Improvement of Bacillus thuringiensis Bacteriocin Production Through Culture Conditions Optimization

Abstract

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.     

Traumatismes fermés des bourses: stratégie de prise en charge

Introduction

Blunt scrotal trauma is increasingly frequent, but surgical exploration of these cases of trauma remains controversial. The objectives of this study were to assess the diagnostic value of clinical examination and ultrasound in testicular trauma and to analyse the complications of the various treatments proposed (surgical and medical treatments), in order to more clearly define the place of medical or surgical treatment in this form of trauma in young adults.

Patients and Methods

130 cases of blunt scrotal trauma were managed between 1993 and 2004. In the absence of clinical and ultrasound criteria of severity (haematocele, very large intratesticular haematoma, rupture of the tunica albuginea), medical treatment consisting of rest, anti-inflammatory drugs, and testicular support was instituted. Surgical exploration was performed when serious lesions of the testis were suspected. Scrotal ultrasound was performed in 68 patients and 46 of them underwent scrotal exploration. The sensitivity and specificity of scrotal ultrasound were determined by comparing radiological findings with definitive intraoperative findings. The immediate morbidity and long-term sequelae were analysed.

Results

The mean age of the patients was 24 years (range: 4 to 73 years). The clinical features were dominated by pain (73.8%) and scrotal swelling (89.2%). The sensitivity of testicular ultrasound was 34.7% for testicular rupture, 100% for testicular contusions, 33.3% for intratesticular hematoma and 76.9% for haematocele. Medical and surgical treatments were instituted in 29.2% and 70.7% of cases, respectively. With a mean follow-up of 8 months, chronic pain and testicular atrophy were observed in 18% and 5.5% of cases, respectively.

Conclusion

In the absence of signs of severity, medical treatment with regular surveillance remains justified. However, in the presence of doubtful clinical or ultrasound findings, surgical exploration must be performed as soon as possible     

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-Tn 10 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-Tn 10 transposition occurred in different sites. Clone MB1, containing a mini-Tn 10 single-copy insertion, lost the BF4 synthesis, but maintained its immunity to BF4. The flanking sequences surrounding the mini-Tn 10 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.     

Iron bioavailability and weight gains to iron-deficient rats fed a commonly consumed tunisian meal ‘bean seeds ragout’ with or without beef and with green or black tea decoction

The Fe bioavailability and the weight gains were evaluated in rats fed a commonly consumed Tunisian meal 'bean seeds ragout' (BSR), with or without beef and with black or green tea decoction. The Fe bioavailability was evaluated in Fe-deficient rats by the hemoglobin repletion method and the Fe stored in the liver. The addition of beef to the BSR significantly increased the Fe bioavailability from this meal by 147% and the reserve of Fe stored in the liver by 77% (P < 0.001). In contrast, both black and green tea decoctions caused a significant decrease of the Fe bioavailability from BSR meal (-19.6 +/- 4.9% and -14.9 +/- 4.1%, respectively). The reserve of Fe stored in the liver was significantly lower in the BSR, the black and the green tea groups than in the positive control group (FeSO4). The weight gains were significantly lower in the black and the green tea groups (3.9 +/- 5.7 g, 13 +/- 1.9 g, respectively) than in the BSR group (24.9 +/- 6 g). The addition of beef to BSR meal counteracted the inhibitory effect of the kidney bean and considerably improved the Fe bioavailability and the Fe stored in the liver of rats. The green tea decoction, which constitutes an important source of antioxidant factors, had the same inhibitory effect as the black tea decoction on the Fe bioavailability from BSR meal. In addition, both black and green teas significantly reduced the weight gains, where the black tea decoction has the most effect.     

Synthesized tyrosyl hydroxyphenylacetate,a novel antioxidant,anti-stress and antibacterial compound

New tyrosyl ester derivative, a naturally occurring phenol with interesting biological properties, has been synthesized in good yield by a direct esterification of tyrosol (Ty) with p-hydroxyphenylacetic acid (p-HPA) using Candida antarctica lipase as a catalyst. The response surface methodology was used to modulate the effects of the enzyme amount (10–50 mg), the tert-butanol/hexane (v/v) ratio (0.16–0.84), the temperature (35–55 °C) and the reaction time (15–45 h) on the tyrosyl hydroxyphenylacetate (Ty-HPA) conversion yield. Under the optimal predicted conditions (enzyme amount: 10 mg, solvents volume ratio 0.16, reaction temperature; 45 °C and 34 h of incubation), a high conversion yield of 79.33 ± 4% was reached. The obtained ester was purified and characterized by NMR, LC/MS and FT-IR methods. ABTS free radical quenching potency demonstrated that the esterified tyrosol (Ty-HPA) was more effective than the natural separated antioxidants: Ty and p-HPA. Furthermore, when used at a non-cytotoxic concentration (100 μM), tyrosyl ester showed significant effectiveness in preventing iron-induced oxidative stress in blood cells compared to the two separated compounds. The antibacterial activity of Ty, p-HPA, mixed solution of Ty + p-HPA and Ty-HPA was performed by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using a micro-well dilution method. Compared to the separated substrates, synthesized ester exhibits the most antibacterial effect mainly against Gram+ bacteria.     

