Self-Resistance Gene-Guided Discovery of the Molecular Basis for Biosynthesis of the Fatty Acid Synthase Inhibitor Cerulenin.

Cerulenin (1) is the first reported natural fatty acid synthase inhibitor and has been intensively researched for its antifungal, anticancer and anti-obesity properties. However, the molecular basis for its biosynthesis has remained a mystery for six decades. Here, we have identified the polyketide biosynthetic gene cluster (cer) responsible for the biosynthesis of 1 from two Sarocladium species using a self-resistance gene mining approach, which we validated via heterologous reconstitution of cer cluster in an Aspergillus nidulans host. Expression of various combinations of cer genes uncovered key pathway intermediates, electrocyclisation products derived from PKS-encoded polyenoic acids, and a suite of 13 new analogues of 1. This enabled us to establish a biosynthetic pathway to 1 that starts with a C12 polyketide precursor with both E and Z double bonds and involves a complex series of epoxidations, double bond shifts, E/Z isomerisation and epoxide reduction. Using in vitro assays, we further validated the roles of amidotransferase CerD in amidation, and oxidase CerF and reductase CerE in the final two-electron oxidation and enone reduction steps towards 1. These findings expand our understanding of complex tailoring modifications in highly reducing PKS pathways and pave the way for the engineered biosynthesis of cerulenin analogues.

Comparative Genotypic Analysis of RAPD and RFLP Markers for Molecular Variation Detection of Methicillin-Resistant Staphylococcus aureus Clinical Isolates.

Background and Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) isolates are associated with various diseases ranged from mild superficial impairments to invasive infections. This study aimed to evaluate the ability of polymerase chain reaction (PCR) based methods namely, restriction fragment length polymorphism (RFLP) of the coa gene and random amplified polymorphic DNA (RAPD), to determine the genetic diversity of MRSA isolates. Materials and Methods: A total of 37 MRSA isolates were conventionally identified depending on their biochemical and microbiological culture characteristics. Genotypic confirmation was based on detection of the associated mecA gene. The genetic variation amongst MRSA isolates was evaluated following the coa gene-based RFLP and RAPD fingerprints. Results: Results illustrated that, the species specific coa gene was detected in all MRSA isolates. The irregular bands intensity, number, and molecular sizes of the PCR amplicons demonstrated the coa gene polymorphism. The incompatible AluI digestion patterns of these amplicons classified the tested MRSA isolates into 20 RFLP patterns which confirm the coa gene polymorphism. Additionally, the PCR-based RAPD analysis showed variable bands number with size range of approximately 130 bp to 4 kbp, which indicated the genetic variation of the tested MRSA isolates as it created 36 variable RAPD banding profiles. Conclusions: coa gene AluI enzymatic restriction sites, amongst the tested MRSA isolates, certify their genetic variation on the basis of the accurate but complicated and relatively expensive coa gene-based RFLP. Conversely, the results verified the excellent ability of the simple and cost-effective PCR-based RAPD analysis to discriminate between MRSA isolates without any preface data about the genome.

Discovery and heterologous biosynthesis of glycosylated polyketide luteodienoside A reveals unprecedented glucinol-mediated product offloading by a fungal carnitine O-acyltransferase domain.

Luteodienoside A is a novel glycosylated polyketide produced by the Australian fungus Aspergillus luteorubrus MST-FP2246, consisting of an unusual 1-O-ß-d-glucopyranosyl-myo-inositol (glucinol) ester of 3-hydroxy-2,2,4-trimethylocta-4,6-dienoic acid. Mining the genome of A. luteorubrus identified a putative gene cluster for luteodienoside A biosynthesis (ltb), harbouring a highly reducing polyketide synthase (HR-PKS, LtbA) fused at its C-terminus to a carnitine O-acyltransferase (cAT) domain. Heterologous pathway reconstitution in Aspergillus nidulans, substrate feeding assays and gene truncation confirmed the identity of the ltb cluster and demonstrated that the cAT domain is essential for offloading luteodienoside A from the upstream HR-PKS. Unlike previously characterised cAT domains, the LtbA cAT domain uses glucinol as an offloading substrate to release the product from the HR-PKS. Furthermore, the PKS methyltransferase (MT) domain is capable of catalysing gem-dimethylation of the 3-hydroxy-2,2,4-trimethylocta-4,6-dienoic acid intermediate, without requiring reversible product release and recapture by the cAT domain. This study expands the repertoire of polyketide modifications known to be catalysed by cAT domains and highlights the potential of mining fungal genomes for this subclass of fungal PKSs to discover new structurally diverse secondary metabolites.

