Isolation and characterization of Bacillus thuringiensis strains from aquatic environments in Spain.

Samples collected from aquatic environments from Spain were analyzed for the occurrence and dipteran toxicity of Bacillus thuringiensis. From a total of 41 samples, 122 isolates were obtained, yielding a B. thuringiensis index of 0.22. Isolates were assigned to 13 different serovars, with serovar thuringiensis (serotype H1) the most frequently found. Toxicity tests carried out revealed that eight isolates (6.6% out of the total) were active against Tipula oleracea larvae. Serological tests assigned these toxic isolates to serovar thuringiensis. The toxicity found in these isolates against the tipulid was approximately seven times lower than that shown by the standard strain B. thuringiensis ser. israelensis IPS-82. Implication of Cry2A protein in toxic activity is hypothesized.

Characterization of Bacillus thuringiensis ser. balearica (Serotype H48) and ser. navarrensis (serotype H50): two novel serovars isolated in Spain.

The novel strains of Bacillus thuringiensis PM9 and NA69, isolated from soil samples in Spain, were classified and characterized in terms of their crystal proteins, plasmid profile, cry genes content, and their toxicological properties against several species of Lepidoptera, Coleoptera, and Diptera. Both strains share morphological and biochemical characteristics with previously described B. thuringiensis strains, although their unique H antigens identify them as two new serotypes. Two new serovar names, B. thuringiensis serovar balearica (H serotype 48) and B. thuringiensis serovar navarrensis (H serotype 50) are proposed for the type strains PM9 and NA69, respectively.

Identification and characterization of the new bacillus thuringiensis serovars pirenaica (serotype H57) and iberica (serotype H59)

Two new Bacillus thuringiensis strains have been classified by the H antigen of the cells and differentiated by their morphological, biochemical and molecular characteristics. The flagellar agglutination showed that both strains bore specific H antigens which allowed their classification as the new serotypes H57 and H59. The serovar names proposed for the type strains characterized in this work are B. thuringiensis ser. pirenaica, for the H serotype 57, and B. thuringiensis ser. iberica, for the H serotype 59. Further characterization of these strains, by means of SDS-PAGE, Western inmunodetection, plasmid profile and cry-gene identification by polymerase chain reaction, confirmed the originality of the two novel serotypes. Toxicity tests carried out against several insect species, belonging to the orders Lepidoptera, Diptera and Coleoptera, showed no detectable insecticidal activity for either of the B. thuringiensis strains.

Characterization of Bacillus thuringiensis serovar bolivia (serotype H63), a novel serovar isolated from the Bolivian high valleys.

The type strain Bacillus thuringiensis var. bolivia (serotype H63), isolated from the Bolivian high valleys, has been characterized at different levels. Its parasporal crystal has an unusual shape and it is composed of a protein of 155 kDa which shows two bands of 75 and 80 kDa after activation. Analysis by PCR shows the presence of cry1 genes, and amplification with specific primers gave products for cry1 E, cry1 D, cry4 A and cry4 B with sizes different to those expected. Immunoblotting tests showed positive reaction for Cry1 E, Cry3 A, Cry4 A and Cry11 A crystal proteins. The plasmid pattern revealed two large and two small plasmids. Toxicity tests were performed against 14 insects and a slight toxicity was found against Plutella xylotella and Trichoplusia ni.

Updating the H-antigen classification of Bacillus thuringiensis.

The classification of Bacillus thuringiensis strains has been revised and updated based on flagellar antigens which have been in use for many years. Sixty-nine serotypes and 13 sub-antigenic groups have now been identified, giving 82 serovars among the 3500 B. thuringiensis isolates of the IEBC Collection. The number of serovars has gradually increased with the total number of strains. The biochemical characters used have also been investigated and their value assessed for identification of B. thuringiensis at the subspecies level. A crystal analysis was carried out in terms of morphology, delta-endotoxin profiles and larvicidal activity for the newly identified serovars. It was found that atypical crystals, some with novel components, are becoming more common. No insect susceptible to these serovars has been discovered among known target species. The number of cross-reacting H-antigens among B. cereus strains is increasing and may be of biological significance.

Genetic diversity of Bacillus cereus/B. thuringiensis isolates from natural sources.

The genetic diversity and relationships among 154 Bacillus cereus/B. thuringiensis isolates recovered from soil samples from five geographic areas in Norway were investigated with multilocus enzyme electrophoresis (MEE). Cluster analysis revealed two major groups (designated cluster I and cluster II) separated at genetic distance greater than 0.55. Cluster I included 62 electrophoretic types (ETs) originating from all five locations, whereas, in cluster II, all but one isolate were from the same location. The isolates were also serotyped with B. thuringiensis flagellar antisera, and 28 distinct serotypes were identified. In general, serotyping did not show correlation to the genetic diversity of the isolates. The presence of IS231- and IS240-like transposable elements was detected in 14% of the strains of cluster II only. Parasporal crystals were observed in three strains; ten other strains were toxic to Trichoplusia ni. We conclude that B. cereus/B. thuringiensis from soil exhibit a high degree of recombination.

Cloning and sequencing of three new putative toxin genes from Clostridium bifermentans CH18.

