Molecular characterization of immune inhibitor A, a secreted virulence protease from Bacillus thuringiensis

The gene for the secreted neutral metalloprotease, immune inhibitor A (InA), from Bacillus thuringiensis var. alesti has been cloned and sequenced. The deduced amino acid sequence has been confirmed by partial amino acid sequencing. The central part of the amino acid sequence showed similarity to the active site in thermolysin. Southern and Western blots show that InA-related sequences are common among other B. thuringiensis subspecies. In Western blots, 17 out of 25 tested species gave a positive signal. Culture filtrates from subspecies expressing InA were toxic when injected in Trichoplusia ni larvae, whereas filtrate from a strain negative in Western blot had no effect when injected. The LD50 dose of purified InA protein injected in T. ni larvae was 12.5 +/- 2.5 ng per mg of larval body weight.     

Higher production of rabies virus in serum-free medium cell cultures on microcarriers

The aprA gene encoding alkaline protease A (AprA) was cloned from Bacillus thuringiensis subsp. kurstaki, and the cloned gene was used to construct aprA-deleted (aprA1) strains of B. thuringiensis. An aprA1 strain of B. thuringiensis that contained the wild-type gene for neutral protease A (nprA(+)) displayed levels of extracellular proteolytic activity that were similar to those of an aprA(+)nprA(+) strain. However, when EDTA was included in the protease assay to inhibit NprA activity the aprA1nprA(+) strain displayed only 2% of the extracellular proteolytic activity of the aprA(+)nprA(+) strain. A strain that was deleted for both aprA and nprA (aprA1nprA3 strain) failed to produce detectable levels of proteolytic activity either in the presence or absence of EDTA in the assay. Compared with the aprA(+)nprA(+) strain the aprA1nprA(+) strain yielded 10% more full-length Cry1Bb crystal protein and the aprA1nprA3 strain yielded 25% more full-length Cry1Bb protein. No significant differences were seen in the 50% lethal dose of Cry1Bb protein from aprA(+)nprA(+) and aprA1nprA3 strains against three species of lepidopteran insects. These results suggest that enhanced yield of certain crystal proteins can be obtained by deletion of the genes aprA and nprA which are the major extracellular proteases of B. thuringiensis.     

InhA-like protease secreted by Bacillus sp. S17110 inhabited in turban shell

A strain producing a potent protease was isolated from turban shell. The strain was identified as Bacillus sp. S17110 based on phylogenetic analysis. The enzyme was purified from culture supernatant of Bacillus sp. S17110 to homogeneity by ammonium sulfate precipitation, SP-Sepharose, and DEAE-Sepharose anion exchange chromatography. Protease activity of the purified protein against casein was found to be stable at pH 7 to pH 10 and around 50 degrees . Approximately 70% of proteolytic activity of the enzyme was detected either in the presence of 100 mM SDS or Tween 20. The enzyme activity was enhanced in the presence of Ca2+, Zn2+, Mg2+, but was inhibited by EDTA, indicating that it requires metal for its activity. The purified enzyme was found to be a monomeric protein with a molecular mass of 75 kDa, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. The purified enzyme was analyzed through peptide fingerprint mass spectra generated from matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) and a BLAST search, and identified as immune inhibitor A (inhA) deduced from nucleotide sequence of B. cereus G9241. Since InhA was identified as protease that cleave antibacterial proteins found in insect, inhA-like protease purified from Bacillus sp. S17110 might be pathogenic to sea invertebrates.     

Isolation and characterization of a hemagglutinin from Amphitrite ornata, a polychaetous annelid.

