J Bacteriol, 1996 Jan, 178(2), 377 - 84 Identification of a region of genetic variability among Bacillus anthracis strains and related species; Andersen GL et al.; The identification of a region of sequence variability among individual isolates of Bacillus anthracis as well as the two closely related species, Bacillus cereus and Bacillus mycoides, has made a sequence-based approach for the rapid differentiation among members of this group possible . We have identified this region of sequence divergence by comparison of arbitrarily primed (AP)-PCR "fingerprints" generated by an M13 bacteriophage-derived primer and sequencing the respective forms of the only polymorphic fragment observed . The 1,480-bp fragment derived from genomic DNA of the Sterne strain of B . anthracis contained four consecutive repeats of CAATATCAACAA . The same fragment from the Vollum strain was identical except that two of these repeats were deleted . The Ames strain of B . anthracis differed from the Sterne strain by a single-nucleotide deletion . More than 150 nucleotide differences separated B . cereus and B . mycoides from B . anthracis in pairwise comparisons . The nucleotide sequence of the variable fragment from each species contained one complete open reading frame (ORF) (designated vrrA, for variable region with repetitive sequence), encoding a potential 30-kDa protein located between the carboxy terminus of an upstream ORF (designated orf1) and the amino terminus of a downstream ORF (designated lytB) . The sequence variation was primarily in vrrA, which was glutamine- and proline-rich (30% of total) and contained repetitive regions . A large proportion of the nucleotide substitutions between species were synonymous . vrrA has 35% identity with the microfilarial sheath protein shp2 of the parasitic worm Litomosoides carinii. Vaccine, 1995 Dec, 13(18), 1779 - 84 Experimental anthrax vaccines: efficacy of adjuvants combined with protective antigen against an aerosol Bacillus anthracis spore challenge in guinea pigs; Ivins B et al.; The efficacy of several human anthrax vaccine candidates comprised of different adjuvants together with Bacillus anthracis protective antigen (PA) was evaluated in guinea pigs challenged by an aerosol of virulent B . anthracis spores . The most efficacious vaccines tested were formulated with PA plus monophosphoryl lipid A (MPL) in a squalene/lecithin/Tween 80 emulsion (SLT) and PA plus the saponin QS-21 . The PA+MPL in SLT vaccine, which was lyophilized and then reconstituted before use, demonstrated strong protective immunogenicity, even after storage for 2 years at 4 degrees C . The MPL component was required for maximum efficacy of the vaccine . Eliminating lyophilization of the vaccine did not diminish its protective efficacy . No significant alteration in efficacy was observed when PA was dialyzed against different buffers before preparation of vaccine . PA+MPL in SLT proved superior in efficacy to the licensed United States human anthrax vaccine in the guinea pig model. Res Microbiol, 1995 Nov-Dec, 146(9), 729 - 37 Molecular tools for the study of transcriptional regulation in Bacillus anthracis; Sirard JC et al.; 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. Lab Invest, 1995 Nov, 73(5), 691 - 702 Pathology of experimental inhalation anthrax in the rhesus monkey; Fritz DL et al.; BACKGROUND: The inhalation form of anthrax, although rare, is nearly always fatal because of the rapid progression of the disease with little host response until the terminal stages of the disease . The Gulf War heightened the concern that anthrax could be used as a biologic weapon . Past studies modeling pathologic changes in human inhalation anthrax have used the rhesus monkey . EXPERIMENTAL DESIGN: We studied pathologic changes in the rhesus monkey model of inhalation anthrax . Gross examination as well as light and electron microscopy were used to define pathologic alterations . Immunolabeling techniques were used to identify the anthrax bacillus by light and electron microscopy . RESULTS: Gross changes included hemorrhage in mesenteric (54%) and tracheobronchial (46%) lymph nodes, meninges (38%), lungs (31%), and small intestinal serosa (31%) . Histopathologic changes included suppurative meningitis (77%); hemorrhages in the meninges (54%), neuropil (31%), and pulmonary alveoli (31%); and pneumonia (15%) . Spleens and various lymph nodes from all monkeys had one or more of the following changes: hemorrhage, acute inflammation, extracellular bacilli, lymphocytic depletion, and histiocytosis . Spleens of two monkeys were devoid of extracellular bacilli, but degraded intrahistiocytic bacilli reacted with Ab to Bacillus anthracis cell wall polysaccharide . CONCLUSIONS: In our study, compared with previous reports, meningitis and mesenteric lymph node hemorrhages were more common, whereas mediastinal and tracheobronchial lymph node hemorrhages were less common . Immunostaining highlighted intracellular bacilli that would have been otherwise missed by light microscopic examination. Med Parazitol (Mosk), 1995 Oct-Dec, (4), 35 - 8 {The isolation and purification of the protective antigen and the edema factor from the culture filtrate of Bacillus anthracis STI-1}; Rogov SN et al.; A rapid method for producing a protective antigen (PA) and edema factor (EF), components of anthrax toxin, is described . The specific features of the method are as follows: addition of a mixture of protease inhibitors to the culture fluid; simultaneous concentration on a fiber filter and adsorption on hydroxylapatite, followed by non-linear gradient a phosphate concentration; purification of elution of a phosphate concentration; purification of eluates from salts via electrodialysis . Resultant from protein desorption, PA and EF agents are electrophoretically homogeneous by no less than 80%, 10 liters of filtrate yielded 8 mg of PA and 5 mg of EF . The resultant agents were tested for their biological properties and protective activity. Zentralbl Veterinarmed B, 1995 Aug, 42(6), 361 - 8 Rapid bioassay for detection of Bacillus anthracis in mice; Shlyakhov E et al.; A rapid-bioassay method (RBA) of diagnosing anthrax was developed in mice . A standard RBA consisted of intraperitoneal inoculation of a suspected culture or of a suspension of raw infected animal tissues in four pairs of mice . At 60, 120 and 180 min after inoculation, two mice were killed and smears and impression smears were made from the peritoneal fluid, heart blood, spleen, liver, and kidneys, and then fixed, stained and microscopically examined . Encapsulated rods seen in microscopy confirmed the diagnosis . The remaining pair of mice was kept for a 10-day observation period and served as a control . In the experiments described in this study, RBA showed a high efficiency . A vegetative culture of 18-20 h of Bacillus anthracis growth demonstrated encapsulation in mice as early as 120 min after inoculation; a culture of 3-5 or 6-12 h of growth showed encapsulation at 60 min . Old spores gave encapsulation at 180 min, but, if previously plated for 6 h, encapsulation was observed as early as 60 min after inoculation . Inoculation of raw infected tissues gave positive results at 120 min . As compared with the challenged controls, no deviations between the results of RBA and the final diagnosis of anthrax were observed, but RBA appears to be 7-17 times more rapid . Some variations of the standard RBA are also suggested. Tohoku J Exp Med, 1995 Jul, 176(3), 187 - 90 A case of cutaneous anthrax; Natori N; A 63-year-old man developed black crusts on the parietal scalp that showed mixed infections of dermatophytes and Bacillus anthracis on culture . The lesions improved with bifonazole, griseofulvin and bacampicillin hydrochloride . Although cutaneous anthrax is now a very rare disease, the mortality is high in untreated cases . If a patient has black crusts, anthrax should be differentiated firstly. J Clin Microbiol, 1995 Jul, 33(7), 1847 - 50 Genetic variability of Bacillus anthracis and related species; Harrell LJ et al.; We evaluated the abilities of pulsed-field gel electrophoresis (PFGE) and sequences of intergenic spacer regions (ISRs) between two highly conserved genes, 16S-23S rDNA and gyrB-gyrA ISRs, to detect variation in strains of Bacillus anthracis as well as two closely related species, B . cereus ATCC 14579 and B . mycoides ATCC 6462 . For each restriction enzyme, (NotI, SfiI, and SmaI), the PFGE banding patterns for three B . anthracis strains (Ames, Vollum, and Sterne) were identical . However, closely related species could be differentiated from B . anthracis and from each other . PCR amplification of the 16S-23S rDNA ISR yielded a 143- to 144-bp fragment, showing identical sequences for B . anthracis strains, one nucleotide deletion between B . cerus and B . anthracis, and 13 nucleotide differences between B . mycoides and B . anthracis . The gyrase ISR sequences (121 bp) in B . anthracis strains were also identical, but those in B . cereus and B . mycoides differed from that in B . anthracis by 1 and 2 nucleotides, respectively, and from each other by only 1 nucleotide . Given the diverse geographic origins of these B . anthracis strains, this species is very homogenous . We conclude that methods such as PFGE and sequences of ISRs may be useful in separating B . anthracis from closely related species, but more sensitive methods are needed for strain identification of B . anthracis. Mol Microbiol, 1995 Jun, 16(6), 1171 - 81 The atxA gene product activates transcription of the anthrax toxin genes and is essential for virulence; Dai Z et al.; Bacillus anthracis plasmid pXO1 carries the structural genes for the three anthrax toxin proteins, cya (edema factor), lef (lethal factor), and pag (protective antigen) . Expression of the toxin genes by B . anthracis is enhanced during growth under elevated levels of CO2 . This CO2 effect is observed only in the presence of another pXO1 gene, atxA, which encodes a transactivator of anthrax toxin synthesis . Here we show that transcription of atxA does not appear to differ in cells grown in 5% CO2 compared with cells grown in air . Using a new efficient method for gene replacement in B . anthracis, we constructed an atxA-null mutant in which the atxA-coding sequence on pXO1 is replaced with an omega km-2 cassette . Transcription of all three toxin genes is decreased in the absence of atxA . The pag gene possesses two apparent transcription start sites, P1 and P2; only transcripts with 5' ends mapping to P1 are decreased in the atxA-null mutant . Deletion analysis of the pag promoter region indicates that the 111 bp region upstream of the P1 site is sufficient for atxA-mediated activation of this transcript . The cya and lef genes each have one apparent start site for transcription . Transcripts with 5' ends mapping to these sites are not detected in the atxA-null mutant . The atxA-null mutant is avirulent in mice . Moreover, the antibody response to all three toxin proteins is decreased significantly in atxA-null mutant-infected mice . These data suggest that the atxA gene product also regulates toxin gene expression during infection. Can Vet J, 1995 May, 36(5), 295 - 301 The ecology of anthrax spores: tough but not invincible; Dragon DC et al.; Bacillus anthracis is the causative agent of anthrax, a serious and often fatal disease of wild and domestic animals . Central to the persistence of anthrax in an area is the ability of B . anthracis to form long-lasting, highly resistant spores . Understanding the ecology of anthrax spores is essential if one hopes to control epidemics . Studies on the ecology of anthrax have found a correlation between the disease and specific soil factors, such as alkaline pH, high moisture, and high organic content . Researchers initially suggested that these factors influenced vegetative anthrax bacilli . However, subsequent research has shown that vegetative cells of B . anthracis have very specific nutrient and physiological requirements and are unlikely to survive outside a host . Review of the properties of spores of B . anthracis and other Bacillus species suggests that the specific soil factors linked to epidemic areas reflect important environmental conditions that aid the anthrax spores in causing epidemics . Specifically, high levels of calcium in the soil may help to maintain spore vitality for prolonged periods, thereby increasing the chance of spores encountering and infecting a new host . Cycles of runoff and evaporation may collect spores dispersed from previous epidemics into storage areas, thereby concentrating them . Uptake of large doses of viable spores from storage areas by susceptible animals, via altered feeding or breeding behavior, may then allow the bacterium to establish infection and cause a new epidemic . Literature search for this review was done by scanning the Life Sciences Collection 1982-1994 using the keywords "anthrax" and "calcium