Conjugal transfer between Bacillus thuringiensis and Bacillus cereus strains is not directly correlated with growth of recipient strains

Bacillus thuringiensis and Bacillus cereus belong to the B. cereus species group. The two species share substantial chromosomal similarity and differ mostly in their plasmid content. The phylogenetic relationship between these species remains a matter of debate. There is genetic exchange both within and between these species, and current evidence indicates that insects are a particularly suitable environment for the growth of and genetic exchange between these species. We investigated the conjugation efficiency of B. thuringiensis var. kurstaki KT0 (pHT73-Em^R) as a donor and a B. thuringiensis and several B. cereus strains as recipients; we used one-recipient and two-recipient conjugal transfer systems in vitro (broth and filter) and in Bombyx mori larvae, and assessed multiplication following conjugation between Bacillus strains. The B. thuringiensis KT0 strain did not show preference for genetic exchange with the B. thuringiensis recipient strain over that with the B. cereus recipient strains. However, B. thuringiensis strains germinated and multiplied more efficiently than B. cereus strains in insect larvae and only B. thuringiensis maintained complete spore germination for at least 24 h in B. mori larvae. These findings show that there is no positive association between bacterial multiplication efficiency and conjugation ability in infected insects for the used strains.     

Comparative analysis of the virulence of invertebrate and mammalian pathogenic bacteria in the oral insect infection model Galleria mellonella

Infection of Galleria mellonella by feeding a mixture of Bacillus thuringiensis spores or vegetative bacteria in association with the toxin Cry1C results in high levels of larval mortality. Under these conditions the toxin or bacteria have minimal effects on the larva when inoculated separately. In order to evaluate whether G. mellonella can function as an oral infection model for human and entomo-bacterial pathogens, we tested strains of Bacillus cereus, Bacillus anthracis, Enterococcus faecalis, Listeria monocytogenes, Pseudomonas aeruginosa and a Drosophila targeting Pseudomonas entomophila strain. Six B. cereus strains (5 diarrheal, 1 environmental isolate) were first screened in 2nd instar G. mellonella larvae by free ingestion and four of them were analyzed by force-feeding 5th instar larvae. The virulence of these B. cereus strains did not differ from the B. thuringiensis virulent reference strain 407Cry⁻ with the exception of strain D19 (NVH391/98) that showed a lower virulence. Following force-feeding, 5th instar G. mellonella larvae survived infection with B. anthracis, L. monocytogenes, E. faecalis and P. aeruginosa strains in contrast to the P. entomophila strain which led to high mortality even without Cry1C toxin co-ingestion. Thus, specific virulence factors adapted to the insect intestine might exist in B. thuringiensis/B. cereus and P. entomophila. This suggests a co-evolution between host and pathogens and supports the close links between B. thuringiensis and B. cereus and more distant links to their relative B. anthracis.     

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.     

Correlation between alkaline activation of Bacillus thuringiensis var. kurstaki spores and crystal production

The spores of crystal-forming (Cry⁺) and non-crystal-forming (Cry^-) strains of Bacillus thuringiensis var. kurstaki and Bacillus cereus were tested for the ability to be activated by 0.1 m K₂CO₃ (pH 10). Only the spores of crystal-forming strains could be activated, and this phenotype was independent of whether crystals were present with the spores in the activation solution. The spores of a B. thuringiensis var. kurstaki strain that is temperature sensitive for protoxin accumulation could be activated by the alkaline solution when produced at the permissive temperature, whereas spores produced at the nonpermissive temperature were not activated. The results indicate that protoxin in the spore coat is responsible for the alkaline-activation phenotype and may serve an ecological function for the organism.     

Allelochemical induced stress: Effects of l-canavanine on the pathogenicity of Bacillus thuringiensis in Manduca sexta

Increased susceptibility of Manduca sexta to commercial formulations of the microbial insecticide, Bacillus thuringiensis, as evidenced by lower LD₅₀ and LT₅₀ values, was observed when M. sexta were reared on an artificial diet supplemented with a sublethal concentration (2.5 mm) of l-canavanine. At several dosages of B. thuringiensis, which were administered either by diet contamination or by per os forced feeding, a greater than 70% reduction (P < 0.05) occurred in the LT₅₀ response with canavanine-treated larvae. The LD₅₀ values also were lowered by canavanine treatment. This constitutes the first report of a plant allelochemical enhancing the effect of B. thuringiensis in vivo. It is suggested that canavanine enhances the effect of B. thuringiensis on gut permeability and active transport.     

