Key residues of Bacillus thuringiensis Cry2Ab for oligomerization and pore-formation activity.

As a pore-forming toxin, activation, oligomerization and pore-formation were both required for the mode of action of Cry toxins. Previous results revealed that the helices α4-α5 of Domain I were involved in the oligomerization of Cry2Ab, however, the key residues for Cry2Ab aggregation remained ambiguous. In present studies, we built 20 Cry2Ab alanine mutants site-directed in the helices α4-α5 of Domain I and demonstrated that mutants N151A, T152A, F157A, L183A, L185A and I188A could reduce the assembly of the 250 kDa oligomers, suggesting that these mutation residues might be essential for Cry2Ab oligomerization. As expected, all of these variants showed lower insecticidal activity against P. xylostella. Furthermore, we found that the pore-forming activities of these mutants also decreased when compared to wild-type Cry2Ab. Taken together, our data identified key residues for Cry2Ab oligomerization and emphasized that oligomerization was closely related to the insecticidal activity and pore-forming activity of Cry2Ab.

Synthesis and Characterization of Cry2Ab-AVM Bioconjugate: Enhanced Affinity to Binding Proteins and Insecticidal Activity.

Bacillus thuringiensis insecticidal proteins (Bt toxins) have been widely used in crops for agricultural pest management and to reduce the use of chemical insecticides. Here, we have engineered Bt toxin Cry2Ab30 and bioconjugated it with 4"-O-succinyl avermectin (AVM) to synthesize Cry2Ab-AVM bioconjugate. It was found that Cry2Ab-AVM showed higher insecticidal activity against Plutella xylostella, up to 154.4 times compared to Cry2Ab30. The binding results showed that Cry2Ab-AVM binds to the cadherin-like binding protein fragments, the 10th and 11th cadherin repeat domains in the P. xylostella cadherin (PxCR10-11), with a much higher affinity (dissociation equilibrium constant KD = 3.44 nM) than Cry2Ab30 (KD = 28.7 nM). Molecular docking suggested that the macrolide lactone group of Cry2Ab-AVM ligand docking into the PxCR10-11 is a potential mechanism to enhance the binding affinity of Cry2Ab-AVM to PxCR10-11. These findings offer scope for the engineering of Bt toxins by bioconjugation for improved pest management.

Exposure of helices α4 and α5 is required for insecticidal activity of Cry2Ab by promoting assembly of a prepore oligomeric structure.

Cry2Ab, a pore-forming toxin derived from Bacillus thuringiensis, is widely used as a bio-insecticide to control lepidopteran pests around the world. A previous study revealed that proteolytic activation of Cry2Ab by Plutella xylostella midgut juice was essential for its insecticidal activity against P. xylostella, although the exact molecular mechanism remained unknown. Here, we demonstrated for the first time that proteolysis of Cry2Ab uncovered an active region (the helices α4 and α5 in Domain I), which was required for the mode of action of Cry2Ab. Either the masking or the removal of helices α4 and α5 mediated the pesticidal activity of Cry2Ab. The exposure of helices α4 and α5 did not facilitate the binding of Cry2Ab to P. xylostella midgut receptors but did induce Cry2Ab monomer to aggregate and assemble a 250-kDa prepore oligomer. Site-directed mutagenesis assay was performed to generate Cry2Ab mutants site directed on the helices α4 and α5, and bioassays suggested that some Cry2Ab variants that could not form oligomers had significantly lowered their toxicities against P. xylostella. Taken together, our data highlight the importance of helices α4 and α5 in the mode of action of Cry2Ab and could lead to more detailed studies on the insecticidal activity of Cry2Ab.

Proteolytic activation of Bacillus thuringiensis Vip3Aa protein by Spodoptera exigua midgut protease.

