Multiple linear regression models to simulate spore yields of Bacillus amyloliquefaciens BS13 through optimization of medium composition.

Bacillus amyloliquefaciens is a food spoilage spore-forming bacterium. Its spores are useful for multiple biotechnological applications. Nevertheless, few reports are available regarding the achievement of a high cell density and good sporulation effectiveness under fermentation conditions. Therefore, the current study was designed to optimize a low-cost fermentation medium allowing the highest sporulation yield by B. amyloliquefaciens strain BS13. Our data revealed that tryptone and starch were the best carbon and energy sources. In addition, two nitrogen sources namely, corn steep liquor (CSL) and yeast extract (YE), allowed a significant enhancement of spore production and they were both retained for further optimization. A combination of CaCl2 , MgSO4 , and MnSO4 showed a positive impact on spores' production. The composition of the optimized medium was (in g/L); tryptone 3, starch 15, CSL 13.5, YE 1.5, CaCl2 0.1, MgSO4 ·7H2 O 0.012, and MnSO4 ·7H2 O 0.0012. Such medium was further validated in a 400-L fermentor. The spore yield by B. amyloliquefaciens strain BS13 was enhanced from 3.0 × 1010 spores/mL under flask culture conditions to 6.2 × 1010 spores/mL when cultures were performed on large scale. Therefore, strain BS13 spore preparation could be proposed as a promising probiotic and a biocontrol agent useful for plants, animals, and humans.

Overproduction of the Bacillus thuringiensis Vip3Aa16 toxin and study of its insecticidal activity against the carob moth Ectomyelois ceratoniae.

The vip3Aa16 gene of Bacillus thuringiensis strain BUPM95 was cloned and expressed in Escherichia coli. Optimization of Vip3A16 protein expression was conducted using Plackett-Burman design and response surface methodology. Accordingly, the optimum Vip3A16 toxin production was 170µg/ml at 18h post-induction time and 39°C post-induction temperature. This corresponds to an improvement of 21times compared to the starting conditions. The insecticidal activity, evaluated against Ectomyelois ceratoniae, displayed an LC50 value of 40ng/cm(2) and the midgut histopathology of Vip3Aa16 fed larvae showed vacuolization of the cytoplasm, brush border membrane destruction, vesicle formation in the apical region and cellular disintegration.

Investigation of the steps involved in the difference of susceptibility of Ephestia kuehniella and Spodoptera littoralis to the Bacillus thuringiensis Vip3Aa16 toxin.

BUPM95 is a Bacillus thuringiensis subsp. kurstaki strain producing the Vip3Aa16 toxin with an interesting insecticidal activity against the Lepidopteran larvae Ephestia kuehniella. Study of different steps in the mode of action of this Vegetative Insecticidal Protein on the Mediterranean flour moth (E. kuehniella) was carried out in the aim to investigate the origin of the higher susceptibility of this insect to Vip3Aa16 toxin compared to that of the Egyptian cotton leaf worm Spodoptera littoralis. Using E. kuehniella gut juice, protoxin proteolysis generated a major band corresponding to the active toxin and another band of about 22kDa, whereas the activation of Vip3Aa16 by S. littoralis gut juice proteases generated less amount of the 62kDa active form and three other proteolysis products. As demonstrated by zymogram analysis, the difference in proteolysis products was due to the variability of proteases in the two gut juices larvae. The study of the interaction of E. kuehniella BBMV with biotinylated Vip3Aa16 showed that this toxin bound to a putative receptor of 65kDa compared to the 55 and 100kDa receptors recognized in S. littoralis BBMV. The histopathological observations demonstrated similar damage caused by the toxin in the two larvae midguts. These results demonstrate that the step of activation, mainly, is at the origin of the difference of susceptibility of these two larvae towards B. thuringiensis Vip3Aa16 toxin.

Improvement of Bacillus thuringiensis bacteriocin production through culture conditions optimization.

BUPM4 is a Bacillus thuringiensis subsp. kurstaki strain, isolated from Tunisian soil, producing an original bacteriocin named Bacthuricin F4. The optimization of the latter production conditions was carried out under several physicochemical conditions. It was found that the highest bacteriocin activity was reached at low aeration while bacteriocin synthesis yields were strongly reduced at higher ones. A balance between growth and bacteriocin synthesis, both highly dependent on aeration, was taken into account for the overproduction of bacteriocin. Both glucose and glycerol were shown to be necessary for Bacthuricin F4 maximal synthesis. In addition, the optimal carbon/nitrogen ratio for bacteriocin production is 9. In such optimal conditions, more than 4-fold greater bacteriocin production was obtained than when using TSB medium.

Stability and antibacterial activity of bacteriocins produced by Bacillus thuringiensis and Bacillus thuringiensis ssp. kurstaki.

Bacteriocins are antimicrobial peptides that are produced by bacteria and toxic to bacterial strains closely related to the producer strain. It has previously been reported that Bacillus thuringiensis strain NEB17 and Bacillus thuringiensis subsp. kurstaki BUPM4 produce the bacteriocins thuricin 17 (3,162 Da) and bacthuricin F4 (3,160.05 Da), respectively. Here, we demonstrate that these bacteriocins have functional similarities and show a similar spectrum of antimicrobial activities against indicator strains. We also studied the effects of sterilization methods on the recovery and biological activities of these bacteriocins. They were completely degraded by autoclaving and the two were similarly affected by the tested filter membranes. Polyvinylidene fluoride (PVDF), polyestersulfone (PES), and cellulose acetate (CA) are suitable for filter sterilization of these bacteriocins. The two bacteriocins were stable across a range of storage conditions. These data will facilitate their utilization in food preservation or agricultural applications.

