Optimization of Bacillus amyloliquefaciens BLB369 Culture Medium by Response Surface Methodology for Low Cost Production of Antifungal Activity.

Bacillus amyloliquefaciens BLB369 is an important plant growth-promoting bacterium, which produces antifungal compounds. A statistics-based experimental design was used to optimize a liquid culture medium using inexpensive substrates for increasing its antifungal activity. A Plackett-Burman design was first applied to elucidate medium components having significant effects on antifungal production. Then the steepest ascent method was employed to approach the experimental design space, followed by an application of central composite design. Three factors were retained (candy waste, peptone, and sodium chloride), and polynomial and original trigonometric models fitted the antifungal activity. The trigonometric model ensured a better fit. The contour and surface plots showed concentric increasing levels pointing out an optimized activity. Hence, the polynomial and trigonometric models showed a maximal antifungal activity of 251.9 (AU/mL) and 255.5 (AU/mL) for (19.17, 19.88, 3.75) (g/L) and (19.61, 20, 3.7) (g/L) of candy waste, peptone, and NaCl, respectively. This study provides a potential strategy for improving the fermentation of B. amyloliquefaciens BLB369 in low-cost media for large-scale industrial production.

A novel Vip3Aa16-Cry1Ac chimera toxin: Enhancement of toxicity against Ephestia kuehniella, structural study and molecular docking.

Bacillus thuringiensis Vip3A protein has been widely used for crop protection and for delay resistance to existing insecticidal Cry toxins. During current study, a fusion between vip3Aa16 and the toxic core sequence of cry1Ac was constructed in pHT Blue plasmid. Vip3Aa16-Cry1Ac protein was expressed in the supernatant of B. thuringiensis with a size of about 150 kDa. Bioassays tested on Ephestia kuehniella showed that the use of the chimera toxin as biopesticide improved the toxicity to reach 90% ±â€¯2 with an enhancement of 20% compared to the single Vip3Aa16 protein. The findings indicated that the fusion protein design opens new ways to enhance Vip3A toxicity against lepidopteran species and could avoiding insect tolerance of B. thuringiensis delta-endotoxins. Through computational study, we have predicted for the first time the whole 3D structure of a Vip3A toxin. We showed that Vip3Aa16 structure is composed by three domains like Cry toxins: an N-terminal domain containing hemolysin like fold as well as two others Carbohydrate Binding Module (CBM)-like domains. Molecular docking analysis of the chimera toxin and the single Vip3Aa16 protein against specific insect receptors revealed that residues of CBM like domains are clearly involved in the binding of the toxin to receptors.

Antagonist effects of Bacillus spp. strains against Fusarium graminearum for protection of durum wheat (Triticum turgidum L. subsp. durum).

Bacillus species are attractive due to their potential use in the biological control of fungal diseases. Bacillus amyloliquefaciens strain BLB369, Bacillus subtilis strain BLB277, and Paenibacillus polymyxa strain BLB267 were isolated and identified using biochemical and molecular (16S rDNA, gyrA, and rpoB) approaches. They could produce, respectively, (iturin and surfactin), (surfactin and fengycin), and (fusaricidin and polymyxin) exhibiting broad spectrum against several phytopathogenic fungi. In vivo examination of wheat seed germination, plant height, phenolic compounds, chlorophyll, and carotenoid contents proved the efficiency of the bacterial cells and the secreted antagonist activities to protect Tunisian durum wheat (Triticum turgidum L. subsp. durum) cultivar Om Rabiia against F. graminearum fungus. Application of single bacterial culture medium, particularly that of B. amyloliquefaciens, showed better protection than combinations of various culture media. The tertiary combination of B. amyloliquefaciens, B. subtilis, and P. polymyxa bacterial cells led to the highest protection rate which could be due to strains synergistic or complementary effects. Hence, combination of compatible biocontrol agents could be a strategic approach to control plant diseases.

Localization and in silico study of the vegetative insecticidal proteins Vip2S-Vip1S of Bacillus thuringiensis.

The Bacillus thuringiensis S1/4 strain was previously found to harbour vip1S, vip2S, and vip3 genes. Its plasmid curing led to the obtaining of four partially cured strains S1/4-2, S1/4-3, S1/4-7, and S1/4-9 (vip2S-vip1S (-), vip3 (+)), one strain S1/4-4 (vip2S-vip1S (+), vip3 (-)), and S1/4-0 strain lacking the three genes. Using these derivative strains as templates, PCR amplification and southern blot assay revealed that vip2S-vip1S operon and vip3 gene were localized on two different large plasmids. Bioinformatics studies showed that vip2S (1.356 kb), and vip1S (2.637 kb) genes are encoding by an operon consisting of two ORFs separated by an intergenic spacer of 4bp. Using the InterPro tool, Vip2S was found to belong to the family of Binary exotoxin A and Vip1S to bacterial exotoxin B. In silico modeling indicated that the 3D structure of Vip2S is a mixed α/ß protein and proposed 3D-model of Vip1S. Bioassays of the partially cured strains supernatants showed a weak toxicity of S1/4-4 to the lepidopteran Spodoptera littoralis comparing to a better effect of S1/4-2, S1/4-3, S1/4-7, and S1/4-9, suggesting its eventual contribution to the toxicity. Nevertheless, the concentrated supernatant of S1/4-4 strain was not toxic against the coleopteran Tribolium castaneum.

