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PubMed1983

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

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

Absorption↗
PubMed1991

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

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

Bacillus anthracis↗
PubMed1993

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

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

Bacillus anthracis↗
PubMed2004

Assessment of a new selective chromogenic Bacillus cereus group plating medium and use of enterobacterial autoinducer of growth for cultural identification of Bacillus species.

A new chromogenic Bacillus cereus group plating medium permits differentiation of pathogenic Bacillus species by colony morphology and color. Probiotic B. cereus mutants were distinguished from wild-type strains by their susceptibilities to penicillin G or cefazolin. The enterobacterial autoinducer increased the sensitivity and the speed of enrichment of B. cereus and B. anthracis spores in serum-supplemented minimal salts medium (based on the standard American Petroleum Institute medium) and buffered peptone water.

Bacillus anthracis↗
PubMed1992

Nucleotide sequence of the gene for an alkaline endoglucanase from an alkalophilic Bacillus and its expression in Escherichia coli and Bacillus subtilis.

The gene for an alkaline endoglucanase from the alkalophilic Bacillus sp. KSM-64 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. The nucleotide sequence of a 4.1-kb region of the HindIII insert had two open reading frames, ORF-1 and ORF-2. The protein deduced from ORF-1 was composed of 244 amino acids with an M(r) of 27,865. Subcloning analysis proved that the alkaline endoglucanase was encoded by ORF-2 (822 amino acids with an M(r) of 91,040). Upstream from ORF-2, there were three consensus like sequences of the sigma A-type promoter of Bacillus subtilis, a putative Shine-Dalgarno sequence (AGGAGGT), and a catabolite repression operator-like sequence (TGTAAGCGGTTAACC). The HindIII insert was subcloned into a shuttle vector, pHY300PLK, and the encoded alkaline endoglucanase gene was highly expressed both in E. coli and B. subtilis. One of the three promoter-like sequences in ORF-2 could be suitable for high levels of enzyme expression in both host organisms.

Amino Acid Sequence↗
PubMed1995

Application of the upstream region of a Bacillus endoglucanase gene to high-level expression of foreign genes in Bacillus subtilis.

A 0.4-kb ScaI-HpaI fragment, 199 bp upstream of the structural gene for alkaline endoglucanase, from the alkalophilic Bacillus sp. KSM-64, was found to be essential for the extracellular production of the enzyme by recombinant Bacillus subtilis cells. We constructed a new vector, pHSP64 (5.5 kb), using pHY300PLK and part of the 5' region of the endoglucanase that contained a possible promoter region. Using recombinant B. subtilis cells that carried this vector, very high production of two endoglucanases and of chloramphenicol acetyltransferase was done.

Amino Acid Sequence↗
PubMed2025

Identification of sporulation genes in Bacillus anthracis highlights similarities and significant differences with Bacillus subtilis.

The molecular basis of endospore formation in the model gram-positive bacterium Bacillus subtilis has been investigated for over half a century. Here, using high throughput and classical genetic approaches, we performed a comparative analysis of sporulation in the human pathogen Bacillus anthracis. A transposon-sequencing screen identified >150 genes required for B. anthracis sporulation. As anticipated, many of the genes that are critical for sporulation in B. subtilis were also required for B. anthracis sporulation. However, we identified >50 genes that are important for sporulation in B. anthracis but not in B. subtilis, and 22 B. anthracis sporulation genes that are absent from the B. subtilis genome. To validate the hits from our screen, we generated an ordered transposon-mutant library using Knockout Sudoku. Cytological analysis of a subset of the canonical sporulation-defective mutants revealed similar but not identical phenotypes in the pathogen compared to the model. We investigated several of the newly identified sporulation genes, with an in-depth analysis of one, ORF 04167, renamed ipdA. Sporulating cells lacking ipdA are blocked in the morphological process of engulfment, generating septal bulges. An AlphaFold-Multimer screen and a classical genetic enrichment revealed that IpdA is a secreted inhibitor of the polysaccharide deacetylase PdaN. Our data support a model in which induction of IpdA at the onset of sporulation inhibits deacetylation of the cell wall peptidoglycan (PG), enabling the sporulation-specific PG hydrolases to catalyze engulfment. Altogether, our studies reveal that B. subtilis is an excellent model for endospore formation in B. anthracis, while underscoring the importance of direct analysis in B. anthracis. The suite of tools that we have generated will catalyze the molecular dissection of sporulation and other cell biological processes in this important human pathogen.

