Plasmid-associated sensitivity of Bacillus thuringiensis to UV light.

Spores and vegetative cells of Bacillus thuringiensis were more sensitive to UV light than were spores or cells of plasmid-cured B. thuringiensis strains or of the closely related Bacillus cereus. Introduction of B. thuringiensis plasmids into B. cereus by cell mating increased the UV sensitivity of the cells and spores. Protoxins encoded by one or more B. thuringiensis plasmids were not involved in spore sensitivity, since a B. thuringiensis strain conditional for protoxin accumulation was equally sensitive at the permissive and nonpermissive temperatures. In addition, introduction of either a cloned protoxin gene, the cloning vector, or another plasmid not containing a protoxin gene into a plasmid-cured strain of B. thuringiensis all increased the UV sensitivity of the spores. Although the variety of small, acid-soluble proteins was the same in the spores of all strains examined, the quantity of dipicolinic acid was about twice as high in the plasmid-containing strains, and this may account for the differences in UV sensitivity of the spores. The cells of some strains harboring only B. thuringiensis plasmids were much more sensitive than cells of any of the other strains, and the differences were much greater than observed with spores.     

Regulation of protoxin synthesis in Bacillus thuringiensis.

A derivative of Bacillus thuringiensis subsp. kurstaki (HD-1) formed parasporal inclusions at 25 degrees C, but not at 32 degrees C. This strain differed from the parent only in the loss of a 110-megadalton (Md) plasmid, but plasmid and chromosomal copies of protoxin genes were present in both strains. On the basis of temperature shift experiments, the sensitive period appeared to be during midexponential growth, long before the time of protoxin synthesis at 3 to 4 h after the end of exponential growth. The conditional phenotype could be transferred by cell mating to naturally acrystalliferous Bacillus cereus. In all such cases, a 29-Md protoxin -encoding plasmid was transferred, but this plasmid alone was barely sufficient for protoxin synthesis. Protoxin production increased to detectable levels, but well below those of the parental donor strain, by simultaneous transfer of a 44-Md protoxin -encoding plasmid. Transfer of a 5-Md plasmid with the two larger protoxin -coding plasmids resulted in a protoxin synthesis level approaching that of the donor strain. A role for some of the cryptic plasmids of kurstaki in parasporal body formation was implied. In contrast, a closely related B. thuringiensis strain, HD73 , produced crystals at both 25 and 32 degrees C even when the capacity was transferred on a 50-Md plasmid to B. cereus. The amount of protoxin produced in these B. cereus transcipients , however, was somewhat less than that produced in the parental strain HD73 , implying that catabolic differences, gene dosage, or the presence of a chromosomal gene (or a combination of these) may be necessary for maximum production. A regulatory component of the 29-Md plasmid appeared to be trans-acting and dominant since B. cereus transcipients containing the 29-Md plasmid from kurstaki and the 50-Md plasmid from HD73 produced more protoxin at 25 degrees C than at 30 degrees C. Similar results were obtained when protoxin synthetic capacity was transferred from B. thuringiensis subsp. israelensis to the conditional B. thuringiensis subsp. kurstaki strain.     

Identification of self-transmissible plasmids in four Bacillus thuringiensis subspecies.

The transfer of plasmids by mating from four Bacillus thuringiensis subspecies to Bacillus anthracis and Bacillus cereus recipients was monitored by selecting transcipients which acquired plasmid pBC16 (Tcr). Transcipients also inherited a specific large plasmid from each B. thuringiensis donor at a high frequency along with a random array of smaller plasmids. The large plasmids (ca. 50 to 120 megadaltons), pXO13, pXO14, pXO15, and pXO16, originating from B. thuringiensis subsp. morrisoni, B. thuringiensis subsp. toumanoffi, B. thuringiensis subsp. alesti, and B. thuringiensis subsp. israelensis, respectively, were demonstrated to be responsible for plasmid mobilization. Transcipients containing any of the above plasmids had donor capability, while B. thuringiensis strains cured of each of them were not fertile, indicating that the plasmids confer conjugation functions. Confirmation that pXO13, pXO14, and pXO16 were self-transmissible was obtained by the isolation of fertile B. anthracis and B. cereus transcipients that contained only pBC16 and one of these plasmids. pXO14 was efficient in mobilizing the toxin and capsule plasmids, pXO1 and pXO2, respectively, from B. anthracis transcipients to plasmid-cured B. anthracis or B. cereus recipients. DNA-DNA hybridization experiments suggested that DNA homology exists among pXO13, pXO14, and the B. thuringiensis subsp. thuringiensis conjugative plasmids pXO11 and pXO12. Matings performed between strains which each contained the same conjugative plasmid demonstrated reduced efficiency of pBC16 transfer. However, in many instances when donor and recipient strains contained different conjugative plasmids, the efficiency of pBC16 transfer appeared to be enhanced.     