Three new open reading frames were found downstream from cbm71, a toxin gene from Clostridium bifermentans malaysia (Cbm) strain CH18. The first one (91bp downstream) called cbm72, is 1857bp long and encodes a 71727-Da protein (Cbm72) with a sequence similar to that of Bacillus thuringiensis delta-endotoxins. This protein shows no significant toxicity to mosquito larvae. The two others, cbm17.1 (462bp) and cbm17.2 (459bp), are copies of the same gene encoding Cbm P18 and P16 polypeptides and located 426bp and 1022bp downstream from cbm72, respectively. They encode 17189-Da and 17451-Da proteins with sequences 44.6% similar to that of Aspergillus fumigatus hemolysin; however, they were not hemolytic in the conditions tested.

Distribution of clostridial cry-like genes among Bacillus thuringiensis and Clostridium strains.

The presence of two cry-like genes first identified in Clostridium bifermentans subsp. malaysia CH18 was investigated in Clostridium species including 12 subspecies of Clostridium bifermentans, 13 strains of other members of Clostridia genus, and 13 different subspecies of Bacillus thuringiensis. Oligonucleotides designed to amplify the two toxin genes, cmb71 and cmb72, were used. We found that these genes are present in 80% of the Clostridium bifermentans strains tested and in 8% of the other Clostridium and Bacillus thuringiensis strains.

[Characterization of entomopathogenic Bacillus samples isolated in Senegal and study of their toxicity for malaria vectors]. / Caractérisation de souches de Bacillus entomopathogènes isolées au Sénégal et étude de leur toxicité pour les vecteurs du paludisme.

A screening program developed in Senegal to isolate new strains of entomopathogenic Bacillus has led to the isolation of 194 strains of Bacillus thuringiensis and 9 strains of Bacillus sphaericus from various sites and insect samples. The characterization of these strains regarding their H serotype, their crystal composition and their toxicity against mosquitoes (Culex pipiens, Aedes aegypti and Anopheles stephensi) has led to the isolation of 27 mosquitocidal strains. As malaria is an important public health problem in Senegal, these strains were more completely characterized looking for their toxicity against the two major malaria vectors in Senegal: Anopheles gambiae and Anopheles arabiensis.

A novel insecticidal serotype of Clostridium bifermentans.

A novel Clostridium bifermentans strain toxic to mosquito larvae on ingestion was isolated from a soil sample collected from secondary forest floor. This strain was designated as serovar paraiba (C. b. paraiba) according to its specific H antigen. Clostridium bifermentans paraiba is most toxic to Anopheles maculatus Theobald larvae (LC50 = 0.038 mg/liter), whereas toxicity to Aedes aegypti (Linn.) (LC50 = 0.74 mg/liter) and Culex quinquefasciatus Say (LC50 = 0.11 mg/liter) larvae was 20 and 3 times lower, respectively. The toxicity to An. maculatus larvae is as high as that of Bacillus thuringiensis serovar israelensis. C. b. paraiba was also found to exhibit significant per os insecticidal activity toward adult Musca domestica (Linn.).

The crystal-forming strains of Bacillus laterosporus.

Bacillus laterosporus is a spore-forming bacterium characterized by its ability to produce a canoe-shaped lamellar parasporal inclusion, adjacent to the spore. In some B. laterosporus strains crystalline inclusions of various shapes and sizes, which are released separately from spores during the lysis of the sporangium, were also produced. The morphological characteristics of two crystal-forming strains, B. laterosporus BL 16-92 and LAT 006, were investigated.

Cloning and expression of the first anaerobic toxin gene from Clostridium bifermentans subsp. malaysia, encoding a new mosquitocidal protein with homologies to Bacillus thuringiensis delta-endotoxins.

A gene (cbm71) encoding a 71,128-Da mosquitocidal protein (Cbm71) was obtained by screening a size-fractionated XbaI digest of total genomic DNA from Clostridium bifermentans subsp. malaysia CH18 with two gene-specific oligonucleotide probes. The sequence of the Cbm71 protein, as deduced from the sequence of cbm71, corresponds to that of the 66-kDa protein previously described as one of the mosquitocidal components of C. bifermentans subsp. malaysia. Cbm71 shows limited similarities with Bacillus thuringiensis delta-endotoxins, especially in the four first conserved blocks. However, Cbm71 was not immunologically related to any of the Cry toxins and thus belongs to a novel class of mosquitocidal protein. The cbm71 gene was expressed in a nontoxic strain of B. thuringiensis, and Cbm71 was produced during sporulation and secreted to the supernatant of culture. Trichloroacetic-precipitated supernatant preparations were toxic for mosquito larvae of the species Aedes aegypti, Culex pipiens, and Anopheles stephensi.

A micromethod for serotyping Bacillus thuringiensis.

Serotyping of Bacillus thuringiensis is possible using 96-well microplates instead of tubes. The advantages are a reduction on the incubation time from 120 to 75 min and the amounts of antisera and bacterial suspensions needed 10-fold.

A new serovar of Bacillus thuringiensis possessing 28a28c flagellar antigenic structure: Bacillus thuringiensis serovar jegathesan, selectively toxic against mosquito larvae.

