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Papers by Sameh Sellami

Sellami, S., Cherif, M., Jaoua, S., and Jamoussi, K. 2015. Chimeric vip3Aa16TC gene encoding the ... more Sellami, S., Cherif, M., Jaoua, S., and Jamoussi, K. 2015. Chimeric vip3Aa16TC gene encoding the toxic core of the vegetative insecticidal protein enhanced Bacillus thuringiensis entomopathogenicity. Tunisian Journal of Plant Protection 10: 15-22. Vip3 insecticidal protein is produced by Bacillus thuringiensis during the vegetative stage. Its proteolysis by the midgut juice of susceptible larvae formed four major products of approximately 66, 45, 33 and 22 kDa. In this study, we cloned the vip3Aa16TC DNA encoding the “Vip3Aa16 toxic core (TC)” of 33 kDa corresponding to the Vip3Aa16 region from amino acid 200 to 456. The vip3Aa16TC chimeric gene carried by the pHT-vip3Aa16TC plasmid was under the control of the sporulation dependent promoters (BtI-BtII) and the Shine Dalgarno sequence of cry1Ac gene as well as the cry1Ia gene terminator. Western-blot analysis of the culture supernatants of the recombinant B. thuringiensis strain detected Vip3Aa16TC after growing for 14 to 56 h provi...

La bacterie entomopathogene Bacillus thuringiensis ( Bt ) a ete le premier micro-organisme homolo... more La bacterie entomopathogene Bacillus thuringiensis ( Bt ) a ete le premier micro-organisme homologue dans le monde comme biopesticide a cause de son action specifique et son innocuite pour l’homme et les animaux. L’activite entomopathogene de Bt est principalement due a la synthese des proteines Cry pendant la phase de sporulationqui forment des inclusions cristallines et qui peuvent etre accompagnees par des proteines cytolytiques Cyt. Elle peut produire egalement les bioinsecticides Vip secretes (Vip : VegetativeInsecticidalProteins) durant la phase vegetative, ainsi quedifferents autres facteurs de virulence. Toutes ces toxines synthetisees sont tres actives contre une large gamme d’insectes parmi lesquels les lepidopteres, les dipteres et les coleopteres.Si actuellement, Bt est le microorganisme le plus utilise dans le marche des biopesticides, c'est parce que cette bacterie se multiplie facilement en fermenteurs et ses produits formules se conservent pour de longues durees....

Journal of New Sciences, 2015

Without controlling devastating pests, organisms responsible of plant diseases (fungi, bacteria o... more Without controlling devastating pests, organisms responsible of plant diseases (fungi, bacteria or virus) and weeds, the agricultural and forest losses can be very important. So, it is undeniable that the expansion and the agricultural productivity adopt optimal management strategies of these destructive pests, diseases and weeds to minimize their effects on the environment. For controlling these cultures enemies, the producers used frequently the chemical pesticides which caused many problems like the lack of specificity, and the damage to the health since they are generally corrosive, irritating, and inflammable. They could also lead to many diseases, accumulate and pollute the environment. For these reasons, more safe substituted solutions for the humans and animals were adopted such us the integrated pest management, the physical, the biochemical and the biological pest controls. The biological pest control was defined as a procedure which involves living organisms or active sub...

Journal of Phytopathology, 2017

The endoβ-1,3-1,4-glucanases are glycoside hydrolases involved in the enzymatic depolymerization ... more The endoβ-1,3-1,4-glucanases are glycoside hydrolases involved in the enzymatic depolymerization of 1,3-1,4 β-glucans and showed an antifungal activity against some fungi. Bacillus amyloliquefaciens BLB369 has a high antagonistic activity against phytopathogenic fungi. Its glu369 full-coding sequence of the endoβ-1,3-1,4-glucanase gene (732 bp) was sequenced, cloned and successfully expressed in Escherichia coli Top10. The encoded protein (243 amino acids) has a calculated molecular mass of 27.3 kDa. To simplify the purification procedure, the glu369 coding sequence was cloned into the vector pKJD4. The produced OmpA-His-Glu369 harboured OmpA signal sequence for E. coli periplasmic localization and followed by a 6His residues for its purification. The purified His-tagged proteins revealed two bands on SDS-PAGE analysis with molecular masses of about 30.5 (His-Glu369) and 32.5 kDa (OmpA-His-Glu369). They had the ability to inhibit the growth of phytopathogenic fungus Alternaria alternata. These favourable properties make the endoβ-1,3-1,4-glucanase a good candidate for biotechnological applications.