Proteolysis of Vip3Aa by insect midgut proteases is essential for their toxicity against target insects. In the present study, proteolysis of Vip3Aa was evaluated by Spodoptera exigua midgut proteases (MJ). Trypsin was verified involved in the activation of Vip3Aa and three potential cleavage sites (Lys195, Lys197 and Lys198) were identified. Four Vip3Aa mutants (KKK195197198AAA, KK197198AA, KK195198AA and KK195197AA) were designed and constructed by replacing residues Lys195,197,198, Lys197,198, Lys195,198 and Lys195,197 with Ala, respectively. Proteolytic processing assays revealed that mutants KK197198AA, KK195198AA and KK195197AA could be processed into 66kDa activated toxins by trypsin or MJ while mutant KKK195197198AAA was not cleaved by trypsin and less susceptible to MJ. Bioassays demonstrated that mutants KK197198AA, KK195198AA and KK195197AA were toxic against S. exigua resembled that of wild-type Vip3Aa, however, the LC50 of mutant KKK195197198AAA against S. exigua was higher than wild-type. Those results suggested that proteolysis by MJ was associated with the insecticidal toxicity of Vip3Aa against S. exigua. It also revealed that trypsin played an important role in the formation of Vip3Aa activated toxin. Our studies characterized the proteolytic processing of Vip3Aa and provided new insight into the activation of this novel Bt toxin.

PxAPN5 serves as a functional receptor of Cry2Ab in Plutella xylostella (L.) and its binding domain analysis.

Lepidopteran midgut aminopeptidases N (APNs) are widely studied for their potential roles as one of the receptors for Bacillus thuringiensis (Bt) crystal toxins. In the present study, a loss of function analyses by RNAi (RNA interference) silencing of the Plutella xylostella APN5 (PxAPN5), a binding protein of Bt crystal toxin Cry2Ab, were performed. The knocking down of PxAPN5 in P. xylostella larvae greatly reduced their susceptibility to Cry2Ab and led to a decrease of Cry2Ab binding to P. xylostella midgut. Four truncated fragments of PxAPN5 were further constructed and expressed in Escherichia coli (E.coli) to find the binding region of PxAPN5 to Cry2Ab. The ligand blot result indicated that D1 domain (residues 1-262) and D3 domain (residues 510-620) of PxAPN5 could specially bind to Cry2Ab.

Proteolytic Activation of Bacillus thuringiensis Cry2Ab through a Belt-and-Braces Approach.

Proteolytic processing of Bacillus thuringiensis (Bt) crystal toxins by insect midgut proteases plays an essential role in their insecticidal toxicities against target insects. In the present study, proteolysis of Bt crystal toxin Cry2Ab by Plutella xylostella L. midgut proteases (PxMJ) was evaluated. Both trypsin and chymotrypsin were identified involving the proteolytic activation of Cry2Ab and cleaving Cry2Ab at Arg(139) and Leu(144), respectively. Three Cry2Ab mutants (R139A, L144A, and R139A-L144A) were constructed by replacing residues Arg(139), Leu(144), and Arg(139)-Leu(144) with alanine. Proteolysis assays revealed that mutants R139A and L144A but not R139A-L144A could be cleaved into 50 kDa activated toxins by PxMJ. Bioassays showed that mutants R139A and L144A were highly toxic against P. xylostella larvae, while mutant R139A-L144A was almost non-insecticidal. Those results demonstrated that proteolysis by PxMJ was associated with the toxicity of Cry2Ab against P. xylostella. It also revealed that either trypsin or chymotrypsin was enough to activate Cry2Ab protoxin. This characteristic was regarded as a belt-and-braces approach and might contribute to the control of resistance development in target insects. Our studies characterized the proteolytic processing of Cry2Ab and provided new insight into the activation of this Bt toxin.

Bacillus gobiensis sp. nov., isolated from a soil sample.

