Engineering of Bacillus thuringiensis Cry2Ab toxin for improved insecticidal activity.

Bacillus thuringiensis Cry2Ab toxin was a widely used bioinsecticide to control lepidopteran pests all over the world. In the present study, engineering of Bacillus thuringiensis Cry2Ab toxin was performed for improved insecticidal activity using site-specific saturation mutation. Variants L183I were screened with lower LC50 (0.129 µg/cm2) against P. xylostella when compared to wild-type Cry2Ab (0.267 µg/cm2). To investigate the molecular mechanism behind the enhanced activity of variant L183I, the activation, oligomerization and pore-formation activities of L183I were evaluated, using wild-type Cry2Ab as a control. The results demonstrated that the proteolytic activation of L183I was the same as that of wild-type Cry2Ab. However, variant L183I displayed higher oligomerization and pore-formation activities, which was consistence with its increased insecticidal activity. The current study demonstrated that the insecticidal activity of Cry2Ab toxin could be assessed using oligomerization and pore-formation activities, and the screened variant L183I with improved activity might contribute to Cry2Ab toxin's future application.

Stability is essential for insecticidal activity of Vip3Aa toxin against Spodoptera exigua.

Vegetative insecticidal proteins 3A (Vip3A) were important insecticidal proteins for control of lepidopteran pests. Previous study demonstrated that Vip3Aa and Vip3Ad showed significant difference in insecticidal activities against Spodoptera exigua, while the molecular mechanism remained ambiguous. Here we demonstrated that the difference in insecticidal activities between Vip3Aa and Vip3Ad might be caused by the difference in stability of Vip3Aa and Vip3Ad in S. exigua midgut protease. Vip3Aa was quite stable while Vip3Ad could be further degraded. Molecular dynamics simulation revealed that Vip3Aa was more stable than Vip3Ad, with smaller RMSD and RMSF value. Amino acid sequence alignment indicated that three were three extra prolines (P591, P605 and P779) located on Vip3Aa. We further identified that residue P591 played a crucial role on stability and insecticidal activity of Vip3Aa. Taken together, our study demonstrated that the stability was essential for the insecticidal activity of Vip3A toxins, which might provide new insight into the action mode of Vip3A toxins and contribute to the design Vip3A variants with improved stability and insecticidal activity.

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.

Strong inhibitory activities and action modes of lipopeptides on lipase.

Lipopeptides have been reported to exhibit anti-obesity effects. In this study, we obtained a Bacillus velezensis strain FJAT-52631 that could coproduce iturins, fengycins, and surfactins. Results showed that the FJAT-52631 crude lipopeptide, purified fengycin, iturin, and surfactin standards exhibited strong inhibition activities against lipase with dose-dependence manners (half maximal inhibitory concentration (IC50) = 0.011, 0.005, 0.056, and 0.005 mg/mL, respectively). Moreover, fengycin and surfactin had the comparable activities with orlistat, but iturin not. It was revealed that the inhibition mechanism and type of the lipopeptides were reversible and competitive. The quenching mechanism of lipase was static and only one binding site between lipase and lipopoeptide was inferred from the fluorescence analysis. The docking analysis displayed that fengycin and surfactin could directly interact with the active amino acid residues (Ser or Asp) of lipase, but not with iturin. Our work suggests that the B. velezensis lipopeptides would have great potential to act as lipase inhibitors.

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.

Pd-Catalyzed decarboxylative cross-coupling reactions of epoxides with α,ß-unsaturated carboxylic acids.

A Pd-catalyzed decarboxylative cross-coupling of α,ß-unsaturated carboxylic acids with cyclic and acyclic epoxides has been developed. Both ß-monosubstituted and ß-disubstituted unsaturated carboxylic acids, as well as conjugated diene unsaturated carboxylic acids are suitable reaction substrates. Substituted homoallylic alcohols were obtained in moderate to good yields. The product was obtained as a mixture of diastereomers favoring the anti diastereomer of the cyclic epoxides. This work provides a method for the modification of complex organic molecules containing α,ß-unsaturated carboxylic acids.

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.

Cu-catalyzed cross-coupling reactions of vinyl epoxide with organoboron compounds: access to homoallylic alcohols.

Copper-catalyzed cross-coupling reactions of vinyl epoxide with arylboronates to obtain aryl-substituted homoallylic alcohols are described. The reaction selectivity was different to that of previously reported vinyl epoxide ring-opening reactions using aryl nucleophiles. The reaction proceeded under mild conditions, affording aryl-substituted homoallylic alcohols with high selectivity and in good to excellent yields. The reaction provides convenient access to aryl-substituted homoallylic alcohols from vinyl epoxide.

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.

Bacillus populi sp. nov. isolated from Populus euphratica rhizosphere soil of the Taklamakan desert.

