Deciphering peculiar protein-protein interacting modules in Deinococcus radiodurans

Interactomes of proteins under positive selection from ionizing-radiation-resistant bacteria (IRRB) might be a part of the answer to the question as to how IRRB, particularly Deinococcus radiodurans R₁ (Deira), resist ionizing radiation. Here, using the Database of Interacting Proteins (DIP) and the Protein Structural Interactome (PSI)-base server for PSI map, we have predicted novel interactions of orthologs of the 58 proteins under positive selection in Deira and other IRRB, but which are absent in IRSB. Among these, 18 domains and their interactomes have been identified in DNA checkpoint and repair; kinases pathways; energy and nucleotide metabolisms were the important biological processes that were found to be involved. This finding provides new clues to the cellular pathways that can to be important for ionizing-radiation resistance in Deira.     

Production and optimization of thermophilic alkaline protease in solid-state fermentation by <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CN902

The purpose of the present research is to study the production of thermophilic alkaline protease by a local isolate, Streptomyces sp. CN902, under solid state fermentation (SSF). Optimum SSF parameters for enzyme production have been determined. Various locally available agro-industrial residues have been screened individually or as mixtures for alkaline protease production in SSF. The combination of wheat bran (WB) with chopped date stones (CDS) (5:5) proved to be an efficient mixture for protease production as it gave the highest enzyme activity (90.50 U g⁻¹) when compared to individual WB (74.50 U g⁻¹) or CDS (69.50 U g⁻¹) substrates. This mixed solid substrate was used for the production of protease from Streptomyces sp. CN902 under SSF. Maximal protease production (220.50 U g⁻¹) was obtained with an initial moisture content of 60%, an inoculum level of 1 × 10⁸ (spore g⁻¹ substrate) when incubated at 45°C for 5 days. Supplementation of WB and CDS mixtures with yeast extract as a nitrogen source further increased protease production to 245.50 U g⁻¹ under SSF. Our data demonstrated the usefulness of solid-state fermentation in the production of alkaline protease using WB and CDS mixtures as substrate. Moreover, this approach offered significant benefits due to abundant agro-industrial substrate availability and cheaper cost.     

Enhancement of oxytetracycline production after gamma irradiation-induced mutagenesis of <Emphasis Type="Italic">Streptomyces rimosus</Emphasis> CN08 strain

Streptomyces rimosus CN08 isolated from Tunisian soil produced 8.6 mg l⁻¹ of oxytetracycline (OTC) under submerged fermentation (SmF). Attempts were made for enhancing OTC production after irradiation-induced mutagenesis of Streptomyces rimosus CN08 with Co⁶⁰-γ rays. 125 OTC-producing colonies were obtained after screening on kanamycin containing medium. One mutant called Streptomyces rimosus γ-45 whose OTC production increased 19-fold (165 mg l⁻¹) versus wild-type strain was selected. γ-45 mutant was used for OTC production under solid-state fermentation (SSF). Wheat bran (WB) was used as solid substrate and process parameters influencing OTC production were optimized. Solid-state fermentation increased the yield of antibiotic production (257 mg g⁻¹) when compared with submerged fermentation. Ammonium sulphate as additional nitrogen source enhanced OTC level to 298 mg g⁻¹. Interestingly, OTC production by γ-45 mutant was insensitive to phosphate which opens the way to high OTC production even in medium containing phosphate necessary for optimal mycelia growth.     

