Identification of a gene for Cyt1A-like hemolysin from Bacillus thuringiensis subsp. medellin and expression in a crystal-negative B. thuringiensis strain

Thiéry, Isabelle; Delécluse, Armelle; Tamayo, Martha Claudia; Ordúz, Sergio

doi:10.1128/aem.63.2.468-473.1997

Cited by 40 publications

(13 citation statements)

References 35 publications

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“…The amino acid sequences of six Cyt toxins have been determined [16][17][18][19][20]. Of these toxins, CytA was cloned from B. thuringiensis israelensis [16] and B. thuringiensis morrisoni PG-14 [21], and CytB from B. thuringiensis kyushuensis [17].…”

Section: Introductionmentioning

confidence: 99%

Biochemical characterization of Bacillus thuringiensis cytolytic toxins in association with a phospholipid bilayer

Knowles

1999

Biochemical Journal

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The interaction of two Bacillus thuringiensis cytolytic toxins, CytA and CytB, with a phospholipid bilayer and their structure in the membrane-bound state were investigated by proteolysis using phospholipid vesicles as a model system. A toxin conformational change upon membrane binding was detected by comparing the proteolytic profile of membrane-bound toxin and saline-solubilized toxin. When membrane-bound toxin was exposed to protease K or trypsin, novel cleavage sites were found between the alpha-helical N-terminal half and beta-strand C-terminal half of the structure at K154 and N155 in CytA and at I150 and G141 in CytB. N-terminal sequencing of membrane-protected fragments showed that the C-terminal half of the toxin structure comprising mainly beta-strands was inserted into the membrane, whereas the N-terminal half comprising mainly alpha-helices was exposed on the outside of the liposomes and could be removed when liposomes with bound toxin were washed extensively after proteolysis. The C-termini of the membrane-inserted proteolytic fragments were also located by a combination of N-terminal sequencing and measurement of the molecular masses of the fragments by electrospray MS. Using a liposome glucose-release assay, the membrane-inserted structure was seen to retain its function as a membrane pore even after removal of exposed N-terminal segments by proteolysis. These data strongly suggest that the pores for glucose release are assembled from the three major beta-strands (beta-5, beta-6 and beta-7) in the C-terminal half of the toxin.

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Section: Introductionmentioning

confidence: 99%

Biochemical characterization of Bacillus thuringiensis cytolytic toxins in association with a phospholipid bilayer

Knowles

1999

Biochemical Journal

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“…The gene encoding the cytolytic protein Cyt1Ab1 from B. thuringiensis subsp. medellin was isolated by Thiéry et al (23). It is flanked upstream by a p21 gene in the same orientation.…”

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“…This gene encodes a P21 protein with a sequence 84% similar to that of the putative chaperone P20 from B. thuringiensis subsp. israelensis, which is probably responsible for the formation of Cyt1Ab1 crystals in a B. thuringiensis crystalnegative recombinant strain (23).…”

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“…pipiens (strain Montpellier) from susceptible or resistant (Montpellier SPHAE) populations (14,20) and with resistant C. quinquefasciatus (GeoR) larvae (8,13). The bacterial pellet and purified crystals of B. sphaericus strains and of the control strain, B. thuringiensis SPL407(pcytM), were mixed with 10 ml of demineralized water in petri dishes (diameter, 5.5 cm) and tested in duplicate as previously described (23). Each experiment was performed three times.…”

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The Introduction into Bacillus sphaericus of the Bacillus thuringiensis subsp. medellin cyt1Ab1 Gene Results in Higher Susceptibility of Resistant Mosquito Larva Populations to B. sphaericus

Thiéry

Hamon

Delécluse

et al. 1998

Appl Environ Microbiol

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The fragment containing the gene encoding the cytolytic Cyt1Ab1 protein from Bacillus thuringiensis subsp.medellin and its flanking sequences (I. Thiery, A. Delécluse, M. C. Tamayo, and S. Orduz, Appl. Environ. Microbiol. 63:468–473, 1997) was introduced into Bacillus sphaericus toxic strains 2362, 2297, and Iab872 by electroporation with the shuttle vector pMK3. Only small amounts of the protein were produced in recombinant strains 2362 and Iab872. The protein was detected in these strains only by Western blotting and immunodetection with antibody raised against Cyt1Ab1 protein. Large amounts of Cyt1Ab1 protein were produced in B. sphaericus recombinant strain 2297, and there was an additional crystal, other than that of the binary toxin, within the exosporium. The production of the Cyt1Ab1 protein in addition to the binary toxin did not increase the larvicidal activity of theB. sphaericus recombinant strain against susceptible mosquito populations of Culex pipiens orAedes aegypti. However, it partially restored (10 to 20 times) susceptibility of the resistant mosquito populations of C. pipiens (SPHAE) and Culex quinquefasciatus (GeoR) to the binary toxin. The Cyt1Ab1 protein produced in recombinantB. thuringiensis SPL407(pcyt1Ab1) was synthesized in two types of crystal—one round and with various dense areas, surrounded by an envelope, and the other a regular cuboid crystal, very similar to that found in the B. sphaericus recombinant strain.

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“…medellin (serotype H30) which is 86% identical to Cyt1Aa1 from B. thuringiensis subsp. israelensis (31).…”

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Identification and characterization of a previously undescribed cyt gene in Bacillus thuringiensis subsp. israelensis

Guerchicoff

Ugalde

Rubinstein

1997

Appl Environ Microbiol

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Bacillus thuringiensis strains show as a common feature the presence of toxic proteins with cytolytic and hemolytic activities, Cyt1Aa1 being the characteristic cytolytic toxin of Bacillus thuringiensis subsp. israelensis. We have detected the presence of another cyt gene in this subspecies, highly homologous to cyt2Aa1, coding for the 29-kDa cytolytic toxin from B. thuringiensis subsp. kyushuensis. This gene, designated cyt2Ba1, maps upstream of cry4B coding for the 130-kDa crystal toxin, on the 72-MDa plasmid of strain 4Q2-72. Sequence analysis revealed, as a remarkable feature, a 5 mRNA stabilizing region similar to those described for some cry genes. PCR amplification and Southern analysis confirmed the presence of this gene in other mosquitocidal subspecies. Interestingly, anticoleopteran B. thuringiensis subsp. tenebrionis belonging to the morrisoni serovar also showed this gene. On the other hand, negative results were obtained with the antilepidopteran strains B. thuringiensis subsp. kurstaki HD-1 and subsp. aizawai HD-137. Western analysis failed to reveal Cyt2A-related polypeptides in B. thuringiensis subsp. israelensis 4Q2-72. However, B. thuringiensis subsp. israelensis 1884 and B. thuringiensis subsp. tenebrionis did show cross-reactive products, although in very small amounts.

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Identification of a gene for Cyt1A-like hemolysin from Bacillus thuringiensis subsp. medellin and expression in a crystal-negative B. thuringiensis strain

Cited by 40 publications

References 35 publications

Biochemical characterization of Bacillus thuringiensis cytolytic toxins in association with a phospholipid bilayer

Biochemical characterization of Bacillus thuringiensis cytolytic toxins in association with a phospholipid bilayer

The Introduction into Bacillus sphaericus of the Bacillus thuringiensis subsp. medellin cyt1Ab1 Gene Results in Higher Susceptibility of Resistant Mosquito Larva Populations to B. sphaericus

Identification and characterization of a previously undescribed cyt gene in Bacillus thuringiensis subsp. israelensis

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