A 20-kilodalton protein preserves cell viability and promotes CytA crystal formation during sporulation in Bacillus thuringiensis

Wu, Dongrui; Federici, Brian A.

doi:10.1128/jb.175.16.5276-5280.1993

Cited by 121 publications

(128 citation statements)

References 27 publications

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“…Inappropriate proteolysis of CytA (or any other ICP) may result in the release of soluble cytotoxic protein species which could, in turn, impair cellular function and further reduce ICP production. However, the effect of CytA appears to be dependent on the particular host background used for the expression study (Wu and Federici, 1993). Perhaps this is because of host strain differences in protease activity or because of the presence of host genes that can complement the function of the 20 kDa protein.…”

Section: Protein Stability and Crystal Formationmentioning

confidence: 99%

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Regulation of insecticidal crystal protein production in Bacillus thuringiensis

Baum¹,

Malvar

1995

Molecular Microbiology

147

116

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SummaryThe production of insecticidal crystal proteins (ICPs) in Bacillus thuringiensis normally coincides with sporulation, resulting in the appearance of parasporal crystalline inclusions within the mother cell. In most instances, the temporal and spatial regulation of ICP gene expression is determined at the transcriptional level by mother-cell-specific sigma factors that share homology with cr E and eK from Bacillus subtilis. The crylll ICP genes are a notable exception; these genes are transcribed from eA-like promoters during vegetative growth, are induced or derepressed at the onset of stationary phase, and are overexpressed in sporulation mutants of B. thuringiensis blocked in the phosphorylation of Spo0A, a key regulator of sporulation initiation. Transcription alone, however, cannot account for the impressive ability of this bacterium to accumulate insecticidal proteins. A variety of post-transcriptional and post-translational mechanisms also contribute to the efficient production of ICPs in B. thuringiensis, thus making this bacterium a cost-effective biological control agent.

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Section: Protein Stability and Crystal Formationmentioning

confidence: 99%

“…thuringiensis strain cryB (Wu and Federici, 1993). Expression of the cytA gene in the absence of the 20 kDa protein in strain cryB resulted in poor CytA production, inhibition of sporulation, and cell death.…”

Section: Protein Stability and Crystal Formationmentioning

confidence: 99%

Regulation of insecticidal crystal protein production in Bacillus thuringiensis

Baum¹,

Malvar

1995

Molecular Microbiology

147

116

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“…The level of mosquito larvicidal activity of Cyt1Aa by itself is low, but it raises synergistically the activity of Cry4Aa, Cry4Ba or Cry11Aa, to a greater extent than the synergism obtained by combination of the three Cry polypeptides themselves (Crickmore et al, 1995; Khasdan et al, 2001 (Wu & Federici, 1993; Manasherob et al, 2001) and stabilizes Cyt1Aa post-translationally (Adams et al, 1989;Visick & Whiteley, 1991).…”

Section: Introductionmentioning

confidence: 99%

“…kyushuensis has recently been found to have a single pore-forming domain, composed of two outer layers of a-helix hairpins wrapped around mixed b-sheets (Li et al, 1996). Due to high similarity (70 %) in its amino acid sequence to Cyt2Aa, Cyt1Aa is supposed to show a similar folding pattern to Cyt2Aa (Li et al, 1996;Gazit et al, 1997).The level of mosquito larvicidal activity of Cyt1Aa by itself is low, but it raises synergistically the activity of Cry4Aa, Cry4Ba or Cry11Aa, to a greater extent than the synergism obtained by combination of the three Cry polypeptides themselves (Crickmore et al, 1995; Khasdan et al, 2001 (Wu & Federici, 1993; Manasherob et al, 2001) and stabilizes Cyt1Aa post-translationally (Adams et al, 1989;Visick & Whiteley, 1991).The hypothesis that recombinant E. coli cells expressing the cyt1Aa gene die by inhibition of DNA synthesis (Douek et al, 1992) is consistent with the high affinity of Cyt1Aa for phosphatidylethanolamine (Thomas & Ellar, 1983b), the major DNA-bound E. coli phospholipid (Ballesta et al, 1972). This hypothesis predicts that the target is the chromosome replication complex (Douek et al, 1992).…”