Bacillus anthracis↗
PubMed1986

The primary structure of Bacillus cereus neutral proteinase and comparison with thermolysin and Bacillus subtilis neutral proteinase.

The complete amino-acid sequence of a neutral proteinase, produced by Bacillus cereus, was determined by protein sequencing. The neutral proteinase consists of 317 amino-acid residues. The primary structure is 70% homologous to thermolysin, a thermostable neutral proteinase and 45% homologous to Bacillus subtilis neutral proteinase. The zinc-binding site and the hydrophobic pocket of the active site are highly similar in all three proteinases. B. cereus neutral proteinase which is 20 degrees C less thermostable (60 degrees C) than thermolysin (80 degrees C) shows only minor differences in calcium binding sites and salt bridges compared to thermolysin (known from its X-ray diffraction analysis), whereas B. subtilis neutral proteinase (50 degrees C) differs considerably. Therefore it was assumed that the difference in thermostability between B. cereus neutral proteinase and thermolysin is not caused by different metal binding properties, or differences in the active site, but by changes within the rest of the molecule. Calculation of secondary structure potentials according to Chou & Fasman, hydrophobicity and bulkiness of the different structural elements and preferred cold----hot amino-acid residue exchanges indicated, that the thermostability of thermolysin compared to B. cereus neutral proteinase is caused by small effects contributed by numerous amino-acid exchanges distributed over the whole molecule, resulting in increased hydrophobicity of beta-pleated sheet and higher bulkiness of alpha-helical regions.

Amino Acid Sequence↗
PubMed1996

High-level expression of Bacillus thuringiensis phosphatidylinositol-specific phospholipase C by the Bacillus brevis host-vector system.

We succeeded in hyperproduction of Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PIPLC), using a Bacillus brevis 47 expression system. The recombinant B. thuringiensis PIPLC was expressed under the control of the middle wall protein gene promoter in B. brevis expression vector pNU211. A large amount of recombinant PIPLC (0.4 g per liter culture) was secreted into the medium as a mature enzyme, and the enzymatic properties of purified recombinant PIPLC were similar to those of the enzyme from wild-type B. thuringiensis. This system provides a useful approach to the three-dimensional structure-function relationship of PIPLC.

Bacillus↗
PubMed1999

Field evaluation of new water-dispersible granular formulations of Bacillus thuringiensis ssp. israelensis and Bacillus sphaericus against Culex mosquitoes in microcosms.

A variety of formulations of Bacillus thuringiensis var. israelensis de Barjac (B.t.i.) and Bacillus sphaericus Neide (B.s.) have been studied for mosquito control under laboratory and field conditions. High efficacy, specificity, low risk of development of resistance, long shelf-life, and transportability, as well as the safety to nontarget organisms of these 2 microbial agents have been well documented. Some of the currently available formulations of B.t.i. and B.s. have low potency per unit mass. Research and development efforts are focusing on commercializing formulations with high potency and low minimum effective dosage that are suitable for long-distance shipment. To achieve this goal, new water-dispersible granule (WDG) formulations of both microbial agents were prepared and made available by Abbott Laboratories for evaluation. The newly developed WDGs of B.t.i. and B.s. with high potency dispersed readily in water with gentle agitation. These WDGs were evaluated and the minimum effective dosages were determined in microcosms against natural populations of Culex mosquitoes. The minimum effective dosage for B.t.i. WDGs with 4,000 International Toxic Units (ITU)/mg was 0.27-0.53 lb/acre which yielded significant control for up to 7-12 days. The minimum effective dosage for B.s. WDGs with 350-630 ITU/mg was 0.05-0.10 lb/acre, which yielded significant control of immature mosquitoes for up to 14-20 days.

Animals↗
PubMed1999

Mosquito control and bacterial flora in water enriched with organic matter and treated with Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus formulations.

