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D Lereclus

Publications and source records attributed to D Lereclus.

At least 37 records · Page 2Linked to original sources
PubMed1995

Overproduction of encapsulated insecticidal crystal proteins in a Bacillus thuringiensis spo0A mutant.

The spo0A gene of Bacillus subtilis encodes the key factor involved in the initiation of sporulation. It was previously shown that the B. thuringiensis (Bt) cryIIIA gene, encoding a toxin active against coleopteran larvae, is overexpressed in an spo0A mutant of B. subtilis. In this paper we describe the construction of a Bt spo0A mutant strain and its use to produce insecticidal crystal proteins. The spo0A gene of Bt was cloned and identified by its ability to transform a B. subtilis spo0A mutant to prototrophy. Its nucleotide sequence is homologous to the B. subtilis gene. The spo0A gene was replaced in the Bt genome with a disrupted copy to give an Spo- strain unable to initiate sporulation. When the cryIIIA gene was cloned in the Bt spo0A mutant, large amounts of toxins were produced and accumulated to form a large crystal inclusion which remained encapsulated within the ghost cell. These encapsulated toxins were highly active against coleopteran larvae. We anticipate that the cryIIIA expression system and the Bt spo0A mutant will provide a convenient process to generate novel formulations of stabilized and environmentally safe Bt-based biopesticides.

Amino Acid Sequence↗
PubMed1994

Structural and functional analysis of the promoter region involved in full expression of the cryIIIA toxin gene of Bacillus thuringiensis.

The promoter region of the cryIIIA toxin gene of Bacillus thuringiensis is composed of at least three domains: an upstream region extending from nucleotide positions -635 to -553 (with reference to the translational start codon of cryIIIA), an internal region extending from nucleotide positions -553 to -367, and a downstream region extending from nucleotide position -367 to +18. Deletion analysis and transcriptional fusions to the lacZ gene indicate that full expression of cryIIIA requires the association of the upstream and the downstream region. Primer extension experiments reveal a major cryIIIA transcript (designated T-129) starting at nucleotide position -129 and another transcript (designated T-558) starting at nucleotide position -558. Mutation in the -35 region of the promoter responsible for the initiation of T-558 indicates that the upstream promoter is essential for full expression of cryIIIA, although not sufficient. Deletion of the DNA region carrying the previously described cryIIIA promoter does not affect full expression of cryIIIA and does not modify the 5' end of T-129. Taken together, these results indicate that the 5' end of T-129 is not a trnascriptional start site. Therefore, we propose that T-129 results from the processing of the mRNA initiated at the upstream promoter (T-558), generating a stable mRNA with a 5' extremity at nucleotide position -129. From primer extension analysis and transcriptional fusions to lacZ, it appears that the upstream promoter is weakly but significantly expressed during the vegetative phase of growth, is activated at the onset of sporulation and remains active at least until t5. However, unlike the promoters of other cry genes, this promoter is similar to sigma A-dependent promoters rather than sporulation-specific promoters. This promoter may therefore be transcribed by the E sigma A form of RNA polymerase. Activation at the onset of sporulation could result from the disappearance of a repressor, or the appearance of a stationary-phase-specific activator.

Bacillus thuringiensis↗
PubMed1994

Expression in Bacillus subtilis of the Bacillus thuringiensis cryIIIA toxin gene is not dependent on a sporulation-specific sigma factor and is increased in a spo0A mutant.

