The role of the calmodulin-binding and calmodulin-like domains of the epidermal growth factor receptor in tyrosine kinase activation.

The epidermal growth factor receptor (EGFR) harbors a calmodulin (CaM)-binding domain (CaM-BD) and a CaM-like domain (CaM-LD) upstream and downstream, respectively, of the tyrosine kinase (TK) domain. We demonstrate in this paper that deletion of the positively charged CaM-BD (EGFR/CaM-BD∆) inactivated the TK activity of the receptor. Moreover, deletion of the negatively charged CaM-LD (EGFR/CaM-LD∆), leaving a single negative residue (glutamate), reduced the activity of the receptor. In contrast, substituting the CaM-LD with a histidine/valine-rich peptide (EGFR/InvCaM-LD) caused full inactivation. We also demonstrated using confocal microscopy and flow cytometry that the chimera EGFR-green fluorescent protein (GFP)/CaM-BD∆, the EGFR/CaM-LD∆, and EGFR/InvCaM-LD mutants all bind tetramethylrhodamine-labelled EGF. These EGFR mutants were localized at the plasma membrane as the wild-type receptor does. However, only the EGFR/CaM-LD∆ and EGFR/InvCaM-LD mutants appear to undergo ligand-dependent internalization, while the EGFR-GFP/CaM-BD∆ mutant seems to be deficient in this regard. The obtained results and in silico modelling studies of the asymmetric structure of the EGFR kinase dimer support a role of a CaM-BD/CaM-LD electrostatic interaction in the allosteric activation of the EGFR TK.

Towards novel Cry toxins with enhanced toxicity/broader: a new chimeric Cry4Ba / Cry1Ac toxin.

Attempts have been made to express or to merge different Cry proteins in order to enhance toxic effects against various insects. Cry1A proteins of Bacillus thuringiensis form a typical bipyramidal parasporal crystal and their protoxins contain a highly conserved C-terminal region. A chimerical gene, called cry(4Ba-1Ac), formed by a fusion of the N-terminus part of cry4Ba and the C-terminus part of cry1Ac, was constructed. Its transformation to an acrystalliferous B. thuringiensis strain showed that it was expressed as a chimerical protein of 116 kDa, assembled in spherical to amorphous parasporal crystals. The chimerical gene cry(4Ba-1Ac) was introduced in a B. thuringiensis kurstaki strain. In the generated crystals of the recombinant strain, the presence of Cry(4Ba-1Ac) was evidenced by MALDI-TOF. The recombinant strain showed an important increase of the toxicity against Culex pipiens larvae (LC50 = 0.84 mg l-1 ± 0.08) compared to the wild type strain through the synergistic activity of Cry2Aa with Cry(4Ba-1Ac). The enhancement of toxicity of B. thuringiensis kurstaki expressing Cry(4Ba-1Ac) compared to that expressing the native toxin Cry4Ba, might be related to its a typical crystallization properties. The developed fusion protein could serve as a potent toxin against different pests of mosquitoes and major crop plants.

A Bacillus subtilis strain as probiotic in poultry: selection based on in vitro functional properties and enzymatic potentialities.

We have proposed and validate an in vitro probiotic selection, based on enzymatic potentialities associated to well-established probiotic functional properties. A new Bacillus subtilis HB2 isolate, selected based on its high extracellular enzyme production, was chosen as a probiotic candidate for application as animal feed supplement. The HB2 strain showed an excellent acid and bile salts tolerance, a strong adhesion to chick enterocytes and produced antimicrobials against pathogens. An in vivo trial in poultry farming was conducted to evaluate the HB2 probiotic performance. After 35 days, HB2 achieved the higher growth performance than the control groups. The mortality and the feed conversion ratio were significantly decreased. Finally, the HB2 treated group showed wet litter and less severe ammonia odor in the atmosphere. Our study provides new insights into the importance of enzymatic potentialities, associated with the common functional properties, as a novel approach for probiotic selection.

Characterization of thermo-solvent stable protease from Halobacillus sp. CJ4 isolated from Chott Eldjerid hypersaline lake in Tunisia.