Identification of a promoter for the vegetative insecticidal protein-encoding gene vip3LB from Bacillus thuringiensis.

The expression of vip3LB, one of the vegetative insecticidal protein-encoding genes of Bacillus thuringiensis, was studied at the transcriptional level. By primer extension analysis, we have identified, for the first time, the transcription start point of vip3-type gene. Upstream from vip3LB transcription start point, was found a nucleotide sequence partially homologous to the consensus sequence for the E sigma(E) holoenzyme of B. subtilis. Thus, it was strongly suggested that the identified vip3 promoter was under the control of sigma(35)-like enzyme, the B. thuringiensis homolog of sigma(E). The transcriptional activity from the promoter was detected at the vegetative stage of growth starting at mid-log phase as well as during the middle stage of sporulation.

Isolation of the Bacillus thuringiensis plasmid carrying Bacthuricin F4 coding genes and evidence of its conjugative transfer.

INTRODUCTION: Conjugation is an excellent natural mode of DNA transfer in vivo between bacteria, particularly when these conjugative elements carry technological traits such as bacteriocin encoding genes. In the present work, the bacteriocinogenic plasmid pIBF4 from Bacillus thuringiensis responsible of Bacthuricin F4 synthesis was isolated and characterized. METHODOLOGY: To isolate pIBF4, the total plasmid DNA from a non-bacteriocin transposant carrying the mini-Tn10 spectinomycin selective marker was extracted and used to transform Escherichia coli strain Top10. PIBF4 was extracted from the obtained transformant and then subjected to restriction enzyme analysis. Plasmid curing experiments were conducted to test the stability of pIBF4 at a stringent temperature of 42°C. Conjugative behavior of pIBF4 was assessed by mating experiments using the non-bacteriocin transposant mutant as a donor strain and several Bacillus thuringiensis strains as recipients. RESULTS: The pIBF4 plasmid was isolated and had a molecular weight of 19.1 kb. Ninety-five percent of cells retained the pIBF4 plasmid after 200 generations, demonstrating its high stability. PIBF4 was successfully transferred to Bacillus thuringiensis HD1CryB strain with a transfer frequency of 1x10(-8) transconjugants per donor cell. The study of the recipient host range revealed that pIBF4 is specifically transferable to Bacillus thuringiensis strains with variable transfer frequencies depending on the recipient host strain. CONCLUSION: Our results show that pIBF4 is a 19.1 kb highly stable plasmid transferable by conjugation to Bacillus thuringiensis strains with deferent transfer frequencies.

Purification and characterization of a new Bacillus thuringiensis bacteriocin active against Listeria monocytogenes, Bacillus cereus and Agrobacterium tumefaciens.

This study reports on the identification, characterization and purification of a new bacteriocin, named Bacthuricin F103, from a Bacillus thuringiensis strain BUPM103. Bacthuricin F103 production began in the early exponential phase and reached a maximum in the middle of the same phase. Two chromatographic methods based on high performance liquid chromatography and fast protein liquid chromatography systems were used to purify Bacthuricin F103. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that this bacteriocin had a molecular weight of approximately 11 kDa. It also showed a wide range of thermostability of up to 80 °C for 60 min and a broad spectrum of antimicrobial activity over a pH range of 3.0-10.0. This bacteriocin was noted, and for the first time, to exhibit potent antimicrobial activity against Agrobacterium subsp. strains, the major causal agents of crown gall disease in tomato and vineyard crops, and against several challenging organisms in food, such as Listeria monocytogenes and Bacillus cereus. Complete killing with immediate impact on cells was observed within a short period of time. The sequence obtained for Bacthuricin F103 by direct N-terminal sequencing shared considerable homology with hemolysin. Bacthuricin F103 was noted to act through the depletion of intracellular ions, which suggest that the cell membrane was a possible target to Bacthuricin F103.

Generation of Mini-Tn10 transposon insertion mutant library of Bacillus thuringiensis for the investigation of genes required for its bacteriocin production.

Bacillus thuringiensis strain BUPM4 is known for its ability to produce a bacteriocin, called Bacthuricin F4 (BF4), which inhibits the growth of several Gram-positive bacteria and particularly Bacillaceae. This study aimed to use the insertional transposon mutagenesis approach for disrupting and thus identifying genes associated with BF4 synthesis. Here, the mini-Tn10 transposon was used to generate a library of B. thuringiensis mutants. Twenty thousand clones were screened for the search of mutants with affected bacteriocin synthesis. By molecular hybridization, it was demonstrated that the mini-Tn10 transposition occurred in different sites. Clone MB1, containing a mini-Tn10 single-copy insertion, lost the BF4 synthesis, but maintained its immunity to BF4. The flanking sequences surrounding the mini-Tn10 insertion were cloned and sequenced. Homology searches of the surrounding ORFs revealed a strong similarity to a phage tail component, which allowed us to postulate that BUPM4 bacteriocin could be a phage tail-like one.