Bacillus thuringiensis soil populations naturally occurring in the Ryukyus, a subtropic region of Japan

Of 809 soil samples collected from the seven islands of the Ryukyus, Japan, 107 samples (13.2%) contained Bacillus thuringiensis. The frequency of B. thuringiensis among the B. cereus group was 1.1% (235/21842) on the average. The B. thuringiensis soil populations of the Ryukyus consisted of more than 22 H serogroups. The predominant H serotype was the H5ac/21 (serovar canadensis/colmeri), followed by the H3ad (serovar sumiyoshiensis) and H16 (serovar indiana). Geographically, most widely distributed H serogroups were the H16 and H10ac (serovar londrina); the former was recovered from five islands and the latter from three islands. Parasporal inclusions of the isolates were morphologically heterogeneous, roughly grouped into four categories: bipyramidal/cuboidal, spherical/ovoid, irregularly-pointed, and irregular-shaped. About 53% of the isolates formed spherical to ovoid parasporal inclusions. None of the isolates exhibited larvicidal activity against the silkworm, Bombyx mori. Only four isolates belonging to four different serotypes killed larvae of the mosquito, Aedes aegypti. These mosquito-specific isolates all produced spherical parasporal inclusions.     

An extrachromosomal prophage naturally associated with Bacillus thuringiensis serovar israelensis

Bacillus thuringiensis serovar israelensis , an entomopathogen for mosquito larvae, was demonstrated to be lysogenized by temperate phage SU-11 whose genome was located extrachromosomally in the cell. The prophage SU-11 was cured at high frequency from the parental strain by continuous sub-culture at high temperature, but the ability to produce δ-endotoxin remained in the prophage cured strain. Moreover, phage induction was found to occur after mating of serovar israelensis with its prophage cured strain, as well as with B. thuringiensis serovar thuringiensis , B. cereus and B. subtilis .     

Isolation and propagation of phages naturally associated with the aizawai variety of Bacillus thuringiensis

This study describes the isolation of a phage, using mitomycin C and u.v. light, from each of four strains (HD67, HD130, HD228 and HD248) of Bacillus thuringiensis H-serotype 7 (var. aizawai). It also describes the isolation of two indicator strains (12.13 and HD 102) for these phages (φHD67, φHD130, φHD228 and φHD248) and the ideal conditions, using these indicator strains, for maximum phage production.     

Isolation and propagation of phages naturally associated with the aizawai variety of Bacillus thuringiensis

This study describes the isolation of a phage, using mitomycin C and u.v. light, from each of four strains (HD67, HD130, HD228 and HD248) of Bacillus thuringiensis H-serotype 7 (var. aizawai). It also describes the isolation of two indicator strains (12.13 and HD102) for these phages (phi HD67, phi HD130, phi HD228 and phi HD248) and the ideal conditions, using these indicator strains, for maximum phage production.     

Mode of replication, size and distribution of naturally occurring plasmids in Bacillus thuringiensis

Cloned replication origin regions, derived from both small (4.9-7.5 MDa) and large (43-60 MDa) plasmids of Bacillus thuringiensis subspecies kurstaki strains HD73 and HD263 were used as hybridization probes in a Southern-blot analysis to assess both the size and horizontal distribution of native plasmid replicon groups among different subspecies of B. thuringiensis. In general, resident plasmids hybridizing to the replication origin regions derived from strains HD263 and HD73 were more commonly found in kurstaki strains than in non-kurstaki strains, suggesting a non-random distribution of plasmid incompatibility groups. Replication origin regions derived from the large HD263 plasmids (43-60 MDa) hybridized almost exclusively with large plasmids (greater than 30 MDa) of widely varying sizes. In contrast, replication origin regions derived from small plasmids hybridized exclusively with small plasmids (less than 10 MDa) showing little size variation. These results are consistent with previous observations concerning the relationship between plasmid size, mode of replication, and structural stability.     

Phages naturally associated with the aizawai variety of insect pathogen Bacillus thuringiensis and their relevance to strain identification

Of 36 strains of the ' aizawai ' variety of Bacillus thuringiensis (H-serotype 7) 16 were naturally associated with bacteriophages, 11 of which were isolated at titres of 10⁶ plaque-forming units/ml and above. These 11 phages had varied host ranges among Bacillus cereus and five strains of the ' aizawai ' variety of B. thuringiensis. Host range, plaque morphology and differences in cold lability indicated dissimilarities between the phages, which could be used taxonomically to differentiate between strains of this bacillus.     