A novel Bacillus thuringiensis strain highly toxic to mosquitoes was isolated from soil samples in Malaysia. This strain was shown to display a new subfraction of the H-28 flagellar antigen determining a new serovar H28a28c, which was designated serovar jegathesan. Bioassays indicated that Culex quinquefasciatus larvae are the most susceptible to this new isolate, whereas toxicity to Anopheles maculatus and Aedes aegypti larvae was 10 times lower. The potency of this new serotype is also comparable to most of the Malaysian B. thuringiensis H-14 isolates.

New Bacillus bacteriophage species.

Nine new species of tailed Bacillus phages, based on morphological and physicochemical properties, are defined. Phage P10 is one of the largest viruses known. The total number of tailed Bacillus phage species is presently 33.

Full expression of the cryIIIA toxin gene of Bacillus thuringiensis requires a distant upstream DNA sequence affecting transcription.

The cryIIIA gene encoding a coleopteran-specific toxin is poorly expressed in Bacillus thuringiensis when cloned in a low-copy-number plasmid. This weak expression is observed when the gene is cloned only with its promoter and its putative terminator. cryIIIA gene expression was analyzed by using deletion derivatives of a larger DNA fragment carrying the toxin gene and additional adjacent sequences. The results indicate that a 1-kb DNA fragment located 400 bp upstream of the promoter strongly enhances CryIIIA production in B. thuringiensis sporulating cells. Similar results were obtained when the low-copy-number plasmid pHT304 carrying transcriptional fusions between upstream regions of cryIIIA and the lacZ gene was used. Analysis of the start sites, the sizes, and the amounts of cryIIIA-specific mRNAs shows that the enhancement occurs at the transcriptional level by increasing the number of cryIIIA-specific transcripts from the onset of sporulation to about 6 h after the onset of sporulation. The nucleotide sequence of the 1-kb activating fragment and of the 700 bp containing the promoter region and the 5' end of cryIIIA were determined. No potential protein-coding sequences were found upstream of the promoter. The major characteristic of the 1-kb activating fragment is the presence of a 220-bp A + T-rich region.

Construction of Novel Bacillus thuringiensis Strains with Different Insecticidal Activities by Transduction and Transformation.

The shuttle vector pHT3101 and its derivative pHT408, bearing a copy of a cryIA(a) delta-endotoxin gene, were transferred into several Bacillus thuringiensis subspecies through phage CP-54Ber-mediated transduction, with frequencies ranging from 5 x 10 to 2 x 10 transductant per CFU, depending on the strain and on the plasmid. In Cry and Cry native recipients, the introduction of the cryIA(a) gene resulted in the formation of large bipyramidal crystals that were active against the insect Plutella xylostella (order Lepidoptera). In both cases, high levels of gene expression were observed. Transductants displaying a dual specificity were constructed by using as recipients the new isolates LM63 and LM79, which have larvicidal activity against insects of the order Coleoptera. It was not possible, however, to introduce pHT7911 into B. thuringiensis subsp. entomocidus, aizawai, or israelensis by transduction. However, electrotransformation was successful, and transformants expressing the toxin gene cryIIIA, carried by pHT7911, were obtained. Again, high levels of expression of the cloned gene were observed. The results indicate that CP-54Ber-mediated transduction is a useful procedure for introducing cloned crystal protein genes into various B. thuringiensis recipients and thereby creating strains with new combinations of genes. Finally it was also shown that pHT3101 is a very good expression vector for the cloned delta-endotoxin genes in the different recipients.

Structural and genetic organization of IS232, a new insertion sequence of Bacillus thuringiensis.

In the Bacillus thuringiensis strains toxic for the lepidopteran larvae, the delta-endotoxin genes cryIA are frequently found within a composite transposonlike structure flanked by two inverted repeat sequences. We report that these elements are true insertion sequences and designate them IS232. IS232 is a 2,184-bp element and is delimited by two imperfect inverted repeats (28 of 37 bp are identical). Two adjacent open reading frames, overlapping for three codons, span almost the entire sequence of IS232. The potential encoded polypeptides of 50 and 30-kDa are homologous to the IstA and IstB proteins of the gram-negative insertion sequence IS21. The N-terminal part of the 50-kDa polypeptide contains a helix-turn-helix DNA-binding motif. The junctions at the insertion sites of three IS232 elements were analyzed. Each case was different, with 0, 4, or 6 bp of the target DNA being duplicated. Transposition of IS232 in Escherichia coli was demonstrated by using a genetic marker inserted upstream of the two open reading frames.

Transformation and expression of a cloned delta-endotoxin gene in Bacillus thuringiensis.

A shuttle vector containing the replication region of a resident plasmid of B. thuringiensis, was used to determine the conditions allowing efficient transformation of B. thuringiensis by electroporation. Using this plasmid a delta-endotoxin gene was cloned and expressed both in Escherichia coli and B. thuringiensis. It was shown that this gene was poorly expressed in the wild type situation whereas after cloning in acrystalliferous strains of B. thuringiensis large amounts of crystal protein were obtained.