International Journal of Biological Macromolecules, 2018

Bacillus thuringiensis Vip3A protein has been widely used for crop protection and for delay resis... more 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% ± 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.

Toxicon, 2018

LC50 Vip3Aa16 : 92.79 ng/cm 2 LC50 Vip3Aa16 : 9.025 ng/cm 2 Vip3Aa16 Secreted protein from Bacill... more LC50 Vip3Aa16 : 92.79 ng/cm 2 LC50 Vip3Aa16 : 9.025 ng/cm 2 Vip3Aa16 Secreted protein from Bacillus thuringiensis Vip3Aa16 Secreted protein from Bacillus thuringiensis Agrotis segetum Histopathlological effect on Agrotis segetum midgut larvae Insecticidal activity on Agrotis segetum larvae

Journal of Basic Microbiology, 2016

To study the importance of N-and C-terminus of Bacillus thuringiensis Vip3Aa16 (L121I) toxin (88 ... more 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.

Biochemical and biophysical research communications, Jan 22, 2017

The basis of the different susceptibility of Ephestia kuehniella to the Cry1Aa and Cry1Ac δ-endot... more The basis of the different susceptibility of Ephestia kuehniella to the Cry1Aa and Cry1Ac δ-endotoxins from Bacillus thuringiensis kurstaki BNS3 was studied. Both toxins bound specifically to the BBMV of E. kuehniella. The result of the ligand blot showed that Cry1Ac bound to three putative receptors of about 100, 65 and 80 kDa and Cry1Aa interacted only with a 100 kDa protein. Pronase digestion of the BBMV-bound toxins was used to analyze the toxin insertion. Both toxins inserted into the BBMV as monomers however, a 14 kDa peptide of α4-α5 which correspond to the oligomeric form of this peptide was detected in case of Cry1Ac only. Analysis of the in vitro oligomerisation of these toxins in the presence of the BBMV of E. kuehniella showed reduced oligomer formation in case of Cry1Aa in comparison with Cry1Ac. Taken together, these results strongly suggest that the difference of toxicity between Cry1Aa and Cry1Ac to E. kuehniella is due to a deficient oligomerisation of Cry1Aa.

Pesticide Biochemistry and Physiology, 2017

Influence of Ephestia kuehniella stage larvae on the potency of Bacillus thuringiensis Cry1Aa del... more Influence of Ephestia kuehniella stage larvae on the potency of Bacillus thuringiensis Cry1Aa delta-endotoxin

International Journal of Biological Macromolecules, 2016

The Bacillus thuringiensis S1/4 strain was previously found to harbour vip1S, vip2S, and vip3 gen... more 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/β 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.

Microbiological Research, 2016

Bacillus species are attractive due to their potential use in the biological control of fungal di... more 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.

Process Biochemistry, 2014

Abstract A study of the recovery process of Bacillus thuringiensis (Bt) strain BLB1-based bioinse... more Abstract A study of the recovery process of Bacillus thuringiensis (Bt) strain BLB1-based bioinsecticides using ultrafiltration showed that the highest recovery of BLB1 bioactive components was achieved by using a 5 kDa MWCO ultrafiltration membrane to concentrate the supernatant. A bioassay against Ephestia kuehniella showed that the retentate was approximately 49% more toxic than the supernatant, with a lethal concentration (LC50) of 194.00 μg g−1 in the retentate compared to 380.00 μg g−1 in the supernatant. Furthermore, compared to the centrifugate (spore-crystal mixture) mixed with supernatant or saline water, the centrifugate retentate showed 42.7% and 56% higher toxicity against E. kuehniella, respectively. The involvement of proteases, chitinases and Vip3 proteins restored to the retentate suggests synergetic activity with delta-endotoxins and spores, and this activity was confirmed by enzymatic activity and western blot analysis. The formulated centrifugate retentate maintained stability and toxicity after 1 year of storage at 4 °C. These data suggest that the ultrafiltration process enabled significant virulence factors recovery from BLB1 supernatant grown in a complex medium and resulted in a highly toxic and stable formulation.