About 110 newly isolated halophilic and halotolerant bacteria were screened for protease production. A moderately halophilic strain (CJ4), isolated from Chott Eldjerid Hypersaline lake in Tunisia, showed the highest activity on agar plate and was then selected. The biochemical and physiological characterization of the isolate along with the 16S rRNA sequence analysis placed it in the genus Halobacillus. Protease production was maximal at 120 g/L NaCl (2 M) and it started from the post-exponential phase reaching a maximum level at the early decline phase of bacterial growth. Protease activity was optimal at 0.4 M NaCl, pH 9 and 45 °C. It showed an excellent stability over wide ranges of temperatures (30-60 °C), NaCl concentrations (0-5 M), and pH values (5-10), which make it a good candidate for industrial applications at harsh conditions. Crude protease was strongly inhibited by PMSF revealing the dominance of serine proteases. Protease activity exhibited high stability in the presence of several organic solvents and detergent additives. These findings make Halobacillus sp. CJ4 protease with a great interest for many biotechnological applications at high salt or low water content such as peptide synthesis and detergent formulation.

Biopriming of Durum Wheat Seeds with Endophytic Diazotrophic Bacteria Enhances Tolerance to Fusarium Head Blight and Salinity.

There is growing interest in the use of bio inoculants based on plant growth-promoting bacteria (PGPB) to promote plant growth under biotic and abiotic stresses. Here, we provided a detailed account of the effectiveness of a number of endophytic PGPB strains, isolated from the roots of the halophyte Salicornia brachiata in promoting durum wheat growth and enhancing its tolerance to salinity and fusarium head blight (FHB) disease. Bacillus spp. strains MA9, MA14, MA17, and MA19 were found to have PGPB characteristics as they produced indole-3-acetic acid, siderophores, and lytic enzymes, fixed free atmospheric nitrogen, and solubilized inorganic phosphate in vitro. Additionally, the in vivo study that involved in planta inoculation assays under control and stress conditions indicated that all PGPB strains significantly (p < 0.05) increased the total plant length, dry weight, root area, seed weight, and nitrogen, protein, and mineral contents. Particularly, the MA17 strain showed a superior performance since it was the most efficient in reducing disease incidence in wheat explants by 64.5%, in addition to having the strongest plant growth promotion activity under salt stress. Both in vitro and in vivo assays showed that MA9, MA14, MA17, and MA19 strains were able to play significant PGPB roles. However, biopriming with Bacillus subtilis MA17 offered the highest plant growth promotion and salinity tolerance, and bioprotection against FHB. Hence, it would be worth testing the MA17 strain under field conditions as a step towards its commercial production. Moreover, the strain could be further assessed for its plausible role in bioprotection and growth promotion in other crop plants. Thus, it was believed that the strain has the potential to significantly contribute to wheat production in arid and semi-arid regions, especially the salt-affected Middle Eastern Region, in addition to its potential role in improving wheat production under biotic and abiotic stresses in other parts of the world.

Isolation and Characterization of Lytic Pseudomonas aeruginosa Bacteriophages Isolated from Sewage Samples from Tunisia.

Bacteriophages could be a useful adjunct to antibiotics for the treatment of multidrug-resistant Pseudomonas aeruginosa infections. In this study, lytic P. aeruginosa myoviruses PsCh, PsIn, Ps25, and Ps12on-D were isolated from Tunisian sewage samples. Phage Ps12on-D displayed an adsorption time of ~10 min, a short latency period (~10 min), and a large burst size (~115 PFU per infected cell) under standard growth conditions. All phages were active at broad temperature (4 °C to 50 °C) and pH (3.0 to 11.0) ranges and were able to lyse a wide variety of P. aeruginosa strains isolated from clinical and environmental samples worldwide. Illumina sequencing revealed double-stranded DNA genomes ranging from 87,887 and 92,710 bp with high sequence identity to Pseudomonas phage PAK_P1. All four phages based on sequence analysis were assigned to the Pakpunavirus genus. The presented characterization and preclinical assessment are part of an effort to establish phage therapy treatment as an alternative strategy for the management of multidrug-resistant P. aeruginosa infections in Tunisia.

Toward the smallest active subdomain of a TIM-barrel fold: insights from a truncated α-amylase.

