Construction of an <Emphasis Type="Italic">Escherichia coli</Emphasis> to <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> shuttle vector for large DNA fragments

Shuttle vectors for Bacillus thuringiensis or Bacillus cereus usually cannot hold fragments larger than 20 kb. With the development of genome research, shuttle vectors with higher loading capacity are necessary. We constructed an Escherichia coli to B. thuringiensis shuttle vector, pEMB0557, with a large loading capacity. This vector incorporated the ori60 replicon from B. thuringiensis subsp. kurstaki YBT-1520, erythromycin resistance (B. thuringiensis), and chloromycetin resistance (E. coli) genes. A bacterial artificial chromosome library of B. thuringiensis strain CT-43 was constructed and pEMB0557 was able to accommodate at least a 70-kb DNA fragment. Simultaneously, the cry1B gene on a 40-kb fragment could express a 140-kDa protein in plasmid-cured B. thuringiensis BMB171. Due to its high capacity and utility in expressing exogenous genes, pEMB0557 will be useful in cloning (especially silencing genes) and expressing large DNA fragments (e.g., gene clusters) in B. thuringiensis. Plasmid pEMB0557 provides a new tool for B. thuringiensis genome or B. cereus group research.     

Discrimination among <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> H serotypes,serovars and strains based on 16S rRNA, <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">aroE</Emphasis> gene sequence analyses

Our aim was to investigate the capability of each of three genes, 16S rRNA, gyrB and aroE, to discriminate, first, among Bacillus thuringiensis H serotypes; second, among B. thuringiensis serovars from the same H serotype; and third, among B. thuringiensis strains from the same serovar. The 16S rRNA, gyrB and aroE genes were amplified from 21 B. thuringiensis H serotypes and their nucleotide sequences determined. Additional strains from four B. cereus sensu lato species were included for comparison purposes. These sequences were pair-wise compared and phylogenetic relationships were revealed. Each of the three genes under study could discriminate among B. thuringiensis H serotypes. The gyrB and aroE genes showed a discriminatory power among B. thuringiensis H serotypes up to nine fold greater than that of the 16S rRNA gene. The gyrB gene was retained for subsequent analyses to discriminate B. thuringiensis serovars from the same H serotype and to discriminate strains from same serovar. A total of 42 B. thuringiensis strains, which encompassed 25 serovars from 12 H serotypes, were analyzed. The gyrB gene nucleotide sequences were different enough as to be sufficient to discriminate among B. thuringiensis serovars from the same H serotype and among B. thuringiensis strains from the same serovar. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Isolation and Characterization of a Strain of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Subsp. <Emphasis Type="Italic">morrisoni</Emphasis> PG-14 Encoding δ-Endotoxin Cry1Ac

Bacillus thuringiensis 656-3, isolated from a soil sample collected at mushroom houses, showed high toxicity to mushroom flies, Lycoriella mali and Coboldia fuscipes. B. thuringiensis 656-3 produced bipyramidal inclusions and reacted with the H antiserum of B. thuringiensis subsp. morrisoni (H8a8b). The plasmid and protein profiles of B. thuringiensis 656-3 were similar to those of its reference strain, subsp. morrisoni PG-14. However, PCR analysis using cry gene primers showed that B. thuringiensis 656-3, unlike its reference strain, had cry4A, cry4B, cry10A, cry11A, and cry1Ac genes, suggesting that B. thuringiensis 656-3 was a unique strain with respect to gene type. In addition, B. thuringiensis 656-3 showed a high level of toxicity against mushroom flies, L. mali and C. fuscipes.     

Molecular Cloning of a New Crystal Protein Gene cry1Af1 of Bacillus thuringiensis NT0423 from Korean Sericultural Farms

A new cry1Ab-type gene encoding the 130 kDa protein of Bacillus thuringiensis NT0423 bipyramidal crystals was cloned, sequenced, and expressed in a crystal-negative B. thuringiensis host. Hybridization experiments revealed that the crystal protein gene is located on a 44 MDa plasmid of B. thuringiensis NT0423. A strong positive signal detected on the 6.6 kb HindIII fragment from B. thuringiensis NT0423 plasmid DNA was cloned and sequenced. The cry1Ab-type gene, designated cry1Af1, consisted of open reading frame of 3453 bp, encoding a protein of 1151 amino acid residues. The polypeptide has the deduced amino acid sequences predicting molecular masses of 130,215 Da. With both Bt I and Br II promoter sequences were found, the B. thuringiensis NT0423 crystal protein gene promoter closely aligned with those of cry1A-type crystal protein gene. When compared with known sequences of other Cry and Cyt proteins, the Cry1Af1 protein showed maximum 93% sequence identity to Cry1Ab protein of B. thuringiensis subsp. kurstaki. The expressed Cry1Af1 protein in a crystal-negative B. thuringiensis host appears to have strong insecticidal activity against lepidopteran larvae (Plutella xylostella). Crystals containing Cry1Af1 were about six times more toxic than the wild-type crystals of B. thuringiensis NT0423. Received: 20 February 2001 / Accepted: 17 April 2001     