and spore." FEMS Microbiol Lett, 1995 May 1, 128(2), 113 - 8 Differentiation of Bacillus anthracis and other 'Bacillus cereus group' bacteria using IS231-derived sequences; Henderson I et al.; Sequences based on the conserved 20 bp inverted repeat of IS231 variants were used as polymerase chain reaction-based fingerprinting primers of the member species of the Bacillus cereus group (B . anthracis, B . cereus, B . thuringiensis and B . mycoides), because of their close association with transposons, principally Tn4430 in B . thuringiensis . Fingerprints of B . anthracis were simple, and specifically allowed its identification and sub-differentiation from other members of the group . Fingerprints for B . cereus were strain-specific; those for B . thuringensis gave a 1650 bp product, characteristic of IS231 variants A-F . The same reaction conditions gave one or two bands for both B . anthracis and B . cereus that differed by restriction endonuclease mapping from the B . thuringiensis PCR product and established IS231 restriction maps; this does not preclude some kind of relationship between these products and IS231. J Bacteriol, 1995 May, 177(9), 2481 - 9 Purification and characterization of the major surface array protein from the avirulent Bacillus anthracis Delta Sterne-1; Farchaus JW et al.; Many prokaryotic organisms possess surface layer (S-layer) proteins that are components of the outermost cell envelope . With immunogold labeling, it was demonstrated that the protein extractable antigen 1 (EA1) was localized on the outer surface and specifically to cell wall fragments from Bacillus anthracis which retained the S layer . When grown in rich medium under aerobic conditions, the avirulent strain Delta Sterne-1 released large amounts of EA1 into the medium . This EA1 had no higher-order structure initially but formed two-dimensional crystals under defined conditions . The released EA1 was purified in aqueous buffers with a three-step procedure and found to have a mass of 95 kDa when subjected to denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . N-terminal sequence data revealed exact identity to the first eight residues of the S-layer protein from B . thuringiensis 4045 . Gel permeation chromatography of the purified EA1 under nondenaturing conditions revealed a single peak corresponding to a mass of approximately 400 kDa, suggesting that a tetramer or dimer of dimers was the primary species in solution . SDS-PAGE of EA1 purified in the absence of protease inhibitors revealed specific proteolytic processing to an 80-kDa form, which immunoreacted with polyclonal anti-EA1 antibodies . This proteolytic cleavage of EA1 to 80 kDa was duplicated with purified EA1 and the protease trypsin or pronase. Microbiol Res, 1995 May, 150(2), 179 - 86 A nested PCR method for the detection of Bacillus anthracis in environmental samples collected from former tannery sites; Beyer W et al.; A nested PCR has been developed to detect Bacillus anthracis spores in natural soil and waste samples which may be heavily contaminated by organic and inorganic compounds as is the case at former tannery sites . Outer and inner pairs of primers were designed from the protective antigen gene of the plasmid pX01 as well as from the genes B and C of the capsule region of the plasmid pX02 . The DNA was prepared from an enrichment broth after killing the vegetative cells by H2O2 treatment . The method allows the detection of less than 10 spores per 100 g of the original soil sample . This is between 10(4)-fold and 10(7)-fold more sensitive than the conventional culture diagnosis. Infect Immun, 1995 Apr, 63(4), 1369 - 72 Protective immunity induced by Bacillus anthracis toxin-deficient strains; Pezard C et al.; The two toxins secreted by Bacillus anthracis are composed of binary combinations of three proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF) . Six mutant strains that are deficient in the production of one or two of these toxin components have been previously constructed and characterized (C . Pezard, E . Duflot, and M . Mock, J . Gen . Microbiol . 139:2459-2463, 1993) . In this work, we examined the antibody response to the in vivo production of PA, LF, and EF in mice immunized with spores of strains producing these proteins . High titers of antibody to PA were observed after immunization with all strains producing PA, while titers of antibodies to EF and LF were weak in animals immunized with strains producing only EF or LF . In contrast, immunization with strains producing either PA and EF or PA and LF resulted in an increased antibody response to EF or LF, respectively . The differing levels of protection from a lethal anthrax challenge afforded to mice immunized with spores of the mutant strains not only confirm the role of PA as the major protective antigen in the humoral response but also indicate a significant contribution of LF and EF to immunoprotection . We observed, however, that PA-deficient strains were also able to provide some protection, thereby suggesting that immune mechanisms other than the humoral response may be involved in immunity to anthrax . Finally, a control strain lacking the toxin-encoding plasmid was unable to provide protection or elicit an antibody response against bacterial antigens, indicating a possible role for pXO1 in the survival of B . anthracis in a host. Lett Appl Microbiol, 1995 Apr, 20(4), 209 - 11 Evaluation of the Biolog system for the identification of Bacillus anthracis; Baillie LW et al.; The potential of the Biolog system for the identification of Bacillus anthracis was evaluated . In-house generated databases allowed the correct identification of 19 of 20 isolates of B . anthracis within 24 h . Five strains of the closely related B . cereus/thuringiensis group were misidentified as B . anthracis . For this reason the test could only serve as a primary screen with further testing being required to confirm identity . In addition 20% of all the strains of bacilli examined during the study gave unreadable reaction profiles due to false-positive reactions. Appl Environ Microbiol, 1995 Apr, 61(4), 1623 - 6 Comparative analysis of the 16S to 23S ribosomal intergenic spacer sequences of Bacillus thuringiensis strains and subspecies and of closely related species; Bourque SN et al.; Bacillus thuringiensis spacer regions between the 16S and 23S rRNAs were amplified with conserved primers, designated 19-mer and 23-mer primers . A spacer region of 144 bp was determined for all of 6 B . thuringiensis strains, 7 B . thuringiensis subspecies, and 11 B . thuringiensis field isolates, as well as for the closely related species Bacillus cereus and Bacillus anthracis . Computer analysis and alignment of nucleotide sequences identified three mutations and one deletion in the intergenic spacer region (ISR) of B . thuringiensis subsp . kurstaki HD-1 when compared with ISR sequences from other subspecies . The same differences were identified between the ISR of B . thuringiensis strains and the ISR of B . cereus and B . anthracis . These minor differences do not seem to be sufficient to allow the design of a species-specific oligonucleotide probe. J Bacteriol, 1995 Feb, 177(3), 614 - 20 Characterization of the Bacillus anthracis S-layer: cloning and sequencing of the structural gene; Etienne-Toumelin I et al.; Bacillus anthracis, a gram-positive, spore-forming bacterium, is the etiological agent of anthrax . The gene coding for the S-layer protein (sap) was cloned on two contiguous fragments in Escherichia coli, and the complete sequence of the structural gene was determined . The protein, Sap, is composed of 814 residues, including a classical prokaryotic 29-amino-acid signal peptide . The mature form has a calculated molecular mass of 83.7 kDa and a molecular mass of 94 kDa on a sodium dodecyl sulfate-polyacrylamide gel . Sap possesses many charged residues, is weakly acidic, and contains only 0.9% methionine and no cysteine residues . The N-terminal region of Sap shares sequence similarities with the Acetogenium kivui S-layer protein, the Bacillus brevis middle wall protein, the Thermotoga maritima Omp alpha protein, and the Bacillus thuringiensis S-layer protein . Electron microscopy observations showed that this S-layer is not observed on B . anthracis cells in which sap has been deleted. Gene, 1995 Jan 11, 152(1), 75 - 8 The transformation frequency of plasmids into Bacillus anthracis is affected by adenine methylation; Marrero R et al.; Plasmids pLTV1 and pHV33, capable of replicating in both Gram+ and Gram- bacterial hosts (shuttle vectors), when derived from the Escherichia coli strain HB101, were inactive in an electro-transformation assay employing the Bacillus anthracis strains delta Ames-1 and delta V1B-1 as recipients . The same plasmids isolated from the DNA methyltransferase (MTase)-deficient E . coli strain GM2929 (dam, dcm), were able to transform the B . anthracis strains at a frequency of 10(2)-10(3) transformants/micrograms of plasmid DNA . Efficient transformation was also obtained when the plasmids were propagated in strains of B . subtilis 168 (10(2)-10(4) transformants/micrograms of plasmid DNA) . The B . subtilis strains used are known to harbor restriction/modification systems that recognize cytosine as a target for methylation . In contrast, no adenine methylation activities have been reported for these strains . The data presented indicate that DNA containing methylated adenine residues is restricted in the B . anthracis strains studied here, resulting in decreased plasmid DNA-mediated transformation frequencies . This inhibition could be alleviated by propagating plasmid species in MTase-deficient (dam) strains of E . coli or B . subtilis 168, before their introduction into strains of B . anthracis. Gene, 1995 Jan 11, 152(1), 1 - 9 Identification and characterization of a trans-activator involved in the regulation of encapsulation by Bacillus anthracis; Vietri NJ et al.; Production of the plasmid-pXO2-encoded capsule by Bacillus anthracis is required for full virulence of the organism . The induction of capsule synthesis in vitro requires growth in the presence of bicarbonate and CO2; however, little else is known about the regulation of capsule synthesis and the role it plays in the expression of virulence . Recently, transposon Tn917 mutagenesis of B . anthracis plasmid pXO2 identified genes involved in capsule production and genes associated with virulence in inbred mice . One mutant, UUP5, had an 8.2-kb deletion located outside of the capsule structural gene region (cap) . UUP5 was reduced significantly in capsule production and in virulence as compared to the wild-type (wt) parental strain . Using a HindIII-generated pXO2 library, we examined fragments contained in the deleted region and showed that electroporation of the mutant with a cloned 2.3-kb HindIII fragment restored capsule production to wt levels . Sequence analysis of the 2.3-kb fragment revealed a 1449-bp open reading frame (ORF) encoding a 483-amino-acid (57 kDa) protein, in good agreement with the 55-kDa protein detected by in vitro transcription/translation . Construction of a frameshift mutant that replaced the 55-kDa protein with a truncated 34-kDa moiety abrogated the complementing activity of the fragment in UUP5 . mRNAs specific for cap and for the 1449-bp ORF were detected in mutant UUP5 transformed with the unaltered fragment and grown in the presence of bicarbonate, but not in air . No cap-specific mRNA, and very low levels of ORF-specific mRNA, were detected in UUP5 containing the frameshift mutation.