Acta Tropica, 2014

The effectiveness of 10 low-cost UV-absorbers in protecting Bacillus thuringiensis subsp. kurstak... more The effectiveness of 10 low-cost UV-absorbers in protecting Bacillus thuringiensis subsp. kurstaki BLB1 toxins against inactivation by UV-A and UV-B irradiation was evaluated in this study. Among them, two by-products, molasses and olive mill wastewater (OMW) were selected for further studies. They were tested at different concentrations of 0.05, 0.1, 0.15 and 0.2% using the para-aminobenzoic acid (PABA) as a common UV protectant. Interestingly, addition of PABA and OMW to BLB1 formulations was found to be most effective in protecting BLB1 spores at 90.8 and 76.4% respectively and in preserving deltaendotoxin concentration at a level of 81.7 and 72.2%, respectively when used at a concentration of 0.2%. The lowest preserved spores (46.3%) and delta-endotoxin level (12.4%) was found using molasses. In contrast, spore count and delta-endotoxin concentration were completely reduced after an exposure of unprotected Bt strain BLB1 to UV radiations up to 96 h. SDS-PAGE analysis of protected and unprotected samples revealed that delta-endotoxin bands (130, 65-70 kDa) were conserved until 96 h of UV exposure in presence of PABA or OMW compared with their disappearance in presence of molasses after 72 h of exposure and their dramatically decline from 8 h of exposure in unprotected mixture. A complete loss of larvicidal toxicity against Ephestia kuehniella was found after 24 h of exposure in absence of any UVabsorber. Addition of OMW or PABA offered the highest levels of insecticidal activity with 63.2 and 74.7% of residual toxicity, respectively. Whereas, molasses addition, as UV protectant retained only 26.3% of residual activity after 96 h of exposure. Therefore, addition of OMW by-product to Bt formulation may be a suitable alternative to others synthetic chemical compounds. OMW may also provided added value, be environmentally friendly and less hazardous, when used at low concentration.

Applied Biochemistry and Biotechnology, 2014

Tuta absoluta is a destructive moth of Solanaceae plants and especially tomatoes. Here, we consid... more 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.

Molecular Biotechnology, 2009

Photorhabdus temperata and Bacillus thuringiensis are entomopathogenic bacteria exhibiting toxici... more 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.

Molecular Biotechnology, 2009

Vegetative insecticidal protein (Vip) is a class of insecticidal proteins produced by many Bacill... more Vegetative insecticidal protein (Vip) is a class of insecticidal proteins produced by many Bacillus thuringiensis strains during their vegetative growth stage. The vip3LB gene of B. thuringiensis strain BUPM95, which encodes a protein active against the Lepidoptera olive tree pathogenic insect Prays oleae, was cloned into pET-14b vector and overexpressed in Escherichia coli. The expressed Vip3LB protein, found in the E. coli cytoplasmic fraction, was purified and used to produce anti-Vip3LB antibodies. Using the midgut extract of P. oleae, the purified Vip3LB bound to a 65-kDa protein, whereas Cry1Ac toxin bound to a 210-kDa midgut putative receptor. This result justifies the importance of the biological pest control agent Vip3LB that could be used as another alternative particularly in case of resistance to Cry toxins.

Journal of Microbiology and Biotechnology, 2013

Journal of Basic Microbiology, 2012

The bacterium Bacillus thuringiensis was recognized for its entomopathogenic activities related t... more 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/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 didn't 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.

Current Microbiology, 2011

The Vegetative insecticidal Vip3A proteins display a wide range of insecticidal spectrum against ... more 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.