Effect of adding amino acids residues in N- and C-terminus of Vip3Aa16 (L121I) toxin.

To study the importance of N- and C-terminus of Bacillus thuringiensis Vip3Aa16 (L121I) toxin (88 kDa), a number of mutants were generated. The addition of two (2R: RS) or eleven (11R: RSRPGHHHHHH) amino acid residues at the Vip3Aa16 (L121I) C-terminus allowed to an unappropriated folding illustrated by the abundant presence of the 62 kDa proteolytic form. The produced Vip3Aa16 (L121I) full length form was less detected when increasing the number of amino acids residues in the C-terminus. Bioassays demonstrated that the growth of the lepidopteran Ephestia kuehniella was slightly affected by Vip3Aa16 (L121I)-2R and not affected by Vip3Aa16 (L121I)-11R. Additionally, the fusion at the Vip3Aa16 (L121I) N-terminus of 39 amino acids harboring the E. coli OmpA leader peptide and the His-tag sequence allowed to the increase of protease sensitivity of Vip3Aa16 (L121I) full length form, as only the 62 kDa proteolysis form was detected. Remarkably, this fused protein produced in Escherichia coli (E. coli) was biologically inactive toward Ephestia kuehniella larvae. Thus, the N-terminus of the protein is required to the accomplishment of the insecticidal activity of Vip3 proteins. This report serves as guideline for the study of Vip3Aa16 (L121I) protein stability and activity.

Toxicity, activation process, and histopathological effect of Bacillus thuringiensis vegetative insecticidal protein Vip3Aa16 on Tuta absoluta.

Tuta absoluta is a destructive moth of Solanaceae plants and especially tomatoes. Here, we considered the entomopathogenic activity of the Bacillus thuringiensis Vip3Aa16 protein heterologously produced by Escherichia coli against T. absoluta. Purified Vip3Aa16 showed lower lethal concentration 50 % against third instar larvae (Toxin/tomato leaf) (335 ± 17 ng/cm(2)) compared to that of B. thuringiensis kurstaki HD1 δ-endotoxins (955 ± 4 ng/cm(2)) (P < 0.05). Action mode examination showed that Vip3Aa16 (88 kDa) was more sensitive to proteolysis activation by the chymotrypsin than the trypsin or the larvae gut soluble proteases, yielding derivative proteins essentially of about 62 and 33 kDa. The gut-soluble proteases could contain trypsin-like enzymes implicated in Vip3Aa16 activation since the proteolysis was inhibited using specific protease inhibitors. Additionally, we showed that the histopathological effect of Vip3Aa16 on T. absoluta larva midguts consisted on a microvillus damage and an epithelial cell rupture.

Efficiency and midgut histopathological effect of the newly isolated Bacillus thuringiensis KS δ-endotoxins on the emergent pest Tuta absoluta.

Tuta absoluta (Povolny, 1994) is a devastating moth to the Solanaceae plants. It is a challenging pest to control, especially on tomatoes. In this work, we studied the entomopathogenic activity of the Cry-forming δ-endotoxins produced by Bacillus thuringiensis strain KS and B. thuringiensis kurstaki reference strain HD1 against T. absoluta. These strains carried the cry2, cry1Ab, cry1Aa / cry1Ac, and cry1I genes, and KS also carried a cry1C gene. The δ-endotoxins of KS were approximately twofold more toxic against the third instar larvae than those of HD1, as they showed lower 50% and 90% lethal concentrations (0.80 and 2.70 µg/cm² (δ-endotoxins/tomato leaf)) compared with those of HD1 (1.70 and 4.50 µg/cm²) (p < 0.05). Additionally, the larvae protease extract showed at least six caseinolytic activities, which activated the KS and HD1 δ- endotoxins, yielding the active toxins of about 65 kDa and the protease-resistant core of about 58 kDa. Moreover, the histopathological effects of KS and HD1 δ-endotoxins on the larvae midgut consisted of an apical columnar cell vacuolization, microvillus damage, and epithelial cell disruption. These results showed that the KS strain could be a candidate for T. absoluta control.

Screening and identification of a Bacillus thuringiensis strain S1/4 with large and efficient insecticidal activities.