(ABSTRACT TRUNCATED AT 250 WORDS) Arch Biochem Biophys, 1995 Jan 10, 316(1), 5 - 13 In vitro processing of anthrax toxin protective antigen by recombinant PC1 (SPC3) and bovine intermediate lobe secretory vesicle membranes; Friedman TC et al.; Protective antigen (PA), an 83-kDa protein produced by Bacillus anthracis, requires proteolytic activation at a tetrabasic site (RKKR167) before it can combine with either edema factor or lethal factor on the cell surface . The complex is then endocytosed and the target cell intoxicated . Previous work has demonstrated that furin, a ubiquitously distributed, subtilisin-like protease, can perform this cleavage . In this study, another member of the furin family, PC1 (SPC3), was tested as a putative processing enzyme for PA . Recombinant PC1, partially purified from the medium of stably transfected L-cells, cleaved PA to a 63-kDa fragment (PA63) and a 20-kDa fragment (PA20) . Amino-terminal sequence analysis of the 63 kDa product demonstrated that cleavage occurred between Arg167 and Ser168 . The pH optimum for in vitro PA cleavage was 6.0 and the enzymatic activity was calcium-dependent . Medium from untransfected L-cells did not cleave PA . Site-directed mutagenesis of the tetrabasic cleavage site revealed that PC1 preferred to cleave sequences containing basic residues at positions -1 and -4 relative to the wild-type cleavage site, demonstrating that PC1 can cleave substrates at a monobasic residue site in vitro . Substrates having basic residues at the -1 and -2 positions were cleaved with approximately twofold less efficiency than wild-type PA . Mutants of PA containing basic residues in positions -1 and either -2 or -4 of the cleavage site were predicted to be substrates for PC1 and were more toxic to L-cells expressing PC1 than to untransfected L-cells . These results demonstrate that PA is cleaved by PC1 in vivo . Membranes from bovine intermediate lobe secretory vesicles which contain both prohormone convertases, PC1 and PC2, also cleaved PA to PA63 with a pH optimum of 5.5 . Immunodepletion studies using antisera against PC1 and PC2 showed that these are the enzymes primarily responsible for the cleavage of PA in the membrane preparation . Thus, both recombinant PC1 and a membrane preparation containing endogenous PC1 can activate PA. Biosens Bioelectron, 1995 Summer, 10(6-7), 501 - 7 Sensitive detection of biotoxoids and bacterial spores using an immunomagnetic electrochemiluminescence sensor; Gatto-Menking DL et al.; Extremely sensitive detection of various biotoxoids and bacterial spores using the commercial ORIGEN analyzer was achieved by capture on antibody-conjugated micron sized magnetic beads (MBs) followed by binding of ruthenium (II) trisbipyridal chelate (Ru(bpy)2+3-labelled reporter antibodies . Immunomagnetically captured target materials were collected on a magnet . Electrochemiluminescence (ECL) was evoked from the Ru(bpy)3(2+)-tagged reporter antibodies by application of an electrical potential . Femtogram sensitivity levels were obtained for all biotoxoids tested including botulinus A, cholera beta subunit, ricin and staphylococcal enterotoxoid B by this immunomagnetic (IM)-ECL approach . An IM-ECL assay for Bacillus anthracis spores yielded a detection limit of at least 100 spores . The ECL signal was a function of analyte quantity over several orders of magnitude, but the immunological 'hook' effect at high antigen loads made quantitation impossible over a broader range . All assays were performed with a maximum combined incubation and assay time of approximately 40 min . This work demonstrates the extreme sensitivity of the IM-ECL approach for soluble and particulate antigens. FEMS Microbiol Lett, 1994 Dec 15, 124(3), 343 - 8 Zinc content of the Bacillus anthracis lethal factor; Kochi SK et al.; We present evidence that the anthrax toxin lethal factor binds multiple zinc atoms . Results from atomic adsorption spectroscopy indicate that lethal factor contains approximately three zinc atoms per toxin molecule . Lethal factor treated with EDTA and o-phenanthroline contained a similar number of zinc atoms, indicating that all three zinc atoms are tightly bound to the protein . Lethal factor contains the highly conserved zinc-binding consensus sequence, HExxH, that is present in all known zinc metalloproteases . In addition, lethal factor contains an inverted form of this motif, HxxDH, which may also be involved in zinc binding . Using a large array of protease model substrates, however, we were unable to detect an endogenous protease activity for lethal factor. Epidemiol Infect, 1994 Dec, 113(3), 479 - 90 Information on which to base assessments of risk from environments contaminated with anthrax spores; Watson A et al.; Although there has been a considerable amount of research conducted into Bacillus anthracis, the causative agent of anthrax, the data are widely disseminated in the scientific literature and are therefore not always easy to assimilate . In view of continuing concern about potential anthrax contamination in environmental materials and sites, this review brings together the currently available information relating to the health hazards from B . anthracis . The relevance of the available information for risk assessment purposes is assessed. J Biol Chem, 1994 Nov 18, 269(46), 29039 - 46 The chymotrypsin-sensitive site, FFD315, in anthrax toxin protective antigen is required for translocation of lethal factor; Singh Y et al.; The protective antigen (PA) component of anthrax toxin contains two sites that are uniquely sensitive to proteolytic cleavage . Cleavage at the sequence RKKR167 by the cellular protease furin is absolutely required for toxicity, whereas cleavage by chymotrypsin or thermolysin at the sequence FFD315 inactivates the protein, apparently by blocking the ability of PA to translocate the catalytic moieties of the toxins, lethal factor (LF) and edema factor (EF), to the cytosol of eukaryotic cells . To specify the role of the chymotrypsin-sensitive site of PA in the translocation of LF, we altered residues 313-315 . None of the mutations in this region interfered with the ability of PA to bind to its cellular receptor, be cleaved by cell surface furin, and bind LF . Substitution of Ala for Asp315 or for both Phe313 and Phe314 reduced the ability of PA to intoxicate cells in the presence of LF by 3- and 7-fold, respectively . Substitution of Phe313 by Cys greatly reduced the rate of LF translocation and delayed toxicity . The rate at which the Cys-substituted PA killed cells was increased significantly by blocking the sulfhydryl group with iodoacetamide, suggesting that this added Cys interacts with cellular proteins and slows translocation of LF . Deletion of the 2 Phe rendered PA completely non-toxic . This deleted PA protein lacked the ability shown by native PA to form oligomers on cells and in solution and to induce release of 86Rb from Chinese hamster ovary cells . These results suggest that the chymotrypsin-sensitive site in PA is required for membrane channel formation and translocation of LF into the cytosol . PA double mutants were constructed that cannot be cleaved at either the furin or chymotrypsin sites . These PA proteins were more stable in Bacillus anthracis culture supernatants and may therefore be useful as a replacement for PA in anthrax vaccines. Mol Med, 1994 Nov, 1(1), 7 - 18 Role of macrophage oxidative burst in the action of anthrax lethal toxin; Hanna PC et al.; BACKGROUND: Major symptoms and death from systemic Bacillus anthracis infections are mediated by the action of the pathogen's lethal toxin on host macrophages . High levels of the toxin are cytolytic to macrophages, whereas low levels stimulate these cells to produce cytokines (interleukin-1 beta and tumor necrosis factor-alpha), which induce systemic shock and death . MATERIALS AND METHODS: Experiments were performed to assess the possibility that the oxidative burst may be involved in one or both of lethal toxin's effects on macrophages . Toximediated cell lysis, superoxide anion and cytokine production were measured . Effects of antioxidants and macrophage mutations were examined . RESULTS: RAW264.7 murine macrophages treated with high levels of toxin released large amounts of superoxide anion, beginning at about 1 hr, which correlates with the onset of cytolysis . Cytolysis could be blocked with various exogenous antioxidants or with N-acetyl-L-cysteine and methionine, which promote production of the endogenous antioxidant, glutathione . Mutant murine macrophage lines deficient in production of reactive oxygen intermediates (ROIs) were relatively insensitive to the lytic effects of the toxin, whereas a line with increased oxidative burst potential showed elevated sensitivity . Also, cultured blood monocyte-derived macrophages from a patient with Chronic Granulomatous Disease, a disorder in which the phagocyte's oxidative burst is disabled, were totally resistant to toxin, in contrast to control monocytes . CONCLUSIONS: These results imply that the cytolytic effect of the toxin is mediated by ROIs . Additionally, cytokine production and consequent pathologies showed partial dependence on macrophage ROIs . Antioxidants moderately inhibited toxin-induced cytokine production in vitro, and BALB/c mice pretreated with N-acetyl-L-cysteine or mepacrine showed partial protection against lethal toxin . Thus ROIs are involved in both the cytolytic action of anthrax lethal toxin and the overall pathologic process in vivo. Vaccine, 1994 Nov, 12(15), 1395 - 401 Bacillus anthracis protective antigen, expressed in Salmonella typhimurium SL 3261, affords protection against anthrax spore challenge; Coulson NM et al.; The protective antigen (PA) gene from Bacillus anthracis has been expressed in Salmonella typhimurium SL 3261 (aroA) . Expression was achieved by cloning the gene after the plac promoter in a high copy number plasmid . The recombinant PA was exported into the periplasm . This construct was unstable in vivo and also reduced the colonization ability of the host S . typhimurium . Mouse-passaging of the recombinant Salmonella resulted in a strain with enhanced colonization ability and increased stability of the plasmid in vivo . This effect appeared to be due to a reduction in copy number of the PA-encoding plasmid . Mice were vaccinated with recombinant S . typhimurium and adjuvanted PA and challenged with virulent B . anthracis . Only mice vaccinated with adjuvanted PA or orally with the mouse-passaged recombinant showed partial protection . The degree of protection observed after oral vaccination with the recombinant S . typhimurium was similar to the degree of protection afforded by adjuvanted PA and suggested that the use of S . typhimurium to deliver PA is an effective approach for inducing protection against B . anthracis . The results presented also suggest that the degree of protection demonstrated in the mouse may not fully indicate the potential of the recombinant Salmonella as an effective vaccine in other species. Mol Microbiol, 1994 Nov, 14(3), 411 - 26 Characterization of spo0A homologues in diverse Bacillus and Clostridium species identifies a probable DNA-binding domain; Brown DP et al.; Spo0A is a phosphorylation-activated transcription factor of Bacillus subtilis . It is a member of the response regulator superfamily of bacterial signal transduction proteins and controls many of the changes in gene expression that occur during the transition into stationary phase and during the initiation of sporulation . To identify the domains of Spo0A most critical for determining its structural and functional features, presumptive homologues of the spo0A gene were characterized in a collection of eight Bacillus species and six Clostridium species representing phylogenetically diverse members of these genera . An alignment of the partial or complete DNA sequences of these homologues revealed three regions of especially high conservation in the effector domain . We speculate that the most highly conserved of these corresponds to the recognition helix of a putative helix-turn-helix motif, and, therefore, represents the actual DNA-containing surface of the protein . In the case of homologues identified in Bacillus anthracis and Clostridium acetobutylicum and retrieved by polymerase chain reaction amplification, we confirmed by gene-disruption analysis that the homologue actually is required for initiation of sporulation . Apparent homologues of the B . subtilis spoIVB gene were also discovered immediately upstream from the spo0A homologues in all Bacillus and Clostridium species examined . The discovery of homologues of B . subtilis sporulation genes in these diverse species implies that the gene products required for specifying pathways of sporulation-specific gene activation and for determining key morphogenetic changes may be highly conserved and suggests that an approach similar to that undertaken here might be used as a general strategy to retrieve and compare their gene sequences . Exhaustive efforts to detect a spo0A-like gene in non-endospore formers, including close relatives of Bacillus such as Listeria and Staphylococcus, were uniformly unsuccessful, suggesting that regulation of gene activity during the transition into stationary phase mediated by Spo0A-like proteins may be exclusive to the endospore-forming bacteria. Infect Immun, 1994 Oct, 62(10), 4432 - 9 Anthrax edema toxin differentially regulates lipopolysaccharide-induced monocyte production of tumor necrosis factor alpha and interleukin-6 by increasing intracellular cyclic AMP; Hoover DL et al.; Bacillus anthracis exotoxins mediate most of the symptomatology of severe anthrax . In addition to a clinical syndrome reminiscent of septic shock, which may be mediated by cytokines produced by macrophages stimulated with lethal toxin, infected patients show profound edema at sites of infection . Edema is mediated by edema toxin (ET), which comprises of a binding molecule, protective antigen, and an active moiety, edema factor, which possesses intrinsic adenylyl cyclase activity . Intracellular cyclic AMP (cAMP) regulates the production of several cytokines that modulate edema formation and play important roles in host defense against invading bacteria . To determine whether ET enhanced the accumulation of cAMP in