AmyTM is a truncated mutant of the α-amylase of Bacillus stearothermophilus US100. It has been derived from the wild type amylase gene via a reading frame shift, following a tandem duplication of the mutant primer, associated to an Adenine base deletion. AmyTM was composed of 720 nucleotides encoding 240 amino acid residues out of 549 of the wild type. The AmyTM protein was devoided of the three catalytic residues but still retains catalytic activity. It is Ca-independent maltotetraose producing amylase, optimally active at pH 6 and 60°C, under monomeric or multimeric forms. AmyTM is the smallest functional truncated TIM barrel. It contains the ßαßα unit as the minimal subdomain associated to an enzymatic function. The enzymatic activity can, until now, be attributed to the presence of the whole domain B, in the structure of AmyTM. This mutant revealed, for the first time, the regeneration of a catalytic site after its abolition. This fact may be considered as the restoration of a primitive active site, which was lost in the course of evolution toward more stable domains.

First Report of Filamentous Phages Isolated from Tunisian Orchards to Control Erwinia amylovora.

Newly discovered Erwinia amylovora phages PEar1, PEar2, PEar4 and PEar6 were isolated from three different orchards in North Tunisia to study their potential as biocontrol agents. Illumina sequencing revealed that the PEar viruses carry a single-strand DNA genome between 6608 and 6801 nucleotides and belong to the Inoviridae, making them the first described filamentous phages of E. amylovora. Interestingly, phage-infected cells show a decreased swimming and swarming motility and a cocktail of the four phages can significantly reduce infection of E. amylovora in a pear bioassay, potentially making them suitable candidates for phage biocontrol.

The importance of an extra loop in the B-domain of an alpha-amylase from B. stearothermophilus US100.

To provide insight into the potential role of a loop in domain B of several bacterial alpha-amylases, molecular and structural investigation of Bacillus stearothermophilus alpha-amylase (Amy US100) was used as a model. Combination deletion mutants of G(213), I(214) and G(215), described as a loop-forming on the surface bacterial amylases, were subjected to biochemical and structural investigation. Thermoactivity, thermostability as well calcium requirement were studied for each mutant. Thus, deletion of one residue differently affects only the thermostability. Shortening the loop by deletion of G(213)-I(214) or I(214)-G(215) improved the thermostability and reduces calcium requirement. However, the deletion of three residues has a negative effect on thermostability and reduces the optimal temperature by 17 degrees C. The structural investigation showed that stabilizing deletions contribute to reinforce the architecture of domain B and the active site conformation. The deletion of three residues reduces the flexibility of this region and abolishes a denser hydrogen bond network.

Structural investigation and homology modeling studies of native and truncated forms of alpha-amylases from Sclerotinia sclerotiorum.

The filamentous ascomycete Sclerotinia sclerotiorum is well known for its ability to produce a large variety of hydrolytic enzymes for the degradation of plant polysaccharide material. Two alpha-amylases designated as ScAmy54 and ScAmy43 were biochemically characterized and predicted to play an important role in starch degradation. Those enzymes produce specific oligosaccharides, essentially maltotriose, that have a considerable commercial interest. The primary structures of the two enzymes were analyzed by N-terminal sequencing, MALDI-TOF mass spectrometry, and cDNA cloning, and implied that the two proteins have the same N-terminal catalytic domain and ScAmy43 was produced from ScAmy54 by truncation of 96 amino acids at the carboxyl-terminal region. The result of genomic analysis suggested that the two enzymes originated from the same alpha-amylase gene and that truncation of ScAmy54 to ScAmy43 occurred probably during the S. sclerotiorum cultivation. The structural gene of ScAmy54 consisted of 9 exons and 8 introns, containing a single 1,500-bp open reading frame encoding 499 amino acids including a signal peptide of 21 amino acids. ScAmy54 exhibited high amino acid identity to other liquefying fungal alpha-amylases, essentially in the four conserved regions and in the putative catalytic triad. A 3D structure model of ScAmy54 and ScAmy43 was built using the 3D structure of 2guy from A. niger as template. ScAmy54 with three domains A, B, and C, including the well-known (beta/alpha)8-barrel motif in domain A, has a typical structure of the alpha-amylase family. ScAmy43 composed only of domains A and B constitutes a smallest fungal alpha-amylase with only a catalytic domain.

Evidence of the importance of the Met115 for Bacillus thuringiensis subsp. israelensis Cyt1Aa protein cytolytic activity in Escherichia coli.