Extracts of the marine polychaetous annelid, Amphitrite ornata, agglutinate rat, rabbit, chicken and human erythrocytes and in other work have been shown to inhibit the growth of Ehrlich ascites tumors in mice. Fractionation of extracts on Sephadex G-100 gave three active fractions with molecular weights of 30 000, 54 000 and 100 000. The 30 000 dalton fraction (B) was purified 72-fold by ammonium sulfate precipitation, gel filtration and preparative disc gel electrophoresis. The purified hemagglutinin, amphitritin, was homogenous on analytical disc gel electrophoresis at four different pH values and gave a sharp boundary in sedimentation velocity ultracentrifugation. The three fractions showed paralled specificity toward rat and chicken erythrocytes, the former giving the higher titer. The purified agglutinin was active toward human blood groups A, B and O and exhibited 4-fold higher activity toward group A. The hemagglutinin titer against rat red blood cells was lowered only by N-acetylgalactosamine, the terminal sugar residue of the group A determinant. None of the saccharides tested inhibited agglutination of chicken erythrocytes. Hemagglutinin activity was insensitive to dialysis or treatment with EDTA. The activity was not affected by digestion with trypsin or pronase, but was destroyed by phenol extraction. Analytical disc gel electrophoresis showed one protein band with high anodal mobility at pH 8.5, which was not affected by proteolytic enzymes but was removed by phenol. Activity was unaffected by heating at 70 degrees C for 30 min but was destroyed by similar treatemtn at 85 degrees C. Activity was at a maximum at pH 7-9 and decreased reversibly down to pH 4 at which point it was irreversibly inactivated. The higher molecular weight agglutinin (A1) could be dissociated to give amphitritin by treatment with 6M urea of precipitation in 55% (NH4)2SO4. This dissociation was not reversed by dialysis. Amphitritin is a glycoprotein with a molecular weight determined by gel filtration of 30 000 and by approach to equilibrium sedimentation of 32 000. Amino acid analysis showed a preponderance of aspartic and glutamic acids and relatively large amounts of glycine, proline, alanine, valine and cysteine. The carbohydrate moeity which represented 12.8% of the molecule, contained mannose, galactose, glucosamine and sialic acid. Amphitritin is the first hemagglutinin to be isolated from a polychaetous annelid.     

A Bacillus thuringiensis S-layer protein involved in toxicity against Epilachna varivestis (Coleoptera: Coccinellidae)

The use of Bacillus thuringiensis as a biopesticide is a viable alternative for insect control since the insecticidal Cry proteins produced by these bacteria are highly specific; harmless to humans, vertebrates, and plants; and completely biodegradable. In addition to Cry proteins, B. thuringiensis produces a number of extracellular compounds, including S-layer proteins (SLP), that contribute to virulence. The S layer is an ordered structure representing a proteinaceous paracrystalline array which completely covers the surfaces of many pathogenic bacteria. In this work, we report the identification of an S-layer protein by the screening of B. thuringiensis strains for activity against the coleopteran pest Epilachna varivestis (Mexican bean beetle; Coleoptera: Coccinellidae). We screened two B. thuringiensis strain collections containing unidentified Cry proteins and also strains isolated from dead insects. Some of the B. thuringiensis strains assayed against E. varivestis showed moderate toxicity. However, a B. thuringiensis strain (GP1) that was isolated from a dead insect showed a remarkably high insecticidal activity. The parasporal crystal produced by the GP1 strain was purified and shown to have insecticidal activity against E. varivestis but not against the lepidopteran Manduca sexta or Spodoptera frugiperda or against the dipteran Aedes aegypti. The gene encoding this protein was cloned and sequenced. It corresponded to an S-layer protein highly similar to previously described SLP in Bacillus anthracis (EA1) and Bacillus licheniformis (OlpA). The phylogenetic relationships among SLP from different bacteria showed that these proteins from Bacillus cereus, Bacillus sphaericus, B. anthracis, B. licheniformis, and B. thuringiensis are arranged in the same main group, suggesting similar origins. This is the first report that demonstrates that an S-layer protein is directly involved in toxicity to a coleopteran pest.     

Biological activities of lipopolysaccharides from Pectinatus cerevisiophilus

Abstract A procedure is described in which the protein crystals produced by Bacillus thuringiensis var. israelensis were solubilized in 50 mM NaOH with 10 mM EDTA at pH 11.7. This solubilization procedure gave protein gel profiles identical with those for intact crystals while maintaining full biological activity in the form of erythrocyte lysis capability. Crystals with and without protease activity were equally toxic to Aedes aegypti larvae.     