Identification of Bacillus anthracis by polyclonal antibodies against extracted vegetative cell antigens

The extractable protein antigens EA1 and EA2 of Bacillus anthracis were prepared from electrophoresis transblots of SDS extracts of vegetative bacteria of the Sterne strain. Hyperimmune guinea-pig antiserum against EA2 failed to react with B. anthracis cells in immunofluorescence (IF) tests. Guinea-pig antiserum against EA1 (anti-EA1) reacted strongly in IF tests with non-encapsulated vegetative cell of 10 of 12 strains of B. anthracis and with cells of strains of B. cereus and B. thuringiensis. The unreactive B. anthracis strains were delta-Vollum-1B-1 and Texas. Encapsulated cells of B. anthracis stained poorly except for small bright regions. Absorption of anti-EA1 with cells of B. cereus NCTC 8035 and NCTC 9946 removed activity towards all B. cereus strains tested, but only partly reduced cross-reaction with B. thuringiensis strains. Absorption of anti-EA1 with B. thuringiensis 4041 removed activity towards this strain and B. cereus strains. Evidence is produced that B. thuringiensis cells grown on nutrient agar possess more cross-reacting antigens than cells grown in nutrient broth. The reaction of anti-EA1 with Bacillus spores immobilized in clumps on microscope slides was attributed to contaminating vegetative debris because well-separated individual spores failed to react. A rapid IF test was developed allowing identification of B. anthracis sampled from overnight cultures on blood plates. When sodium dodecyl sulphate extracts of B. anthracis vegetative cells were analysed on immunoblots (Western blots) by reaction with anti-EA1, a number of bands were visualized in addition to the expected 91 kiloDalton EA1 band. Prior absorption of anti-EA1 with B. cereus or B. thuringiensis cells resulted in the disappearance of most or all of the brands in blots of these species, but had less effect on blots of the B. anthracis strains. All six B. anthracis strains that were blotted including delta-Vollum-1B-1 and Texas, could thus be distinguished from B. cereus and B. thuringiensis by their differential reaction with unabsorbed and absorbed anti-EA1.     

Transfer of chromosomal genes and plasmids in Bacillus thuringiensis.

A low frequency of chromosomal gene transfer from Bacillus thuringiensis to Bacillus cereus was detected by cell mating, with a tryptophan marker being the most frequently transferred gene among four that were tested. The process was resistant to DNase and was not mediated by cell filtrates. Among several B. thuringiensis subspecies tested, transfer was best with a derivative of B. thuringiensis subsp. kurstaki HD1, which lost several plasmids. All of the B. cereus recombinants contained at least one plasmid from the donor B. thuringiensis; frequently, it was a plasmid that encoded a protoxin gene. In matings with B. thuringiensis subsp. kurstaki HD1, a 29-megadalton plasmid that contained a ca. 2.5-kilobase region of homology with the chromosome was always transferred. No detectable transfer of chromosomal genes was found in B. thuringiensis subsp. kurstaki HD1 strains lacking this plasmid, suggesting that there may be chromosome mobilization.     

Transfer of chromosomal genes and plasmids in Bacillus thuringiensis

A low frequency of chromosomal gene transfer from Bacillus thuringiensis to Bacillus cereus was detected by cell mating, with a tryptophan marker being the most frequently transferred gene among four that were tested. The process was resistant to DNase and was not mediated by cell filtrates. Among several B. thuringiensis subspecies tested, transfer was best with a derivative of B. thuringiensis subsp. kurstaki HD1, which lost several plasmids. All of the B. cereus recombinants contained at least one plasmid from the donor B. thuringiensis; frequently, it was a plasmid that encoded a protoxin gene. In matings with B. thuringiensis subsp. kurstaki HD1, a 29-megadalton plasmid that contained a ca. 2.5-kilobase region of homology with the chromosome was always transferred. No detectable transfer of chromosomal genes was found in B. thuringiensis subsp. kurstaki HD1 strains lacking this plasmid, suggesting that there may be chromosome mobilization.     

The solubility of inclusion proteins from Bacillus thuringiensis is dependent upon protoxin composition and is a factor in toxicity to insects.