monocytes and thereby influenced cytokine production, we cultured human monocytes with endotoxin (lipopolysaccharide {LPS}) and dilutions of ET and determined the levels of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) in culture supernatant fluids . We further estimated cytokine-specific mRNA accumulation in monocytes by reverse transcription PCR and examined intracellular cAMP concentrations following treatment with ET . ET and LPS each induced monocytes to secrete comparable amounts of IL-6 . ET did not inhibit and in most experiments modestly enhanced LPS-induced IL-6 production . In contrast to this stimulatory effect on IL-6 production, ET induced little or no TNF-alpha production . Moreover, ET profoundly inhibited LPS-induced TNF-alpha synthesis . These regulatory phenomena were also observed at the mRNA level in association with dose-related enhancement of intracellular cAMP in ET-treated monocytes . Monocytes treated with dibutyryl cAMP, an active analog of cAMP, produced cytokines in a pattern identical to that of cells treated with ET . The disruption of cytokine networks as a consequence of unregulated, ET-induced cAMP accumulation in human monocytes may impair cellular antimicrobial responses and contribute to clinical signs and symptoms. Lett Appl Microbiol, 1994 Oct, 19(4), 225 - 7 Evaluation of Bacillus subtilis strain IS53 for the production of Bacillus anthracis protective antigen; Baillie LW et al.; An asporogenous strain of Bacillus subtilis, IS53, transformed with plasmid pPA102, produces the protective antigen (PA) component of the tripartite toxin of B . anthracis . Addition of yeast extract was required to support growth and PA production in all the media examined . Protective antigen expression was down-regulated during exponential growth and extracellular proteases caused marked degradation of the mature protein. J Bacteriol, 1994 Aug, 176(16), 5188 - 92 The three Bacillus anthracis toxin genes are coordinately regulated by bicarbonate and temperature; Sirard JC et al.; The two Bacillus anthracis toxins are composed of three proteins, protective antigen, lethal factor, and edema factor . The structural genes for these three components are located on the virulence plasmid pXO1 . We constructed transcriptional fusions between the regulatory region of each of these genes and lacZ . Each construct was then inserted as a single copy at the corresponding toxin gene locus on pXO1, resulting in three isogenic strains . Two environmental factors, bicarbonate and temperature, were found to induce beta-galactosidase synthesis in each recombinant strain . Furthermore, the transcription of the three toxin genes appears to be coordinately regulated. Vaccine, 1994 Aug, 12(10), 872 - 4 Efficacy of a standard human anthrax vaccine against Bacillus anthracis spore challenge in guinea-pigs; Ivins BE et al.; The efficacy of an anthrax vaccine licensed for human use, MDPH-PA, was tested in guinea-pigs intramuscularly challenged with 10, 100 or 1000 LD50 of spores from two virulent strains of Bacillus anthracis, Vollum 1B and Ames . As demonstrated in other investigations, immunization with MDPH-PA provided better protection against challenge with the Vollum 1B strain than with the Ames strain, although vaccine efficacy against the Ames strain was better than previously reported . Enzyme-linked immunosorbent assay of serum antibody titres to B . anthracis protective antigen showed that there was no significant correlation between survival and antibody titres. Infection, 1994 Jul-Aug, 22(4), 281 - 2 Impaired neutrophil function in the cutaneous form of anthrax; Alexeyev OA et al.; Spontaneous and Bacillus anthracis induced luminol-dependent chemiluminescence of neutrophils was studied in six patients with the cutaneous form of anthrax . Spontaneous chemiluminescence in anthrax was decreased compared to the healthy controls (p < 0.05) . B . anthracis, opsonized by complement-containing sera from patients, induced chemiluminescence in neutrophils from control donors but not from patients . B . anthracis, opsonized by complement-free sera from the patients, did not cause an increase in chemiluminescence response in either the patient or the control neutrophils . Also, B . anthracis, opsonized with normal sera and nonopsonized, did not induce chemiluminescence in either patient or control neutrophils . It was concluded that oxidative metabolism by neutrophils is impaired in anthrax, whereas the functional capacity of antibodies seems to be unaffected. Antibiot Khimioter, 1994 Jun, 39(6), 15 - 9 {Comparative evaluation of the effectiveness of fluoroquinolones in experimental anthrax infection}; D'iakov SI et al.; Comparative antibacterial activity and protective efficacy of ciprofloxacin, pefloxacin and lomefloxacin were estimated in a model of anthrax . The MICs of the three drugs determined by the method of serial dilutions for three vaccinal strains of Bacillus anthracis were 0.5 to 1.0 microgram/ml . The protective efficacy of the chemotherapeutics in experimental anthrax induced by the spores of the vaccinal strain 71/12, Tsenkovsky was evaluated in mathematically designed four-factor experiments . It depended on the infective dose and the chemotherapy term and amounted in the protective use of the drugs in the daily doses, equivalent to those for humans, to 80-100-percent protection of the animals infected with 10 LD50 of the biological agent, to 50-80-percent protection with the use of 100 LD50, to 40-70-percent protection with the use of 1000 LD50 and to 50-90-percent protection in the therapeutic use of the fluoroquinolones . This was indicative of the fact that the fluoroquinolones were chemotherapeutically highly active in the treatment of experimental anthrax . The marked therapeutic efficacy of the fluoroquinolones and the high percentage of the animal protection after their urgent prophylactic use in a single dose are their obvious merits . The total values of the protective efficacy of the three fluoroquinolones with respect to anthrax were practically the same. Appl Environ Microbiol, 1994 May, 60(5), 1622 - 5 Identification of capsule-forming Bacillus anthracis spores with the PCR and a novel dual-probe hybridization format; Reif TC et al.; Anthrax is a fatal infection of humans and livestock that is caused by the gram-positive bacterium Bacillus anthracis . The virulent strains of B . anthracis are encapsulated and toxigenic . In this paper we describe the development of a PCR technique for identifying spores of B . anthracis . Two 20-mer oligonucleotide primers specific for the capB region of 60-MDa plasmid pXO2 were used for amplification . The amplification products were detected by using biotin- and fluorescein-labeled probes in a novel dual-probe hybridization format . Using the combination of PCR amplification and dual-probe hybridization, we detected two copies of the bacterial genome . Because the PCR assay could detect a minimum of 100 unprocessed spores per PCR mixture, we attempted to facilitate the release of DNA by comparing the effect of limited spore germination with the effect of mechanical spore disruption prior to PCR amplification . The two methods were equally effective and allowed us to identify single spores of B . anthracis in PCR mixtures. Microb Pathog, 1994 Apr, 16(4), 305 - 11 Effect of different plasmids on colonization of mouse tissues by the aromatic amino acid dependent Salmonella typhimurium SL 3261; Coulson NM et al.; The stability of plasmids pBR322, pUC19 and pBluescript and their effect on bacterial colonization of mice was determined . S . typhimurium SL 3261 carrying high copy number plasmids colonized spleen and liver tissues poorly compared to low copy number plasmids . After inoculation into mice, the stability of the plasmids appeared to be inversely related to the plasmid's size and complexity . Mouse-passaging a pBluescript-based recombinant plasmid expressing the Protective Antigen of Bacillus anthracis selected for a mutant S . typhimurium strain (designated G3) that colonized at high levels and more stably maintained plasmids than S . typhimurium SL 3261 . S . typhimurium G3 down-regulated the copy number of ColE1 plasmids . The significance of these data for vaccine design is discussed. Biochem Biophys Res Commun, 1994 Mar 15, 199(2), 676 - 82 Structure-function analysis of Bacillus anthracis edema factor by using monoclonal antibodies; Little SF et al.; Edema toxin of Bacillus anthracis is composed of protective antigen (PA) and edema factor (EF), a calcium- and calmodulin-dependent adenylate cyclase . At least five different antigenic regions of EF were identified using a competitive-binding, enzyme-linked immunosorbent assay of paired monoclonal antibodies (mAbs) . Two mAbs, 9F5 and 7G10, inhibited the binding of 125I-EF to cell-bound PA . However, only 9F5 inhibited the elongation response of Chinese hamster ovary cells in the presence of edema toxin . Cleavage of EF at the two aspartic acid-proline residues by acid hydrolysis resulted in three fragments: a C-terminal 17 kDa fragment, a central 53 kDa fragment, and an N-terminal 18 kDa fragment . Immunoblots of EF cleaved by formic acid mapped mAbs 9F5 and 7G10 to the N-terminal 18 kDa fragment, mAb 1E6 to the C-terminal 17 kDa fragment, and the remaining 7 mAbs to the central 53 kDa fragment . mAbs 7G10 and 9F5 defined an antigenic region within amino acids 1-156 of EF which is involved in interaction with PA in forming edema toxin. Presse Med, 1994 Mar 12, 23(10), 477 - 8 {Visceral form of human anthrax imported from Africa}; Paulet R et al.; Widespread vaccination has largely eliminated anthrax in Europe (the last case was reported in France in 1972) but the disease remains endemic in many developing countries . The usual cutaneous presentation (malignant pustules) is much more familiar than the various visceral manifestations including digestive tract, pulmonary or meningeal signs . We report a case of a 33-year-old immigrant living in France who was hospitalized for asthenia, dyspnoea, mucopurulant expectoration and moderate diarrhoea 3 days after a 3-month stay in Senegal and Gambia . The temperature was 39 degrees C at admission and blood pressure 110/70 mmHg . Crepitants were heard at the base of the right lung and the rest of the physical examination was normal . Blood was drawn for culture . Laboratory tests and the chest X-ray led to the diagnosis of pneumopathy and a treatment of amoxicillin and clavulanic acid was given with oxygenotherapy . The patient's temperature returned to normal but over the next 48 hours the dyspnoea worsened together with the black diarrhoea . The abdomen was painful . There were no skin lesions . The chest X-ray revealed an extension of the bilateral pulmonary images and bilateral pleural effusion . Laboratory tests revealed thrombopenia (platelet count 38,000/mm3) hyperleukocytosis (WBC 48,000/mm3) and haemolysis (Hb 4 milligrams) . The diagnosis was made on the basis of the initial blood cultures which were positive for Bacillus anthracis . All other samples were negative, including HIV serology . Despite adapted antibiotic therapy (penicillin G, 8MU/day, was initiated on day 2), multiple organ failure occurred with septic shock and pulmonary oedema . The patient died in the intensive care unit on day 7 . Fatal outcome due to anthrax is described in 25% of the visceral forms but reaches 100% in cases of septicaemia . The haemolysis observed in this case is not mentioned in the classical descriptions of anthrax . When treating septic syndromes in patients who have returned from endemic zones, clinicians should entertain the diagnosis of anthrax since the risk of fatal outcome is increased greatly in case of delayed diagnosis. Biochemistry, 1994 Mar 8, 33(9), 2604 - 9 The effects of pH on the interaction of anthrax toxin lethal and edema factors with phospholipid vesicles; Kochi SK et al.; Bacillus anthracis secretes three distinct proteins which interact in binary combinations to produce two toxins . The two effector moieties, edema factor (EF) and lethal factor (LF), interact competitively with the cell receptor-binding moiety, protective antigen (PA), to produce biologically distinct effects . The passage of the toxins through an acidified endosomal compartment is an essential step in the intoxication process, and it has been shown that low pH triggers the insertion of the activated form of PA, PA63, into model lipid bilayers . In this study, we have examined the effects of pH on the interaction of LF and EF with a model membrane system . Protein labeling by radioactive phospholipid probes indicated that both LF and EF are able to insert into asolectin lipid bilayers in a pH-dependent manner . For LF, the extent of insertion into the bilayer was accompanied in parallel by the release of calcein from preloaded LUV (large unilamellar vesicles) . The transition pH for protein insertion, however, was somewhat higher