New findings on an anti-proteinase in human plasma: l'inter-alpha-trypsin inhibitor]

Inter-alpha-trypsin inhibitor (I alpha I) has been purified from C.N.T.S. fraction III as starting material. The purification procedure includes D.E.A.E. cellulose chromatography and gel filtration on G 150 Sephadex in the presence of EDTA. The purified protein gives one precipitation line in immunoelectrophoresis against anti-whole human sérum. It reacts only with an anti I alpha I immune serum and possesses a strong antitryptic activity. When studied in starch or polyacrylamide gel electrophoresis 2 components are observed, each of them having the same antigenic structure and the same antitryptic activity as the crude preparation. The slower and less important component is dissociated by 0,1% SDS. The molecular weight estimation of I alpha I BY PAA/SDS is about 180,000. This result is not modified by the presence of 1% beta mercaptoethanol indicating that I alpha I consists of one polypeptide chain. Crude preparation reveals under the same electrophoretical conditions small amounts of low molecular weight components (135,000 52,000 and 26,000) which can be due to a proteolytic action on I alpha I. Indeed plasmin is able to produce such fragments having an antitryptic activity as shown by fibrin/polyacrylamide gel electrophoresis. The relationship between small molecular weight inhibitors of human serum and bronchial secretions and those obtained after degradation of I alpha I by plasmin is discussed.     

Biochemical and immunological characterization of the secreted forms of human neutrophil gelatinase

Human neutrophils contain a neutral metalloproteinase which degrades denatured collagens and potentiates the action of interstitial collagenase. This gelatinase is rapidly secreted from neutrophils stimulated with phorbol myristate acetate. The secreted enzyme has been purified by a combination of chromatography on DEAE-cellulose and gelatin-Sepharose. The purified enzyme was latent and had a specific activity of 24,000 units. Estimated molecular weight obtained by gel filtration was 150,000-180,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed three bands with relative molecular weights of 225,000, 130,000, and 92,000. Electrophoresis in the presence of a reducing agent revealed a single band of Mr = 92,000. All the proteins seen on the unreduced gel were found to contain proteolytic activity against gelatin and native type V collagen. Polyclonal antibodies were prepared against the Mr = 130,000 and 92,000 proteins. When analyzed by immunoblotting, both antibodies recognized all three proteins. Furthermore, the identical three proteins were identified by the antibodies when crude culture medium was immunoblotted. The purified enzyme was inhibited by EDTA and 1,10-phenanthroline but not by serine or thiol proteinase inhibitors, suggesting that the enzyme is a metalloendoproteinase. The enzyme had little or no activity against common protein substrates such as bovine serum albumin or casein. Native type I collagen was not cleaved under conditions where native type V collagen was extensively degraded.     

Thuricin: the bacteriocin produced by Bacillus thuringiensis

Bacillus thuringiensis serovar, thuringiensis (HD-2) demonstrated antibacterial activity against 48 of 56 strains of B. thuringiensis and against some other Gram-positive species but not against Gram-negative species. The antibacterial activity was not inducible by mitomycin C or by ultraviolet irradiation, and additional activity was not liberated from cells by sonication. Upon dilution of the antibacterial substance, zones of inhibition diminished without the appearance of plaques. Gel filtration chromatography indicated an Mr greater than 950,000 for the bacteriocin (thuricin) in its native form. The native thuricin was sedimented by ultracentrifugation, but electron microscopy of the pellet failed to reveal phage particles or phage components. Nondenaturing polyacrylamide gel electrophoresis (PAGE) of thuricin demonstrated the association of bacteriocin activity with a protein band which migrated only slightly into a 5% gel. Sodium dodecyl sulfate (SDS)-PAGE of partially purified thuricin revealed five major bands. Thuricin activity was substantially reduced by treatment with chymotrypsin, pronase, subtilisin, trypsin, and heat at 96 degrees C but not by treatment with lysozyme, phospholipase C, papain, peptidase, or organic solvents. It exhibited a bactericidal and bacteriolytic effect on a sensitive strain, B. thuringiensis serovar, canadensis (MF4). Partially purified preparations of thuricin had phospholipase A activity which was adsorbed by sensitive cells but not by cells which were insensitive to thuricin. Antibacterial activity was blocked by preincubation of thuricin with phospholipid. Loss of a 150-mDa plasmid was correlated with loss of thuricin production.     

