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.     

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.     

Five unique temperate phages from a polylysogenic strain of Bacillus thuringiensis subsp. aizawai

Five temperate phages were isolated from strain 4042B of Bacillus thuringiensis subsp. aizawai. The phages, which were heteroimmune, could also be distinguished by their host ranges, plaque and particle morphologies, serological specificities, and locations of restriction endonuclease cleavage sites on their chromosomes. Besides maintaining a stable lysogenic relationship with the 4042B host strain, each phage formed a stable lysogen with Bacillus cereus.     

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 .     

Generalized transduction in Bacillus thuringiensis var. aizawai

J.R.M. INAL. V. KARUNAKARAN AND H.D. BURGES. 1992. Four temperate phages, ØHD67, ØHD130, ØHD228 and ØHD248, which were isolated from Bacillus thuringiensis H-serotype 7 strains, were tested for their ability to mediate transduction within H-serotype 7 and also between H-serotype 7 and 3a3b. The optimum conditions to facilitate transduction were determined. The four phages successfully mediated transduction within serotype 7 of all five auxotrophic markers tested and thus seem to have the ability to mediate generalized transduction. The phages also mediated transduction between H-serotype 7 and H-serotype 3a3b of 10 of 11 auxotrophic markers tested.     

Isolation and characterization of a novel insecticidal crystal protein gene from Bacillus thuringiensis subsp. aizawai.

Bacillus thuringiensis subsp. aizawai EG6346, a novel grain dust isolate, was analyzed by Southern blot hybridization for its insecticidal crystal protein (ICP) gene profile. Strain EG6346 lacks previously characterized cryIA ICP genes yet does possess novel cryI-related gene sequences. A recombinant genomic plasmid library was constructed for strain EG6346 in Escherichia coli. One recombinant plasmid, pEG640, isolated from the library contained a novel ICP gene on a 5.7-kb Sau3A insert. The sequence of this gene, designated cryIF, was related to, but distinct from, the published sequences for other cryI genes. A second novel cryI-related sequence was also located on pEG640, approximately 500 bp downstream from cryIF. Introduction of cryIF into a Cry- B. thuringiensis recipient strain via electroporation enabled sufficient production of CryIF protein for quantitative bioassay analyses of insecticidal specificity. The CryIF crystal protein was selectively toxic to a subset of lepidopteran insects tested, including the larvae of Ostrinia nubilalis and Spodoptera exigua.     

Structural characteristics of DNA of Bacillus thuringiensis phages

The spectral characteristics of DNA from two phages of the polylysogenic Bacillus thuringiensis subsp. galleriae culture 1-97 were studied. The typical parameters of melting and the negative reaction with formaldehyde are indicative of the double-helical structure of these DNAs. The phage DNAs differ in the molar content of nitrogen bases (32 and 38 mole% of GC) and in their distribution along the molecule. This distribution is uniform in the DNA of one phage whereas the other phage DNA is composed of heterological segments with a different nucleotide composition.     

Physico-chemical properties of several phages of Bacillus thuringiensis

A study was made of biological and physico-chemical properties of phages of Bac. thuringiensis as well as of a number of parameters of nucleic acids isolated from these phages. The phages contain double-stranded DNA. Molecular weights of DNA from three phages--Tg9, Tg10 and Tg13 have been determined by two independent methods: by measuring the contour length of DNA, from the sedimentation constant and for DNA of phage Tg10 also by endonuclease EcRI hydrolysis. These methods gave similar results. On the basis of the temperature of DNA melting the content of GC pairs was found equal to 37.9, 33.4 and 35.1 mole% for DNA's of phage Tg9, Tg10 and Tg13, respectively. On the basis of measuring the intervals of DNA melting a conclusion was made that DNA of the Tg9 and Tg13 phage has a random distribution of base pairs, while DNA of phage Tg10 displays some clustering of base distribution along the molecule. It has been shown that restrictase EcoRI hydrolyses phage Tg10 DNA into 6 fragments of different molecular weights; DNA's of Tg9 and Tg13 phages are not hydrolyzed. A possibility of existance of phage Tg10 DNA in linear and ring forms has been established. The characteristics of phage particles have been determined by electron microscopy.     

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.     

Characteristics of Bacillus thuringiensis phages with circular permutation in the DNA molecule

Bacteriophages Tm2 and Tg27 of different origins but identical in biological properties have been compared. Physicochemical characteristics of bacteriophages have revealed the existence of end repeats and circular permutation of phage DNA. Phages Tm2 and Tg27 share the same dimensions of incapsulated DNA, differing in the sizes of phage genome and end repeats. Bacteriophage Tm2 genome is 45.2 kb. long with the end repeats containing 5.7%. The genome of Tg27 is 42.53 kb and 11.8% of end repeat. The bacteriophages relation has been confirmed by heteroduplex and restriction analysis. Tm2 and Tg27 share 84% of homology. Two regions of nonhomology are found representing a single-stranded loop and equishouldered vesicle with the sizes 2.19 kb and 5.03 kb, respectively.     