Sellami, S., Cherif, M., Jaoua, S., and Jamoussi, K. 2015. Chimeric vip3Aa16TC gene encoding the ... more Sellami, S., Cherif, M., Jaoua, S., and Jamoussi, K. 2015. Chimeric vip3Aa16TC gene encoding the toxic core of the vegetative insecticidal protein enhanced Bacillus thuringiensis entomopathogenicity. Tunisian Journal of Plant Protection 10: 15-22. Vip3 insecticidal protein is produced by Bacillus thuringiensis during the vegetative stage. Its proteolysis by the midgut juice of susceptible larvae formed four major products of approximately 66, 45, 33 and 22 kDa. In this study, we cloned the vip3Aa16TC DNA encoding the “Vip3Aa16 toxic core (TC)” of 33 kDa corresponding to the Vip3Aa16 region from amino acid 200 to 456. The vip3Aa16TC chimeric gene carried by the pHT-vip3Aa16TC plasmid was under the control of the sporulation dependent promoters (BtI-BtII) and the Shine Dalgarno sequence of cry1Ac gene as well as the cry1Ia gene terminator. Western-blot analysis of the culture supernatants of the recombinant B. thuringiensis strain detected Vip3Aa16TC after growing for 14 to 56 h provi...

La bacterie entomopathogene Bacillus thuringiensis ( Bt ) a ete le premier micro-organisme homolo... more La bacterie entomopathogene Bacillus thuringiensis ( Bt ) a ete le premier micro-organisme homologue dans le monde comme biopesticide a cause de son action specifique et son innocuite pour l’homme et les animaux. L’activite entomopathogene de Bt est principalement due a la synthese des proteines Cry pendant la phase de sporulationqui forment des inclusions cristallines et qui peuvent etre accompagnees par des proteines cytolytiques Cyt. Elle peut produire egalement les bioinsecticides Vip secretes (Vip : VegetativeInsecticidalProteins) durant la phase vegetative, ainsi quedifferents autres facteurs de virulence. Toutes ces toxines synthetisees sont tres actives contre une large gamme d’insectes parmi lesquels les lepidopteres, les dipteres et les coleopteres.Si actuellement, Bt est le microorganisme le plus utilise dans le marche des biopesticides, c'est parce que cette bacterie se multiplie facilement en fermenteurs et ses produits formules se conservent pour de longues durees....

Journal of New Sciences, 2015

Without controlling devastating pests, organisms responsible of plant diseases (fungi, bacteria o... more Without controlling devastating pests, organisms responsible of plant diseases (fungi, bacteria or virus) and weeds, the agricultural and forest losses can be very important. So, it is undeniable that the expansion and the agricultural productivity adopt optimal management strategies of these destructive pests, diseases and weeds to minimize their effects on the environment. For controlling these cultures enemies, the producers used frequently the chemical pesticides which caused many problems like the lack of specificity, and the damage to the health since they are generally corrosive, irritating, and inflammable. They could also lead to many diseases, accumulate and pollute the environment. For these reasons, more safe substituted solutions for the humans and animals were adopted such us the integrated pest management, the physical, the biochemical and the biological pest controls. The biological pest control was defined as a procedure which involves living organisms or active sub...

Journal of Phytopathology, 2017

The endoβ-1,3-1,4-glucanases are glycoside hydrolases involved in the enzymatic depolymerization ... more The endoβ-1,3-1,4-glucanases are glycoside hydrolases involved in the enzymatic depolymerization of 1,3-1,4 β-glucans and showed an antifungal activity against some fungi. Bacillus amyloliquefaciens BLB369 has a high antagonistic activity against phytopathogenic fungi. Its glu369 full-coding sequence of the endoβ-1,3-1,4-glucanase gene (732 bp) was sequenced, cloned and successfully expressed in Escherichia coli Top10. The encoded protein (243 amino acids) has a calculated molecular mass of 27.3 kDa. To simplify the purification procedure, the glu369 coding sequence was cloned into the vector pKJD4. The produced OmpA-His-Glu369 harboured OmpA signal sequence for E. coli periplasmic localization and followed by a 6His residues for its purification. The purified His-tagged proteins revealed two bands on SDS-PAGE analysis with molecular masses of about 30.5 (His-Glu369) and 32.5 kDa (OmpA-His-Glu369). They had the ability to inhibit the growth of phytopathogenic fungus Alternaria alternata. These favourable properties make the endoβ-1,3-1,4-glucanase a good candidate for biotechnological applications.