Cyt1Aa is a cytolytic toxin, found together with the delta-endotoxins in Bacillus thuringiensis subsp. israelensis parasporal insecticidal crystals. The latter are used as an environmental friendly insecticide against mosquitoes and black flies. Contrary to Cry delta-endotoxin, the mode of action of Cyt1Aa is not completely understood. In the absence of direct structural data, a novel mutated cyt1Aa gene was used to obtain indirect informations on Cyt1Aa conformation changes in the lipid membrane environment. A mutated cyt1Aa gene named cyt1A97 has been isolated from a B. thuringiensis israelensis strain named BUPM97. The nucleotide sequence predicted a protein of 249 amino acids residues with a calculated molecular mass of 27 kDa. Both nucleotide and amino acid sequences similarity analysis revealed that cyt1A97 presents one amino acid different from the native cyt1Aa gene. This mutation was located in the helix alpha C corresponding to a substitution of Met(115) by a Thr. The heterologous expression of the cyt1A97 and another cyt1Aa-type gene called cyt1A98, not affected by such mutation used as control, was performed in Escherichia coli. It revealed that the mutated Cyt1A97 protein was over produced as inclusion bodies showing a very weak toxicity to E. coli contrarily to Cyt1A98 that stopped E. coli growth. Hence, hydrophobic residue Met at position 115 of Cyt1Aa should play a very important role for the maintenance of the structure and cytolytic functions of Cyt1Aa.

Thermostability enhancement and change in starch hydrolysis profile of the maltohexaose-forming amylase of Bacillus stearothermophilus US100 strain.

The implications of Asn315 and Val450 in the atypical starch hydrolysis profile of Bacillus stearothermophilus Amy (a-amylase) US100 have been suggested previously [Ben Ali, Mhiri, Mezghani and Bejar (2001) Enzyme Microb. Tech. 28, 537-542]. In order to confirm this hypothesis, three mutants were generated. Of these two have a single mutation, N315D or V450G, whereas the third contains both mutations. Analysis of the starch breakdown-profile of these three mutants, as well as of the wild-type, allowed us to conclude that each single mutation induces a small variation in the hydrolysis product. However, the major end product produced by the double mutant shifts from maltopentaose/maltohexaose to maltose/maltotriose, confirming the involvement of these two residues in starch hydrolysis. The superimposition of AmyUS100 model with that of Bacillus licheniformis shows in AmyUS100 an additional loop containing residues Ile214 and Gly215. Remarkably, the deletion of these two residues increases the half-life at 100 degrees C from 15 min to approx. 70 min. Moreover, this engineered amylase requires less calcium, 25 p.p.m. instead of 100 p.p.m., to reach maximal thermostability.

Acute and delayed responses of C-reactive protein, malondialdehyde and antioxidant markers after resistance training session in elite weightlifters: Effect of time of day.

The aim of this study was to investigate the effect of an Olympic-Weightlifting-session followed by 48-h recovery period on the oxidative and antioxidant parameters' diurnal variation. Nine weightlifters (21 ± 0.5 years) performed, in randomized order, three Olympic-Weightlifting-sessions at 08 h:00, 14 h:00 and 18 h:00. Blood samples were collected: at rest and 3 min and 48 h after each session. C-reactive protein (CRP), rate of lipid peroxidation and antioxidant activities were assessed. At rest, analysis of variance showed a significant time of day (TOD) effect (p < 0.05) for uric acid, catalase and glutathione peroxidase with higher values at 14 h:00 and 18 h:00 compared with 08 h:00. However, no significant TOD effect for malondialdehyde, total bilirubin and CRP was observed. Given the profound changes (p < 0.001) in the post-training session values, these diurnal variations have been altered immediately and even 48 h after the training sessions. Despite the significant decreases in the post-training values after the 48-h recovery period (p < 0.05), levels of lipid peroxidation and enzymatic defense remained elevated (p < 0.05) 48 h after the morning training session. However, after the afternoon and evening sessions, the same period was sufficient to return values to the baseline levels. In conclusion, the morning session seems to generate the most important acute and delayed lipid peroxidation responses. Therefore, weightlifting coaches should avoid scheduling their training sessions in the morning-hours.

Glucose isomerase of the Streptomyces sp. SK strain: purification, sequence analysis and implication of alanine 103 residue in the enzyme thermostability and acidotolerance.