Genetic differentiation between sympatric populations of Bacillus cereus and Bacillus thuringiensis

Little is known about genetic exchanges in natural populations of bacteria of the spore-forming Bacillus cereus group, because no population genetics studies have been performed with local sympatric populations. We isolated strains of Bacillus thuringiensis and B. cereus from small samples of soil collected at the same time from two separate geographical sites, one within the forest and the other at the edge of the forest. A total of 100 B. cereus and 98 B. thuringiensis strains were isolated and characterized by electrophoresis to determine allelic composition at nine enzymatic loci. We observed genetic differentiation between populations of B. cereus and B. thuringiensis. Populations of a given Bacillus species--B. thuringiensis or B. cereus--were genetically more similar to each other than to populations of the other Bacillus species. Hemolytic activity provided further evidence of this genetic divergence, which remained evident even if putative clones were removed from the data set. Our results suggest that the rate of gene flow was higher between strains of the same species, but that exchanges between B. cereus and B. thuringiensis were nonetheless possible. Linkage disequilibrium analysis revealed sufficient recombination for B. cereus populations to be considered panmictic units. In B. thuringiensis, the balance between clonal proliferation and recombination seemed to depend on location. Overall, our data indicate that it is not important for risk assessment purposes to determine whether B. cereus and B. thuringiensis belong to a single or two species. Assessment of the biosafety of pest control based on B. thuringiensis requires evaluation of the extent of genetic exchange between strains in realistic natural conditions.     

The field persistence of Bacillus thuringiensis spores on Cercis occidentalis leaves

The field persistence of viable spores of four Bacillus thuringiensis formulations, Amdal^®, Biotrol^® BTB 183, Thuricide^® HP, and Thuricide^® 90TS, was measured and compared on leaves of Cercis occidentalis. For Amdal, Biotrol BTB 183, and Thuricide 90TS the field persistence was compared also at two locations, Auburn and Sacramento, California, which differed in altitude and climate. The comparisons of field persistence were based on a segmented linear model of the decay of average viable spore count on a logarithmic scale, because much of the field data strongly rejected the simple log linear model.No significant difference in field persistence of viable spores was found between the two locations. Significant differences were found in both magnitude and pattern of field persistence compared to previously reported measurements of Thuricide 90TS, where leaves of Quercus agrifolia were the substrate, and a log linear pattern of decay of viable spore count was found. The persistence half-life of Thuricide 90TS on Q. agrifolia leaves at Monterey, California, was 3.9 days, compared to a persistence half-life during the first 3 days of 0.63 day on leaves of C. occidentalis with the pooled Auburn/Sacramento data (two-sided P < 0.001).The persistence half-life for Thuricide HP during the first 3 days was 1.85 days, which was significantly different from the corresponding result of 0.58 day for Amdal and 0.63 day for Thuricide 90TS (P < 0.04).     

Hypocomplementemia associated with naturally occurring lymphosarcoma in pet cats.

Eighty cats were classified by indirect immunofluorescence and histologic diagnosis into four categories: normal, feline leukemia virus (FeLV) infected; normal noninfected; lymphosarcoma-FeLV infected; lymphosarcoma, no FeLV present. All viremic cats with lymphosarcoma were found to be hypocomplementemic and activation of the complement system had occurred via the classical pathway. Sera of cats with lymphosarcoma in the absence of FeLV had varying levels of total hemolytic complement (TCH50) ranging from normal to hypocomplementemic. Approximately 50% of the cats that were viremic but histologically and clinically free of disease had TCH50 levels within normal range, and the remainder exhibited varying degrees of hypocomplementemia.     

Metallic-Oxide encrustations of the nonprosthecate stalks of naturally occurring populations ofPlanctomyces bekefii

Naturally occurring metallic-oxide encrustations of the multifibrillar stalks of morphotype Ia (Planctomyces bekefii) of theBlastocaulis-Planctomyces group of budding and nonprosthecately appendaged prokaryotes from two sources-a pond in Budapest, Hungary (containing topotypical material) and Gebharts Teich, a fishpond in Austria-have been examined by energy-dispersive X-ray spectroscopy. Some rosettes of morphotype Ib (Planctomyces crassus) also occurred in the Budapest pond; only morphotype Ia was found in Gebharts Teich. Manganese was the major metallic element in the morphotype Ia stalk encrustations from the Budapest pond; iron was not detected. However, morphotype Ib rosettes occurring in the same Budapest pond sampes contained both manganese and iron in their stalk encrustations. Both iron and manganese were present in the morphotype Ia stalk encrustation of rosettes from Gebharts Teich.     