Expression of the Bacillus thuringiensis cryIIIA gene encoding a Coleoptera-specific toxin is weak during vegetative growth and is activated at the onset of the stationary phase. cryIIIA'-'lacZ fusions and primer extension analysis show that the regulation of cryIIIA expression is similar in Bacillus subtilis and in B. thuringiensis. Activation of cryIIIA expression was not altered in B. subtilis mutant strains deficient for the sigma H and sigma E sporulation-specific sigma factors or for minor sigma factors such as sigma B, sigma D, or sigma L. This result and the nucleotide sequence of the -35 and -10 regions of the cryIIIA promoter suggest that cryIIIA expression might be directed by the E sigma A form of RNA polymerase. Expression of the cryIIIA'-'lacZ fusion is shut off after t2 (2 h after time zero) of sporulation in the B. subtilis wild-type strain grown on nutrient broth sporulation medium. However, no decrease in cryIIIA-directed beta-galactosidase activity occurred in sigma H, kinA, or spo0A mutant strains. Moreover, beta-galactosidase activity was higher and remained elevated after t2 in the spo0A mutant strain. beta-Galactosidase activity was weak in abrB and spo0A abrB mutant strains, suggesting that AbrB is responsible for the higher level of cryIIIA expression observed in a spo0A mutant. However, both in spo0A and spo0A abrB mutant strains, beta-galactosidase activity remained elevated after t2, suggesting that even in the absence of AbrB, cryIIIA expression is controlled through modulation of the phosphorylated form of Spo0A. When the cryIIIA gene is expressed in a B. subtilis spo0A mutant strain or in the 168 wild-type strain, large amounts of toxins are produced and accumulate to form a flat rectangular crystal characteristic of the coleopteran-specific B. thuringiensis strains.

Bacillus subtilis↗
PubMed1993

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.

Bacillus thuringiensis↗
PubMed1992

Expansion of insecticidal host range of Bacillus thuringiensis by in vivo genetic recombination.

We describe a novel approach for the insertion of an insecticidal toxin gene into a resident plasmid in Bacillus thuringiensis (Bt). A gene encoding a coleopteran-specific toxin was cloned within a fragment of IS232 and inserted into a plasmid thermosensitive for replication in Bt. The plasmid was used to transform a Bt strain toxic to lepidoptera, and the transformants were then selected at non-permissive temperature for clones in which the vector had integrated into a copy of IS232 present on a resident plasmid. A second recombination event was selected such that the vector was eliminated and the newly introduced toxin gene was conserved. The resulting strain contained only DNA of Bt origin, and displayed insecticidal activity against both lepidoptera and coleoptera.

Bacillus thuringiensis↗
PubMed1992

spbA locus ensures the segregational stability of pTH1030, a novel type of gram-positive replicon.

The replication region of the plasmid pHT1030 of Bacillus thuringiensis was previously mapped to a 2.9 kb DNA fragment. The DNA sequence was analysed and it was shown that the minimal replicon resides within a 1 kb fragment of DNA carrying no potential protein coding sequence. Moreover, no production of single-stranded DNA intermediates was detected in the plasmid-containing cells. pHT1030 therefore belongs to a class of replicons not previously described in Gram-positive bacteria. Examination of the segregational stability of deletion derivatives of pHT1030 in bacilli defined two stability regions. One is located within the minimal replicon of pHT1030, whereas the second (spbA) is not required for replication. spbA encodes a 15 kDa protein and ensures the segregational stability of the plasmid. This effect of spbA is particularly highlighted in sporulation. The absence of the spbA locus gives rise to plasmid-free spores at high frequency, whereas the spbA+ plasmids are stably maintained. The stability of the plasmids during sporulation seems to be correlated with an unequal division of the cell by the sporulation septum.

Amino Acid Sequence↗
PubMed1992

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.

Journal Article↗
PubMed1991

Construction of cloning vectors for Bacillus thuringiensis.

The replication region of the Bacillus thuringiensis plasmid, pHT1030, was treated with hydroxylamine. Various copy-number mutants were selected and subsequently used to construct shuttle vectors with multiple cloning sites. These recombinant plasmids are very stable and allowed the cloning of a delta-endotoxin-encoding gene in B. thuringiensis. Comparison between gene expression level and vector copy-number indicated that a plateau in delta-endotoxin production is reached with a copy-number of about fifteen per equivalent chromosome.

Bacillus thuringiensis↗
PubMed1990

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.

Amino Acid Sequence↗
PubMed1989

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.

Bacillus thuringiensis↗
PubMed1989

Nucleotide sequence and analysis of the N-terminal coding region of the Spodoptera-active delta-endotoxin gene of Bacillus thuringiensis aizawai 7.29.