Differential specificity of two insecticidal toxins from Bacillus thuringiensis var. aizawai

Bacillus thuringiensis var. aizawai HD-249 produces more than one protein of 130-135 kD in its insecticidal crystal delta-endotoxin. We describe an indirect method of assessing the relative contribution to toxicity of two of these protoxins using monospecific antibodies directed against their active proteolytic products. Our results show that one toxin is active against Spodoptera frugiperda but not Choristoneura fumiferana cells in vitro, while the other lyses C. fumiferana but not S. frugiperda cells. There is no indication of synergism between these toxins in vitro.     

Mosquitocidal activity of the CryIC delta-endotoxin from Bacillus thuringiensis subsp. aizawai.

The cloned 135-kDa CryIC delta-endotoxin from Bacillus thuringiensis is a lepidopteran-active toxin, displaying high activity in vivo against Spodoptera litoralis and Spodoptera frugiperda larvae and in vitro against the S. frugiperda Sf9 cell line. Here, we report that the CryIC delta-endotoxin cloned from B. thuringienesis subsp. aizawai HD-229 and expressed in an acrystalliferous B. thuringiensis strain is also toxic to Aedes aegypti, Anophles gambiae, and Culex quinquefasciatus mosquito larvae. Furthermore, when solubilized and proteolytically activated by insect gut extracts, CryIC is cytotoxic to cell lines derived from the first two of these dipteran insects. This activity was not observed for two other lepidopteran-active delta-endotoxins, CryIA(a) and CryIA(c). However, in contrast to the case with a lepidopteran and dipteran delta-endotoxin cloned from B. thuringiensis subsp. aizawai IC1 (M.Z. Haider, B. H. Knowles, and D. J. Ellar, Eur. J. Biochem. 156:531-540, 1986), no differences in the in vitro specificity or processing of CryIC were found when it was activated by lepidopteran or dipteran gut extract. The recombinant CryIC delta-endotoxin expressed in Escherichia coli was also toxic to A. aegypti larvae. By contrast, a second cryIC gene cloned from B. thuringiensis subsp. aizawai 7.29 (V. Sanchis, D. Lereclus, G. Menou, J. Chaufaux, S. Guo, and M. M. Lecadet, Mol. Microbiol. 3:229-238, 1989) was nontoxic. DNA sequencing showed that the two genes were identical. However, CryIC from B. thuringiensis subsp. aizawai 7.29 had been cloned with a truncated C terminus, and when it was compared with the full-length CryIC delta-endotoxin, it was found to be insoluble under alkaline reducing conditions. These results show that CryIC from B. thuringiensis subsp. aizawai is a dually active delta-endotoxin.     

Isolation and characterization of phages infecting Bacillus cereus

Aim: To isolate and characterize bacteriophages (phages) that infect the foodborne pathogen Bacillus cereus. Methods and Results: Two phages were isolated from soil based on their ability to form plaques on four indicator hosts including Bacillus thuringiensis subsp. israelensis, and three isolates of B. cereus. The purified phages were characterized by morphology, host range, single‐step growth curves and restriction enzyme digestion profiles. The phages appeared to be of the Myoviridae family based on their structure in electron micrographs. The phages lysed bacteria of several species, produced average burst sizes of 322 and 300 phages per infected cell, and both had genomes over 90 kb. The phages were chloroform‐resistant and stable at 4°C. They reduced the concentration of B. cereus in mashed potatoes by >6 log₁₀ CFU ml^?1 within 24 h at room temperature, when applied at a high concentration. Conclusions: The relatively narrow host range within B. cereus might mean that these phages need to be used as part of a ‘cocktail’ of phages for biocontrol, but their efficacy for the control of their host in food was demonstrated. Significance and Impact of the Study: This is the first report of biocontrol by phages of B. cereus in food.     

Comparative study of the physicochemical and biological properties of Bacillus thuringiensis phages

Two phages of the polylysogenic Bacillus thuringiensis subsp. galleriae 1-97 culture differ in the morphology of their particles. A comparative study of their antigenic properties has shown the possibility of cross serological reactions of neutralization with antiphagal sera. The results imply that there is an antigenic relationship between these phages. The data indicate that the nucleic acid component of the both phages is DNA. The sensitivity of phage 1-97 A to some physical factors has been studied.     