A Gram-stain-positive, rod-shaped, endospore-forming, aerobic bacterium designated FJAT-4402T, was isolated from the weed rhizosphere soil of the Gobi desert in the Xinjiang Autonomous Region in the north-west of China. Isolate FJAT-4402T grew at 15-40 °C (optimum 30 °C), pH 5-10 (optimum pH 7) and in 0-3 % (w/v) NaCl (optimum 0 %). Phylogenetic analyses, based on 16S rRNA gene sequences, showed that isolate FJAT-4402T was a member of the genus Bacillus and was most closely related to Bacillus licheniformis DSM 13T (96.2 %). The isolate showed 33.3 % DNA-DNA relatedness to the closest reference isolate, B. licheniformis DSM 13T. The diagnostic diamino acid of the peptidoglycan of isolate FJAT-4402T was meso-diaminopimelic acid and the predominant isoprenoid quinone was MK-7. The major cellular fatty acids were anteiso-C15 : 0 (28.5 %), iso-C15 : 0 (20.1 %), anteiso-C17 : 0 (14.3 %), iso-C16 : 0 (9.6 %), C16 : 0 (8.4 %), iso-C17 : 0 (6.2 %) and iso-C14 : 0 (4.7 %) and the DNA G+C content was 42.0 mol%. The phenotypic, chemotaxonomic and genotypic properties indicated that strain FJAT-4402T represents a novel species within the genus Bacillus, for which the name Bacillus gobiensis sp. nov. is proposed. The type strain is FJAT-4402T ( = DSM 29500T = CGMCC 1.12902T).

A protein engineering of Bacillus thuringiensis δ-endotoxin by conjugating with 4"-O-succinoyl abamectin.

Conjugation of Bacillus thuringiensis δ-endotoxin (Bt toxin) with other toxins for insect pest control has been proposed as a new efficient strategy with increasing insecticidal toxicity and target range and delay the onset of insect resistance. A modified method was investigated by conjugating Bt toxin with 4"-O-succinoyl abamectin to form a new biocide which was named as BtA. 'Zero-length' cross-linker EDC in combination with NHS activated 4"-O-succinoyl abamectin and extended half-life period of active intermediate for binding to Bt toxin. The dissociation constant for 4"-O-succinoyl abamectin binding to Bt toxin was 6.44 µM by fluorescence quenching analysis. BtA showed a higher insecticidal toxicity against Plutella xylostella, while the relative-toxicity multiple of BtA to Bt toxin was calculated as 5.6. The interaction between Bt toxins with their receptors played a key role in toxicity of Bt toxins. The binding analysis showed the dissociation rate for the binding of BtA to its receptors (7.495 × 10(-3) S(-1)) was twice slower than that of Bt toxin (1.695 × 10(-2) S(-1)). The relative dissociation constant of BtA to Bt toxin was only 29% for the binding to the receptors. These results demonstrated that BtA bound to the receptor in BBMV with significantly higher affinity compared with Bt toxin.

Synthesis of 4'-thiosemicarbazonegriseofulvin and its effects on the control of enzymatic browning and postharvest disease of fruits.

4'-Thiosemicarbazonegriseofulvin, a new thiosemicarbazide derivative of griseofulvin, was synthesized and evaluated for its potential in the control of enzymatic browning and postharvest disease of fruits. Browning on fruits is mainly due to the enzymatic oxidation of phenolic compounds catalyzed by tyrosinase. 4'-Thiosemicarbazonegriseofulvin could effectively inhibit the activity of tyrosinase, and its 50% inhibitory concentration (IC(50)) against tyrosinase was determined to be 37.8 µM. It was a reversible and noncompetitive inhibitor of tyrosinase, and its inhibition constant (K(I)) was determined to be 38.42 µM. The antifungal activity of 4'-thiosemicarbazonegriseofulvin was studied against four fungi (Fusarium oxysporum, Fusarium moniliforme, Fusarium solani, and Colletotrichum truncatum) that often cause postharvest diseases of fruits. The results showed that 4'-thiosemicarbazonegriseofulvin could also strongly inhibit the mycelial growth of the four target fungi; the 50% lethal concentration (LC(50)) values were 5.4, 7.0, 15.3, and 1.5 mM, respectively.

Synthesis and antityrosinase activities of alkyl 3,4-dihydroxybenzoates.

In insects, tyrosinase plays important roles in normal developmental processes, such as cuticular tanning, scleration, wound healing, production of opsonins, encapsulation and nodule formation for defense against foreign pathogens. Thus, tyrosinase may be regarded as a potential candidate for novel bioinsecticide development. A family of alkyl 3,4-dihydroxybenzoates (C6-C9), new tyrosinsase inhibitors, were synthesized. Their inhibitory effects on the activity of tyrosinase have been investigated. The results showed all of them could inhibit the activity of tyrosianse effectively. The order of potency was nonyl 3,4-dihydroxybenzoate (C9DB) > octyl 3,4-dihydroxybenzoate(C8DB) > heptyl 3,4-dihydroxybenzoate(C7DB) > hexyl 3,4-dihydroxybenzoate (C6DB). The kinetic analysis of these four compounds on tyrosinase was taken to expound their inhibitory mechanism. The research of the control of insects in agriculture was taken as C6DB for example. C6DB could inhibit the development and molting of Plutella xylostella effectively. To clarify its insecticidal mechanism, we researched the expression of tyrosinase in the P. xylostella treated with C6DB by real-time quantitative PCR. The results showed C6DB could inhibit the expression of tyrosinase in the P. xylostella as expected.

