Probing the essential catalytic residues and substrate affinity in the thermoactive Bacillus stearothermophilus US100 L-arabinose isomerase by site-directed mutagenesis

The L-arabinose isomerase (L-AI) from Bacillus stearothermophilus US100 is characterized by its high thermoactivity and catalytic efficiency. Furthermore, as opposed to the majority of l-arabinose isomerases, this enzyme requires metallic ions for its thermostability rather than for its activity. These features make US100 L-AI attractive as a template for industrial use. Based on previously solved crystal structures and sequence alignments, we identified amino acids that are putatively important for the US100 L-AI isomerization reaction. Among these, E306, E331, H348, and H447, which correspond to the suggested essential catalytic amino acids of the L-fucose isomerase and the L-arabinose isomerase from Escherichia coli, are presumed to be the active-site residues of US100 L-AI. Site-directed mutagenesis confirmed that the mutation of these residues resulted in totally inactive proteins, thus demonstrating their critical role in the enzyme activity. A homology model of US100 L-AI was constructed, and its analysis highlighted another set of residues which may be crucial for the recognition and processing of substrates; hence, these residues were subjected to mutagenesis studies. The replacement of the D308, F329, E351, and H446 amino acids with alanine seriously affected the enzyme activities, and suggestions about the roles of these residues in the catalytic mechanism are given. The mutation F279Q strongly increased the enzyme's affinity for L-fucose and decreased the affinity for L-arabinose compared to that of the wild-type enzyme, showing the implication of this amino acid in substrate recognition.     

Plant regeneration via somatic embryogenesis from in vitro tissue culture in two Tunisian Cucumis melo cultivars Maazoun and Beji

Hypocotyl, cotyledon and zygotic embryo explants from two Tunisian Cucumis melo L. cultivars Beji and Maazoun, cultured on the MS medium added with 2,4-D (0.25–1 mg l⁻¹) and BA (0.10–0.50 mg l⁻¹), produce calluses with somatic embryos after 3 weeks of culture. For Beji c.v. the highest percentage (62.50%) of embryogenesis was observed for cotyledons. The average embryo number per callus was 10.40. Embryogenesis induction for zygotic embryos reached 33.50% with 29 embryos per callus. The embryogenesis ability of hypocotyls did not exceed 12.50% (2.50 embryos per callus). Somatic embryogenesis for Maazoun c.v. explants was less efficient. Embryos formation was observed only for cotyledons (29%) and zygotic embryos (25%). Cotyledonary staged embryos, when transferred to hormone free MS medium, germinated. The maximum germination rates were 51.50 and 44.50%, respectively for Maazoun and Beji c.v. The highest percentage (36.50%) of survival plants was noted for Beji c.v. Regenerants were diploids (2n = 2x = 24) and morphologically similar to their parents issued from seeds.     

Comparison of disc diffusion, Etest and agar dilution for susceptibility testing of colistin against Enterobacteriaceae

Aims: In this study, we compared different methods of colistin susceptibility testing, disc diffusion, agar dilution and Etest using a set of Enterobacteriaceae isolates that included colistin‐resistant strains. Methods and results: Susceptibility of 200 clinical isolates of Enterobacteriaceae to colistin was tested to compare agar dilution (reference method), disc diffusion (50 and 10 μg) and Etest. MICs (minimum inhibitory concentrations) were interpreted using the criteria established by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Colistin exhibited excellent activity against Escherichia coli and E. cloacae (MIC₉₀ = 0·5 mg l^?1). In contrast, colistin was less active against Klebsiella pneumoniae (MIC₉₀ = 16 mg l^?1). Resistance rates varied from 0% in E. coli to 1·8% in E. cloacae and 13% in K. pneumoniae. High rates of very major errors were observed in the disc diffusion test using either the criteria of the Comité de l’antibiogramme de la Société Française de Microbiologie (CA‐SFM) or the criteria of the Clinical and Laboratory Standards Institute (CLSI), respectively, 3·5 and 2·5%. When the criteria of Gales et al. were applied, the number of very major errors was reduced to one (0·5%). The Etest showed good concordance with agar dilution method. Conclusion: Disc susceptibility testing methods are unreliable on detecting colistin resistance. MIC should be determined to confirm the susceptibility results by disc diffusion. Significance and Impact of the study: We recommend the determination of MIC by Etest for all multidrug‐resistant Enterobacteriaceae when colistin is required for the treatment.     