Silymarin, a natural antioxidant, protects cerebral cortex against manganese-induced neurotoxicity in adult rats

Manganese (Mn) is an essential element for biological systems, nevertheless occupational exposure to high levels of Mn can lead to neurodegenerative disorders, characterized by serious oxidative and neurotoxic effects with similarities to Parkinson’s disease. The aim of this study was to investigate the potential effects of silymarin (SIL), an antioxidant flavonoid, against manganese chloride induced neurotoxicity both in vivo (cerebral cortex of rats) and in vitro (Neuro2a cells). Twenty-eight male Wistar rats were randomly divided into four groups: the first group (C) received vehicle solution (i.p.) served as controls. The second group (Mn) received orally manganese chloride (20 mg/ml). The third group (Mn + SIL) received both Mn and SIL. The fourth group (SIL) received only SIL (100 mg/kg/day, i.p.). Animals exposed to Manganese chloride showed a significant increase in TBARS, NO, AOPP and PCO levels in cerebral cortex. These changes were accompanied by a decrease of enzymatic (SOD, CAT, GPx) and non-enzymatic (GSH, NpSH, Vit C) antioxidants. Co-administration of silymarin to Mn-treated rats significantly improved antioxidant enzyme activities and attenuated oxidative damages observed in brain tissue. The potential effect of SIL to prevent Mn induced neurotoxicity was also reflected by the microscopic study, indicative of its neuroprotective effects. We concluded that silymarin possesses neuroprotective potential, thus validating its use in alleviating manganese-induced neurodegenerative effects.     

Genetic Diversity of Food-Isolated Salmonella Strains through Pulsed Field Gel Electrophoresis (PFGE) and Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR)

All over the world, the incidence of Salmonella spp contamination on different food sources like broilers, clams and cow milk has increased rapidly in recent years. The multifaceted properties of Salomnella serovars allow the microorganism to grow and multiply in various food matrices, even under adverse conditions. Therefore, methods are needed to detect and trace this pathogen along the entire food supply network. In the present work, PFGE and ERIC-PCR were used to subtype 45 Salmonella isolates belonging to different serovars and derived from different food origins. Among these isolates, S. Enteritidis and S. Kentucky were found to be the most predominant serovars. The Discrimination Index obtained by ERIC-PCR (0.85) was slightly below the acceptable confidence value. The best discriminatory ability was observed when PFGE typing method was used alone (DI = 0.94) or combined with ERIC-PCR (DI = 0.93). A wide variety of profiles was observed between the different serovars using PFGE or/and ERIC-PCR. This diversity is particularly important when the sample origins are varied and even within the same sampling origin.     

Biochemical detection of a metallo-β-lactamase in carbapenem resistant strain ofStreptomyces sp. CN229 isolated from soil

Streptomyces sp. CN229 was isolated from Tunisia soil. This strain displayed antimicrobial activity against Gram positive and Gram negative bacteria. In addition it is resistant to most β-lactam antibiotics including imipenem and meropenem (MIC imipenem >70 μg/ml). Metallo-β-lactamase (MβL) production was confirmed by either imipenem MIC decrease in the presence of ethylene diamine tetraactic acid (EDTA) or the inhibition zone enhancement around EDTA-impregnated imipenem, or meropenem discs. Isolectric focusing analysis demonstrated the production of β-lactamase with pI of 5.8 that is inhibited by EDTA.Streptomyces sp. CN229 was screened for the imipenem resistance genes,bla _VIM andbla _IMP previously identified inPseudomonas aeruginosa. The presence of these genes was not confirmed by specific PCR analysis. We concluded that carbapenem resistance inStreptomyces sp. CN229 strain is mainly due to production of a novel carbapenemase. Our data show for the first time that MβL is produced byStreptomyces sp. MβL-mediated imipenem and meropenem resistance inStreptomyces is a cause for concern in the study of resistance evolution and antibiotic cluster biosynthetic genes.     

Detection of WWE2-related Lentisphaerae by 16S rRNA gene sequencing and fluorescence in situ hybridization in landfill leachate

We collected samples of anaerobic landfill leachate from municipal solid waste landfill (Vert-le-Grand, France) and constructed 16S rRNA clone libraries using primers targeting Planctomycetes and relatives (Pla46F and 1390R). Analyses of 16S rRNA gene sequences resulted in the abundant representation of WWE2-related Lentisphaerae, members of the phylum Lentisphaerae, in the clone library (98% of the retrieved sequences). Although the sequences that are phylogenetically affiliated with the cultured isolate Victivallis vadensis were identified (WWE2 subgroup II), the majority of the sequences were affiliated with an uncultured Lentisphaerae lineage (WWE2 subgroup I). We designed oligonucleotides probes targeting the specific 16S rRNA gene regions of those 2 subgroups. Fluorescence in situ hybridization confirmed the abundance of the uncultivated WWE2 subgroup I in our leachate samples.     