Inhibitory effects of fatty acids on the activity of mushroom tyrosinase.

The effects of fatty acids, octanoic acid, (2E, 4E)-hexa-2,4-dienoic acid, hexanoic acid, (2E)-but-2-enoic acid, and butyric acid on the activities of mushroom tyrosinase have been investigated. The results showed that the fatty acids can potently inhibit both monophenolase activity and diphenolase activity of tyrosinase, and that the unsaturated fatty acids exhibited stronger inhibitory effect against tyrosinase than the corresponding saturated fatty acids, and the inhibitory effects were enhanced with the extendability of the fatty acid chain. For the monophenolase activity, the fatty acids could not only lengthen the lag period, but also decrease the steady-state activities. For the diphenolase activity, fatty acids displayed reversible inhibition. Kinetic analyses showed that octanoic acid and hexanoic acid were mixed-type inhibitors and (2E,4E)-hexa-2,4-dienoic acid and (2E)-but-2-enoic acid were noncompetitive inhibitors. The inhibition constants have been determined and compared.

Inactivation kinetics of polyphenol oxidase from pupae of blowfly (Sarcophaga bullata) in the dimethyl sulfoxide solution.

The effects of dimethyl sulfoxide (DMSO) on the activity of polyphenol oxidase (PPO, EC 1.14.18.1) from blowfly pupae for the oxidation of L-3,4-dihydroxyphenylalanine were studied. The results showed that low concentrations of DMSO could lead to reversible inactivation to the enzyme. The IC(50) value, the inactivator concentration leading to 50% activity lost, was estimated to be 2.35 M. Inactivation of the enzyme by DMSO was classified as mixed type. The kinetics of inactivation of PPO from blowfly pupae in the low concentrations of DMSO solution was studied using the kinetic method of the substrate reaction. The rate constants of inactivation were determined. The results show that k(+0) was much larger than k'(+0), indicating that the free enzyme molecule was more fragile than the enzyme-substrate complex in the DMSO solution. It was suggested that the presence of the substrate offers marked protection of this enzyme against inactivation by DMSO.

Antityrosinase and antimicrobial activities of trans-cinnamaldehyde thiosemicarbazone.

Tyrosinase (EC 1.14.18.1) is a key enzyme in pigment biosynthesis of organisms. trans-Cinnamaldehyde thiosemicarbazone, a derivative of benzaldehyde thiosemicarbazone, was synthesized as an inhibitor of tyrosinase. The inhibitory effects of this compound on the activity of mushroom tyrosinase were investigated. The results showed that trans-cinnamaldehyde thiosemicarbazone could potently inhibit both monophenolase activity and diphenolase activity of tyrosinase. For monophenolase activity, trans-cinnamaldehyde thiosemicarbazone could not only lengthen the lag time but also decrease the steady-state rate. For diphenolase activity, the IC(50) value was determined to be 5.72 microM. Kinetic analyses showed that trans-cinnamaldehyde thiosemicarbazone was a reversible and mixed type inhibitor on this enzyme. The inhibition constants (K(I) and K(IS)) were determined to be 4.45 and 8.85 muM, respectively. Furthermore, the antibacterial activity against Bacillus subtilis, Escherichia coli, Staphyloccocus aureus, Pseudomonas aeruginosa, Klebsiella pneumonia, and Agrobacterium tumefaciens was investigated. The results showed that trans-cinnamaldehyde thiosemicarbazone was more effective against B. subtilis and S. aureus with the same minimum inhibitory concentration (MIC) of 50 microg/mL and with the same minimum bactericidal concentration (MBC) of 50 microg/mL.