A rod-shaped, endospore-forming, aerobic bacterium, designated FJAT-45347T, was isolated from rhizosphere soil collected from the Taklamakan desert in Xinjiang (PR China). Growth was observed at 15-35 °C (optimum 25 °C), in 0 % and 20.0 % NaCl (optimum 8.0 %) and at pH 7.5-12.0 (optimum 8.0), respectively. The cell-wall peptidoglycan contained meso-diaminopimelic acid and the isoprenoid quinone was MK-7. The main fatty acids were iso-C15 : 0, anteiso-C15 : 0 and anteiso-C17 : 0. The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Phylogenetic analysis based on 16S rRNA gene sequences affiliated FJAT-45347T to the genus Bacillus, and it showed the highest sequence similarities to Bacillus clarkii DSM 8720T (96.1 %). The average nucleotide identity and in silico DNA-DNA hybridization values between FJAT-45347T and the most closely related species were 68.5 and 26.2 %, respectively, which were lower than the thresholds commonly used to define species (96 and 70 %, respectively), indicating that it represented a member of a different taxon. The DNA G+C content was 40.6 mol%. The phenotypic characters and taxono-genomics study revealed that FJAT-45347T represents a novel species of the genus Bacillus, for which the name Bacilluspopuli sp. nov. is proposed. The type strain is FJAT-45347T (=DSM 104632T=CCTCC AB 2016257T).

Bacillus praedii sp. nov., isolated from purplish paddy soil.

A Gram-stain-positive, rod-shaped, endospore-forming, aerobic bacterium, designated strain FJAT-25547T, was isolated from the purplish paddy soil collected from Linshan Township, Yanting Prefecture of Sichuan Province in PR China (31° 16' N 105° 27' E). Growth was achieved aerobically at temperatures between 15 and 40 °C (optimum 30 °C), with between 0 and 10.0 % NaCl (w/v) (optimum 4 %) and in the range of pH 5.0-12.0 (optimum pH 9.0). The cell-wall peptidoglycan contained meso-diaminopimelic acid, and the main isoprenoid quinone was MK-7. The major fatty acids were iso-C15 : 0 (55.4 %), anteiso-C15 : 0 (22.2 %), iso-C16 : 0 (5.1 %) and iso-C14 : 0 (6.5 %). The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain FJAT-25547T was a member of the genus Bacillus and was most closely related to Bacillus horneckiae DSM 23495T (97.7 % similarity), Bacillus eiseniae A1-2T (97.5 %), Bacillus mesophilum IITR-54T (97.2 %) and Bacillus kochii WCC 4582T (97.0 %). The average nucleotide identity value between strain FJAT-25547T and the type strain of the most closely related species, B. horneckiae DSM 23495T, was 77.7 %, less than the proposed cut-off value of 96.0 % for differentiating species within the genus. The in silico DNA-DNA hybridization value of strain FJAT-25547T with the most closely related species was 22.7 %, <70 %, again indicating they belong to different taxa. The DNA G+C content of strain FJAT-25547T was 39.1 mol%. This taxono-genomics study revealed that strain FJAT-25547T represents a novel species of the genus Bacillus for which the name Bacillus praedii sp. nov. (type strain FJAT-25547T=CCTCC AB 2015208T=DSM 101002T) is proposed.

Bacillus wudalianchiensis sp. nov., isolated from grass soils of the Wudalianchi scenic area.

A Gram-stain-positive, rod-shaped, endospore-forming, aerobic bacterium, designated FJAT-27215T, was isolated from grass soil collected from Wudalianchi in the Heilongjiang Province of China. Growth was observed at 10-60 °C (optimum 30 °C), in 0 and 3.0 % NaCl (optimum 0 %) and at pH 5.0-10.0 (optimum 7.0), respectively. The cell-wall peptidoglycan contained meso-diaminopimelic acid and the isoprenoid quinone was MK7. The main fatty acids were iso-C15 : 0, anteiso-C15 : 0, anteiso-C17 : 0, and iso-C16 : 0. The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidyl ethanolamine. Phylogenetic analysis based on 16S rRNA gene sequences affiliated strain FJAT-27215T to the genus Bacillus. Strain FJAT-27215T showed high sequence similarities to Bacillus encimensis SGD-V-25T (98.6 %), Bacillus badius NBRC 15713T (98.6 %), Domibacillus indicus SD111T (96.9 %) and Bacillus thermotolerans SgZ-8T (96.5 %). The average nucleotide identity values between strain FJAT-27215T and the type strains of closely related species were much lower than the 96 % threshold value for delineation of genomic prokaryotic species. The in silico DNA-DNA hybridization values between strain FJAT-27215T and the most closely related strain B. encimensis SGD-V-25T showed a similarity of 22.4 % and lower than 70 %, indicating that they belong to different taxa. The phenotypic characters and taxono-genomics study revealed that strain FJAT-27215T represents a novel Bacillus species, for which the name Bacillus wudalianchiensis sp. nov. is proposed. The type strain is FJAT-27215T (=CCTCC AB 2015266T=DSM 100757T).