EFFECT OF TANNIC ACID ON THE EFFECTIVENESS OF BACILLUlS THURINGIENSIS VAR. KURSTAKI AGAINST HELICOVERPA ARMIGERA (HÜBNER)*

Abstract The role of tannic acid in increasing effectiveness of Bacillus thuringiensis var. kurstaki (HD-1) against Helicover pa armigera was examined in bioassays on semisynthetic diet. Concentrations of B. thuringiensis (0 %, 0.005 %, 0. 01 %, 0.015 %, 0.02 %, 0.025 % wet weight) were incorporated into the diet containing 0. 025% tannic acid and tannic acid-free diet. LD₅₀ of B. thuringiensis with tannic acid were 0.006% but that without tannic acid was 0.011%. Both B. thuringiensis and tannic acid retarded growth of H. armigera significantly, but there was no synergetic effect between them. Choice tests showed that B, thuringiensis deterred feeding of the fifth instar larvae of H. armigera but tannic acid had no such effect. Experiments on colony growth of B. thuringiensis on NBA media containing tannic acid (0, 1, 3, 6, 9, 12, 15, 18, 21 mg/100 ml) demonstrated that tannic acid reduced colony growth of B. thuringiensis, and inhibited sporulation above 15 mg/100 ml.     

Cloning,production and expression of the bacteriocin enterocin A produced by <Emphasis Type="Italic">Enterococcus faecium</Emphasis> PLBC21 in <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Two genes, ctc and ctc2, responsible for surface layer (S-layer) protein synthesis in Bacillus thuringiensis CTC, were mutated and resulted in B. thuringiensis Tr5. To synthesize and express the N-acyl-homoserine lactonase (AHL-lactonase) in the extracellular space of B. thuringiensis, the aiiA _4Q7 gene (an AHL-lactonase gene from B. thuringiensis 4Q7), which confers the ability to inhibit plant soft rot disease in B. thuringiensis 4Q7, was fused with the upstream sequence of the ctc gene, which in turn is essential for S-layer protein secretion and anchoring on the cell surface. The resulting fusion gene, slh-aiiA, was expressed in B. thuringiensis Tr5 to avoid competition for the extracellular space with the native S-layer protein. Our results indicate that B. thuringiensis Tr5 containing the fusion gene slh-aiiA displayed high extracellular AHL-degrading activity. When compared with wild-type B. thuringiensis strains, the ability of the constructed strain to inhibit soft rot disease caused by Erwinia carotovora SCG1 was markedly increased. These findings provide evidence for a significant advance in our ability to inhibit soft rot disease caused by E. carotovora.     

Biological, Immunological, and Genetic Analysis of Bacillus thuringiensis Isolated from Granary in Korea

To isolate a naturally occurring novel Bacillus thuringiensis strain, we investigated the distribution, toxicity, morphology, H serotype, and gene type of B. thuringiensis from residue samples of granary in Korea. A total of 163 B. thuringiensis isolates out of 411 samples producing spore and crystal were obtained. In toxicity tests, 80% of all isolates were toxic to lepidoptera, and 12% were not toxic to any of tested insects. And dipteran-active and lepidopteran/dipteran-active isolates were rare (2% and 6%, respectively). 152 B. thuringiensis isolates produced typical rhomboidal crystals, and the remainder produced parasporal inclusions with various morphologies. Serological test showed that B. thuringiensis isolates in granary represented 12 H serotypes, indicating varied distribution of B. thuringiensis. Of these, the serotype 3ab predominated, followed by the serotype 7 and 4ac. B. thuringiensis isolates of the serotype 3ab, 4ac, 5ab, 7, 8ab, 9, and 23 were toxic to lepidoptera, and the serotype 8bd, 12, 18, and 20ac were nontoxic, while 14 isolates were untypable by 33 B. thuringiensis H antisera. The frequency of toxicity against lepidoptera and diptera was primarily highly toxic. PCR analysis using cryI gene type-specific primers showed that cryIA(b) genes are frequently found and cryIE gene exists in only one isolate. Analysis of B. thuringiensis crystals and plasmid DNAs indicated a diversity of crystal and gene types. Received: 15 January 1998 / Accepted: 18 February 1998     

