antibodies against the tetrasaccharide may have potential as immuno-capturing components for a highly sensitive spore detection system.