Limites de la scintigraphie osseuse dans le suivi des métastases osseuses du carcinome mammaire : étude du centre tunisien

Bone metastases, a relatively frequent complication of breast carcinoma, are more and more associated with better survival. This in part through a specific detection and monitoring. In Sousse (Tunisia), bone scan is the exam most frequently performed for monitoring bone metastases, which is also the only functional exam available until December 2011. We studied the place of bone scan in the monitoring of these bone metastases. We followed 115 patients between 2000 and 2004 with a follow-up of 7 to 11 years. We used Measurable Criteria Disease response of Anderson (MDA) scintigraphic criteria. Complete remission was rare. Partial remission rate decreased gradually over time. Partial remission rate was 22.6% at the first evaluation and became equal to 10% at the fourth assessment. Stability is a false reassuring category since among the 27 cases judged to be stable after the first evaluation, only nine continued to be stable at the fourth evaluation and one at the sixth. In conclusion, bone scan is reliable only in cases of complete or partial remission. For other stages bone scintigraphy had many limitations since this examination informs about the osteoblastic reaction around metastases and not the tumour itself. ¹⁸F-FDG-PET/CT (¹⁸Fluoro-deoxy-glucose) indeed shows the metastasis metabolism itself, so is better positioned to evaluate the therapeutic response.     

Gene cloning and molecular characterization of the Talaromyces thermophilus lipase catalyzed efficient hydrolysis and synthesis of esters

A genomic bank from Talaromyces thermophilus fungus was constructed and screened using a previously isolated fragment lipase gene as probe. From several clones isolated, the nucleotide sequence of the lipase gene (TTL gene) was completed and sequenced. The TTL coding gene consists of an open reading frame (ORF) of 1083 bp encoding a protein of 269 Aa with an estimated molecular mass of 30 kDa. The TTL belongs to the same gene family as Thermomyces lanuginosus lipase (TLL, Lipolase®), a well known lipase with multiple applications. The promoter sequence of the TTL gene showed the conservation of known consensus sequences PacC, CreA, Hap2-3-4 and the existence of a particular sequence like the binding sites of Oleate Response Element (ORE) and Fatty acids Responsis Element (FARE) which are similar to that already found to be specific of lipolytic genes in Candida and Fusarium, respectively. Northern blot analysis showed that the TTL expression was much higher on wheat bran than on olive oil as sole carbon source. Compared to the Lipolase®, this enzyme was found to be more efficient for the hydrolysis and the synthesis of esters; and its synthetic efficiency even reached 91.6% from Waste Cooking Oil triglycerides.     

Kinetic and structural characterization of triacylglycerol lipases possessing phospholipase A1 activity

The pancreatic lipase gene family displays various substrate selectivities for triglycerides and phospholipids. The structural basis for this difference in substrate specificity has not been definitively established. Based on a kinetic comparative study between various pancreatic lipase family members, we showed here that porcine pancreatic lipase (PPL), which was so far classified as “classical lipase”, was able to hydrolyze phosphatidylcholine (PC). Amino acid sequence alignments revealed that Val260 residue in PPL lid could be critical for the interaction with lipid substrate. Molecular dynamics was applied to investigate PC binding modes within the catalytic cavity of PPL and human pancreatic lipase (HPL), aiming to explain the difference of specificity of these enzymes towards phospholipids. Results showed that with HPL, the oxyanion hole was not able to accommodate the PC molecule, suggesting that no activity could be obtained. With PPL, the formation of a large pocket involving Val260 allowed the PC molecule to come near the catalytic residues, suggesting that it could be hydrolyzed. One more interesting finding is that human pancreatic lipase related protein 2 could hydrolyze phospholipids through its PLA₁ and PLA₂ activities. Overall, our study shed the light on new structural features of the phospholipase activity of pancreatic lipase family members.