Strategy for orchard use of Bacillus thuringiensis while minimizing impact on Choristoneura rosaceana parasitoids

Trials were conducted to study how spring Bacillus thuringiensis Berliner subsp. kurstaki treatments on apple may be timed to maximize the survival of parasitoids of the obliquebanded leafroller, Choristoneura rosaceana (Harris) (Lepidoptera: Tortricidae), found in the southern interior of British Columbia, Canada. Orchard collections verified that second through fourth instar obliquebanded leafrollers were found in varying proportions from pink through the petal fall stage of apple development when spring B. thuringiensis treatments are applied vs. lepidopteran pests. Laboratory‐reared second through fourth instar obliquebanded leafrollers, unparasitized and parasitized by one of three native parasitoid species, were fed untreated apple leaves or leaves treated with B. thuringiensis. The highest mortality of unparasitized obliquebanded leafrollers occurred when fourth instars were exposed to B. thuringiensis‐treated leaves; B. thuringiensis‐induced mortality in the unparasitized second and third instars was less than 50%. The consumption of B. thuringiensis‐treated leaves by host larvae significantly increased the percentage of dead host larvae in all parasitized and unparasitized treatments. However, because of the low susceptibility of this leafroller species to B. thuringiensis, relatively high numbers (38–43%) of three obliquebanded leafroller parasitoid species were able to survive the consumption of B. thuringiensis by second and third instar host larvae. Fourth instar obliquebanded leafrollers were found at the full bloom and petal fall stage of apple development in the orchard, at which time B. thuringiensis treatments are recommended for optimal leafroller control. The highest parasitoid mortality due to host mortality was recorded in Apophua simplicipes Cresson (Hymenoptera: Ichneumonidae) and Macrocentrus linearis (Nees) (Hymenoptera: Braconidae), when the hosts were treated as fourth instars. Both of these parasitoids emerge from fifth and sixth instar obliquebanded leafrollers. Bacillus thuringiensis did not have as negative an impact on Apanteles polychrosidis Viereck (Hymenoptera: Braconidae), which emerges when the host is in the fourth instar. When leafroller mortality and parasitism were combined, the B. thuringiensis treatment did not significantly increase host elimination above that of parasitism alone, except for larvae parasitized by A. simplicipes that were in the fourth instar. The consumption of B. thuringiensis by unparasitized larvae was shown to slow larval development.     

UBER DEN BIOTEST VON BACILLUS THURINGIENSIS-EXOTOXIN MIT DROSOPHILA MELANOGASTER

Summary Larvae of Drosophila melanogaster (Meigen), placed as eggs on the substrate, are suitable for bioassay of the toxic qualities of the supernatant from culture media of Bacillus thuringiensis Berliner. Only supernatants of active strains of B. thuringiensis var. thuringiensis contained a thermostable exotoxin and, therefore, caused significant mortality. Supernatants prepared from cultures of other varieties of B. thuringiensis, however, as well as from cultures of Bacillus cereus had no effect (Tab. I). Pure preparations of the spore-endotoxin-complex of B. thuringiensis ded not kill larvae of Drosophila melanogaster even in doses of 0.16 per cent of the medium (Tab. II). According to the qualitative classification of commercial B. thuringiensis-preparations by Burgerjon, Biospor 2802 (Farbwerke Hoechst, Germany), belongs to group I, whereas Bakthane L-69 (Rohm & Haas, USA) is representative of group II.