Electron microscope study of the serological relations of Bacillus thuringiensis phages

The kinetics of neutralization cross-reactions was studied with Bacillus thuringiensis subsp. galleriae 1-97 phages, and serological complexes were investigated using immunoelectron microscopy. The results of these studies have made it possible to locate both common and specific antigenic components of the phages.     

Electron microscopic study of the interaction between phages and Bacillus thuringiensis cells

The interaction of phages belonging to different morphological groups with the cells of Bacillus thuringiensis var. galleriae R and S variants was studied. No adsorption of phages Tg11 and Tg18 on the cells of R variant was found upon infection in a liquid medium. What is characteristic of phage Tg11 is that it is predominantly adsorbed at the poles of S variant cells. Phage Tg18 particles are uniformly distributed along the perimeter of S variant cells. Phage Tg13 is adsorbed on the both variant cells. Phage aggregates with the elements of cell walls having a tetrahonal assembly of the subunits can be revealed in phage Tg13 lysates. The size of the subunits is 7 nm and the distance between their centers is 11 nm. A structured element, apparently the T-layer, is involved in the adsorption of phage Tg13 on the cells.     

Assessment of cone-damaging insects in a Swedish spruce seed orchard and the efficacy of large-scale application of Bacillus thuringiensis variety aizawai x kurstaki against lepidopterans

In a 2-yr study, we investigated the efficacy of large-scale application of the biological insecticide Bacillus thuringiensis variety aizawai x kurstaki (Btk) in a Swedish spruce, Picea abies L. (Karst.), seed orchard for controlling damage caused by four lepidopteran species: Dioryctria abietella Den et. Schiff. (Pyralidae), Eupithecia abietaria G?tze, E. analoga Djakonov (Geometridae), and Cydia strobilella (L.) (Tortricidae). The frequencies of these species, and Strobilomyia anthracina Czerny (Diptera: Anthomyiidae), were regularly monitored throughout the vegetative growth season to map their temporal distribution patterns and to quantify occurrences of species that may have been present in the cones at some stage during the season but migrated before the final sampling. This investigation revealed that E. abietaria occurred in similar numbers to D. abietella and has probably been overlooked as a potentially serious pest in spruce seed orchards in Sweden. To determine the number, timing, and rate of Btk required to control the lepidopterans, spraying was conducted at different phases of flowering and cone development, and three rates of Btk were applied. The Btk treatment reduced cone damage caused by D. abietella and Eupithecia spp. by one-half in 2002, a year with an intermediate number of cones, but the effect was weaker in 2003, when the cone crop was low. Damage caused by C. strobilella was not affected by the treatment. The different rates of Btk application did not affect the results, and repeated spraying seemed to be effective during 2002 but not in 2003.     

Relationship between endospore viability and insecticidal potency of Bacillus thuringiensis subsp. aizawai NT0423

Bioassay can be used for analysis of the biological potency of Bacillus thuringiensis (Bt) in fermentation and formulation but requires precise scheduling and several repetitions. Alternatively, this work explored if the endospore counting could be used to predict the potency of Bt technical powder. Analyses of Bt technical powers provided a strong linear relationship (r = 0.971) between the number of viable endospores and the potency of the technical powder against second instar Plutella xylostella (L.) larvae. Next, a Bt wettable powder formulation was stored at 25 and 40 °C for 12 weeks to investigate the influence of storage temperature on the prediction of insecticidal potency based on the counting. At 25 °C storage, the insecticidal potency could be predicted based on the counting, but at 40 °C the predicted insecticidal potency was much lower than the measured potency. These results suggest that the NT0423 endospore viability can be used to predict its potency in production, but the relationship may not be the same following the storage at high temperature.     

Specific phages used for differentiation of the entomopathogenic bacterium Bacillus thuringiensis]

The sensitivity to specific phages and morphological, physiological, and antigenic properties were compared among several strains of Bacillus thuringiensis isolated from insects inhabiting various geographical zones. All 43 cultures assigned to Bac. thuringiensis var. sotto and 198 among 170 cultures classed as Bac. thuringiensis var. dendrolimus were found to belong to Bac. thuringiensis var. dendrolimus. None of these cultures was resistant to its specific phage. The same was true of 22 studied cultures of Bac. thuringiensis var. galleriae. Only two among studied 45 cultures of Bac. thuringiensis var. thuringiensis were resistant to phages specific for this variety. Therefore, the abundance of variants resistant to specific phages in natural conditions differs among the varieties of Bac. thuringiensis. In most cases, cultures of the same variety of Bac. thuringiensis isolated from various insects inhabiting different geographical zones are identical by their sensitivity to specific phages and by other important characteristics.