than that for membrane destabilization . The extent of protein radiolabeling and the release of calcein from LUV incubated with EF was similar to that seen with LF; however, the pH dependency was significantly less . Low pH-induced membrane insertion by both proteins was accompanied by only a minimal change in conformation . These results suggest that LF and EF may be actively involved in the process of toxin translocation. J Bacteriol, 1994 Feb, 176(3), 586 - 95 Regulation of the Bacillus anthracis protective antigen gene: CO2 and a trans-acting element activate transcription from one of two promoters; Koehler TM et al.; The pag gene of Bacillus anthracis, located on plasmid pXO1 (185 kb), encodes protective antigen, a component of the anthrax lethal and edema toxins . Synthesis of protective antigen is enhanced during growth of the organism with elevated levels of CO2 . The CO2 effect is at the level of transcription, and pXO1-encoded regulatory factors have been implicated in control of pag expression . We used a Tn917-LTV3 insertion mutant of B . anthracis in which the wild-type pag gene on pXO1 was replaced with a pag-lacZ transcriptional fusion to monitor pag promoter activity . Expression of the pag-lacZ fusion is induced five- to eightfold during growth in 5% CO2 compared with growth in air . Growth in 20% CO2 increases transcription up to 19-fold . By monitoring pag-lacZ expression in atmospheres with different O2 and CO2 concentrations, we demonstrated definitively that the CO2 effect is specific and not simply a result of increased anaerobiosis . The results of 5' end mapping of pag transcripts indicate multiple sites of transcript initiation . We have determined two major apparent start sites, designated P1 and P2, located at positions -58 and -26 relative to the translation initiation codon, respectively . Analysis of total RNA from late-log-phase cells shows comparable initiation from P1 and P2 in wild-type strains grown in aerobic conditions . However, initiation from P1 is increased approximately 10-fold in cultures grown with an elevated level (5%) of CO2 . We have identified a locus on pXO1, more than 13 kb upstream from the pag gene, which enhances pag transcription . When added in trans, this locus increases the level of transcripts with 5' ends mapping to P1 but has no effect on the level of transcripts with 5' ends mapping to P2 . The CO2 effect on P1 is observed only in the presence of the activator locus. Mol Microbiol, 1994 Feb, 11(3), 471 - 9 Bacillus anthracis pXO1 virulence plasmid encodes a type 1 DNA topoisomerase; Fouet A et al.; The virulence plasmid pXO1 is responsible for toxin production in Bacillus anthracis . A DNA fragment from pXO1 was isolated and was shown, by sequence analysis, to contain part of a type 1 DNA topoisomerase gene . Attempts to clone the entire wild-type gene, designated topX, in Escherichia coli, were unsuccessful . In order to obtain the complete gene, it was first insertionally inactivated and then cloned in the mutated form . The deduced amino acid sequence of Topo X1 shows similarities to that of the two E . coli type 1 DNA topoisomerases . The N-terminal two-thirds of the putative B . anthracis protein exhibits strongest sequence similarity to topoisomerase III, whereas the C-terminal portion contains cysteine residues that could form three zinc-binding domains, as they do in topoisomerase I . The suggested active-site tyrosine is conserved in all three proteins . The regulation of expression from the topX promoter is modified by addition of a gyrase inhibiting antibiotic . The Topo X1 protein is likely to be involved in the stability of pXO1. Med Trop (Mars), 1994, 54(1), 33 - 7 {Delayed hypersensitivity after anthrax vaccination . I--Study of guinea pigs vaccinated against anthrax}; Shlyakhov E et al.; To evaluate delayed hypersensitivity after anthrax vaccination, an Anthraxin skin test was performed in 682 guinea pigs at various times after immunization with veterinary unencapsulated active anthrax vaccine . Results were compared with those obtained in unimmunized control guinea pigs (n = 216), in guinea pigs that received a non-immunizing dose of live vaccine (n = 183) and in guinea pigs inoculated with inactivated vaccine (n = 120) . Anthraxin skin tests were positive in the first postvaccination days . The incidence and intensity of positive tests peaked between two weeks and one month after vaccination and then gradually decreased during the first year . Study of resistance of guinea pigs to an inoculum at a lethal dose of a virulent strain of Bacillus anthracis showed a close correlation between positive tests and resistance . These findings demonstrate development of cell-mediated immunity after anthrax vaccination . The Anthraxin skin test should have practical applications for the production of vaccines and for evaluation of the immune status of vaccinated livestock {corrected}. Mol Gen Mikrobiol Virusol, 1994 Jan-Feb, (1), 30 - 3 {A species-specific DNA probe for identifying toxic strains of anthrax pathogens}; Timoshin VB et al.; On the plasmid DNA pOX01 of the anthrax pathogen two BamHI fragments were localized which facilitate detection of the Bacillus anthracis strains carrying pXO1 replicon . These fragments, after complete hydrolysis of plasmid DNA by HindIII, were cloned on the vector plasmids pUC19 and pBR322 by the "shot-gun" method in Escherichia coli cells . It is shown that the 900 bp BamHI/HindIII fragment from the pZAT1 recombinant plasmid has an ability for specific hybridization with DNA of toxigenic strains of B . anthracis and could be used as species-specific anthracic DNA probe which identifies toxigenic strains of the anthrax pathogen differentiating it from the other species of Bacillus genus as well as from the bacteria of other taxonomy groups. Int J Syst Bacteriol, 1994 Jan, 44(1), 99 - 105 Differentiation of Bacillus anthracis from other Bacillus cereus group bacteria with the PCR; Henderson I et al.; Variation among isolates of Bacillus anthracis was examined by using restriction fragmentation patterns and the PCR performed with arbitrary and sequence-specific oligonucleotide primers . The patterns were compared with the patterns generated from strains of closely related species belonging to the "Bacillus cereus group" of bacteria, including B . cereus, Bacillus thuringiensis, and Bacillus mycoides . All B . anthracis profiles were identical for each of 18 restriction enzymes, each of 10 arbitrary PCR primers, and a repetitive extragenic palindrome-specific PCR primer . The PCR profiles generated with a coliphage M13-based primer exhibited slight pattern variation in a 400- to 500-bp band region . The B . anthracis profiles were unique compared with the profiles of the other species examined . In these other species, strain-to-strain variations were observed . Our results showed that isolates of B . anthracis are almost completely homogeneous, indicating a clonal lineage, and are distinct from other members of the B . cereus group and that B . anthracis, as a species in its own right, may have evolved only relatively recently. Clin Diagn Lab Immunol, 1994 Jan, 1(1), 78 - 82 Enzyme-linked immunosorbent assay using a recombinant baculovirus-expressed Bacillus anthracis protective antigen (PA): measurement of human anti-PA antibodies; Iacono-Connors LC et al.; We developed an antigen capture enzyme-linked immunosorbent assay (ELISA) which does not require purified protective antigen (PA) for detection of human antibodies to Bacillus anthracis PA . Lysates of Spodoptera frugiperda (Sf-9) cells infected with recombinant baculovirus containing the PA gene were used as the source of PA to develop the ELISA . Recombinant PA from crude Sf-9 cell lysates or PA purified from B . anthracis Sterne strain was captured by an anti-PA monoclonal antibody coated onto microtiter plates . We demonstrated that human serum antibody titers to PA were identical in the ELISA whether we used crude Sf-9 cell lysates containing recombinant baculovirus-expressed PA or purified Sterne PA . Finally, false-positive results observed in a direct ELISA were eliminated with this antigen capture ELISA . Thus, the antigen capture ELISA with crude preparations of baculovirus-expressed PA is reliable, safe, and inexpensive for determining anti-PA antibody levels in human sera. Mol Gen Mikrobiol Virusol, 1993 Nov-Dec, (6), 27 - 9 Quantitation of Bacillus anthracis by using of soybean agglutinin conjugates; Kalinin NL et al.; We examine the possibility of using the soybean agglutinin (SBA) marked by peroxidase (HRP), biotin, FITC, or gold in order to determine the number of Bacillus anthracis cells of vaccine strain STI . It was shown that the technique based on interaction between the lectin and microbial cell walls likely are not inferior in sensitivity to traditional ELISA variants . The sensitivities of methods were 10(4) cells/ml in the case of SBA-biotin, 10(5) cells/ml in the case of SBA-HRP, or 10(6) cells/ml in the cases of SBA-gold and SBA-FITC. J Appl Bacteriol, 1993 Nov, 75(5), 463 - 72 The development and assessment of DNA and oligonucleotide probes for the specific detection of Bacillus anthracis; Hutson RA et al.; Two DNA probes and a number of oligonucleotide probes were designed from the virulence factor genes of Bacillus anthracis . These probes were tested for specificity against 52 B . anthracis strains and 233 Bacillus strains encompassing 23 other species . A rapid slot blotting technique was used for screening the large numbers of isolates involved . All probes tested appeared to be specific for B . anthracis under high stringency conditions . These probes could differentiate between virulent and avirulent strains . The probes were also applied to the detection of B . anthracis in routine environmental and clinical samples . A non-radioactive hybridization and detection system based on digoxigenin-11-dUTP was developed. Proc Natl Acad Sci U S A, 1993 Nov 1, 90(21), 10198 - 201 On the role of macrophages in anthrax; Hanna PC et al.; Bacillus anthracis, the causative agent of anthrax, produces systemic shock and death in susceptible animals, primarily through the action of its lethal toxin . This toxin, at high concentrations, induces lysis of macrophages in vitro but shows little or no effect on other cells . We found that when mice were specifically depleted of macrophages by silica injections, they became resistant to the toxin . Sensitivity could be restored by coinjection of toxin-sensitive cultured macrophages (RAW 264.7 cells) but not by coinjection of other cell lines tested . These results implied that macrophages mediate the action of lethal toxin in vivo and led us to investigate their role in death of the mammalian host . Sublytic concentrations of lethal toxin, orders of magnitude lower than those required to induce lysis of RAW 264.7 cells, were found to induce these cells to express interleukin 1 (IL-1) and tumor necrosis factor in vitro . Passive immunization against IL-1 or injection of an IL-1 receptor antagonist protected mice from toxin challenge, whereas anti-tumor necrosis factor provided little, if any, protection . These results imply that systemic shock and death from anthrax result primarily from the effects of high levels of cytokines, principally IL-1, produced by macrophages that have been stimulated by the anthrax lethal toxin. Clin Microbiol Rev, 1993 Oct, 6(4), 324 - 38 Bacillus cereus and related species; Drobniewski FA; Bacillus cereus is a gram-positive aerobic or facultatively anaerobic spore-forming rod . It is a cause of food poisoning, which is frequently associated with the consumption of rice-based dishes . The organism produces an emetic or diarrheal syndrome induced by an emetic toxin and enterotoxin, respectively . Other toxins are produced during growth, including phospholipases, proteases, and hemolysins, one of which, cereolysin, is a thiol-activated hemolysin . These toxins may contribute to the pathogenicity of B . cereus in nongastrointestinal disease . B . cereus isolated from clinical material other than feces or vomitus was commonly dismissed as a contaminant, but increasingly it is being recognized as a species with pathogenic potential . It is now recognized as an infrequent cause of serious nongastrointestinal infection, particularly in drug addicts, the immunosuppressed, neonates, and postsurgical patients, especially when prosthetic implants such as ventricular shunts are inserted . Ocular infections are the commonest types of severe infection, including endophthalmitis, panophthalmitis, and keratitis, usually with the characteristic formation of corneal ring abscesses . Even with prompt surgical and antimicrobial agent treatment, enucleation of the eye and blindness are common sequelae . Septicemia, meningitis, endocarditis, osteomyelitis, and surgical and traumatic wound infections are other manifestations of severe disease . B . cereus produces