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Über den Biotest von Bacillus thuringiensis-Exotoxin mit drosophila melanogaster

Zusammenfassung Zur Prüfung der toxischen Qualitäten von Kulturüberständen von Bacillus thuringiensis-Kulturen dienten Larven von Drosophila melanogaster. Es wurden abgezählte mengen von Eiern auf das kontaminierte Substrat aufgebracht und am Ende des Versuchs die entwickelten Imagines registriert. Nur Überstände aktiver Stämme von B. thuringiensis var. thuringingiensis enthielten ein thermostabiles Exotoxin und bewirkten eine entsprechende Mortalität. Kultur-überstände anderer Varietäten von B. thuringiensis wie auch von Bacillus cereus hatten keine Wirkung (Tab. I). Gereinigte Präparate des Sporen-Endotoxin-Komplexes von B. thuringiensis waren selbst in Dosen von 0,16% des Mediums unwirksam (Tab. II). Entsprechend der qualitativen Klassifikation von industriellen B. thuringiensis-Präparaten nach Burgerjon gehört Biospor 2802 (Farbwerke Hoechst, Deutschland) zur Gruppe I, während Bakthane L-69 (Rohm & Haas, USA) für die Gruppe II repräsentativ ist.

     

B. thuringiensis is a Poor Colonist of Leaf Surfaces

The ability of several Bacillus thuringiensis strains to colonize plant surfaces was assessed and compared with that of more common epiphytic bacteria. While all B. thuringiensis strains multiplied to some extent after inoculation on bean plants, their maximum epiphytic population sizes of 10⁶ cfu/g of leaf were always much less than that achieved by other resident epiphytic bacteria or an epiphytically fit Pseudomonas fluorescens strain, which attained population sizes of about 10⁷ cfu/g of leaf. However B. thuringiensis strains exhibited much less decline in culturable populations upon imposition of desiccation stress than did other resident bacteria or an inoculated P. fluorescens strain, and most cells were in a spore form soon after inoculation onto plants. B. thuringiensis strains produced commercially for insect control were not less epiphytically fit than strains recently isolated from leaf surfaces. The growth of B. thuringiensis was not affected by the presence of Pseudomonas syringae when co-inoculated, and vice versa. B. thuringiensis strains harboring a green fluorescent protein marker gene did not form large cell aggregates, were not associated with other epiphytic bacteria, and were not found associated with leaf structures, such as stomata, trichomes, or veins when directly observed on bean leaves by epifluorescent microscopy. Thus, B. thuringiensis appears unable to grow extensively on leaves and its common isolation from plants may reflect immigration from more abundant reservoirs elsewhere.     

Hemolysin II is more characteristic of Bacillus thuringiensis than Bacillus cereus

To investigate the distribution of the hemolysin II determinant among strains of Bacillus cereus and Bacillus thuringiensis, thirteen strains of B. cereus and fourteen strains of B. thuringiensis strains were tested for hybridization of their chromosomal DNAs with a DNA probe containing the B. cereus hemolysin II gene. In addition, the production of hemolysin II, whose activity is not inhibited by cholesterol, was tested. The presence (absence) of the hybridization response in the microorganism's genome correlated with the presence (absence) of cholesterol-unaffected hemolysin production. Only four out of thirteen B. cereus strains were found to give a positive response in hybridization experiments, whereas thirteen out of fourteen B. thuringiensis strains responded positively. DNAs from ten B. thuringiensis strains contained a 3.5 kb EcoRV fragment, which hybridized with the B. cereus hemolysin II gene probe. The 3.5 kb EcoRV DNA fragment from one of these strains (B. thuringiensis VKM-B1555) was cloned and expressed in Escherichia coli cells. The hemolysin encoded by the cloned DNA fragment was not inhibited by cholesterol and possessed all other properties of B. cereus hemolysin II. The obtained data clearly show limited distribution of hemolysin II among B. cereus strains and demonstrate that hemolysin II is more characteristic of B. thuringiensis than B. cereus.     

Occurrence of Bacillus thuringiensis on Cured Tobacco Leaves

A worldwide survey was conducted to evaluate the frequency and distribution of Bacillus thuringiensis populations on cured tobacco leaves during post-harvest storage. In total, 133 tobacco samples of different types and origins were analyzed. Nine percent of the samples showed the presence of B. thuringiensis, and 24 B. thuringiensis strains were isolated and characterized. The majority of the isolates produced bipyramidal crystals, and three fourths of them showed a second type of crystal protein (cuboidal or heterogeneous crystals). Only three isolates showed the rhomboidal crystal morphology characteristic of the anti-coleopteran B. thuringiensis subsp. tenebrionis. PCR analysis with primers specific for cry1 and cry3 genes revealed eight distinct cry gene profiles. The results of this study indicate that B. thuringiensis is naturally present at low frequency on the phylloplane of cured tobacco leaves and that its distribution is worldwide. Received: 26 August 1999 / Accepted: 5 October 1999     