An activated by cobalt alkaline aminopeptidase from Bacillus mycoides

An intracellular arginine--specific aminopeptidase synthesized by Bacillus mycoides was purified and characterized. The purification procedure for studied aminopeptidase consisted of ammonium sulphate precipitation and three chromatographic steps: anion exchange chromatography and gel permeation chromatography. A molecular weight of -50 kDa was estimated for the aminopeptidase by gel permeation chromatography and SDS-PAGE. The optimal activity of the enzyme on arginyl-beta-naphthylamide as a substrate was at 37 degrees C and pH 9.0. The enzyme showed maximum specificity for basic amino acids: such as Arg and Lys but was also able to hydrolyze aromatic amino acids: Trp, Tyr, and Phe. Co2+ ions activated the enzyme, while Zn2+, Cu2+, Hg2+ and Mn2+ inhibited it. The enzyme is a metalloaminopeptidase whose activity is inhibited by typical metalloaminopeptidase inhibitors: EDTA and 1,10-phenanthroline. Analysis of fragments of the amino acid sequence of the purified enzyme demonstrated high similarity to AmpS of Bacillus cereus and AP II of B. thuringensis.     

Benzylideneacetone, an immunosuppressant, enhances virulence of Bacillus thuringiensis against beet armyworm (Lepidoptera: Noctuidae)

Benzylideneacetone (BZA) is a metabolite of gram-negative entomopathogenic bacterium Xenorhabdus nematophila, and it acts as an enzyme inhibitor against phospholipase A2 (PLA2). PLA2 catalyzes a committed biosynthetic step of eicosanoids, which mediate insect immune reactions to infection by microbial pathogens. This study tested a hypothesis that a putative immunosuppressive activity of BZA may enhance virulence of Bacillus thuringiensis against the fifth instars of Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae). In in vitro conditions, BZA significantly inhibited hemocyte microaggregation induced by B. thuringiensis and impaired hemocyte-spreading behavior of S. exigua in a dose-dependent manner. Oral administration of BZA gave similar immunosuppressive effect on the hemocytes of the fifth instars. Although BZA itself did not possess any insecticidal activity on oral administration, when BZA was treated in a mixture with a low dose of B. thuringiensis spp. aizawai to fifth instars, the bacterial virulence was significantly enhanced. BZA also enhanced virulence of B. thuringiensis spp. kurstaki, which alone was of limited effectiveness against S. exigua. This study suggests that an immunosuppression by BZA is positively linked to potentiation of B. thuringiensis.     

Characterization of an inhibitor of the intracellular protease from Bacillus subtilis.

A specific inhibitor of intracellular serylprotease from Bacillus subtilis has been isolated from both growing and sporulating cells. Like other protease inhibitors isolated from eukaryotic cells, the inhibitor from B. subtilis is a thermostable protein. A purification method is described. The molecular weight estimated by Biogel filtration and SDS gel electrophoresis is about 15,500. Both proteolytic and esterolytic activities of intracellular protease are equally sensitive to inhibition. With azocoll or Z-tyrosine p-nitrophenylester as substrates, noncompetitive inhibition patterns are observed. The inhibitor has no effect on the proteolytic or esterolytic activities of the extracellular serylprotease. A similar thermostable inhibitor is also present in Bacillus megaterium.     

Purification, amino acid sequence and characterization of Bacthuricin F4, a new bacteriocin produced by Bacillus thuringiensis

AIMS: Purification and characterization of a new bacteriocin, Bacthuricin F4 of Bacillus thuringiensis. METHODS AND RESULTS: A newly isolated B. thuringiensis subsp. kurstaki strain BUPM4, was shown to produce a novel bacteriocin named Bacthuricin F4. The highest bacteriocin activity was found in the growth medium and evidenced in the late exponential growth phase. Bacthuricin F4 could be purified by a two-step procedure: ammonium sulphate precipitation of protein from culture supernatant followed by a reverse phase chromatography. Upon purification, the specific activity was increased 100-fold. This bacteriocin was heat-stable up to 70 degrees C and resisted up to pH 3.0. Bacthuricin F4 was sensitive to proteases demonstrating its proteinaceous nature. Its molecular mass, determined by mass spectrometry was 3160.05 Da. Direct N-terminal sequencing of Bacthuricin F4 revealed the following sequence: DWTXWSXL. The latter was unique in the databases. Bacthuricin F4 was active against Bacillus species while it had little or no effect on Gram-negative bacteria. CONCLUSIONS: A strain BUPM4 of B. thuringiensis subsp. kurstaki, was shown to produce a new bacteriocin named Bacthuricin F4 of both new molecular mass (3160.05 Da) and new amino acid terminal sequence. This is, to our knowledge, the first bacteriocin exhibiting such characteristics reported to be produced by B. thuringiensis. SIGNIFICANCE AND IMPACT OF THE STUDY: The bacteriocin produced by the B. thuringiensis strain BUPM4 respond to both criteria of thermostability and stability to low pHs. Thus, it could be used for the control of the related species of Bacillus harmful for agricultural products.     

