Cry1Ac Protoxin and Its Activated Toxin from Bacillus thuringiensis Act Differentially during the Pathogenic Process.

Although the new dual model of the Bacillus thuringiensis insecticidal mechamism indicated that both Cry1A protoxin and activated toxin have the potency to kill insects, the difference in the toxic pathways elicited by the protoxin and activated toxin was less understood at the molecular level. Through utilizing the CF-203 cell line derived from the midgut of Choristoneura fumiferana, we found that there existed obvious differences in the binding sites and endocytosis pathways for the two forms of Cry1Ac. In addition, it was revealed that Cry1Ac protoxin existed predominantly in the midgut of Plutella xylostella at the early stage after ingesting Cry1Ac crystals, which brought about obvious damage to the midgut epithelium and exhibited different binding sites on the brush border membrane vesicle compared to the toxin. These findings supported the dual mode of action of B. thuringiensis Cry1A proteins and improved our understanding of the molecular features that contribute to the protoxin toxicity.

The conserved cysteine residues in Bacillus thuringiensis Cry1Ac protoxin are not essential for the bipyramidal crystal formation.

To evaluate the function of conserved cysteine residues in Cry1Ac protoxin, we constructed a series of Cry1Ac mutants in which single or multiple cysteine residues were replaced with serine. It was found that cysteine substitution had little effect on the protoxin expression and bipyramidal crystal formation. Bioassays using Plutella xylostella larvae showed that two mutants with fourteen cysteine residues in the C-terminal half and all sixteen residues replaced had similar toxicity as wildtype Cry1Ac protoxin. Our study suggests that the conserved cysteine resudues in the Cry1Ac protoxin are not essential for deposition into a bipyramidal crystal even though the C-terminal half was directly involved in crystal formation.

Solubility enhancement of Cry2Aa crystal through carboxy-terminal extension and synergism between the chimeric protein and Cry1Ac.

It was reported that the highly conserved C-terminal region of Bacillus thuringiensis Cry1A protoxins was very important for parasporal crystal formation and solubility feature in alkaline environment. In order to improve the solubilization efficiency of Cry2Aa crystal, the coding sequences of Cry2Aa protein and the C-terminal half of Cry1Ac were fused seamlessly through Red/ET homologous recombination and expressed in an acrystalliferous B. thuringiensis strain under the control of the cry1Ac promoter and terminator. Microscopic observation revealed that the recombinant strain containing the chimeric gene cry2Aa-1Ac produced distinct parasporal inclusion with semispherical to approximately cuboidal shape during sporulation. SDS-PAGE analysis showed that this strain expressed stable 130-kDa Cry2Aa-1Ac chimeric protein, which was confirmed to be the correctly expressed product by LC-MS/MS. The chimeric protein inclusion could be effectively dissolved at pH 10.5 and activated by trypsin like the parental Cry1Ac crystal. While, the parental Cry2Aa crystal exhibited very low solubility under this condition. Bioassays against third-instar larvae of Helicoverpa armigera proved that the chimeric protein was more toxic than Cry2Aa. Additionally, synergistic effect was clearly detected between the chimeric protein and Cry1Ac against H. armigera, while there was only additive effect for the combination of wild Cry2Aa and Cry1Ac. These results indicated that the developed chimeric protein might serve as a potent insecticidal toxin used in the field against lepidopteran pests.

The full-length Cry1Ac protoxin without proteolytic activation exhibits toxicity against insect cell line CF-203.

The new dual model for Bacillus thuringiensis insecticidal mechanism proposed that Cry1A protoxins without proteolytic activation could bind to insect midgut receptors to exert toxicity. To evaluate insecticidal potency of Cry1Ac protoxin at precluding interference of midgut proteases, the cytotoxicity of Cry1Ac protoxin against midgut cell line CF-203 derived from Choristoneura fumiferana was analyzed. It was revealed that Cry1Ac protoxin was toxic to CF-203 cells and there existed certain differences in the cytological changes when treated with protoxin and toxin. Our cell-based study provided direct evidence for the proposed dual model and shed light on exploring the difference between two toxic pathways elicited by intact protoxin and activated toxin.

[Online aeration backwashing in the inverse direction of effluent applied in dynamic membrane bio-reactor].

Aeration in the inverse direction of effluent was applied as the measure of online backwashing to control membrane pollution in the dynamic membrane bio-reactor treating sewage wastewater. When the intensity of aeration was 5.6 m3/(m2 x h) and the aeration time was 5 min, it took 45 min for the dynamic membrane to recover filtration capacity. With the recovery of dynamic membrane filterability, effluent turbidity was below 5 NTU. The backwashing interval of the reactor could maintain about 15 h. SEM pictures showed that online aeration backwashing in the inverse direction of effluent could efficiently destroy part of dynamic membrane layer. After the dynamic membrane recovery, dynamic membrane could check more than 50% TOC of various molecule weights range > 3 x 10(4). Aeration in the inverse direction of effluent was an economical and effective means of online backwashing in the dynamic membrane bio-reactor.