Extrachromosomal DNA in Bacillus thuringiensis var. kurstaki, var. finitimus, var. sotto, and in Bacillus popilliae

Four entomopathogenic bacteria contained extrachromosomal deoxyribonucleic acid (DNA) molecules of various sizes. Bacillus thuringiensis var. kurstaki contained twelve elements banding on agarose gels that ranged from 0.74 to > 50 × 10⁶ daltons, three of which were giant extrachromosomal DNA elements. B. thuringiensis var. sotto contained one giant extrachromosomal DNA element with a molecular size of about 23.5 × 10⁶ daltons and two lesser elements of 0.80 and 0.62 × 10⁶ daltons. B. thuringiensis var. finitimus harbored two giant DNA elements corresponding to >50 × 10⁶ daltons and two lesser bands with relative small size (0.98 and 0.97 × 10⁶ daltons). B. popilliae contained no giant extrachromosomal DNA elements but did contain two smaller elements corresponding to 4.45 and 0.58 × 10⁶ daltons. The possible use of extrachromosomal DNA elements that prove to be autonomous replicons for recombinant DNA studies is discussed.     

Pathogenesis of Bacillus sphaericus strain SSII-1 infections in Culex pipiens quinquefasciatus ( = C. pipiens fatigans) larvae

Numbers of viable bacteria in second instar Culex pipiens quinquefasciatus larvae were determined following ingestion of pathogenic strain SSII-1 and nonpathogenic Bacillus sphaericus. Numbers of nonpathogenic B. sphaericus recovered from larvae declined rapidly after cessation of feeding, as did numbers of pathogenic SSII-1 cells fed at LD₂₀ dosage. When pathogenic cells were fed at LD₇₀ dosage, the number of B. sphaericus in larvae increased following initial decline. When chloroformtreated SSII-1 cultures, in which all bacteria except spores were dead, were fed at LD₁₀ and LD₉₈ dosages, no viable B. sphaericus were recovered from larvae. In all SSII-1 treatments, other bacterial flora multiplied rapidly in larvae following onset of mortality; the role of this multiplication in the pathogenesis was not determined. It is proposed that toxic material is released when SSII-1 cells are digested and that multiplication of B. sphaericus in the larval gut is not essential in the pathogenesis. There appears to be no difference in the pathogenesis when differing numbers of B. sphaericus. i.e., LD_10–20 or LD_70–98 dosages, are ingested. Possible nature of the toxic material is discussed.     

Description of Bacillus toyonensis sp. nov., a novel species of the Bacillus cereus group,and pairwise genome comparisons of the species of the group by means of ANI calculations

Strain BCT-7112^T was isolated in 1966 in Japan from a survey designed to obtain naturally occurring microorganisms as pure cultures in the laboratory for use as probiotics in animal nutrition. This strain, which was primarily identified as Bacillus cereus var toyoi, has been in use for more than 30 years as the active ingredient of the preparation TOYOCERIN^®, an additive for use in animal nutrition (e.g. swine, poultry, cattle, rabbits and aquaculture). Despite the fact that the strain was initially classified as B. cereus, it showed significant genomic differences from the type strains of the B. cereus group that were large enough (ANI values below 92%) to allow it to be considered as a different species within the group. The polyphasic taxonomic study presented here provides sufficient discriminative parameters to classify BCT-7112^T as a new species for which the name Bacillus toyonensis sp. nov. is proposed, with BCT-7112^T (=CECT 876^T; =NCIMB 14858^T) being designated as the type strain. In addition, a pairwise comparison between the available genomes of the whole B. cereus group by means of average nucleotide identity (ANI) calculations indicated that besides the eight classified species (including B. toyonensis), additional genomospecies could be detected, and most of them also had ANI values below 94%. ANI values were on the borderline of a species definition only in the cases of representatives of B. cereus versus B. thuringiensis, and B. mycoides and B. weihenstephanensis.     

Electron microscopy of the surfaces of bacillus spores

The surface structures of the spores of Bacillus cereus, Bacillus thuringiensis, and Brevibacillus laterosporus were studied by transmission and scanning electron microscopy. Platinum deposition and negative staining with uranyl acetate revealed appendages and exosporium in B. thuringiensis and B. cereus. The exosporium structure was visualized by negative staining and ultrathin sectioning. For staining the exosporium polysaccharide, Alcian blue was used during fixation. The results obtained show the differences in structural organization of appendages and exosporium in different strains. Canoe-shaped inclusions were revealed in all Br. laterosporus strains, while strain IGM16-92 had a fibrillar capsule as well. Electron microscopy using a dual beam scanning electron microscope Quanta 200 3D provided the information of the spore surface relief without sample treatment (fixation and dehydration). The spores of Br. laterosporus strains had folded surface, unlike the smooth surface of B. cereus and B. thuringiensis spores. The diversity of external spore structures was shown within a species, which may be used for detection of bacteria at the strain level. Optimized procedures for visualization of spore surface by different electron microscopic techniques were discussed.     