The glucose isomerase gene (xylA) from the Streptomyces sp. SK strain encodes a 386-amino-acid protein (42.7 kDa) showing extensive identities with many other bacterial glucose isomerases. We have shown by gel filtration chromatography and SDS-PAGE analysis that the purified recombinant glucose isomerase (SKGI) is a 180 kDa tetramer of four 43 kDa subunits. Sequence inspection revealed that this protein, present some special characteristics like the abundance of hydrophobic residues and some original amino-acid substitutions, which distinguish SKGI from the other GIs previously reported. The presence of an Ala residue at position 103 in SKGI is especially remarkable, since the same amino-acid was found at the equivalent position in the extremely thermostable GIs from Thermus thermophilus and Thermotoga neapolitana; whereas a Gly was found in the majority of less thermostable GIs from Streptomyces. The Ala103Gly mutation, introduced in SKGI, significantly decreases the half-life time at 90 degrees C from 80 to 50 min and also shifts the optimum pH from 6.5 to 7.5. This confirms the implication of the Ala103 residue on SKGI thermostability and activity at low pH. A homology model of SKGI based on the SOGI (that of Streptomyces olivochromogenes) crystal structure has been constructed in order to understand the mutational effects on a molecular scale. Hence, the Ala103Gly mutation, affecting enzyme properties, is presumed to increase molecular flexibility and to destabilize, in particular at elevated temperature, the 91-109 loop that includes the important catalytic residue, Phe94.

Enhancement of the thermostability of the maltogenic amylase MAUS149 by Gly312Ala and Lys436Arg substitutions.

Based on sequence alignments and homology modeling, Gly 312 and Lys 436 of the maltogenic amylase from Bacillus sp. US149 (MAUS149) were selected as targets for site-directed mutagenesis to improve the thermostability of the enzyme. Variants of MAUS149 with amino acid substitutions G312A, K436R and G312A-K436R had substrate specificities, kinetic parameters and pH optima similar to those of the wild-type enzyme; however, the enzymes with substitutions K436R and G312A-K436R, had an optimal temperature of 45 °C instead of the 40 °C for the wild-type enzyme. The half-life time at 55 °C increased from 15 to 25 min for the double mutant. Molecular modeling suggests that the increase in thermostability was due to new hydrophobic interactions and the formation of a salt bridge and hydrogen bond in the G312A and K436R variants, respectively. The double mutant could be a potential candidate for application in the bread industry.

Molecular cloning, structural analysis and modelling of the AcAFP antifungal peptide from Aspergillus clavatus.

An abundantly secreted thermostable peptide (designed AcAFP) with a molecular mass of 5777 Da was isolated and purified in a previous work from a local strain of A. clavatus (VR1). Based on the N-terminal amino acid (aa) sequence of the AcAFP peptide, an oligonucleotide probe was derived and allowed the amplification of the encoding cDNA by RT-PCR. This cDNA fragment encodes a pre-pro-protein of 94 aa which appears to be processed to a mature product of 51 aa cys-rich protein. The deduced aa sequence of the pre-pro-sequence reveals high similarity with ascomycetes antifungal peptide. Comparison of the nucleotide sequence of the genomic fragment and the cDNA clone revealed the presence of an open reading frame of 282 bp interrupted by two small introns of 89 and 56 bp with conserved splice site. The three-dimensional (3D) structure modeling of AcAFP exhibits a compact structure consisting of five anti-parallel beta barrel stabilized by four internal disulfide bridges. The folding pattern revealed also a cationic site and spatially adjacent hydrophobic stretch. The antifungal mechanism was investigated by transmission and confocal microscopy. AcAFP cause cell wall altering in a dose-dependent manner against the phytopathogenic fungus Fusarium oxysporum.

Heterologous expression, secretion and characterization of the Geobacillus thermoleovorans US105 type I pullulanase.

Pullulanase type I of Geobacillus thermoleovorans US105 strain (PUL US105) was produced and secreted efficiently in the E. coli periplasmic or extracellular fraction using two different signal peptides. Hence, the open reading frame was connected downstream of the lipase A signal peptide of Bacillus subtilis strain leading to an efficient secretion of an active form enzyme on the periplasmic fraction. In addition, pul US105 was fused to the alpha-amylase signal sequence of the Bacillus stearothermophilus US100 strain. The monitoring of the pullulanase activity and Western blot analysis for this last construction showed that the most activity was found in the supernatant culture, proving the efficient secretion of this natively cytoplasmic enzyme as an active form. The PUL US105 was purified to homogeneity from the periplasmic fraction, using heat treatment, size exclusion, and anion-exchange chromatography. The native pullulanase has a molecular mass of 160 kDa and is composed of two identical subunits of 80 kDa each. It was independent for metallic ions for its activity, while its thermostability was obviously improved in presence of only 0.1 mM CaCl2.