Bacillus thuringiensis subsp. aizawai HD133 is one of several strains particularly effective against Plodia interpunctella selected for resistance to B. thuringiensis subsp. kurstaki HD1 (Dipel). B. thuringiensis subsp. aizawai HD133 produces inclusions containing three protoxins, CryIA(b), CryIC, and CryID, and the CryIC protoxin has been shown to be active on resistant P. interpunctella as well as on Spodoptera larvae. The CryIA(b) protoxin is very similar to the major one in B. thuringiensis subsp. kurstaki HD1, and as expected, this protoxin was inactive on resistant P. interpunctella. A derivative of B. thuringiensis subsp. aizawai HD133 which had been cured of a 68-kb plasmid containing the cryIA(b) gene produced inclusions comprising only the CryIC and CryID protoxins. Surprisingly, these inclusions were much less toxic for resistant P. interpunctella and two other Lepidoptera than those produced by the parental strain, whereas the soluble protoxins from these strains were equally effective. In contrast, inclusions from the two strains were about as active as soluble protoxins for Spodoptera frugiperda larvae, so toxicity differences between inclusions may be due to the solubilizing conditions within particular larval guts. Consistent with this hypothesis, it was found that a higher pH was required to solubilize protoxins from inclusions from the plasmid-cured strain than from B. thuringiensis subsp. aizawai HD133, a difference which is probably attributable to the absence of the CryIA(b) protoxin in the former. The interactions of structurally related protoxins within an inclusion are probably important for solubility and are thus another factor in the effectiveness of B. thuringiensis isolates for particular insect larvae.     

Virulent spores of Bacillus anthracis and other Bacillus species deposited on solid surfaces have similar sensitivity to chemical decontaminants

AIMS: To compare the relative sensitivity of Bacillus anthracis and spores of other Bacillus spp. deposited on different solid surfaces to inactivation by liquid chemical disinfecting agents. METHODS AND RESULTS: We prepared under similar conditions spores from five different virulent and three attenuated strains of B. anthracis, as well as spores of Bacillus subtilis, Bacillus atrophaeus (previously known as Bacillus globigii), Bacillus cereus, Bacillus thuringiensis and Bacillus megaterium. As spore-surface interactions may bias inactivation experiments, we evaluated the relative binding of different spores to carrier materials. The survival of spores deposited on glass, metallic or polymeric surfaces were quantitatively measured by ASTM standard method E-2414-05 which recovers spores from surfaces by increasing stringency. The number of spores inactivated by each decontaminant was similar and generally within 1 log among the 12 different Bacillus strains tested. This similarity among Bacillus strains and species was observed through a range of sporicidal efficacy on spores deposited on painted metal, polymeric rubber or glass. CONCLUSIONS: The data obtained indicate that the sensitivity of common simulants (B. atrophaeus and B. subtilis), as well as spores of B. cereus, B. thuringiensis, and B. megaterium, to inactivation by products that contain either: peroxide, chlorine or oxidants is similar to that shown by spores from all eight B. anthracis strains studied. SIGNIFICANCE AND IMPACT OF THE STUDY: The comparative results of the present study suggest that decontamination and sterilization data obtained with simulants can be safely extrapolated to virulent spores of B. anthracis. Thus, valid conclusions on sporicidal efficacy could be drawn from safer and less costly experiments employing non-pathogenic spore simulants.     

Genetic relationships between sympatric populations of Bacillus cereus and Bacillus thuringiensis, as revealed by rep-PCR genomic fingerprinting

The bacterial strain Bacillus cereus is closely related to Bacillus thuringiensis, although any genetic relationship between the two strains is still in debate. Using rep-PCR genomic fingerprinting, we established the genetic relationships between Brazilian sympatric populations of B. cereus and B. thuringiensis simultaneously collected from two geographically separate sites. We observed the formation of both B. thuringiensis and B. cereus clusters, as well as strains of B. cereus that are more closely related to B. thuringiensis than to other B. cereus strains. In addition, lower genetic variability was observed among B. thuringiensis clusters compared to B. cereus clusters, indicating that either the two species should be categorized as separate or that B. thuringiensis may represent a clone from a B. cereus background.     

The protoxin composition of Bacillus thuringiensis insecticidal inclusions affects solubility and toxicity.