beta-lactamases, unlike Bacillus anthracis, and so is resistant to beta-lactam antibiotics; it is usually susceptible to treatment with clindamycin, vancomycin, gentamicin, chloramphenicol, and erythromycin . Simultaneous therapy via multiple routes may be required. J Gen Microbiol, 1993 Oct, 139 ( Pt 10), 2459 - 63 Construction of Bacillus anthracis mutant strains producing a single toxin component; Pezard C et al.; The two protein exotoxins secreted by Bacillus anthracis are composed of three distinct components: protective antigen (PA), lethal factor (LF), and (o)edema factor (EF) . We have developed a genetic strategy that permits us selectively to inactivate each of the genes coding for PA, EF or LF . This strategy involved the deletion of a portion of the structural gene and the insertion of an antibiotic resistance cassette . With this technique, double mutant strains of B . anthracis producing only one toxin component have been constructed . Characterization of the mutant strains indicated that they produced the expected single toxin protein . Using a simple, two-step protocol, we have purified PA, LF and EF to homogeneity from culture supernatants . These three mutant strains are potentially powerful tools for studying the individual effect of each toxin component in vitro and in vivo. J Bacteriol, 1993 Sep, 175(17), 5329 - 38 Cloning and characterization of a gene whose product is a trans-activator of anthrax toxin synthesis; Uchida I et al.; The 184-kb Bacillus anthracis plasmid pXO1, which is required for virulence, contains three genes encoding the protein components of anthrax toxin, cya (edema factor gene), lef (lethal factor gene), and pag (protective antigen gene) . Expression of the three proteins is induced by bicarbonate or serum . Using a pag-lacZ transcriptional construct to measure pag promoter activity, we cloned in Bacillus subtilis a gene (atxA) whose product acts in trans to stimulate anthrax toxin expression . Deletion analysis located atxA on a 2.0-kb fragment between cya and pag . DNA sequencing identified one open reading frame encoding 476 amino acids with a predicted M(r) of 55,673, in good agreement with the value of 53 kDa obtained by in vitro transcription-translation analysis . The cloned atxA gene complemented previously characterized Tn917 insertion mutants UM23 tp29 and UM23 tp32 (J . M . Hornung and C . B . Thorne, Abstr . 91st Gen . Meet . Am . Soc . Microbiol . 1991, abstr . D-121, p . 98), which are deficient in synthesis of all three toxin proteins . These results demonstrate that the atxA product activates not only transcription of pag but also that of cya and lef . beta-Galactosidase synthesis from the pag-lacZ transcriptional fusion construct introduced into an insertion mutant (UM23 tp62) which does not require bicarbonate for toxin synthesis indicated that additional regulatory genes other than atxA play a role in the induction of anthrax toxin gene expression by bicarbonate. Trends Microbiol, 1993 Aug, 1(5), 187 - 92 Calmodulin-activated bacterial adenylate cyclases as virulence factors; Mock M et al.; Bordetella pertussis and Bacillus anthracis each produce a virulence-associated, calmodulin-dependent adenylate cyclase toxin, which generates increased levels of cyclic AMP in eukaryotic cells . The two proteins share sequence similarities in their catalytic domains . The remaining regions display different structural and functional organizations that account for the differences both in interaction of the two toxins with target cells and in the resulting disease symptoms. Mol Microbiol, 1993 Aug, 9(3), 487 - 96 Identification of a novel gene, dep, associated with depolymerization of the capsular polymer in Bacillus anthracis; Uchida I et al.; 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. Antibiot Khimioter, 1993 Aug-Sep, 38(8-9), 34 - 8 {Characterization of a Rif-R population of Bacillus anthracis}; Pomerantsev AP et al.; Formation of spontaneous RifR mutants was detected in the populations of various strains of Bacillus anthracis (STI-1, Sterne and CH-7) at a rate of 10(-8) per 1 CFU . The levels of the rifampicin resistance in the mutants were different, the MIC ranged from 16 to 512 micrograms/ml . The clones of the RifR population of the virulent strain CH-7 were heterogeneous in the morphological properties of the colonies and cells, the capacity for the synthesis of the toxin and capsule, the sporulation and virulence . The heterogeneity did not correlate with the levels of the antibiotic resistance . Among the clones of the RifR population there were detected deletion variants by the capacity for the synthesis of the toxin and capsule along with the complete ones . The rifampicin therapy of the infection caused by the complete clone was not efficient . The RifR mutation in B . anthracis did not result in cross resistance to penicillins, cephalosporins, tetracyclines, aminoglycosides, macrolides and chloramphenicol. Laryngorhinootologie, 1993 Jul, 72(7), 350 - 1 {Oropharyngeal anthrax}; Onerci M et al.; We report on a 46-year old man suffering from the oropharyngeal form of human anthrax . The patient presented with a sore throat and extensive swelling of the right neck, the oropharynx and especially the right tonsil . The skin of the mandibular and submandibular region showed vesicular lesions followed by ulcerations resulting in a blackish eschar . The diagnosis was verified by bacteriological culture . Thus, in cases of known exposure, in areas where anthrax is endemic or in immigrant workers coming from such areas, infection with bacillus anthracis should be included in the differential diagnosis of inflammatory oedematous lesions of the oropharynx. Antibiot Khimioter, 1993 Jul, 38(7), 30 - 3 {Interaction of bacillus anthracis with benzylpenicillin in vivo and in vitro}; Pomerantsev AP et al.; Interaction of the cells of Bacillus anthracis strain CH-7 with benzylpenicillin was studied . The cells of strain CH-7 were shown to contain the penicillinase gene in the repressed state . Spontaneous derepression of the gene at a rate of 10(-8) resulting in the synthesis of penicillinase was observed . Penicillinase was synthesized constitutionally and its synthesis did not depend on the presence of benzylpenicillin in the cultivation medium . The therapeutic effect of benzylpenicillin in the treatment of the experimental infection induced by the B . anthracis strain producing penicillinase was estimated . The efficacy was shown to depend on the time of the beginning of the antibiotic therapy . When the clinical signs of the infection were evident in the animals contaminated with the penicillinase-producing strain of B . anthracis, their treatment with the mean daily doses of benzylpenicillin failed. J Infect Dis, 1993 May, 167(5), 1239 - 43 Postexposure prophylaxis against experimental inhalation anthrax; Friedlander AM et al.; Inhalation anthrax is a rare disease that is almost invariably fatal . This study determined whether a prolonged course of postexposure antibiotics with or without vaccination would protect monkeys exposed to a lethal aerosol dose of Bacillus anthracis when the antibiotic was discontinued . Beginning 1 day after exposure, groups of 10 animals were given penicillin, ciprofloxacin, doxycycline, doxycycline plus vaccination, vaccination alone, or saline . Antibiotics were administered for 30 days and then discontinued . Vaccine was given on days 1 and 15 . Two animals died of causes other than anthrax and were not included in the statistical analysis . Nine of 10 controls and 8 of 10 animals given only vaccine died . Each antibiotic regimen completely protected animals while on therapy and provided significant long-term protection upon discontinuance of the drug (penicillin, 7 of 10 survived, P < .02; ciprofloxacin, 8 of 9 survived, P < .002; doxycycline, 9 of 10 survived, P < .002; doxycycline plus vaccination, 9 of 9 survived, P < .0002) . Protection against rechallenge was provided by combining postexposure antibiotic treatment with vaccination. Microb Pathog, 1993 May, 14(5), 381 - 8 Non-toxigenic derivatives of the Ames strain of Bacillus anthracis are fully virulent for mice: role of plasmid pX02 and chromosome in strain-dependent virulence; Welkos SL et al.; The toxin-encoding plasmid pX01 and capsule-associated plasmid pX02 are required for full virulence of Bacillus anthracis in some animals . However, the non-toxigenic pX01-cured derivatives of certain anthrax strains are not completely attenuated for mice, and their virulence is strain-dependent . The strain-related differences were partially associated with plasmid pX02 as demonstrated by pX02 transductants of the attenuated vaccine strain UM23-1 cured of pX01 . To determine the virulence of non-toxigenic variants of virulent strains, we isolated pX01- derivatives of the Vollum 1B strain and the more 'vaccine-resistant' Ames strain which carried pX02 from either Ames or Vollum 1B . The 50% lethal dose (LD50) values of the derivatives of both strains which carried the Ames pX02 were not significantly different from the LD50s of the pX01+ pX02+ strains (and were lower than those of pX01+ pX02- strains) . pX02+ derivatives of strain UM23-1 were less virulent than the comparable Ames and Vollum 1B strain derivatives, emphasizing a role for chromosomal loci in the virulence of the latter two strains . Non-toxigenic isolates which carried the Ames pX02 were more virulent for CBA/J mice than those with Vollum 1B pX02, and the differences were mouse strain-dependent . The pX01- pX02+ strains multiplied and achieved high concentrations systemically. J Clin Microbiol, 1993 Apr, 31(4), 887 - 94 Determination of carbohydrate profiles of Bacillus anthracis and Bacillus cereus including identification of O-methyl methylpentoses by using gas chromatography-mass spectrometry; Fox A et al.; 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. Proc Natl Acad Sci U S A, 1993 Mar 15, 90(6), 2291 - 4 Pathology of inhalational anthrax in 42 cases from the Sverdlovsk outbreak of 1979; Abramova FA et al.; A large epidemic of anthrax that occurred in Sverdlovsk (now Ekaterinburg), Russia, in 1979 resulted in the deaths of many persons . A series of 42 necropsies, representing a majority of the fatalities from this outbreak, consistently revealed pathologic lesions diagnostic of inhalational anthrax, namely hemorrhagic necrosis of the thoracic lymph nodes in the lymphatic drainage of the lungs and hemorrhagic mediastinitis . Bacillus anthracis was recovered in bacterial cultures of 20 cases, and organisms were detected microscopically in the infected tissues of nearly all of the cases . A novel observation was primary focal hemorrhagic necrotizing pneumonia at the apparent portal of entry in 11 cases . Mesenteric lymphadenitis occurred in only 9 cases . This remarkably large series demonstrated the full range of effects of anthrax bacteremia and toxemia (edema especially adjacent to sites of extensive infection and pleural effusions) and hematogenously disseminated infection {hemorrhagic meningitis (21 cases) and multiple gastrointestinal submucosal hemorrhagic lesions (39 cases)}. Mol Gen Mikrobiol Virusol, 1993 Mar-Apr, (2), 13 - 6 {Cloning and expression of determinants of Bacillus anthracis protective antigen in Escherichia coli, Bacillus subtilis, and Bacillus anthracis cells}; Tedikov VM et al.; A determinant for a protective antigen (pag) of Bacillus anthracis STI has been cloned . Its expression in Escherichia coli, Bacillus subtilis and Bacillus anthracis cells has been studied . The hybrid plasmids were obtained carrying the different fragments of the gene . The plasmids pPA2 and pPA3 having the 3'-end fragment of pag deleted (the size of 1 kb) still code for a part of protective antigen preserving the immunological and protective properties. J Gen Microbiol, 1993 Mar, 139 ( Pt 3), 601 - 7 A macrolide-lincosamide-streptogramin B resistance determinant from Bacillus anthracis 590: cloning and expression of ermJ; Kim HS et al.; The inducible macrolide-lincosamide-streptogramin B resistance determinant, ermJ, from Bacillus anthracis 590 was cloned in Escherichia coli CSH26 . The DNA sequence of ermJ was similar to that of ermK or ermD from B . licheniformis, suggesting that ermK-like genes have been distributed in Bacillus strains by transposition . Expression of ermJ was achieved in a B . subtilis minicell system, and the rRNA methyltransferase product of ermJ was purified . The molecular mass of the enzyme was 58 kDa, and it was concluded to be a homodimer . Its biochemical characteristics were different from those of ermC methyltransferase. J Clin Microbiol, 1993 Mar, 31(3), 547 - 51 Direct detection of Bacillus anthracis DNA in animals by polymerase chain reaction; Makino SI et al.; Bacillus anthracis is a soil pathogen capable of causing anthrax . To establish a method for specifically detecting B . anthracis for practical applications, such as for