Bacillus thuringiensis: a biotechnology model

This paper is on the different biotechnological approaches that have been used to improve Bacillus thuringiensis (Bt) for the control of agricultural insect pests and have contributed to the successful use of this biological control agent; it describes how a better knowledge of the high diversity of Bt strains and toxins genes together with the development of efficient host-vector systems has made it possible to overcome a number of the problems associated with Bt based insect control measures. First we present an overview of the biology of Bt and of the mode of action of its insecticidal toxins. We then describe some of the progress that has been made in furthering our knowledge of the genetics of Bt and of its insecticidal toxin genes and in the understanding of their regulation. The paper then deals with the use of recombinant DNA technology to develop new Bt strains for more effective pest control or to introduce the genes encoding partial-endotoxins directly into plants to produce insect-resistant trangenic plants. Several examples describing how biotechnology has been used to increase the production of insecticidal proteins in Bt or their persistence in the field by protecting them against UV degradation are presented and discussed. Finally, based on our knowledge of the mechanism of transposition of the Bt transposon Tn4430, we describe the construction of a new generation of recombinant strains of Bt, from which antibiotic resistance genes and other non-Bt DNA sequences were selectively eliminated, using a new generation of site-specific recombination vectors. In the future, continuing improvement of first generation products and research into new sources of resistance is essential to ensure the long-term control of insect pests. Chimeric toxins could also be produced so as to increase toxin activity or direct resistance towards a particular type of insect. The search for new insecticidal toxins, in Bt or other microorganisms, may also provide new weapons for the fight against insect damage.     

Interaction of mannose-binding protein with associated serine proteases: effects of naturally occurring mutations

Mannose-binding protein (MBP; mannose-binding lectin) forms part of the innate immune system. By binding directly to carbohydrates on the surfaces of potential microbial pathogens, MBP and MBP-associated serine proteases (MASPs) can replace antibodies and complement components C1q, C1r, and C1s of the classical complement pathway. In order to investigate the mechanisms of MASP activation by MBP, the cDNAs of rat MASP-1 and -2 have been isolated, and portions encompassing the N-terminal CUB and epidermal growth factor-like domains have been expressed and purified. Biophysical characterization of the purified proteins indicates that each truncated MASP is a Ca(2+)-independent homodimer in solution, in which the interacting modules include the N-terminal two domains. Binding studies reveal that both MASPs associate independently with rat MBP in a Ca(2+)-dependent manner through interactions involving the N-terminal three domains. The biophysical properties of the truncated MASPs indicate that the interactions with MBP leading to complement activation differ significantly from those between components C1q, C1r, and C1s of the classical pathway. Analysis of MASP binding by rat MBP containing naturally occurring mutations equivalent to those associated with human immunodeficiency indicates that binding to both truncated MASP-1 and MASP-2 proteins is defective in such mutants.     

A naturally occurring variant of Hsp90 that is associated with decanalization

The heat shock protein Hsp90 has been the focus of many studies since it was suggested that it acts to mediate the buffering of phenotypic variation. Hsp90-mediated buffering may result in the accumulation of cryptic genetic variation that, when released either as a consequence of environmental or genetic stress, increases the evolvability of a population. Recent studies using laboratory-induced mutations of Hsp90 and/or chemical inhibition to disrupt Hsp90 function confirm that Hsp90 can buffer cryptic genetic variation. We have previously identified a naturally occurring variant in the charged linker region of the Hsp90 gene, and now examine whether this variant is associated with altered levels of trait variability. The variant is associated with the release of cryptic genetic variation for canalized morphological (bristle) traits, but not for uncanalized morphological (wing and bristle) traits, and the effect on canalized traits depends on culture temperature. This suggests that natural genetic variation in Hsp90 may mediate the evolution of canalized morphological traits even if it does not influence the expression of variation for uncanalized traits.     

Isolation and characterization of three autolytic enzymes associated with sporulation of Bacillus thuringiensis var. thuringiensis

Cells of Bacillus thuringiensis containing refractile spores autolyzed readily when suspended in buffer. The autolysate contained enzymes which lysed vegetative cell walls of the organism. Three enzymes were isolated from the autolysate, and each was purified approximately 30-fold. One enzyme, most active near pH 4.0, was found to be an N-acetylmuramidase. The other two enzymes exhibited pH optima at 8.5. One was stimulated by cobalt ions and the other was not. The cobalt-stimulated enzyme was shown to be an N-acetylmuramyl-l-alanine amidase. The cobalt insensitive enzyme exhibited both N-acetylmuramyl-l-alanine amidase and endopeptidase activity. The amidase activity may reflect incomplete separation of the cobalt-stimulated enzyme. The endopeptidase cleaved the peptide bond between l-alanine d-glutamic acid. A cell wall lytic endopeptidase with this specificity has not been previously reported. All three enzymes were extremely limited in the range of bacterial cell walls which they attacked. Except for cell walls of Micrococcus lysodeikticus, which were lysed by the muramidase, only cell walls of members of the genus Bacillus were attacked.     