Three tests were conducted during July 17 to October 30, 1998 to study the impact of two mosquitocidal microbial agents on mosquito larvae and their contribution to bacterial flora in aquatic microcosms. Formulations of Bacillus thuringiensis subsp. israelensis (Bti) and Bacillus sphaericus strain 2362 (Bsph) were applied at various rates to outdoor tubs enriched with rabbit pellets and filled with irrigation water from a reservoir. Mosquito larvae were effectively controlled by all treatments; the magnitude of initial and persistent control depended on materials and dosages applied. Bacterial flora were assessed in the irrigation water as well as water in the enriched tubs before and after treatment with the microbial agents. The irrigation water contained 800-1000 total bacterial cells/ml. The populations of total bacteria and spore formers peaked on day 3 after enriching and filling the tubs, then declined progressively to the low levels at the end of the tests. After treatment, the numbers of Bti and Bsph spores in treated tubs prevailed at a dosage-dependent manner, their populations peaked at three hours after treatment, and declined progressively thereafter. The contribution of Bti and Bsph spores to the total bacterial flora was negligible but significant to the counts of spore-forming bacteria. The gram-negative bacteria made up more than 80% of the total bacterial flora during the test periods; and, of these, gram-negative rods constituted the greatest proportion, gradually increasing from the time of flooding to the end of the tests. Gram-negative cocci also occurred in relatively great proportion, but showed a reverse trend as compared with the gram-negative rods, declining gradually from pretreatment to the end of the tests. Gram-positive rods (spore formers), including Bti and Bsph, occurred in low numbers in all the tests but increased slightly in treated tubs due to the addition of Bti and Bsph spores. Gram-positive cocci occurred occasionally in some water samples.

Animals↗
PubMedDate not supplied

[Duration of the larvicidal effect of spore crystalline mass of bacteria Bacillus thuringiensis spp. israelensis and Bacillus sphaericus in the laboratory setting].

The duration of action of sporocrystalline mass of Bacillus thuringiensis spp. Israelensis (Bti) and Bacillus sphaericus (Bsph) against three-age larvae of An. stephensi mosquitoes was studied in the laboratory setting. The duration of action depends on its initial concentration. The optimal concentration should be at least 10(5) spores/ml for An. stephensi larvae. Decreasing the concentration reduces the duration of larvicidal action. Increasing the number of the larvae placed also diminishes the duration of action shown by Bti. This has no significant influence on the duration of action by Bsph. The presence of dead larvae in the test vessel increases the duration of action by Bti only by 4 times whereas Bsph acts rather long. The follow-ups lasted 60 weeks with full death of the larvae placed again. A significant water resistance of Bti sporocrystalline mixture was found. The mixture retained its larvicidal activity 6 months following its presence in the dechlorinated water without larval placement. Bsph was less resistant and its complete inactivity was noted under the same conditions just following 2 months.

Animals↗
PubMedDate not supplied

[Medium for the production of Bacillus intermedius glutamyl endopeptidase by the recombinant Bacillus subtilis].

A nutrient medium was elaborated for the efficient production of glutamyl endopeptidase by the recombinant Bacillus subtilis strain AJ73 bearing the Bacillus intermedius 3-19 glutamyl endopeptidase gene within a multicopy plasmid. Optimal concentrations of the main nutrients, peptone and inorganic phosphate, were found using a multifactor approach. To provide for active growth and efficient glutamyl endopeptidase production, the cultivation medium of the recombinant strain should be enriched in phosphorus, organic and inorganic nitrogen sources, and yeast extract. Complex protein substrates, such as casein and gelatin, enhanced the biosynthesis of glutamyl endopeptidase. At the same time, easily metabolizable carbon sources suppressed it. The production of glutamyl endopeptidase was stimulated by the bivalent cations Ca2+, Mg2+, and Co2+.

Bacillus↗
PubMed2001

Recovery of commercially produced Bacillus thuringiensis var. israelensis and Bacillus sphaericus from tires and prevalence of bacilli in artificial and natural containers.

We conducted surveys to identify the species of spore-forming bacteria present in natural and artificial containers. Most of our samples came from Illinois. Identification was based on the cellular fatty acid composition of the bacterial cell wall. In addition, we utilized a custom database for commercially produced strains of Bacillus thuringiensis var. israelensis (Bti) and B. sphaericus, to differentiate between larvicidal isolates with commercial or native origin. Native Bti was present at low levels in almost all habitats but was not recovered from bromeliads and metal containers. In temporary woodland pools, 27.9% of the colonies recovered were native Bti. We did not recover larvicidal B. sphaericus in untreated habitats. VectoBac and VectoLex were applied to tires containing water and the tires were sampled 3 months and 9 months after treatment. Isolates of Bti and B. sphaericus with commercial origin were recovered as long as 9 months after application. We noticed numerous cadavers of Aedes triseriatus in several tires 9 months after treatment with VectoBac. We could not determine if this mortality resulted from recycling of Bti in these tires or whether insecticidal crystal proteins from the original treatment were resuspended. Bacillus thuringiensis var. israelensis isolates with commercial ancestry were recovered from untreated tires 9 months after application. Isolates of larvicidal B. sphaericus that differed from the bacteria in VectoLex were also recovered from untreated tires.