the inspection of slaughterhouses, the cap region, which is essential for encapsulation in B . anthracis, was used in a DNA hybridization study by polymerase chain reaction (PCR) . Oligonucleotide primers were designed to amplify a 288-bp DNA fragment within the capA gene by PCR . The amplified DNA sequence specifically hybridized to the DNA of B . anthracis but not to that of other bacterial strains tested . Since this PCR-based method efficiently and specifically detected the capA sequence of bacteria in blood and spleen samples of mice within 8 h after the administration of live B . anthracis, this PCR system could be used for practical applications . By using lysis methods in preparing the samples for PCR, it was possible to amplify the 288-bp DNA segment from samples containing very few bacteria, as few as only 1 sporeforming unit, indicating that the PCR detection method developed in this study will permit the monitoring of B . anthracis contamination in the environment. J Biol Chem, 1993 Jan 25, 268(3), 1695 - 701 Characterization of a synthetic calmodulin-binding peptide derived from Bacillus anthracis adenylate cyclase; Munier H et al.; A 34-amino acid peptide corresponding to residues 532-565 of Bacillus anthracis adenylate cyclase (P532-565), a calmodulin (CaM)-activated enzyme, was synthesized by solid phase method . Although not homologous to any known CaM binding sequence, P532-565 exhibits molecular features characteristic of this class of peptides: a higher proportion of basic and hydrophobic residues, segregated onto the two faces of the alpha-helical structure . Fluorescence measurements and gel retardation analysis showed that P532-565 binds CaM in a Ca(2+)-dependent manner, with a binding energy that represents 80% of the binding energy of the adenylate cyclase-CaM complex . Circular dichroism analysis showed that P532-565 exists in solution as a mixture of random-coil and alpha-helical structures and that trifluoroethanol increases the relative proportion of alpha-helical population . Analysis of proton NMR spectrum in H2O allowed identification of the different amino acid spin systems and complete spectral assignment . The pattern of nuclear Overhauser effect connectivities, intense NN(i,i + 1) and medium range alpha N(i,i + 3) and alpha beta (i,i + 3) indicate the presence of an alpha-helix in the carboxylterminal end (between residues 551 and 563) in fast exchange with extended structures . These data, together with CaM-binding properties of Bordetella pertussis adenylate cyclase, show that despite rather divergent primary structures, the two bacterial enzymes possess similar structural organization of their binding sites for activator protein. South Med J, 1993 Jan, 86(1), 1 - 4 Indigenous human cutaneous anthrax in Texas; Taylor JP et al.; In December 1988 an indigenous case of cutaneous anthrax was identified in Texas . The patient, a 63-year-old male Hispanic from southwest Texas, was a sheep shearer and had a recent history of dissecting sheep that had died suddenly . He experienced an illness characterized by left arm pain and edema . A necrotic lesion developed on his left forearm, with cellulitis and lymphadenopathy . After treatment with oral and intravenous penicillins, the patient fully recovered . Western blot testing revealed a fourfold or greater rise in antibody titer to Bacillus anthracis protective antigen and lethal factor . This represents the first case of indigenous anthrax in Texas in more than 20 years. Ann N Y Acad Sci, 1992 Dec 31, 666, 177 - 90 Narrowing the zone of uncertainty between research and development in biological warfare defense; Huxsoll DL; Although "research" is not prohibited by the Biological Weapons Convention, States Parties to the Convention have maintained the spirit of the Convention in actions relating to research . The confidence-building measures agreed to at RC2 refer to research facilities, publication of research results, and promotion of contacts between scientists engaged in research related to the Convention . However, assessment of basic research on biological agents is not a productive way to distinguish an offensive from a defensive program . Additionally, if a country were to initiate a biological weapons program, basic research on biological agents may not be necessary . For example, the extensive published research on Bacillus anthracis, both as a cause of anthrax in cattle and other species and as a biological-warfare agent, would enable any motivated group or nation to initiate a biological weapons program that could immediately advance to the development and scale-up stages . Research on biological agents for offensive purposes would be characterized by activities such as selection for growth, virulence, and toxin production; improving stability under varying environmental conditions; and selection of strains that might overcome existing means of prophylaxis and treatment . A biological program with an offensive intent would in most cases be characterized by evidence of development efforts in mass production and dissemination, which are often agent-specific . Thus, an assessment of development may distinguish offensive from defensive programs . If a country were to initiate a biological weapons research program, and were willing to risk worldwide condemnation should existence of such a program become known, it is likely that such a program would include development and production capabilities . If a country were not committed to production capability, there would be no rationale for an offensive biological research that would bring worldwide condemnation . Critics of the U.S . Biological Defense Research Program have suggested that the program could easily and quickly be turned into an offensive effort . To accomplish this, however, we have to assume that all military personnel, including the civilians employed by the Department of the Army, are unethical and willing to break the law and run the risk of placing the U.S . in a noncompliance status . The Army is under constant scrutiny by governmental agencies, by visiting scientists, by audiences at scientific meetings, by scientists who review manuscripts for publications, by news media, and by private citizens.(ABSTRACT TRUNCATED AT 400 WORDS) Br J Ophthalmol, 1992 Dec, 76(12), 753 - 4 Anthrax of the eyelids; Amraoui A et al.; Anthrax is a disease caused by Bacillus anthracis . The disease affects primarily herbivores including sheep, cattle, horses, and other domestic animals . Humans may rarely be affected . We examined one male and two female patients with a localised itchy erythematous papule of the eyelid . A necrotising ulcer formed in each of the three cases resulting in a black lesion . Scraping in each case showed Gram positive rods and culture grew Bacillus anthracis . All three patients responded to the intravenous administration of penicillin G, and the lesion resolved leaving scars in two cases . Anthrax is a rare disease but should be considered in the differential diagnosis of ulcers or pustules of the eyelids. FEMS Microbiol Lett, 1992 Nov 1, 77(1-3), 89 - 93 Regulation of pag gene expression in Bacillus anthracis: use of a pag-lacZ transcriptional fusion; Cataldi A et al.; The production of protective antigen (PA), the common component of the two anthrax toxins, is influenced by the environment . In order to examine factors involved in its regulation, a transcriptional fusion between the promoter region of the PA gene (pag) and the lacZ gene was constructed and introduced into Bacillus anthracis Sterne . Activity of the pag promoter was followed by measuring beta-galactosidase activities under various growth and medium conditions . Expression from the pag promoter was observed throughout exponential-phase and was maximal in early stationary phase . The activity of the pag promoter was stimulated by the addition of glucose in the medium. Mol Biol Cell, 1992 Nov, 3(11), 1269 - 77 Biochemical and physiological changes induced by anthrax lethal toxin in J774 macrophage-like cells; Hanna PC et al.; Experiments were performed to probe the mechanism by which Bacillus anthracis Lethal Toxin (LeTx) causes lysis of J774 macrophage-like cells . After incubation of cells with saturating concentrations of the toxin, two categories of effects were found, which were distinguishable on the basis of chronology, Ca(2+)-dependence, and sensitivity to osmolarity . The earliest events (category I), beginning 45 min postchallenge, were an increase in permeability to 22Na and 86Rb and a rapid conversion of ATP to ADP and AMP . Later events (category II) included alterations in membrane permeability to 45Ca, 51Cr, 36Cl, 35SO4, 3H-amino acids, and 3H-uridine, beginning at 60 min; inhibition of macromolecular synthesis, leakage of cellular lactate dehydrogenase and onset of gross morphological changes, at approximately 75 min; and cell lysis, beginning at 90 min . Category II events exhibited an absolute requirement for extracellular Ca2+ and were blocked by addition of 0.3 M sucrose to the medium, whereas category I events were attenuated, but not blocked, by either of these conditions . On the other hand, both ATP depletion and the category II events were blocked in osmotically stabilized medium that was also isoionic for Na+ and K+ . This suggests that permeabilization of the plasma membrane to monovalent cations and water may be the earliest of the physiological changes described here . The resulting influx of Na+ and efflux of K+ would be expected to cause depletion of ATP, via increased activity of the Na+/K+ pump . Subsequently the influx of Ca2+, induced by depletion of ATP, imbalances in monovalent cautions, and/or more dramatic changes in permeability due to influx of water, would be expected to trigger widespread changes leading ultimately to cytolysis. J Infect Dis, 1992 Nov, 166(5), 1184 - 7 Serum concentrations of penicillin, doxycycline, and ciprofloxacin during prolonged therapy in rhesus monkeys; Kelly DJ et al.; Concentrations of penicillin, doxycycline, and ciprofloxacin were measured by bioassay in sera of rhesus monkeys treated with these drugs for inhalation anthrax . Antibiotic doses were determined on the basis of published serum concentration data from humans and comparative body surface area calculations for humans and rhesus monkeys . The antibiotics were well tolerated . Serum peak and trough concentrations of penicillin, doxycycline, and ciprofloxacin, respectively, averaged 2.7 and 0.8, 1.31 and 0.26, and 1.22 and 0.14 microgram/mL . These were within the range usually observed with standard oral doses in humans, and peak concentrations in all monkeys exceeded the MICs for 90% of Bacillus anthracis strains. Eur J Clin Microbiol Infect Dis, 1992 Sep, 11(9), 839 - 42 Familial outbreak of agricultural anthrax in an area of northern Italy; de Lalla F et al.; Three cases of cutaneous anthrax are reported which occurred in a farming family in northern Italy . Epidemiological studies revealed contact with an infected cow (delivery of a stillborn fetus and slaughter) . The cow was slaughtered soon after the delivery; cultures of carcass specimens yielded growth of Bacillus anthracis . The origin of the animal infection was not known . Serum samples were obtained from all 11 members of the family group and randomly from 10 of the 75 cows on the farm, which appeared to be in good health . Tests for antibodies against protective antigen and lethal factor using EIA and Western blot techniques were positive in three subjects (in paired sera) with cutaneous anthrax and in one subject who neither had had direct contact with the infected cow nor showed any sign of anthrax. J Biol Chem, 1992 Aug 25, 267(24), 17186 - 93 Functional characterization of protease-treated Bacillus anthracis protective antigen; Novak JM et al.; Characterization of the functional domains of Bacillus anthracis protective antigen (PA, 83-kDa), the common cellular binding molecule for both anthrax edema toxin and anthrax lethal toxin, is important for understanding the mechanism of entry and action of the anthrax toxins . In this study, we generated both biologically active (facilitates killing of J774A.1 cells in combination with lethal factor, LF) and inactive preparations of PA by protease treatment . Limited proteolytic digestion of PA in vitro with trypsin generated a 20-kDa fragment and a biologically active 63-kDa fragment . In contrast, limited digestion of PA with chymotrypsin yielded a preparation containing 37- and 47-kDa fragments defective for biological activity . Treatment with both chymotrypsin and trypsin generated three major fragments, 20, "17," and 47 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . This PA preparation was also biologically inactive . To investigate the nature of the defect resulting from chymotrypsin treatment, we assayed PA preparations for the ability to bind to the cellular receptor and to bind and internalize 125I-LF . All