International Journal of Biological Macromolecules, 2018

Bacillus thuringiensis Vip3A protein has been widely used for crop protection and for delay resis... more 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% ± 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.

Toxicon, 2018

LC50 Vip3Aa16 : 92.79 ng/cm 2 LC50 Vip3Aa16 : 9.025 ng/cm 2 Vip3Aa16 Secreted protein from Bacill... more LC50 Vip3Aa16 : 92.79 ng/cm 2 LC50 Vip3Aa16 : 9.025 ng/cm 2 Vip3Aa16 Secreted protein from Bacillus thuringiensis Vip3Aa16 Secreted protein from Bacillus thuringiensis Agrotis segetum Histopathlological effect on Agrotis segetum midgut larvae Insecticidal activity on Agrotis segetum larvae

Journal of Basic Microbiology, 2016

To study the importance of N-and C-terminus of Bacillus thuringiensis Vip3Aa16 (L121I) toxin (88 ... more 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.

Biochemical and biophysical research communications, Jan 22, 2017

The basis of the different susceptibility of Ephestia kuehniella to the Cry1Aa and Cry1Ac δ-endot... more The basis of the different susceptibility of Ephestia kuehniella to the Cry1Aa and Cry1Ac δ-endotoxins from Bacillus thuringiensis kurstaki BNS3 was studied. Both toxins bound specifically to the BBMV of E. kuehniella. The result of the ligand blot showed that Cry1Ac bound to three putative receptors of about 100, 65 and 80 kDa and Cry1Aa interacted only with a 100 kDa protein. Pronase digestion of the BBMV-bound toxins was used to analyze the toxin insertion. Both toxins inserted into the BBMV as monomers however, a 14 kDa peptide of α4-α5 which correspond to the oligomeric form of this peptide was detected in case of Cry1Ac only. Analysis of the in vitro oligomerisation of these toxins in the presence of the BBMV of E. kuehniella showed reduced oligomer formation in case of Cry1Aa in comparison with Cry1Ac. Taken together, these results strongly suggest that the difference of toxicity between Cry1Aa and Cry1Ac to E. kuehniella is due to a deficient oligomerisation of Cry1Aa.

Pesticide Biochemistry and Physiology, 2017

Influence of Ephestia kuehniella stage larvae on the potency of Bacillus thuringiensis Cry1Aa del... more Influence of Ephestia kuehniella stage larvae on the potency of Bacillus thuringiensis Cry1Aa delta-endotoxin

International Journal of Biological Macromolecules, 2016

The Bacillus thuringiensis S1/4 strain was previously found to harbour vip1S, vip2S, and vip3 gen... more 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/β 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.

Microbiological Research, 2016

Bacillus species are attractive due to their potential use in the biological control of fungal di... more 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.

Process Biochemistry, 2014

Abstract A study of the recovery process of Bacillus thuringiensis (Bt) strain BLB1-based bioinse... more Abstract A study of the recovery process of Bacillus thuringiensis (Bt) strain BLB1-based bioinsecticides using ultrafiltration showed that the highest recovery of BLB1 bioactive components was achieved by using a 5 kDa MWCO ultrafiltration membrane to concentrate the supernatant. A bioassay against Ephestia kuehniella showed that the retentate was approximately 49% more toxic than the supernatant, with a lethal concentration (LC50) of 194.00 μg g−1 in the retentate compared to 380.00 μg g−1 in the supernatant. Furthermore, compared to the centrifugate (spore-crystal mixture) mixed with supernatant or saline water, the centrifugate retentate showed 42.7% and 56% higher toxicity against E. kuehniella, respectively. The involvement of proteases, chitinases and Vip3 proteins restored to the retentate suggests synergetic activity with delta-endotoxins and spores, and this activity was confirmed by enzymatic activity and western blot analysis. The formulated centrifugate retentate maintained stability and toxicity after 1 year of storage at 4 °C. These data suggest that the ultrafiltration process enabled significant virulence factors recovery from BLB1 supernatant grown in a complex medium and resulted in a highly toxic and stable formulation.