Most Bacillus thuringiensis strains producing toxins active on lepidoptera contain several plasmid-encoded delta-endotoxin genes and package related protoxins into a single inclusion. It was previously found that in B. thuringiensis subsp. aizawai HD133, which produces an inclusion comprising the CryIAb, CryIC, and CryID protoxins, there is a spontaneous loss in about 1% of the cells of a 45-mDa plasmid containing the cryIAb gene. As a result, inclusions produced by the cured strain were less readily solubilized at pH 9.2 or 9.5 and had a decreased toxicity for Plodia interpunctella, despite the presence of the CryIC protoxin, which was active when solubilized. These results suggested that protoxin composition was a factor in inclusion solubility and toxicity and that the cryIAb gene, which is also present on an unstable plasmid in several other subspecies, may have a unique role in inclusion solubility and toxicity. Introduction of a cloned copy of this gene into the plasmid-cured derivative of B. thuringiensis subsp. aizawai HD133 resulted in an increase in the solubility at pH 9.2 of all of the inclusion proteins from less than 20% to greater than 45% and a lowering of the 50% lethal concentration (LC50, in micrograms [dry weight] per square centimeter) of inclusions for Spodoptera frugiperda from 35 to 10. These values are the same as those found with inclusions from B. thuringiensis subsp. aizawai HD133, and in all cases, the LC50 of the solubilized protoxins was 10. Transformants containing related cryIA genes produced inclusions which were more than 95% solubilized at pH 9.2 but also had LC50 of 10.(ABSTRACT TRUNCATED AT 250 WORDS)     

Corrected Version Distribution of Heterogeneous and Homologous Plasmids in Bacillus spp

A total of 75 strains (including 5 reference strains) of Bacillus amyloliquefaciens, B. cereus, B. circulans, B. licheniformis, B. megaterium, B. pumilus, B. sphaericus, B. subtilis, and B. thuringiensis and 36 species-unidentified Bacillus strains were surveyed for plasmids by cesium chloride-ethidium bromide equilibrium centrifugation of cell lysates in a study of antibiotic resistance in host cells. Of the 111 strains, 13 (including 3 reference strains) were found to harbor plasmids, and 5 of the 13 showed antibiotic resistance. This antibiotic resistance appeared not to be due to the plasmids, however, because the trait was not cured by cultivation of cells in nutrient medium containing ethidium bromide (1 mug/ml), sodium dodecyl sulfate (0.2 mug/ml), or novobiocin (1 mug/ml), except in one strain, in which kanamycin and streptomycin resistances were cured by novobiocin. One strain of B. amyloliquefaciens, S294, was found to harbor a plasmid, pFTB14, which differed from the plasmid species of classes 1 to 6 in B. subtilis and B. amyloliquefaciens, as determined by restriction analysis and DNA contour length determination. However, in DNA-DNA hybridization on a filter after Southern blotting from an agarose gel, the pFTB14 DNA hybridized with plasmids of classes 1 to 5. Three strains of B. thuringiensis each carried at least 4 to 11 plasmid species, whereas no plasmids were detected in four strains of B. cereus, which, in relation to B. thuringiensis, is closely related taxonomically and has highly homologous DNA sequences. The plasmid DNAs prepared from species other than B. subtilis and B. amyloliquefaciens did not hybridize with that of pFTB14.     

4种细菌芽胞中α/β-SASP分子结构对比分析

认识和描述不同细菌芽胞α/β-SASP的分子结构特征,为深入开展以α/β-SASP为靶向修饰的应用技术提供科学依据.运用生物信息学方法和技术,比对分析4种菌株,炭疽芽胞杆菌Ames 株、苏云金芽胞杆菌serovar konkukian 97-27 株、腊样芽胞杆菌ATCC 10987株、枯草芽胞杆菌168 株的α/β-SASP基因及蛋白质一、二、三级结构的异同.基因-ClustalW2;一级结构-ClustalW2和ProtParam tool;二级结构-SOPMA;三级结构-SWISS-MODEL和Swiss-Pdbviewer4.0.1.4种菌株的α/β-SASP基因及蛋白质一、二、三级结构有明显的同源性,炭疽芽胞、苏云金芽胞和腊样芽胞的生物学特征非常相似.在开展细菌芽胞的α/β-SASP基因及生物效应研究时,可以首选苏云金杆菌芽胞或腊样杆菌芽胞作为炭疽杆菌芽胞的试验菌,其次可以选择枯草杆菌芽胞.     

Insights into the genetic organization of the Bacillus mycoides cryptic plasmids pDx14.2 and pSin9.7 deduced from their complete nucleotide sequence

Bacillus mycoides, a member of the Bacillus cereus group of bacteria, can be easily distinguished from close species because of colony shape, made by filaments of cells, resembling fungal hyphae, curving clock- or counterclockwise depending on the strain. Two plasmids, one from a strain curving to the right (pDx14.2), the other from a strain curving to the left (pSin9.7), were sequenced and analyzed for gene content and replication mode. Rolling-circle replication modules and mobilization proteins were found, very similar to those of other plasmids of the B. cereus group bacilli, mostly Bacillus thuringiensis living in the same ecosystem, suggesting active plasmid exchange in nature.     