Chemistry and biology of the ramoplanin family of peptide antibiotics

The peptide antibiotic ramoplanin factor A2 is a promising clinical candidate for treatment of Gram-positive bacterial infections that are resistant to antibiotics such as glycopeptides, macrolides, and penicillins. Since its discovery in 1984, no clinical or laboratory-generated resistance to this antibiotic has been reported. The mechanism of action of ramoplanin involves sequestration of peptidoglycan biosynthesis Lipid intermediates, thus physically occluding these substrates from proper utilization by the late-stage peptidoglycan biosynthesis enzymes MurG and the transglycosylases (TGases). Ramoplanin is structurally related to two cell wall active lipodepsipeptide antibiotics, janiemycin, and enduracidin, and is functionally related to members of the lantibiotic class of antimicrobial peptides (mersacidin, actagardine, nisin, and epidermin) and glycopeptide antibiotics (vancomycin and teicoplanin). Peptidomimetic chemotherapeutics derived from the ramoplanin sequence may find future use as antibiotics against vancomycin-resistant Enterococcus faecium (VRE), methicillin-resistant Staphylococcus aureus (MRSA), and related pathogens. Here we review the chemistry and biology of the ramoplanins including its discovery, structure elucidation, biosynthesis, antimicrobial activity, mechanism of action, and total synthesis.     

Inhibition of the Staphylococcus aureus sortase transpeptidase SrtA by phosphinic peptidomimetics

During pathogenesis, Gram-positive bacteria utilize surface protein virulence factors such as the MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) to aid the initiation and propagation of infection through adherence to host endothelial tissue and immune system evasion. These virulence-associated proteins generally contain a C-terminal LPXTG motif that becomes covalently anchored to the peptidoglycan biosynthesis intermediate lipid II. In Staphylococcus aureus, deletion of the sortase isoform SrtA results in marked reduction in virulence and infection potential, making it an important antivirulence target. Here we describe the chemical synthesis and kinetic characterization of a nonhydrolyzable phosphinic peptidomimetic inhibitor of SrtA derived from the LPXTG substrate sequence.     

Development and validation of a new real-time assay for the quantification of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in the soil: a comparison with conventional soil plating

Verticillium wilt of olive, caused by the soil-borne fungus Verticillium dahliae, is one of the most serious diseases of olive tree. In this study, a SYBR Green-based quantitative polymerase chain reaction (Q-PCR) assay targeting the intergenic spacer (IGS) region of the ribosomal DNA (rDNA) was developed to quantify V. dahliae microsclerotia (MS) in soils cropped with olive tree. In order to make the assay quantitative, the number of rDNA units in the genome was estimated using Q-PCR and fixed at 25 copies/genome. The assay was highly specific for V. dahliae, with no cross-amplification with other soil-borne pathogens. The sensitivity analysis showed similar slopes and efficiency, from both fungal DNA (slope?=??3.405, r²?=?0.976, E?=?96.64 %) and the positive recombinant plasmid (y?=??3.36, r²?=?0.989, E = 98.43 %), thus indicating a high accuracy of the assay. The assay exhibits a high intra- and inter-run reproducibility at a very low concentration of 10² copies/μL (CV%?≈?1 %). When the real-time PCR assay was applied to quantify MS in five naturally infested soil samples, it was able to detect V. dahliae in as few as two MS g^?1 of soil. Q-PCR estimates of pathogen DNA were significantly correlated with disease severity (r²?=?0.944) and with the soil plating method (r²?=?0.845). This new assay will be a valuable tool and can be applied for disease risk prediction before installing new plantations, and provides a more complete and rapid examination for soils subjected to such a treatment program.