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.

Draft Genome Sequences of Type Strains Bacillus drentensis DSM 15600T and Bacillus novalis DSM 15603T.

Here, we report the draft genome sequences of Bacillus drentensis DSM 15600T and Bacillus novalis DSM 15603T with 5,305,306 bp and 5,667,584 bp, respectively, which will provide useful information for the functional gene mining and application of these two species. The average DNA G+C contents were 38.91% and 40.01%, respectively.

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.

Draft Genome Sequence of Bacillus mesonae FJAT-13985T (=DSM 25968T) for Setting Up Phylogenomics in Genomic Taxonomy of the Bacillus-Like Bacteria.

Bacillus mesonae FJAT-13985(T) is a Gram-positive, spore-forming, and aerobic bacterium. Here, we report the draft genome sequence of B. mesonae FJAT-13985(T) with 5,807,726 bp, which will provide useful information for setting up phylogenomics in the genomic taxonomy of the Bacillus-like bacteria, as well as for the functional gene mining and application of B. mesonae FJAT-13985(T).

Bacillus loiseleuriae sp. nov., isolated from rhizosphere soil from a loiseleuria plant.

A Gram-stain-positive, rod-shaped, endospore-forming, aerobic bacterium, designated strain FJAT-27997T, was isolated from the rhizosphere soil of a Loiseleuria plant collected from Sichuan province in China. Growth was observed aerobically between 20 and 35 °C (optimum 30 °C), between 0 and 3.0 % (w/v) NaCl (optimum at 0 %) concentration and pH in the range 6.0-9.0 (optimum at pH 7.0). The cell-wall peptidoglycan contained meso-diaminopimelic acid and the major isoprenoid quinone was menaquinone MK-7. The main fatty acids were iso-C15 : 0, anteiso-C15 : 0, iso-C14 : 0, C16 : 0 and C14 : 0. The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Phylogenetic analyses based on 16S rRNA gene sequences showed that isolate FJAT-27997T was a member of the genus Bacillus and was related most closely to Bacillus simplex DSM 1321T (97.95 % similarity), followed by Bacillushuizhouensis GSS03T (97.9 %). The average nucleotide identity value between strain FJAT-27997T and the most closely related species, B. simplex DSM 1321T, was 71.60 % (JSpecies), less than the previously proposed cut-off value of 96 % for differentiating species within the genus. The in silico DNA-DNA hybridization values between strain FJAT-27997T and its most closely related species were <70 %, again indicating they belong to different taxa. The main fatty acids were iso-C15 : 0 and anteiso-C15 : 0. The novel strain could be differentiated from other known Bacillus species on the basis of several phenotypic characters and fatty acid profiles. This taxononomic/genomic study revealed that strain FJAT-27997T represents a novel Bacillus species, for which the name Bacillus loiseleuriae sp. nov. (type strain FJAT-27997T =CCTCC AB 2015285T=DSM 101776T) is proposed.

Draft Genome Sequence of Bacillus marisflavi TF-11T (JCM 11544), a Carotenoid-Producing Bacterium Isolated from Seawater from a Tidal Flat in the Yellow Sea.

Bacillus marisflavi TF-11(T) (JCM 11544) is a Gram-positive, spore-forming, and carotenoid-producing bacterium isolated from seawater from a tidal flat in the Yellow Sea. Here, we report the first draft genome sequence of B. marisflavi TF-11(T), which comprises 4.31 Mb in 11 scaffolds with a G+C content of 48.57%.

Genome Sequence of Paenibacillus sp. Strain FJAT-28004 for the Genome Sequencing Project for Genomic Taxonomy and Phylogenomics of Bacillus-Like Bacteria.

Paenibacillus sp. strain FJAT-28004 is a spore forming and strictly aerobic bacterium. Here, we report the draft 7,479,858-bp genome sequence of Paenibacillus sp. FJAT-28004, which will provide useful information for genomic taxonomy and phylogenomics of the genus Paenibacillus, as well as for the functional gene mining and application of Paenibacillus sp. FJAT-28004.

Draft Genome Sequence of Bacillus sp. FJAT-27238 for Setting up Phylogenomic Analysis of Genomic Taxonomy of Bacillus-Like Bacteria.

Bacillus sp. FJAT-27238 is a Gram-positive, spore-forming, and aerobic bacterium. Here, we report the draft genome sequence of Bacillus sp. FJAT-27238, with 6,134,829 bp, which will provide useful information for setting up phylogenomic analysis of the genomic taxonomy of Bacillus-like bacteria as well as for the functional gene mining and application of strain FJAT-27238. The genomic DNA G+C content was 47.37%.