radiolabeled PA preparations bound with specificity to J774A.1 cells and exhibited affinities similar to native 83-kDa PA . Once bound to the cell surface receptor, both trypsin-treated PA and chymotrypsin/trypsin-treated PA specifically bound 125I-LF with high affinity . Finally, these PA preparations delivered 125I-LF to a Pronase-resistant cellular compartment in a time- and temperature-dependent fashion . Thus, the biological defect exhibited by chymotrypsin-treated PA is not at the level of cell binding or internalization but at a step later, such as toxin routing or processing by J774A.1 cells . These protease-treated preparations of PA should prove useful in both elucidating the intracellular processing of anthrax lethal toxin and determining the structure-function relationship of PA and LF. FEMS Microbiol Lett, 1992 Jul 1, 73(1-2), 75 - 80 Comparative analysis of 23S ribosomal RNA gene sequences of Bacillus anthracis and emetic Bacillus cereus determined by PCR-direct sequencing; Ash C et al.; The primary structures of the 23S ribosomal RNA genes of Bacillus anthracis and an emetic strain of Bacillus cereus were determined by direct sequencing of enzymatically amplified chromosomal DNA . The 23S rRNA gene sequences of B . anthracis and B . cereus were found to be almost identical and showed only two differences (a single nucleotide change, and a single base insertion in B . cereus) . The feasibility of using PCR-direct sequencing for the rapid sequence determination of large-subunit rRNA genes is demonstrated. J Infect Dis, 1992 Jun, 165(6), 1145 - 8 Detection of spores of Bacillus anthracis using the polymerase chain reaction; Carl M et al.; The polymerase chain reaction (PCR) was used to identify spores of Bacillus anthracis . By using an assay capable of amplifying a 1247-bp fragment from the gene that encodes the edema factor of B . anthracis, as few as 10(3) copies of a plasmid containing the edema factor gene and as few as 2 x 10(4) spores were detected . Subjecting the product of this PCR to a second PCR designed to amplify a 208-bp fragment nested within the 1247-bp product improved detection to a single plasmid copy per PCR and to two spores of B . anthracis per PCR. J Biol Chem, 1992 May 15, 267(14), 9816 - 20 The role of histidine 63 in the catalytic mechanism of Bordetella pertussis adenylate cyclase; Munier H et al.; Of the 9 histidines located in the catalytic domain of Bordetella pertussis adenylate cyclase, three (His63, His106, and His298) were found to be conserved in the adenylate cyclase of Bacillus anthracis, another calmodulin-dependent enzyme . Substitution of His63 with Arg, Glu, Gln, or Val decreased the catalytic efficiency of adenylate cyclase between 2 and 3 orders of magnitude and altered the kinetic properties of the enzyme . These effects varied in relation to the nature of the substituting residue, pH, and direction of the reaction, i.e . ATP cyclization (forward) or ATP synthesis (reverse) . Arg was the best substituent for His63 as catalyst in the forward reaction, with shift of the optimum pH to the alkaline side, whereas Glu was the best substituent for His63 in the reverse reaction, with shift of the optimum pH to the acidic side . Diethyl pyrocarbonate, which had a deleterious effect on wild-type adenylate cyclase was ineffective on His63 mutants . From these results we conclude that His63 is involved in the reaction mechanism of adenylate cyclase, which requires a general acid/base catalyst, most probably as an intermediate in a charge-relay system. Antibiot Khimioter, 1992 Apr, 37(4), 31 - 4 {Comparison of therapeutic effects of antibiotics of the tetracycline group in the treatment of anthrax caused by a strain inheriting tet-gene of plasmid pBC16}; Pomerantsev AP et al.; In vivo and in vitro efficacy of tetracyclines was studied with respect to anthracic infection induced by a tetracycline-resistant resistant strain containing plasmid pBC16 . The plasmid-containing strain was resistant to tetracycline, doxycycline and minocycline, the MICs exceeding those for the initial strain 500, 640 and 80 times, respectively . There was no therapeutic effect of tetracycline and doxycycline in the treatment and urgent prophylaxis of anthracic infection caused by the tetracycline-resistant strain of Bacillus anthracis . High therapeutic efficacy of minocycline in the average therapeutic concentrations was shown irrespective of the contaminating doses and strains . Minocycline was recommended for treatment and urgent prophylaxis of anthracic infection caused by tetracycline-resistant B . anthracis strains. Biochemistry, 1992 Mar 31, 31(12), 3215 - 22 A monoclonal antibody directed against the catalytic site of Bacillus anthracis adenylyl cyclase identifies a novel mammalian brain catalytic subunit; Orlando C et al.; A brain adenylyl cyclase was shown to contain an epitope closely related to that specified by a conserved sequence containing a nucleotide-binding consensus sequence GXXXXGKS and located in the catalytic sites of bacterial, calmodulin-dependent adenylyl cyclases {Goyard, S., Orlando, C., Sabatier, J.-M., Labruyere, E., d'Alayer, J., Fontan, G., van Rietschoten, J., Mock, M., Danchin, A., Ullmann, A., & Monneron, A . (1989) Biochemistry 28, 1964-1967} . A monoclonal antibody, mab 164, produced against a peptide corresponding to this conserved sequence specifically inhibited the Bordetella pertussis adenylyl cyclase . It also specifically inhibited rat and rabbit brain synaptosomal adenylyl cyclases . The extent of inhibition depended upon the type of enzyme purification, reaching 90% for the calmodulin-sensitive species of enzyme and 20-35% for the forskolin-agarose-retained species . The extent of inhibition in a given fraction also depended upon the effector present . mab 164 reacted on Western blots of forskolin-agarose-retained fractions with a 175-kDa component and did not recognize the Gs alpha stimulatory subunit . Consequently, the 175-kDa protein was considered as a good candidate for an adenylyl cyclase catalyst . The adenylyl cyclase activity contained in forskolin-agarose-retained fractions was further purified on calmodulin-Sepharose . On Western blots of such fractions, mab 164 reacted with a 140-kDa protein, a component that appeared to derive from the 175-kDa protein enriched in the previous step . The kcat of this 140-kDa presumptive adenylyl cyclase was estimated to be of the order of 600 s-1.(ABSTRACT TRUNCATED AT 250 WORDS) J Gen Microbiol, 1992 Mar, 138 ( Pt 3), 543 - 9 Serum protease cleavage of Bacillus anthracis protective antigen; Ezzell JW Jr et al.; The protective antigen component of anthrax lethal toxin, produced in vitro, has a molecular mass of 83 kDa . Cell-culture studies by others have demonstrated that upon binding of the 83 kDa protective antigen to cell-surface receptors, the protein is cleaved by an unidentified cell-associated protease activity . The resultant 63 kDa protein then binds lethal factor to form lethal toxin, which has been proposed to be internalized by endocytosis . We found that, in the blood of infected animals, the protective antigen exists primarily as a 63 kDa protein and appears to be complexed with the lethal factor component of the toxin . Conversion of protective antigen from 83 to 63 kDa was catalysed by a calcium-dependent, heat-labile serum protease . Except for being complexed to protective antigen, there was no apparent alteration of lethal factor during the course of anthrax infection . The protective antigen-cleaving protease appeared to be ubiquitous among a wide range of animal species, including primates, horses, goats, sheep, dogs, cats and rodents. Infect Immun, 1992 Feb, 60(2), 662 - 8 Immunization against anthrax with Bacillus anthracis protective antigen combined with adjuvants; Ivins BE et al.; The protective efficacy of immunization against anthrax with Bacillus anthracis protective antigen (PA) combined with different adjuvants was tested in Hartley guinea pigs and CBA/J and A/J mice . Adjuvant components derived from microbial products that were tested included threonyl-muramyl dipeptide (threonyl-MDP); monophosphoryl lipid A (MPL); trehalose dimycolate (TDM); and the delipidated, deproteinized, cell wall skeleton (CWS) from either Mycobacterium phlei or the BCG strain of Mycobacterium bovis . Non-microbially derived adjuvants tested included aluminum hydroxide and the lipid amine CP-20,961 . In guinea pigs, all adjuvants and adjuvant mixtures enhanced antibody titers to PA as well as survival after a parenteral challenge of virulent B . anthracis Ames spores . In contrast, PA alone or combined with either aluminum hydroxide or CP-20,961 failed to protect mice . Vaccines containing PA combined with threonyl-MDP or MPL-TDM-CWS protected a majority of female CBA/J mice . Statistical analysis of survival data in the guinea pigs indicated that PA-MPL-CWS and PA-MPL-TDM-CWS were more efficacious than the currently licensed human anthrax vaccine. J Appl Bacteriol, 1992 Jan, 72(1), 21 - 8 Bacillus anthracis but not always anthrax; Turnbull PC et al.; Gram-positive bacilli isolated during epidemiological investigations which, on the basis of conventional tests, resemble Bacillus anthracis but which fail to produce the capsule or to induce anthrax in test animals have long been dismissed in clinical and veterinary laboratories as B . cereus or simply as unidentified Bacillus spp . and thereupon discarded as inconsequential . In this study, the application of newly available DNA probe, polymerase chain reaction and specific toxin antigen detection technology has revealed that a proportion of such strains are B . anthracis which lack the plasmid carrying the capsule gene (pXO2) . While these techniques cannot, of course, be used to confirm the identities of strains resembling B . anthracis but which also lack the plasmid carrying the toxin genes (pXO1), the likelihood that these also are bona fide B . anthracis becomes more acceptable . (As yet no naturally occurring pXO1-/2+ strains have been found.) At this point, the significance of the presence of such avirulent forms of B . anthracis in specimens can only be a subject for speculation, but the possibility that they may be indicators of virulent parents somewhere in the system being examined must be considered. Biochem Biophys Res Commun, 1991 Oct 31, 180(2), 531 - 7 Location of receptor-binding region of protective antigen from Bacillus anthracis; Little SF et al.; The pag gene, which codes for protective antigen (PA), a common component of the lethal and edema toxins of Bacillus anthracis, was cloned and expressed in Escherichia coli . Nested deletions of pag were generated into the C-terminus coding region . Recombinant proteins were analyzed by Western blot with either an anti-PA polyclonal antisera or two monoclonal antibodies that neutralized lethal toxin and edema toxin activities by inhibiting the binding of PA to cell receptors . Localization of the receptor binding domain within the C-terminal region of PA was suggested by the inability of the monoclonal antibodies 3B6 and 14B7 to recognize the recombinant proteins expressed by C-terminal deletions of the pag gene. J Biol Chem, 1991 Oct 25, 266(30), 20124 - 30 Functional mapping of anthrax toxin lethal factor by in-frame insertion mutagenesis; Quinn CP et al.; Linker insertion mutagenesis was employed to create structural disruptions of the lethal factor (LF) protein of anthrax toxin to map functional domains . A dodecameric linker was inserted at 17 blunt end restriction enzyme sites throughout the gene . Paired MluI restriction sites within the linker allowed the inserts to be reduced from four to two amino acids . Shuttle vectors containing the mutated genes were transformed into the avirulent Bacillus anthracis UM23C1-1 for expression and secretion of the gene products . Mutations at five sites in the central one-third of the sequence made the protein unstable, and purified protein could not be obtained . Mutated LF proteins with insertions at the other sites were purified and assessed for toxic activity in a macrophage lysis assay and for their ability to bind to the protective antigen (PA) component of anthrax toxin, the receptor binding moiety . Most insertions located in the NH2-terminal one-third of the LF protein eliminated both toxicity and binding to PA, while all four insertions in the COOH-terminal one-third of the protein eliminated toxicity without affecting binding to PA . These data support the hypothesis that the NH2-terminal domain contains the structures required for binding to PA and the COOH-terminal domain contains the putative catalytic domain of LF. Infect Immun, 1991 Oct, 59(10), 3472 - 7 Contribution of individual toxin components to virulence of Bacillus anthracis; Pezard C et al.; Three proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF; a calmodulin-dependent adenylate cyclase), compose the lethal (PA + LF) and edema