Acta Tropica, 2014

The effectiveness of 10 low-cost UV-absorbers in protecting Bacillus thuringiensis subsp. kurstak... more The effectiveness of 10 low-cost UV-absorbers in protecting Bacillus thuringiensis subsp. kurstaki BLB1 toxins against inactivation by UV-A and UV-B irradiation was evaluated in this study. Among them, two by-products, molasses and olive mill wastewater (OMW) were selected for further studies. They were tested at different concentrations of 0.05, 0.1, 0.15 and 0.2% using the para-aminobenzoic acid (PABA) as a common UV protectant. Interestingly, addition of PABA and OMW to BLB1 formulations was found to be most effective in protecting BLB1 spores at 90.8 and 76.4% respectively and in preserving deltaendotoxin concentration at a level of 81.7 and 72.2%, respectively when used at a concentration of 0.2%. The lowest preserved spores (46.3%) and delta-endotoxin level (12.4%) was found using molasses. In contrast, spore count and delta-endotoxin concentration were completely reduced after an exposure of unprotected Bt strain BLB1 to UV radiations up to 96 h. SDS-PAGE analysis of protected and unprotected samples revealed that delta-endotoxin bands (130, 65-70 kDa) were conserved until 96 h of UV exposure in presence of PABA or OMW compared with their disappearance in presence of molasses after 72 h of exposure and their dramatically decline from 8 h of exposure in unprotected mixture. A complete loss of larvicidal toxicity against Ephestia kuehniella was found after 24 h of exposure in absence of any UVabsorber. Addition of OMW or PABA offered the highest levels of insecticidal activity with 63.2 and 74.7% of residual toxicity, respectively. Whereas, molasses addition, as UV protectant retained only 26.3% of residual activity after 96 h of exposure. Therefore, addition of OMW by-product to Bt formulation may be a suitable alternative to others synthetic chemical compounds. OMW may also provided added value, be environmentally friendly and less hazardous, when used at low concentration.

Applied Biochemistry and Biotechnology, 2014

Tuta absoluta is a destructive moth of Solanaceae plants and especially tomatoes. Here, we consid... more 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.

Molecular Biotechnology, 2009

Photorhabdus temperata and Bacillus thuringiensis are entomopathogenic bacteria exhibiting toxici... more 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.

Molecular Biotechnology, 2009

Vegetative insecticidal protein (Vip) is a class of insecticidal proteins produced by many Bacill... more Vegetative insecticidal protein (Vip) is a class of insecticidal proteins produced by many Bacillus thuringiensis strains during their vegetative growth stage. The vip3LB gene of B. thuringiensis strain BUPM95, which encodes a protein active against the Lepidoptera olive tree pathogenic insect Prays oleae, was cloned into pET-14b vector and overexpressed in Escherichia coli. The expressed Vip3LB protein, found in the E. coli cytoplasmic fraction, was purified and used to produce anti-Vip3LB antibodies. Using the midgut extract of P. oleae, the purified Vip3LB bound to a 65-kDa protein, whereas Cry1Ac toxin bound to a 210-kDa midgut putative receptor. This result justifies the importance of the biological pest control agent Vip3LB that could be used as another alternative particularly in case of resistance to Cry toxins.

Journal of Microbiology and Biotechnology, 2013

Journal of Basic Microbiology, 2012

The bacterium Bacillus thuringiensis was recognized for its entomopathogenic activities related t... more 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/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 didn't 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.

Current Microbiology, 2011

The Vegetative insecticidal Vip3A proteins display a wide range of insecticidal spectrum against ... more 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.