Animals↗
PubMedDate not supplied

[Physiological activity of mixed cultures of Methylcoccus capsulatus UKM B-3030 with Bacillus megaterium UKM B-5723T and Bacillus subtilis VKPM B-1489 on solid surface colonization].

Physiological activity of monoculture of Methylococcus capsulatus UCM B-3030 and its mixed cultures with two bacilli species distinguished by proteolytical properties--Bacillus megaterium UCM B-5723T and Bacillus subtilis BK[symbol: see text]M B-4189 in terms of long-duration cultivation is investigated. It is shown, that the most active methane consumption by methanotrophic bacteria and their mixed cultures with bacilli under solid surface colonization occurred on the second-eighth day of cultivation. The obtained results permit one to evaluate intensity of CH4 oxidation process by typical microflora of some econiches (depleted coal mines, in particular).

Bacillus megaterium↗
PubMedDate not supplied

[Culture media for growth and spore formation of Bacillus subtilis and Bacillus licheniformis].

The nutrient media for deep cultivation of Bacillus subtilis 3 and Bacillus licheniformis 31 have been optimized. It has been shown, that the media caused much higher biomass production from 2.0 x 10(10) CFU/ml media for B. subtilis 3 to 5.1 x 10(10) CFU/ml for B. licheniformis 31 and 50% production of spores by both cultures under such cultivation conditions.

Bacillus↗
PubMedDate not supplied

[The regulation of Bacillus intermedius glutamyl endopeptidase biosynthesis in the recombinant Bacillus subtilis strain during sporulation].

The growth of the recombinant Bacillus subtilis strain AJ73 carrying the Bacillus intermedius 3-19 glutamyl endopeptidase gene on a multicopy plasmid and the effect of some nutrients on the efficiency of extracellular glutamyl endopeptidase production in the stationary growth phase were studied. In this phase, the concentration of glutamyl endopeptidase in the culture liquid peaked at the 48th and 78th h of cultivation and depended on the composition of the cultivation medium. Unlike the synthesis of glutamyl endopeptidase in the trophophase (i.e., during vegetative growth), which was suppressed by glucose, the synthesis of this enzyme during sporulation was resistant to glucose present in the cultivation medium. A multifactorial experimental design allowed optimal proportions between the concentrations of major nutrients (peptone and inorganic phosphate) to be determined. Inorganic phosphate and ammonium ions augmented the production of glutamyl endopeptidase by 30-150%, and complex organic substrates, such as casein and gelatin, enhanced the production of glutamyl endopeptidase by 50-100%. During sporulation, the production of glutamyl endopeptidase was stimulated by some bivalent cations (Ca2+, Mg2+, and Co2+) and inhibited by others (Zn2+, Fe2+, and Cu2+). The inference is drawn that the regulatory mechanisms of glutamyl endopeptidase synthesis during vegetative growth and sporulation are different.

Bacillus↗
PubMedDate not supplied

[Biosynthesis of Bacillus intermedius glutamyl endopeptidase in recombinant Bacillus subtilis cells during the stationary growth phase].

We studied the biosynthesis of Bacillus intermedius glutamyl endopeptidase in the recombinant Bacillus subtilis strain AJ73 delta58.21 during the stationary growth phase. We optimized the composition of the culture medium to favor effective enzyme production during the stationary growth phase, and found that the nutritional requirements for glutamyl endopeptidase synthesis were different in the stationary phase and growth retardation phase. Proteinase accumulation was activated by complex organic substrates (casein and gelatin). During final stages of the culture growth, the enzyme production was stimulated by Ca2+, Mn2+, and Co2+ and inhibited by Zn2+, Fe2+, and Cu2+. The synthesis of glutamyl endopeptidase in the late stationary phase was not inhibited by glucose, unlike that in the trophophase during proliferation. We conclude that the regulatory mechanisms of proteinase synthesis during vegetative growth and sporulation are different.

Bacillus↗