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confidence: 99%

Compaction of the Escherichia coli nucleoid caused by Cyt1Aa

et al. 2003

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Compaction of the Escherichia coli nucleoid in the cell's centre was associated with the loss of colony-forming ability; these effects were caused by induction of Cyt1Aa, the cytotoxic 27 kDa protein from Bacillus thuringiensis subsp. israelensis. Cyt1Aa-affected compaction of the nucleoids was delayed but eventually more intense than compaction caused by chloramphenicol. The possibility that small, compact nucleoids in Cyt1Aa-expressing cells resulted in DNA replication run-out and segregation following cell division was ruled out by measuring relative nucleoid length. Treatments with membrane-perforating substances other than Cyt1Aa did not cause such compaction of the nucleoids, but rather the nucleoids overexpanded to occupy nearly all of the cell volume. These findings support the suggestion that, in addition to its perforating ability, Cyt1Aa causes specific disruption of nucleoid associations with the cytoplasmic membrane. In situ immunofluorescence labelling with Alexa did not demonstrate a great amount of Cyt1Aa associated with the membrane. Clear separation between Alexa-labelled Cyt1Aa and 49,6-diamidino-2-phenylindole (DAPI)-stained DNA indicates that the nucleoid does not bind Cyt1Aa. Around 2 h after induction, nucleoids in Cyt1Aa-expressing cells started to decompact and expanded to fill the whole cell volume, most likely due to partial cell lysis without massive peptidoglycan destruction. INTRODUCTIONDuring sporulation, various subspecies of the Grampositive soil bacterium Bacillus thuringiensis produce large amounts of insecticidal crystal proteins (ICP), the so-called d-endotoxins , each toxic against larvae of a different group of insects. The ICP of B. thuringiensis subsp. israelensis is specific against the larvae of mosquitoes and black flies (Goldberg & Margalit, 1977;Margalith & Ben-Dov, 2000), vectors of many human infectious diseases (Service, 1986). The crystal is composed of four major polypeptides, Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa (of 125, 135, 68 and 28 kDa, respectively), which are encoded by genes carried on the~130 kDa plasmid nicknamed pBtoxis (Ben-Dov et al., 1999). Cyt1Aa, which is not homologous to any of the known Cry toxins , is the most prominent of the four polypeptides, but it is less specific than Cry4Aa, Cry4Ba and Cry11Aa, haemolytic and cytotoxic in vitro (Thomas & Ellar, 1983b;Drobniewski & Ellar, 1988;Hofte & Whiteley, 1989). The broad cytolytic activity of Cyt1Aa has been attributed to its hydrophobicity and ability to bind zwitterionic phospholipids (Thomas & Ellar, 1983a, b). Cyt1Aa is inserted into the membrane to create cation-selective single channels of 1-2 nm in diameter, leading to colloid-osmotic lysis (Knowles & Ellar, 1987;Knowles et al., 1989), and induces leakage of low-molecular-mass substances from lipid vesicles (Drobniewski & Ellar, 1988.Seven cytolytic, mosquitocidal-specific toxins are currently known (Earp & Ellar, 1987;Drobniewski & Ellar, 1989;Yu et al., 1991;Koni & Ellar, 1993;Cheong & Gill, 1997;Guerchicoff et al., 1997;Thiery et al., 199...

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“…3). Moreover, the accessory protein P20, known to raise the levels of Cyt1Aa (Wu & Federici, 1993) and of Cyt2Ba (Nisnevitch et al, 2006) in acrystalliferous strains of B. thuringiensis subsp. israelensis, did not assist in cyt1Ca expression (Fig.…”

Section: Interactions Of Different Cyt Toxins With Cry4aamentioning

confidence: 99%

Cyt1Ca from Bacillus thuringiensis subsp. israelensis: production in Escherichia coli and comparison of its biological activities with those of other Cyt-like proteins

et al. 2006

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The larvicidal activity of Bacillus thuringiensis subsp. israelensis against dipteran larvae is determined by four major polypeptides of the parasporal crystalline body produced during sporulation. Cyt1Aa shows the lowest toxicity when used alone but is the most synergistic with any of the other proteins. The sequence of the plasmid pBtoxis, which contains all the toxin genes in this subspecies, revealed a new cyt-like coding sequence named cyt1Ca. In addition to the Cyt-like region, the predicted Cyt1Ca contained an extra domain at the C terminus, which appeared to be a β-trefoil carbohydrate-binding motif, as found in several ricin-like toxins. The gene was PCR-amplified from pBtoxis and cloned in several vectors, allowing high-level expression in Escherichia coli. Cyt1Ca was purified by nickel-nitrilotriacetic acid affinity chromatography, characterized, and its biological activity was determined. Toxicity against larvae of Aedes aegypti of Cyt1Ca in recombinant E. coli cells was compared with that of Cyt1Aa and Cyt2Ba, and the ability of these proteins to enhance the activity of Cry4Aa was assessed. Although Cyt2Ba appeared able to interact with Cry4Aa, no activity for Cyt1Ca was observed, even when produced in truncated form. Furthermore, in contrast to Cyt1Aa, Cyt1Ca did not lyse sheep erythrocytes, and it was not bactericidal to the host cell.

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A 20-kilodalton protein preserves cell viability and promotes CytA crystal formation during sporulation in Bacillus thuringiensis

Cited by 121 publications

References 27 publications

Regulation of insecticidal crystal protein production in Bacillus thuringiensis

Regulation of insecticidal crystal protein production in Bacillus thuringiensis

Compaction of the Escherichia coli nucleoid caused by Cyt1Aa

Cyt1Ca from Bacillus thuringiensis subsp. israelensis: production in Escherichia coli and comparison of its biological activities with those of other Cyt-like proteins

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