The bacterium Bacillus thuringiensis was recognized for its entomopathogenic activities related to Cry and Cyt proteins forming the δ-endotoxins and some extracellular activities like the vegetative insecticidal proteins (VIP) and Cry1I. These activities may act specifically against diverse organisms and some of them typically characterize each strain. Here, we screened a set of 212 B. thuringiensis strains to search the higher insecticidal activities. These strains had bipyramidal and cubic crystal morphologies and 30% of them showed PCR amplification of vip3 internal region, from which five isolates (S1/4, S17, S122, S123, and S144) showed plasmid profile variability. These five strains contained the cry1I, cry1Aa and/or cry1Ac, cry1Ab and cry2 genes, and S1/4 harbored in addition the cry1C, vip1, and vip2 genes. They produced from 25 to 46 µg δ-endotoxin per 10(7) spores. Their δ-endotoxins displayed distinct lethal concentrations 50% against either Spodoptera littoralis or Ephestia kuehniella larvae with the lowest one for S1/4, which was also active against Tuta absoluta. Fortunately, the analysis of the culture supernatants revealed that S1/4 had the higher toxicity towards these lepidopteron but it did not show any toxicity against the Tribolium castaneum coleopteran larvae; additionally, S1/4 displayed an antibacterial activity. S1/4 is a good candidate for agricultural pest control, as it is more efficient than the reference strain HD1.

Increase of the Bacillus thuringiensis secreted toxicity against lepidopteron larvae by homologous expression of the vip3LB gene during sporulation stage.

The Vegetative insecticidal Vip3A proteins display a wide range of insecticidal spectrum against several agricultural insect pests. The fact that the expression of vip3 genes occurs only during the vegetative growth phase of Bacillus thuringiensis is a limiting factor in term of production level. Therefore, extending the synthesis of the Vip proteins to the sporulation phase is a good alternative to reach high levels of toxin synthesis. In this study, we have demonstrated that the maximal production of the secreted Vip3LB (also called Vip3Aa16) during the growth of the wild-type strain B. thuringiensis BUPM 95 is reached at the end of the vegetative growth phase, and that the protein remains relatively stable in the culture supernatant during the late sporulation stages. The vip3LB gene was cloned and expressed under the control of the sporulation dependant promoters BtI and BtII in B. thuringiensis BUPM 106 (Vip3(-)) and BUPM 95 (Vip3(+)) strains. The examination of the culture supernatants during the sporulation phase evidenced the synthesis of Vip3LB and its toxicity against the second-instars larvae of the Lepidopteron insect Spodoptera littoralis for the recombinant BUPM 106. Moreover, there was an increase of the Vip3LB synthesis level and an enhancement of the oral toxicity for the recombinant BUPM 95 resulting from the expression of the vip3LB gene during both the vegetative and sporulation phases and the relative stability of the Vip3LB protein.

Heterologous expression of Bacillus thuringiensis vegetative insecticidal protein-encoding gene vip3LB in Photorhabdus temperata strain K122 and oral toxicity against the lepidoptera Ephestia kuehniella and Spodoptera littoralis.

Photorhabdus temperata and Bacillus thuringiensis are entomopathogenic bacteria exhibiting toxicities against different insect larvae. Vegetative Insecticidal Protein Vip3LB is a Bacillus thuringiensis insecticidal protein secreted during the vegetative growth stage exhibiting lepidopteran specificity. In this study, we focused for the first time on the heterologous expression of vip3LB gene in Photorhabdus temperata strain K122. Firstly, Western blot analyses of whole cultures of recombinant Photorhabdus temperata showed that Vip3LB was produced and appeared lightly proteolysed. Cellular fractionation and proteinase K proteolysis showed that in vitro-cultured recombinant Photorhabdus temperata K122 accumulated Vip3LB in the cell and appeared not to secrete this protein. Oral toxicity of whole cultures of recombinant Photorhabdus temperata K122 strains was assayed on second-instar larvae of Ephestia kuehniella, a laboratory model insect, and the cutworm Spodoptera littoralis, one of the major pests of many important crop plants. Unlike the wild strain K122, which has no effect on the larval growth, the recombinant bacteria expressing vip3LB gene reduced or stopped the larval growth. These results demonstrate that the heterologous expression of Bacillus thuringiensis vegetative insecticidal protein-encoding gene vip3LB in Photorhabdus temperata could be considered as an excellent tool for improving Photorhabdus insecticidal activities.

A secreted anti-activator, OspD1, and its chaperone, Spa15, are involved in the control of transcription by the type III secretion apparatus activity in Shigella flexneri.

Bacteria of Shigella spp. are responsible for shigellosis in humans and use a type III secretion (TTS) system to enter epithelial cells and trigger apoptosis in macrophages. Transit of translocator and effector proteins through the TTS apparatus is activated upon contact of bacteria with host cells. Transcription of approximately 15 genes encoding effectors is regulated by the TTS apparatus activity and controlled by MxiE, an AraC family activator, and its coactivator IpgC, the chaperone of IpaB and IpaC translocators. Using a genetic screen, we identified ospD1 as a gene whose product negatively controls expression of genes regulated by secretion activity. OspD1 associates with the chaperone Spa15 and the activator MxiE and acts as an anti-activator until it is secreted. The mechanism regulating transcription in response to secretion activity involves an activator (MxiE), an anti-activator (OspD1), a co-anti-activator (Spa15), a coactivator (IpgC) and two anti-coactivators (IpaB and IpaC) whose alternative and mutually exclusive interactions are controlled by the duration of the TTS apparatus activity.