Purification and characterization of extracellular proteinases excreted by a strain of Bacillus subtilis BS2, isolated from fermented African locust bean 'iru'

Proteinases were excreted by strains of Bacillus subtilis during fermentation of African locust bean cotyledons. Those excreted by one strain were purified and characterized by ammonium sulphate precipitation, ion-exchange chromatography (IEC), gel filtration, inhibition tests and polyacrylamide gel electrophoresis (PAGE). Three proteinases and an esterase without proteolytic activity were identified. A serine proteinase which showed a high degree of hydrophobicity and a neutral proteinase were present. The third proteinase showed both proteolytic and esterolytic activities, and had multiple electrophoretic mobilities on polyacrylamide gel.     

Development of a Bacillus thuringiensis-based assay on Lygus hesperus

Lygus hesperus Knight (Hemiptera: Miridae) is an economically important insect pest controlled primarily by chemical pesticides. Bacillus thuringiensis Berliner is a gram-positive bacterium, which upon sporulation produces a parasporal inclusion body, the crystal. The latter, in some strains, exhibits specific insecticidal activities against lepidopteran, coleopteran, or dipteran pests. The aim of the present work was to develop a Bacillus thuringiensis-based assay on L. hesperus. Several factors involved in the expression of the B. thuringiensis insecticidal activity were assayed for their effects on L. hesperus mortality. We show that the choice of the L. hesperus diet type, the L. hesperus developmental stages, the B. thuringiensis crystal alkaline solubilization buffers, the dialysis buffers, the dialyzed solubilization buffers, and the proteolytic activation can all significantly affect the L. hesperus viability. This work provides essential information in pinpointing key steps before the establishment of a thorough screening program for B. thuringiensis strains expressing antihemipteran activity.     

Possible involvement of gut-lumen bacteria in in-vivo proteolytic degradation of Bacillus thuringiensisδ-endotoxins

Larval gu juice from aseptically reared beet armyworm, Spodoptera exigua , exhibited lowered processing activity towards the parasporal inclusion proteins of Bacillus thuringiensis as compared with that from normal larvae. Of the seven aerobic bacteria predominantly found in guts of normal larvae, a Bacillus isolate showed marked proteolytic activity against inclusion proteins. The results suggest the possible participation of gut-lumen micro-organisms in in-vivo degradation and processing of B. thuringiensis δ-endotoxins.     

Purification and characterization of UDP-N-acetyl-D-glucosamine:dolichol phosphate N-acetyl-D-glucosamine-1-phosphate transferase involved in the biosynthesis of asparagine-linked glycoproteins in the mammary gland