The nucleotide sequence of a 2711bp DNA segment which contains the N-terminal coding sequence and the 5' flanking region of a crystal protein gene (bta) from Bacillus thuringiensis subsp. aizawai 7.29 has been determined. The coding region encodes an 824 amino-acid polypeptide corresponding to a carboxy-terminally truncated delta-endotoxin specifically active against the cotton leaf worm Spodoptera littoralis. Comparison of the deduced amino acid sequence of the bta gene with that of the 4.5, 5.3 and 6.6 kb classes of lepidopteran-active delta-endotoxins revealed that the Bta sequence contains a very high level of amino acid substitutions in the N-terminal part of the protoxin molecule. The substitutions are grouped in several highly variable segments separated by highly conserved regions. These conserved domains are also present in the dipteran- and coleopteran-active delta-endotoxins. The control region of the bta gene shows considerable DNA identity with the control regions of the other lepidopteran-active genes. Deletions of the 3' region of the gene were carried out and the toxic fraction of the bta delta-endotoxin was identified with the N-terminal half of the molecule.

Amino Acid Sequence↗
PubMed1988

Structural and functional analysis of Tn4430: identification of an integrase-like protein involved in the co-integrate-resolution process.

The 4149-bp transposon Tn4430 from Bacillus thuringiensis is delineated by 38-bp inverted repeats and codes for a 113-kd protein that shares homology with the transposases (TnpA) of Tn3, Tn21 and Tn501. Through transpositional recombination, this protein generates the formation of co-integrates between both donor and target replicons, with duplication of Tn4430 molecules. These features are characteristic of transposons of the Tn3 family (class II elements). The second step of the transposition process, the co-integrate resolution, is mediated by a 32-kd protein. This protein (TnpI) displays regional similarities with site-specific recombinases of the integrase family, such as Int of bacteriophage lambda, Cre of bacteriophage P1 or TnpA and TnpB of the Tn554 transposon. Moreover, the 250-bp sequence upstream to the tnpI gene contains several structural features that are reminiscent of the attP attachment site of phage lambda. This unique association between the integrase-like TnpI recombinase and the TnpA transposase qualifies Tn4430 as a member of a new group within the class II mobile genetic elements.

Amino Acid Sequence↗
PubMed1988

Multiplicity of delta-endotoxin genes with different insecticidal specificities in Bacillus thuringiensis aizawai 7.29.

The hypothesis according to which multiple and different delta-endotoxin genes could determine the host-range specificity of the lepidopteran strains of Bacillus thuringiensis is being checked in the case of strains aizawai 7.29 (serotype 7) and entomocidus 601 (serotype 6). From these strains, several crystal protein genes, belonging to different structural types, have been isolated. One of the cloned genes that is not present in strain entomocidus 601 is duplicated in strain aizawai 7.29. This gene belongs to a previously characterized type of crystal protein gene and encodes a protein preferentially active against Pieris brassicae. Two other genes, of presumed chromosomal location, are present in both strains and each displays a unique physical map. In both strains the two genes are in close proximity and in the same orientation. The first, which belongs to a new type of crystal protein gene, encodes a 130-140 kD protein that is not significantly active against the two insect species tested. The other new type of crystal protein gene directs the synthesis of a polypeptide preferentially active against Spodoptera littoralis.

Animals↗
PubMed1988

Identification of a delta-Endotoxin Gene Product Specifically Active against Spodoptera littoralis Bdv. among Proteolysed Fractions of the Insecticidal Crystals of Bacillus thuringiensis subsp. aizawai 7.29.