Characterization of the adenine nucleoside specific phosphorylase of Bacillus cereus

Adenosine phosphorylase, a purine nucleoside phosphorylase endowed with high specificity for adenine nucleosides, was purified 117-fold from vegetative forms of Bacillus cereus. The purification procedure included ammonium sulphate fractionation, pH 4 treatment, ion exchange chromatography on DEAE-Sephacel, gel filtration on Sephacryl S-300 HR and affinity chromatography on N⁶-adenosyl agarose. The enzyme shows a good stability to both temperature and pH. It appears to be a homohexamer of 164 ± 5 kDa. Kinetic characterization confirmed the specificity of this phosphorylase for 6-aminopurine nucleosides. Adenosine was the preferred substrate for nucleoside phosphorolysis (k_cat/K_m 2.1 × 10⁶ s^− 1 M^− 1), followed by 2′-deoxyadenosine (k_cat/K_m 4.2 × 10⁵ s^− 1 M^− 1). Apparently, the low specificity of adenosine phosphorylase towards 6-oxopurine nucleosides is due to a slow catalytic rate rather than to poor substrate binding.     

The control of Monomorium pharaonis (Hymenoptera: Formicidae) with Bacillus thuringiensis

In this study, the effect of different preparations made from Bacillus thuringiensis var. thuringiensis (strains: CCEB 555 and CCEB 058) on ants, Monomorium pharaonis, under laboratory conditions is reported. The different preparations tested consisted of (1) a liquid culture of the strain B. thuringiensis CCEB 555 (containing spores and exotoxin), (2) the supernatant of the culture broth of strain CCEB 555 (containing exotoxin), and (3) the biological preparation “Bathurin” prepared from the strain B. thuringiensis CCEB 058 (containing spores and inclusions, without exotoxin). The preparations were used either pure or in alternation with borax, i.e., 1 wk borax, 3 wk the respective preparation for several months. All preparations were found to be toxic to M. pharaonis and their effect was characterized by a slow extinction of the ant colony. Administration of “Bathurin” (1.3%) yielded a 100% mortality after 20 wk. Using a liquid culture of B. thuringiensis var. thuringiensis, 100% mortality was recorded after 21 wk, a period of time which did not differ from that obtained with the supernatant of the culture containing exotoxin. The alternation with borax was found to accelerate ant mortality by 9–10 wk after administration. In all experiments, the worker ants died first, the queen ants surviving them by 1–3 wk.In experiments employing worker ants only, a 100 and 98% mortality, respectively, occurred within 3 wk after administration of a liquid culture of B. thuringiensis and “Bathurin” supplemented with borax.     

Dosage-mortality studies with commercial Bacillus thuringiensis sprayed in a modified Potter's tower against some forest insects

Dosage-mortality tests were carried out with commercial Bacillus thuringiensis (B.T.) (Dipel)^® against various instars of the spruce budworm, Choristoneura fumiferana, the white-marked and Douglas fir tussock moths, Hemerocampa leucostigmata and Orgyia pseudotsugata, and the gypsy moth, Porthetria dispar. Dipel was applied as a dilute (10^?2) molasses suspension onto artificial diet surface in a spray tower designed to simulate aerial application. Probit analysis of the results showed that LD₅₀s expressed both in terms of gallons deposited per acre and as spores and crystals deposited per cm² increased with larval age for all species. The spruce budworm was the most sensitive to the bacteria, followed in decreasing order of sensitivity by the white-marked tussock moth, Douglas fir tussock moth, and the gypsy moth. The mean slopes for all instars of the four species were 1.6, 3.1, 2.6, and 2.2, respectively, indicating that precision-wise, the assay of B.T. on artificial medium was good. The relatively low slope for spruce budworm is explained by its peculiar feeding habit. Among all species tested, bacteria-treated larvae gained weight at a considerably reduced rate compared with untreated ones. Reduction in weight resulting from lowered feeding activity intensified as dosage rates increased. The implication of this in terms of mortality assessments in microbial control operations is discussed.It is suggested that 0.02 gallon (4 × 10⁶ International Units) of Dipel Molasses deposited per acre may achieve economic control of fourth- to sixth-instar budworm and first-to second-instar gypsy moths. A deposit rate for second- to fifth-instar white-marked or Douglas fir tussock moths appears to be in the vicinity of 0.01 gallon (2 × 10⁶ IU) per acre.     