Distribution Analysis of IS<Emphasis Type="Italic">231</Emphasis>-like Sequences Among <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Serovars Inferred from Restriction Fragment Length Polymorphisms

The distribution of IS231 has been analyzed in Bacillus thuringiensis serovars. A 723-bp HaeII conserved fragment from IS231M has been used as a probe against EcoRI-digested B. thuringiensis total DNA to yield serovar-specific hybridization profiles. The approach was useful at revealing the extent of distribution of IS231-like sequences between and within strains. Of the 88 B. thuringiensis strains tested, 70 showed hybridization banding patterns that comprised between one and 20 distinct bands. These 70 B. thuringiensis strains were grouped based on banding pattern similarities. Interestingly, intraserovar strains did not necessarily cluster together.     

Germination and conjugation of Bacillus thuringiensis subsp. israelensis in the intestine of gnotobiotic rats

Aims: To study the ability of Bacillus thuringiensis subsp. israelensis spores to germinate and subsequently transfer a conjugative plasmid in the intestinal tract of gnotobiotic rats. Methods and Results: Germination was studied by feeding germ-free rats with spores of a B. thuringiensis strain harbouring a plasmid encoding green fluorescent protein (GFP), which enabled quantification of germinated bacteria by flow cytometry. To study in vivo conjugation, germ-free rats were first associated with a B. thuringiensis recipient strain and after 1 week an isogenic donor strain harbouring the conjugative plasmid pXO16 was introduced. Both strains were given as spores and transfer of pXO16 was observed from the donor to the recipient strain. Conclusions: Bacillus thuringiensis is able to have a full life cycle in the intestine of gnotobiotic rats including germination of spores, several cycles of growth and sporulation of vegetative cells. For the first time conjugative plasmid transfer in a mammalian intestinal tract was shown between two B. thuringiensis strains. Significance and Impact of the Study: Strains of B. thuringiensis are used worldwide to combat insect pests, and this study brings new insights into the nature of B. thuringiensis showing the potential of the bacteria to germinate and transfer DNA in the mammalian intestinal tract.     

Isolation and Characterization of a Bacillus thuringiensis ssp. kurstaki Strain Toxic to Spodoptera exigua and Culex pipiens

A strain of Bacillus thuringiensis with dual toxicity was isolated from Korean soil samples and named K2. K2 was determined as ssp. kurstaki (H3a3b3c) by serological test and produced bipyramidal-shaped parasporal inclusions. The plasmid and protein profiles of B. thuringiensis K2 were different from those of the reference strain, ssp. kurstaki HD-1. To verify gene type of B. thuringiensis K2, PCR analysis with specific cry gene primers was performed. The result showed that B. thuringiensis K2 had cry1Aa, cry1Ab, cry1C, and cry1D type genes, whereas ssp. kurstaki HD-1 had cry1Aa, cry1Ab, cry1Ac, and cry2 type genes. In addition, B. thuringiensis K2 had high toxicity against Spodoptera exigua and Culex pipiens, whereas B. thuringiensis ssp. kurstaki HD-1 does not have high toxicity against these two insect species. Received: 19 January 2001 / Accepted: 21 February 2001     

Bacteriocin-like inhibitor substances produced by Mexican strains of Bacillus thuringiensis

Bacteriocins are antimicrobial peptides synthesized and secreted by bacteria and could potentially be used as natural food preservatives. Here, we report the production of bacteriocin-like inhibitor substances (Bt-BLIS) by five Mexican strains of Bacillus thuringiensis. Bacillus thuringiensis subsp. morrisoni (LBIT 269), B. thuringiensis subsp. kurstaki (LBIT 287), B. thuringiensis subsp kenyae (LBIT 404), B. thuringiensis subsp. entomocidus (LBIT 420) and B. thuringiensis subsp. tolworthi (LBIT 524) produced proteinaceous Bt-BLIS with high levels of activity against Bacillus cereus and other gram-positive bacteria. Although none was active against the gram-negative bacteria, Escherichia coli, Shigella species and Pseudomonas aeruginosa, the five Bt-BLIS demonstrated antimicrobial activity against Vibrio cholerae, the etiologic agent of cholera. Biochemical and biophysical studies demonstrated that the five Bt-BLIS could be categorized into two groups, those produced by LBIT 269 and 287 (Group A) and LBIT 404, 420, 524 (Group B), based on relative time of peptide synthesis, distinctive bacterial target specificity and stability in a wide range of temperatures and pH. Because of their stability and bactericidal activities against B. cereus and V. cholerae agents of emetic, diarrheal and lethal syndromes in humans, these Bt-BLIS could potentially be used as biodegradable preservatives in the food industry.     