Comparative evaluation of E-test and CLSI methods for Itraconazole,Fluconazole and Ketoconazole susceptibilities of Microsporum canis strains

The incidence of resistance to antifungal agents for dermatophytes is increasing, but most of the methods currently available to test the antifungal susceptibility of Microsporum canis still require standardization. The aims of this study were: (i) to evaluate the antifungal susceptibility of M. canis strains recovered from animals to ketoconazole (KTZ), fluconazole (FLZ) and itraconazole (ITZ) using a modified CLSI broth microdilution (CLSI M38-A2-BMD) and the E-test® protocols and (ii) to estimate the agreement between the methods. Tentative azole epidemiological cutoff values (ECVs) were also proposed in order to interpret the results of in vitro susceptibility tests and to establish the agreement between the E-test and CLSI BMD methods. A total of forty clinical M. canis strains from animals with skin lesions were tested, and the essential (EA) and categorical agreement (CA) between the two methods were determined. KTZ displayed the lowest MIC values, while ITZ and FLZ the highest. The ECV for KTZ and ITZ were 4 μg/ml, while those of FLZ was 64 μg/ml. Based on ECVs, about 88% of M. canis strains were susceptible to all azoles being a cross-resistance with ITZ-FLZ registered for one strain. A total of five M. canis strains showed MIC?>?ECV for FLZ using CLSI, while one strain showed MIC?>?ECV for ITZ using both tests. KTZ, ITZ and FLZ showed EA ranging from 92.5 to 95%, for all azoles and CA?>?97% except for FLZ (87.5%). The good CA between the E-test and the CLSI BMD provides evidence of the reliability of the former method to test the antifungal susceptibility of M. canis for ITZ and KTZ and not for FLZ.

     

Analysis of bacterial DNA in synovial tissue of Tunisian patients with reactive and undifferentiated arthritis by broad-range PCR, cloning and sequencing

Introduction

Bacteria and/or their antigens have been implicated in the pathogenesis of reactive arthritis (ReA). Several studies have reported the presence of bacterial antigens and nucleic acids of bacteria other than those specified by diagnostic criteria for ReA in joint specimens from patients with ReA and various arthritides. The present study was conducted to detect any bacterial DNA and identify bacterial species that are present in the synovial tissue of Tunisian patients with reactive arthritis and undifferentiated arthritis (UA) using PCR, cloning and sequencing.

Methods

We examined synovial tissue samples from 28 patients: six patients with ReA and nine with UA, and a control group consisting of seven patients with rheumatoid arthritis and six with osteoarthritis (OA). Using broad-range bacterial PCR producing a 1,400-base-pair fragment from the 16S rRNA gene, at least 24 clones were sequenced for each synovial tissue sample. To identify the corresponding bacteria, DNA sequences were compared with sequences from the EMBL (European Molecular Biology Laboratory) database.

Results

Bacterial DNA was detected in 75% of the 28 synovial tissue samples. DNA from 68 various bacterial species were found in ReA and UA samples, whereas DNA from 12 bacteria were detected in control group samples. Most of the bacterial DNAs detected were from skin or intestinal bacteria. DNA from bacteria known to trigger ReA, such as Shigella flexneri and Shigella sonnei, were detected in ReA and UA samples of synovial tissue and not in control samples. DNA from various bacterial species detected in this study have not previously been found in synovial samples.

Conclusion

This study is the first to use broad-range PCR targeting the full 16S rRNA gene for detection of bacterial DNA in synovial tissue. We detected DNA from a wide spectrum of bacterial species, including those known to be involved in ReA and others not previously associated with ReA or related arthritis. The pathogenic significance of some of these intrasynovial bacterial DNAs remains unclear.     

The Attractive Serratia plymuthica BMA1 Strain With High Rock Phosphate-Solubilizing Activity and Its Effect on the Growth and Phosphorus Uptake by Vicia faba L. Plants

Abstract

The present work aims to investigate the attractive ability of the newly isolated bacterium Serratia plymuthica BMA1, to release phosphorus (P) from rock phosphate (RP) and also to assess its beneficial effect in promoting the growth of Vicia faba. This strain exhibited the highest RP-solubilization activity ever reported, with 450?mg l^?1 of soluble P at 30?°C. At 10 and 20?°C, its RP-solubilization was estimated at 100 and 200?mg l^?1, respectively. HPLC analysis revealed that RP-solubilizing activity was associated with the release of gluconic acid. The hydroxyapatite (HA) solubilization activity concomitantly occurred with the secretion of gluconic acid and lactic acid. Under greenhouse conditions, application of BMA1 strain as an inoculant in presence of RP as the sole P source, considerably increased phosphorus uptake by V. faba L. and upgraded its overall growth in terms of dry weight and length by 76% and 39%, respectively. This growth promoting effect was accompanied by a substantial increase in chlorophyll contents. Additionally, phosphorus levels within roots and shoots of S. plymuthica BMA1-treated plants were approximately three times greater, compared to the non-inoculated control plants. When HA was used instead of RP, bacterization with BMA1 strain also enhanced the plant growth parameters and P contents, but significantly less than RP. These findings revealed that S. plymuthica BMA1 could be a potential candidate to improve the agronomic effectiveness of RP, toward a clean P-nutrition through the formulation of bio-phosphate fertilizers for plant growth promotion.     

Bacterial sucrose isomerases: properties and structural studies

Due to their significant role in food industry, sucrose isomerases are good candidates for rational protein engineering. Hence, specific modifications in order to modify substrate affinity and selectivity, product specificity but also to adapt their catalytic properties to particular industrial process conditions, is interesting. Our work on the structural studies of the sucrose isomerase, MutB, which presents the first structural data available on a trehalulose synthase and the first experimental data on complexed forms of sucrose isomerases represents a significant advance in the understanding of these enzymes. In this review we give an overview of what is known on biochemical properties and structural aspects of different sucrose isomerases in particular those reported from bacteria.