Effect of the chitin binding domain deletion from Bacillus thuringiensis subsp. kurstaki chitinase Chi255 on its stability in Escherichia coli.

Bacillus thuringiensis subsp. kurstaki BUPM255 secretes a chitobiosidase Chi255 having an expected molecular weight of 70.665 kDa. When the corresponding gene, chi255, was expressed in E. coli, the active form, extracted from the periplasmic fraction of E. coli/pBADchi255, was of about 54 kDa, which suggested that Chi255 was excessively degraded by the action of E. coli proteases. Therefore, in vitro progressive C-terminal Chi255 deleted derivatives were constructed in order to study their stability and their activity in E. coli. Interestingly, when the chitin binding domain (CBD) was deleted from Chi255, an active form (Chi2555Delta5) of expected size of about 60 kDa was extracted from the E. coli periplasmic fraction, without the observation of any proteolytic degradation. Compared to Chi255, Chi255Delta5 exhibited a higher chitinase activity on colloidal chitin. Both of the enzymes exhibit activities at broad pH and temperature ranges with maximal enzyme activities at pH 5 and pH 6 and at temperatures 50 degrees C and 40 degrees C, respectively for Chi255 and Chi255Delta5. Thus, it was concluded that the C-terminal deletion of Chi255 CBD might be a nice tool for avoiding the excessive chitinase degradation, observed in the native chitinase, and for improving its activity.

Proteolysis, histopathological effects, and immunohistopathological localization of delta-endotoxins of Bacillus thuringiensis subsp. kurstaki in the midgut of lepidopteran olive tree pathogenic insect Prays oleae.

Considering the fact that Prays oleae is one of the most pathogenic insects to the olive tree in the Mediterranean basin, particularly in Tunisia, the mode of action of Cry insecticidal toxins of Bacillus thuringiensis kurstaki in Prays oleae midgut was investigated. The proteolysis of Bacillus thuringiensis delta-endotoxins in the midgut was a key step in determining their potency against Prays oleae. The latter's proteases activated the delta-endotoxins early, yielding stable toxins. The in vitro and in vivo binding of these toxins to Prays oleae larvae midgut was studied immunohistochemically, evidencing a midgut columnar cell vacuolization, microvilli damage, and then a pass of epithelium cell content into the larvae midgut. Moreover, Bacillus thuringiensis toxins were shown to bind to the apical microvilli of the midgut epithelial cells. The in vitro study of the interaction of Prays oleae midgut proteins with biotinylated Bacillus thuringiensis toxins allowed the prediction of four suitable receptor proteins in Prays oleae.

Morphological and physiological changes of Staphylococcus aureus exposed to hypochlorous acid.

AIMS: To characterize hypochlorous acid (HOCl) stress resistance of Staphylococcus aureus and to assess physiological state and changes in cell morphology. METHODS AND RESULTS: Clinical wild-type strain of S. aureus was used in the stress with HOCl at concentrations ranging from 0 to 4 mg l(-1). Concentrations below 1.5 mg l(-1) caused no significant drop in viability. During 2 h of HOCl stress at 2 mg l(-1), there was appearance of minicells capable of passing through the 0.45 microm pores of filtration membranes. Intracellular proteins increased gradually to reach a level of 51% of dry weight and an enhanced synthesis of at least two proteins of 23 and 220 kDa was concluded. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Staphylococcus aureus can undergo morphological and physiological changes during 2 h of exposure to 2 mg l(-1) of HOCl, which represents an adaptative response towards the hypochlorous acid stress. This evolution limits the use of 0.45 microm pores size membrane filters for research on S. aureus in waters and the clinical environment.

In situ immunocytochemical detection of potyviral proteins in plant cells.

A method for in situ protein immunodetection using a peroxidase labeling system is described for detecting functional and structural proteins encoded by potato virus Y (Tunisian isolate) in plant tissues. Such Potyviruses are characterized by the accumulation of inclusion bodies containing viral encoded proteins other than coat protein. These proteins are functional at early stages of infection, making them easy to detect. Data are compared to those obtained by immunofluorescence techniques. Our technique can be used as a preliminary method for rapid detection of virus infection using antibodies directed against functional proteins.

Inhibition of potato virus Y NIa activity: preparation of monoclonal antibody directed against PVY NI protein that inhibits cleavage of PVY polyprotein.

A partially purified nuclear inclusion (NI) fraction was obtained from tobacco plants infected by potato virus Y (PVY). Four monoclonal antibodies (MAbs) were produced and characterized using this semipurified fraction as antigen. Data showed that only one was directed against NIa whereas two were directed against cytoplasmic inclusion (CI) protein and the last one against coat protein (CP). These results were due to the fact that the semipurified NI fraction was usually contaminated with CI and CP proteins. When used on in situ immunofluorescence method the anti-NIa MAb showed accumulation of the NIa protein in both nucleus and cytoplasm. In vivo, this MAb was able to detect different forms of the NIa protein including precursors and cleavage products. It was also able to inhibit the cleavage of the polyprotein detected in the semipurified NI.

[Determination of the frequency of the A2m (2) allele of immunoglobin A in the Tunisian population by RFLP analysis]. / Détermination de la fréquence de l'allèle A2M (2) des immunoglobulines A dans la population tunisienne par la technique de RFLP.

The human IgA2 subclass has two allotypes A2m(1) and A2m (2). Using the RFLP technique, we determined the A2m(2) frequency in a sample of 29 tunisian individuals. In accordance with Lefranc and all. [6], the presence of a polymorphic EcoRI site of the A2m(2) allele was confirmed in our RFLP study whereas no BamHI nor HindIII polymorphisms were shown. Among the 29 cases studies, 11 were heterozygous A2m2-1, 17 homozygous A2m1-1 and 1 homozygous A2m2-2. The A2m2 allele frequency in the Tunisian population (0,22) is intermediate between the European frequency and the African one.