Recovery of Bacillus thuringiensis from Marine Sediments of Japan

Marine sediments from a Japanese bay were examined for the occurrence of Bacillus thuringiensis. Of 1313 colonies belonging to the Bacillus cereus/B. thuringiensis group, 22 (1.7%) were allocated to B. thuringiensis. Marine isolates of B. thuringiensis consisted of heterogeneous multiple H serogroups; 10 isolates were assigned to the eight serovars (kurstaki, sumiyoshiensis, sotto, aizawai, darmstadiensis, thompsoni, neoleonensis, and higo); two motile isolates failed to react with the reference antisera; and the others were serologically untestable. Insecticidal activities were associated with two kurstaki isolates (toxic to both Lepidoptera and Diptera) and a higo isolate (Diptera-specific). None of the parasporal inclusion proteins of the 22 isolates exhibited in vitro cytotoxic activity against two vertebrate cells, sheep erythrocytes and HeLa cells. All B. thuringiensis isolates had no halophilism, although seawater-based medium supported their growth, sporulation, and formation of parasporal inclusions. Received: 29 November 1999 / Accepted: 10 January 2000     

Simple and Rapid Method for PCR Characterization of Large <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strain Collections

Polymerase chain reaction (PCR)-based identification of Bacillus thuringiensis toxin genes has become a routine step in most B. thuringiensis isolation and characterization initiatives. In the present study we propose a simplified method for extensive PCR analysis of B. thuringiensis cry and cyt genes of particular interest in large-scale screening programs. Fifty B. thuringiensis strains were screened for the presence of genes of the cry1 subfamily. Identical results were obtained when our method was used in comparison to other methodology based on a standard alkaline lysis preparation of plasmid DNA. Additional tests evidenced the fitness of our method in a particular multiplex–PCR analysis. The main advantages of the proposed methodology are that it is simpler and quicker than commonly used protocols (as DNA template preparation is substituted by direct addition of small amounts of B. thuringiensis liquid cell suspension to the reaction mixture) and that it allows simultaneous handling (bacterial growth and PCR) of up to 96 strains per round.     

Phylogenetic relationships among <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> (Bacillaceae: Bacillales) strains based on a comparison of SSU rRNA sequences and genome profiling

Bacillus thuringiensis Berliner has previously been classified via the serological identification of flagellar antigens. However, the phylogenetic relationships among strains of B. thuringiensis cannot be investigated by serotyping. Furthermore, high levels of homology have been found in gene sequences among various strains, complicating the determination of their evolutionary relationships. In order to elucidate the phylogenetic relationships within B. thuringiensis, we analyzed 40 strains belonging to typical serotypes using two approaches: an analysis of small subunit (SSU) rRNA sequences and genome profiling (GP) based on temperature gradient gel electrophoresis of random PCR products. The SSU rRNA analysis resulted in all 40 strains forming a single cluster with Bacillus cereus Frankland & Frankland. The distances among the subclusters were too small to further classify the strains. On the other hand, the phylogenetic analysis based on GP resulted in three clusters of B. thuringiensis strains. These results suggest that GP is a better method for the determination of phylogenetic relationships within B. thuringiensis.     

Contributions of gut bacteria to Bacillus thuringiensis-induced mortality vary across a range of Lepidoptera

Background  

Gut microbiota contribute to the health of their hosts, and alterations in the composition of this microbiota can lead to disease. Previously, we demonstrated that indigenous gut bacteria were required for the insecticidal toxin of Bacillus thuringiensis to kill the gypsy moth, Lymantria dispar. B. thuringiensis and its associated insecticidal toxins are commonly used for the control of lepidopteran pests. A variety of factors associated with the insect host, B. thuringiensis strain, and environment affect the wide range of susceptibilities among Lepidoptera, but the interaction of gut bacteria with these factors is not understood. To assess the contribution of gut bacteria to B. thuringiensis susceptibility across a range of Lepidoptera we examined larval mortality of six species in the presence and absence of their indigenous gut bacteria. We then assessed the effect of feeding an enteric bacterium isolated from L. dispar on larval mortality following ingestion of B. thuringiensis toxin.