Microbial ecology of Bacillus thuringiensis: fecal populations recovered from wildlife in Korea

A total of 34 fecal samples, collected from 14 species of wild mammals in Korea, were examined for the occurrence of Bacillus thuringiensis. The organism was detected in 18 (53%) samples. Among the three food-habit groups, herbivorous animals yielded the highest frequency (69%) of samples positive for B. thuringiensis, followed by omnivorous animals (50%). Of the six fecal samples from carnivorous animals, only one sample contained B. thurin giensis. Among 527 isolates belonging to the Bacillus cereus - B. thuringiensis group, 43 (8%) were assigned to B. thurin giensis on the basis of the formation of parasporal inclusions. Of the 43 isolates, 13 were serologically allocated to the nine H-antigenic serotypes: H3ad (serovar sumiyoshiensis), H15 (dakota), H17/27 (tohokuensis/ mexicanensis), H19 (tochigiensis), H21 (colmeri), H29 (amagiensis), H31/49 (toguchini/muju), H42 (jinghongiensis), and H44 (higo). Other isolates were untestable or untypable by the 55 reference H antisera available. Insecticidal activity was associated with 23% of the fecal populations: three isolates killed larvae of the silkworm, Bombyx mori (Lepidoptera), and seven exhibited larvicidal activity against the mosquito, Aedes aegypti (Diptera). There was no larvicidal activity against the three lepidopterous insects: Plutella xylostella, Spodoptera exigua, and Spodoptera litura. The overall results suggest that wild animals in Korea are in contact with naturally occurring B. thuringiensis at high frequencies through the daily food intake of plants.     

Tracking the source of Bacillus thuringiensis Cry1Ab endotoxin in the environment

The application of Bacillus thuringiensis (Bt) and the growing of genetically-modified crops are currently practised to control infestations of crop-eating insects. The increasing use of these biopesticides could lead to an increase in Cry1Ab endotoxin in both terrestrial and aquatic environments. The aim of this study was to quantify levels of Cry1Ab endotoxin and locate its source in the environment. Agricultural soils and surface waters were spiked with crystals (biopesticide-Dipel^®) or with pure Bt-corn endotoxin. Cry1Ab concentrations were then determined with immunoassays. Additionally, surface water, soils and sediments were sampled in an area sprayed with Bt kurstaki and at a site where genetically-modified corn expressing Cry1Ab is grown. Isotopic analysis was performed on the endotoxin from Bt and Bt corn to characterize the proportions of ¹³C/¹²C and ¹⁵N/¹⁴N. The results showed that Bt-corn endotoxin is degraded more rapidly in water than in soils (t_1/2: 4 and 9 days, respectively), while crystals appeared to be more resilient, as expected. The isotopic patterns of ¹³C and ¹⁵N in Bt-corn endotoxin differed markedly from Bt, making it possible to track the source of Cry1Ab in the environment. Preliminary field surveys indicate that Cry1Ab is fairly uncommon in aquatic environments, being found only at trace concentrations when it is detected.     

Fermentation broth of Bacillus thuringiensis as a source of precursors for production of nikkomycins

Production of nikkomycins (nucleoside antibiotics inhibiting chitin synthesis) by Streptomyces tendae (ATCC 31160) was considerably enhanced by addition of fermentation wastes of Bacillus thuringiensis var. kurstaki (HD 263) to the basal soymannitol medium. Analysis of this fermentation broth for nucleic acid derivatives showed that they contained about 0·8 g/l of various nucleosides and bases (uracil 0·43 g/l; hypoxanthine 0·21 g/l; as well as uridine, cytosine and adenine).     

Cyclophellitol: a naturally occurring mechanism-based inactivator of beta-glucosidases

The natural product cyclophellitol, isolated from the culture filtrate of a mushroom, Phellinus sp. is found to be a highly specific and effective irreversible inactivator of beta-glucosidases. It inactivates the beta-glucosidases from both almond emulsin and Agrobacter sp. according to pseudo-first order kinetics with inactivation constants of Ki = 0.34 mM, ki = 2.38 min-1, and Ki = 0.055 mM, ki = 1.26 min-1 respectively. No reactivation of the inactivated enzyme is seen upon dialysis, thus providing evidence for the irreversibility of the inactivation. The high specificity of this inactivator is evidenced by the fact that even at very high (12 mM) concentrations of cyclophellitol, no inactivation of yeast alpha-glucosidase was observed, and only extremely slow (t1/2 greater than 5 hours) inactivation of E. coli beta-galactosidase could be detected.