Homologous and heterologous transcription of the Cry+-plasmid in Bacillus thuringiensis

The possibility of homologous and heterologous transception of Cry+ plasmids in Bacillus thuringiensis is demonstrated. Cry+ plasmids from crystal bearing strain of Bacillus thuringiensis were transferred into acrystalline strain belonging to H5 serotype by mutual incubation. The donor strain was previously marked by the transmissive plasmid pAM beta 1 coding for erythromycin and lincomycin resistance. The transcipients having acquired the ability to synthesize delta-endotoxin were referred to H5 serotype due to their phenotype. By analogous method Cry+ plasmid was transferred from Bacillus thuringiensis to Bacillus cereus. Bacillus cereus strain GP7 was used as a recipient strain resistant to tetracycline. The presence of delta-endotoxin in transcipients was confirmed by bioprobes and immunoenzyme assay. To prove the transfer of Cry+ plasmid the plasmid profiles of the parent strains and transcipients have been analyzed. The formation of cellular contacts during mutual incubation of Bacillus thuringiensis and Bacillus cereus strains was demonstrated by electron microscopic study of ultrafine cuts.     

Rapid characterization of spores of Bacillus cereus group bacteria by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption-ionization (MALDI) time-of-flight mass spectrometry was used to characterize the spores of 14 microorganisms of the Bacillus cereus group. This group includes the four Bacillus species B. anthracis, B. cereus, B. mycoides, and B. thuringiensis. MALDI mass spectra obtained from whole bacterial spores showed many similarities between the species, except for B. mycoides. At the same time, unique mass spectra could be obtained for the different B. cereus and B. thuringiensis strains, allowing for differentiation at the strain level. To increase the number of detectable biomarkers in the usually peak-poor MALDI spectra of spores, the spores were treated by corona plasma discharge (CPD) or sonicated prior to MALDI analysis. Spectra of sonicated or CPD-treated spores displayed an ensemble of biomarkers common for B. cereus group bacteria. Based on the spectra available, these biomarkers differentiate B. cereus group spores from those of Bacillus subtilis and Bacillus globigii. The effect of growth medium on MALDI spectra of spores was also explored.     

Efficacy of a Fluorescent-Antibody Procedure for Identifying Bacillus cereus in Foods

One hundred and seventeen strains of Bacillus were examined by the fluorescent-antibody technique by using the globulin fraction of serum prepared against spores of B. cereus T. All but one strain of the 59 B. cereus tested fluoresced at the exosporium surface. Fluorescent staining of B. anthracis, B. thuringiensis, and B. mycoides was also observed. Absorption of the globulin fraction with B. anthracis and B. mycoides resulted in the elimination of staining of these organisms. Absorption with B. thuringiensis ATCC 10792 removed antibodies reacting with 6 of the strains of B. thuringiensis tested. Absorption with B. thuringiensis var. galleriae removed antibodies against B. cereus to such a degree that the globulin fraction was unusable.     

Mating system for transfer of plasmids among Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis.

To facilitate the analysis of genetic determinants carried by large resident plasmids of Bacillus anthracis, a mating system was developed which promotes plasmid transfer among strains of B. anthracis, B. cereus, and B. thuringiensis. Transfer of the selectable tetracycline resistance plasmid pBC16 and other plasmids from B. thuringiensis to B. anthracis and B. cereus recipients occurred during mixed incubation in broth. Two plasmids, pXO11 and pXO12, found in B. thuringiensis were responsible for plasmid mobilization. B. anthracis and B. cereus transcipients inheriting either pXO11 or pXO12 were, in turn, effective donors. Transcipients harboring pXO12 were more efficient donors than those harboring pXO11; transfer frequencies ranged from 10(-4) to 10(-1) and from 10(-8) to 10(-5), respectively. Cell-to-cell contact was necessary for plasmid transfer, and the addition of DNase had no effect. The high frequencies of transfer, along with the fact that cell-free filtrates of donor cultures were ineffective, suggested that transfer was not phage mediated. B. anthracis and B. cereus transcipients which inherited pXO12 also acquired the ability to produce parasporal crystals (Cry+) resembling those produced by B. thuringiensis, indicating that pXO12 carries a gene(s) involved in crystal formation. Transcipients which inherited pXO11 were Cry-. This mating system provides an efficient method for interspecies transfer of a large range of Bacillus plasmids by a conjugation-like process.