At least three different insecticidal crystal protein genes were shown to be expressed in Bacillus thuringiensis subsp. aizawai 7.29, a strain that is potentially active against the cotton leafworm Spodoptera littoralis Bdv. Among crude K-60 fractions (60- to 70-kilodalton [kDa] molecules) that were products of proteolysed crystals containing the active domains of the protoxin molecules, we were able to distinguish several distinct components on the basis of their antigenic relationship and their larvicidal properties. A purified fraction designated SF2 was a 61-kDa component specifically active against Pieris brassicae L. and homologous to the B. thuringiensis subsp. berliner 1715 plasmid-encoded crystal protein. A second fraction designated SF1 was composed of 63- and 65-kDa polypeptides and was specifically active against S. littoralis. The SF1 fraction and particularly the 65-kDa component were not antigenically related to the 61-kDa component. The purified fractions were compared with the products of three different crystal protein genes we previously cloned from total DNA of B. thuringiensis subsp. aizawai, among them a new type of crystal protein gene encoding a protein that is specifically active against S. littoralis and other insects of the Noctuidae family. This approach led us to consider the 65-kDa component as a minimum active part of a delta-endotoxin that is encoded by this new gene. Products of the two other cloned genes can be correlated with the 61- and 63-kDa components, respectively. Thus, in B. thuringiensis subsp. aizawai 7.29, multiple delta-endotoxin genes of different structural types direct the synthesis of several delta-endotoxins with different host specificities which were identified as components of the insecticidal crystals.

Journal Article↗
PubMed1986

Identification of Tn4430, a transposon of Bacillus thuringiensis functional in Escherichia coli.

The mobile genetic element Tn4430, originating from the gram-positive bacterium, Bacillus thuringiensis, and previously described as the Th-sequence, is the first transposon isolated from the genus Bacillus. In the present work a gene (APH-III) conferring resistance to kanamycin was inserted into this 4.2 kb transposon. Transposition experiments showed that Tn4430 omega APH-III could transpose in the gram-negative host Escherichia coli when its insertion functions were supplied by an intact copy of Tn4430. By transposing Tn4430 omega APH-III directly onto pBR322, it was possible to determine the nucleotide sequence of the terminal inverted repeats of Tn4430 and of the target DNA site. Identical 38 bp in inverted orientation are situated at each end of the transposon and there is a direct duplication of 5 bp at the insertion site. Thus, it is clear that Tn4430 is closely related to the transposons belonging to the Tn3 family (class II elements).

Bacillus thuringiensis↗
PubMed1985

Recent aspects of genetic manipulation in Bacillus thuringiensis.

The conjugative plasmid pAM beta 1 was transferred from Streptococcus faecalis to several strains of Bacillus thuringiensis by a filter-mating process. From a transconjugant clone of B. thuringiensis a hybrid plasmid resulting from an in vivo insertion into pAM beta 1 of a 3 Md DNA sequence was isolated. This 3 Md DNA molecule (Th sequence) is related to several host plasmids found in different serotypes of B. thuringiensis. A reciprocal conjugation-like process involving the transfer of pAM beta 1 from B. thuringiensis to S. faecalis was also demonstrated. The comparison of the restriction maps of the crystal genes from plasmid and chromosomal origins of different serotypes, six of which having been cloned in E. coli, revealed the existence of two classes of genes which are very similar in the map corresponding to the N-terminal part of the protein, and which differ essentially in the 3' region. The presence of the transposon-like Th sequence was found in several cases associated with the crystal gene in the same host plasmid, and a model for their structural organization is proposed.

Bacillus thuringiensis↗
PubMed1984

A transposon-like structure related to the delta-endotoxin gene of Bacillus thuringiensis.

A DNA segment (Th-sequence) has been found in several strains of Bacillus thuringiensis. This Th-sequence [3 megadaltons (Md)] induces adjacent deletions when it is located in the pAM beta 1 plasmid derived from Streptococcus faecalis. Electron microscopic examination of reannealed single strands of one plasmid (pMT9) carrying such a deletion revealed that the Th-sequence corresponds to a single-stranded loop (2.8 Md) bounded by a short double-stranded stem (less than 0.2 Md). Southern blotting experiments established that in B. thuringiensis the Th-sequence was generally located on the large plasmid which also harbours the gene coding for the delta-endotoxin (crystal protein). Hybridization and heteroduplex analysis of the extrachromosomal DNA from the berliner 1715 strain demonstrated that the crystal gene and the Th-sequence are located in close vicinity on a 42-Md plasmid and that they are separated by a 1.3-Md DNA segment. This DNA segment is repeated in inverted orientation, once immediately adjacent to the Th-sequence and once 1.8 Md beyond the crystal gene. A model for the organization of these DNA sequences inside a transposon-like structure is proposed.

Bacillus thuringiensis↗