The GlcNAc-1-P-transferase that initiates the dolichol cycle for the biosynthesis of asparagine-linked glycoproteins has been purified from the lactating bovine mammary gland. After solubilization from microsomes with 0.25% Nonidet P-40, the enzyme activity was stabilized with 20% glycerol, 20 micrograms/ml phosphatidylglycerol, 5 microM dolichol phosphate, and 2.5 microM UDP-GlcNAc. The purification protocol involved (NH4)2SO4 precipitation, gel filtration on Sephacryl S-300, DEAE-TSK, and hydroxylapatite chromatography. The purified enzyme was devoid of several readily detectable glycosyltransferases of the dolichol cycle. It showed two bands (A, 50 kDa and B, 46 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after either Coomassie Blue or silver staining. Antisera (anti-A and anti-B) raised against individual bands A and B inhibited the enzyme activity in solubilized microsomes. Each of the partially purified antibodies recognizes both bands A and B on Western blots of the enzyme; with the solubilized microsomes, the antibodies also recognize an additional polypeptide of approximately 70 kDa. When radioiodinated microsomes were immunoprecipitated with anti-B and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, again bands of 46, 50, and 70 kDa were observed. The peptide mapping of 50 and 46 kDa bands of the purified enzyme by chemical cleavage with N-chlorosuccinimide gave similar fragmentation patterns. The results indicate that either 70 kDa band is a precursor form of the enzyme or this polypeptide, representing the native enzyme or its subunit, is proteolyzed to smaller, enzymatically active peptide(s) of 50 and 46 kDa during purification despite the inclusion of several inhibitors against serine-proteases in all buffers used for tissue homogenization and enzyme purification. A number of properties of the purified enzyme, including its specific activation by Man-P-Dol were also characterized.     

Isolation of Two Acetyl Esterases from Extracts of Bacillus subtilis

Acrylamide gel electrophoresis of crude cellular extracts of Bacillus subtilis revealed the presence of two acetyl esterases. Esterase A, the slower migrating enzyme, was found to be present in both vegetative and sporulating cells, whereas esterase B activity was more abundant after exponential growth ceased. Both esterases were present in the supernatant fraction of lysed spheroplasts and in a disrupted spore preparation. Of four pleiotropic asporogenous mutants tested, three exhibited decreased esterase B activity. Esterases A and B were partially purified by differential precipitation and co-chromatographed on diethylaminoethyl (DEAE)-cellulose (pH 7.5) and DEAE-Sephadex (pH 8.5). By employing gel filtration chromatography, the two esterases were separated, and molecular weights of 160,000 and 51,000 were estimated for esterases A and B, respectively. Esterase A was further purified to electrophoretic homogeneity by differential heating and preparative starch block electrophoresis. Sodium dodecyl sulfate-acrylamide gel electrophoresis of purified esterase A yielded a single protein band with a molecular weight of 31,000. The pI values of esterases A and B were determined to be 6.4 and 5.4, respectively.     

Screening of strains identified as extremely thermophilic bacilli for extracellular proteolytic activity and general properties of the proteinases from two of the strains

T. COOLBEAR, C.W. EAMES, Y. CASEY, R.M. DANIEL AND H.W. MORGAN. 1991. Forty-one strains isolated from thermal areas in New Zealand, Fiji and Antarctica were shown to be extremely thermophilic Bacillus spp. (growth optima > 65^.C) by comparison with reference strains with a series of standard tests. Some morphological and physiological variation between strains was noted. Various assay procedures were employed to assess the strains for their ability to produce extracellular proteolytic activity. The strain EA. 1 gave the highest yield of proteolytic activity under the conditions imposed. A second strain, OK3A.1, also gave high yields of activity but differed from the EA.1 activity in that it was more tolerant to both high pH and EDTA. The proteinases from these two strains were purified and characterized. Maximum activity was given by EA.1 proteinase over a narrow pH range with an optimum at pH 6.7 and 50% activity limits at pH 5.6 and 7.5. OK3A.1 had a similar pH optimum but was active over a broader range with 50% activity limits at pH 5.2 and 8.5. Both enzymes were endo-acting proteinases; neither showed activity against two small synthetic peptides. By SDS-polyacrylamide gel electrophoresis the molecular masses for EA.1 proteinase and OK3A.1 proteinase were 42 000 Da and 32 000 Da respectively. Both enzymes were resistant to 10 mmol/1 phenylmethylsulphonylfluoride and iodoacetic acid, but were deactivated by EDTA. Whereas EA.1 proteinase was inhibited by o -phenanthroline and activated by zinc ions, OK3A.1 proteinase was unaffected by either agent although some dependence on divalent metal ions for activity was apparent. The enzymes were stabilized by calcium ions, EA.1 proteinase exhibiting a half-life of 2 h at 85.C whilst OK3A.1 proteinase was less stable with a half-life of 40 min at this temperature.     

Purification and characterization of the major surface array protein from the avirulent Bacillus anthracis Delta Sterne-1.

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.