Application of different downstream processing methods and their comparison for the large-scale preparation of Bacillus thuringiensis var. israelensis after fermentation for mosquito control

Bacillus thuringiensis var. israelensis, a gram positive, spore-forming bacillus, produces parasporal crystal protein during sporulation, which is toxic in the mosquito larvae gut. An efficient downstream processing method for separating the spore crystal complex (SCC) from the fermented broth of B. thuringiensis var. israelensis is required to achieve maximum mosquitocidal activity. The different downstream processing methods, viz., tangential flow ultra-filtration, continuous centrifugation and acid precipitation were compared for their efficiency in separating SCC from broth obtained from a pilot-scale fermentor (100 l capacity). Among the three downstream processing methods, tangential flow ultra-filtration yielded the maximum amount of biomass (53.3 g/l), maximum number of spores (2.30 × 10¹⁸ CFU/ml) and highest level of larvicidal activity (LC₅₀ 28 nl/ml) against Aedes aegypti Bora-Bora strain followed by continuous centrifugation and acid precipitation methods.     

Asporogenous Bacillus thuringiensis Mutant Producing High Yields of δ-Endotoxin

An asporogenous Bacillus thuringiensis subsp. kurstaki strain IK mutant, strain 290-1, which produced high yields of δ-endotoxin, was obtained by ethyl methane sulfonate treatment of a spore suspension. The mutant strain produced about the same amount of δ-endotoxin as that produced by the parent strain, but 10¹⁰ to 10¹¹ cells did not form any detectable dormant spores.     

Persistence ofBacillus thuringiensis andBacillus cereus in soil supplemented with grass or manure

Summary Persistance of inocula ofBacillus thuringiensis spores, parasporal crystals, andBacillus cereus spores in soil supplemented with dried-grass or partly composted, dried-chicken manure (100 mg supplement per 900 mg soil,^–0.01 MPa water availability, 25°C) were monitored over a period of up to 64 days by dilution plating and bioassay with larvae ofPieris brassicae. The inoculantB. thuringiensis population increased 22 x in level in grass-supplemented soil, but declined in manure-supplemented soil to 0.22 x the original level. TheB. cereus inocula declined in both soil treatments to approximately 0.1 x the original level. Insecticidal activity of theB. thuringiensis parasporal crystal decreased exponentially in grass and manuresupplemented soils, with half-lives of approximately 9.5 and 8.5 days respectively.     

Infection of Tribolium castaneum with Bacillus thuringiensis: Quantification of Bacterial Replication within Cadavers,Transmission via Cannibalism,and Inhibition of Spore Germination

Reproduction within a host and transmission to the next host are crucial for the virulence and fitness of pathogens. Nevertheless, basic knowledge about such parameters is often missing from the literature, even for well-studied bacteria, such as Bacillus thuringiensis, an endospore-forming insect pathogen, which infects its hosts via the oral route. To characterize bacterial replication success, we made use of an experimental oral infection system for the red flour beetle Tribolium castaneum and developed a flow cytometric assay for the quantification of both spore ingestion by the individual beetle larvae and the resulting spore load after bacterial replication and resporulation within cadavers. On average, spore numbers increased 460-fold, showing that Bacillus thuringiensis grows and replicates successfully in insect cadavers. By inoculating cadaver-derived spores and spores from bacterial stock cultures into nutrient medium, we next investigated outgrowth characteristics of vegetative cells and found that cadaver-derived bacteria showed reduced growth compared to bacteria from the stock cultures. Interestingly, this reduced growth was a consequence of inhibited spore germination, probably originating from the host and resulting in reduced host mortality in subsequent infections by cadaver-derived spores. Nevertheless, we further showed that Bacillus thuringiensis transmission was possible via larval cannibalism when no other food was offered. These results contribute to our understanding of the ecology of Bacillus thuringiensis as an insect pathogen.     

Production of heat-stable exotoxin by Bacillus thuringiensis and related bacteria

Heat-stable exotoxin production by 740 strains of Bacillus thuringiensis and related bacteria was investigated using the housefly, Musca domestica, from the following viewpoints: (1) the relation-ship between B. thuringiensis flagellar (H) serotypes and exotoxin production and (2) the exotoxin production by Bacillus species other than B. thuringiensis. Of 437 isolates belonging to 11 serotypes of B. thuringiensis which had been confirmed to produce parasporal inclusions, 35 isolates belonging to serotypes 1, 3a:3b, 4a:4c, and 10 produced heat-stable exotoxin. Exotoxin was not detected in the isolates of serotypes 3a, 4a:4b, 5a:5b, 5a:5c, 6, 7, and 8a:8b. No heat-stable exotoxin was demonstrated in 28 acrystalliferous isolates which possessed H antigens of B. thuringiensis serotypes 1, 3a, 4a:4b, 4a:4c, 5a:5c, 6, 7, 10, 11a:11c, and 12. A total of 270 B. cereus isolates which did not possess B. thuringiensis H antigen were examined and three isolates were found to produce heat-stable exotoxin. No heat-stable exotoxin was produced by B. subtilis (two strains), B. natto (one strain), and B. megaterium (two strains). These results indicate that the heat-stable exotoxin production in B. thuringiensis is a strain-specific property rather than a serotype(subspecies)-specific property.     

Effect of high concentration of nutrients onBacillus thuringiensis cultures

Summary Bacterial insecticide production using a strain ofBacillus thuringiensis var. kurstaki was studied in batch culture considering the influence of increasing concentration of components of a glucose — yeast extract — mineral salts medium.It was found that spore counts were increased from 1.08×10¹² spores. 1^–1 to 7.36×10¹² spores. 1^–1 and toxin level from 1.05 mg.ml^–1 to 6.85 mg.ml^–1, when the concentration of glucose was increased from 8 to 56 (g 1^–1), with the corresponding increase in the rest of medium components. Higher concentration of nutrients inhibit either spore count or toxin production.Preliminary experiments of fed-batch cultures which allows the use of high amounts of nutrients were also carried out. In this case spore counts of 1.2×10¹³ spores.1^–1 were achieved.     

Polyphosphate Storage during Sporulation in the Gram-Negative Bacterium Acetonema longum

Using electron cryotomography, we show that the Gram-negative sporulating bacterium Acetonema longum synthesizes high-density storage granules at the leading edges of engulfing membranes. The granules appear in the prespore and increase in size and number as engulfment proceeds. Typically, a cluster of 8 to 12 storage granules closely associates with the inner spore membrane and ultimately accounts for ∼7% of the total volume in mature spores. Energy-dispersive X-ray spectroscopy (EDX) analyses show that the granules contain high levels of phosphorus, oxygen, and magnesium and therefore are likely composed of polyphosphate (poly-P). Unlike the Gram-positive Bacilli and Clostridia, A. longum spores retain their outer spore membrane upon germination. To explore the possibility that the granules in A. longum may be involved in this unique process, we imaged purified Bacillus cereus, Bacillus thuringiensis, Bacillus subtilis, and Clostridium sporogenes spores. Even though B. cereus and B. thuringiensis contain the ppk and ppx genes, none of the spores from Gram-positive bacteria had granules. We speculate that poly-P in A. longum may provide either the energy or phosphate metabolites needed for outgrowth while retaining an outer membrane.     

Recovery of Bacillus thuringiensis and other bacteria from larvae of Spodoptera littoralis previously fed B. thuringiensis-treated leaves

Exposure of a spore-crystal suspension of Bacillus thuringiensis to UV irradiation for (200 lx) 8.5 min killed most of the spores ($\text{P}\text{P}{}_0\text{= 2.6 × 10}{}^{\mathrm{?4}}$), while the insecticidal activity of the suspension to larvae of Spodoptera littoralis was only slightly affected. Numbers of colony-forming units (CFU) of B. thuringiensis recovered from larvae after ingestion of spores decreased with time as long as the larvae lived and several hours after larval death. Only 3–6 hr after larval death, the spores germinated and multiplied, reaching up to 100-fold after 24 hr. When UV-irradiated suspensions were used, numbers of CFU per larva were too scarce to be recovered from living larvae. However, 1.5 × 10⁶ CFU/larva were recovered 24 hr after death. It seems that the disruption of the gut epithelium by the endotoxin caused a change in the unfavorable conditions for endospore germination, thus providing the suitable ambient for germination and multiplication of B. thuringiensis. Numbers of other bacteria present per milligram of healthy larva increased with larval weight, predominantly Streptococcus sp. and Erwinia sp. In dead larvae, the increase of Erwinia sp. was higher than that of Streptococcus sp. Other bacterial species isolated were: Corynebacterium sp., Micrococcus sp., Serratia marcescens, and Bacillus sp.