Trichoderma hamatum Strain Th23 Promotes Tomato Growth and Induces Systemic Resistance against Tobacco Mosaic Virus.

Trichoderma hamatum strain Th23, isolated from tomato roots, was molecularly identified using phylogenetic analysis based on ITS, tef1, and rpb2 gene sequences and evaluated for its efficiency in suppressing tobacco mosaic virus (TMV) infection for the first time. Under greenhouse conditions, the application of Th23 promoted tomato growth with significant increases in shoot and root parameters as well as improved total chlorophyll content. Compared to the nontreated tomato plants, the soil pretreatment of tomato plants 48 h before TMV inoculation produced a significant reduction in the TMV accumulation level by 84.69% and enhanced different growth parameters. In contrast, TMV had a deleterious impact on fresh and dry matter accumulation and inhibited photosynthetic capacity. Furthermore, the protective activity of Th23 was associated with a significant increase in reactive oxygen species scavenging enzymes (PPO, CAT, and SOD) as well as decreased nonenzymatic oxidative stress markers (H2O2 and MDA) compared to the TMV treatment at 15 days post-viral inoculation (dpi). In addition, considerable increases in the transcriptional levels of polyphenolic genes (HQT and CHS) and pathogenesis-related proteins (PR-1 and PR-7) were shown to induce systemic resistance against TMV. Consequently, the ability of T. hamatum strain Th23 to promote plant growth, induce systemic resistance, and boost innate immunity against TMV infestation supported the incorporation of Th23 as a potential biocontrol agent for managing plant viral infections. To the best of our knowledge, this is the first report of the antiviral activity of T. hamatum against plant viral infection.

Synthesis of Silver Nanocomposite Based on Carboxymethyl Cellulose: Antibacterial, Antifungal and Anticancer Activities.

Traditional cancer treatments include surgery, radiation, and chemotherapy. According to medical sources, chemotherapy is still the primary method for curing or treating cancer today and has been a major contributor to the recent decline in cancer mortality. Nanocomposites based on polymers and metal nanoparticles have recently received the attention of researchers. In the current study, a nanocomposite was fabricated based on carboxymethyl cellulose and silver nanoparticles (CMC-AgNPs) and their antibacterial, antifungal, and anticancer activities were evaluated. The antibacterial results revealed that CMC-AgNPs have promising antibacterial activity against Gram-negative (Klebsiella oxytoca and Escherichia coli) and Gram-positive bacteria (Bacillus cereus and Staphylococcus aureus). Moreover, CMC-AgNPs exhibited antifungal activity against filamentous fungi such as Aspergillus fumigatus, A. niger, and A. terreus. Concerning the HepG2 hepatocellular cancer cell line, the lowest IC50 values (7.9 ± 0.41 µg/mL) were recorded for CMC-AgNPs, suggesting a strong cytotoxic effect on liver cancer cells. As a result, our findings suggest that the antitumor effect of these CMC-Ag nanoparticles is due to the induction of apoptosis and necrosis in hepatic cancer cells via increased caspase-8 and -9 activities and diminished levels of VEGFR-2. In conclusion, CMC-AgNPs exhibited antibacterial, antifungal, and anticancer activities, which can be used in the pharmaceutical and medical fields.

Foliar Application of Nanoclay Promotes Potato (Solanum tuberosum L.) Growth and Induces Systemic Resistance against Potato Virus Y.

Potato virus Y (PVY) is one of the most harmful phytopathogens. It causes big problems for potatoes and other important crops around the world. Nanoclays have been extensively studied for various biomedical applications. However, reports on their interactions with phytopathogens, particularly viral infections, are still limited. In this study, the protective activity of Egyptian nanoclay (CE) and standard nanoclay (CS) against PVY was evaluated on potato (Solanum tuberosum L.) plants. Their physicochemical and morphological properties were examined with scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), and energy dispersive spectrometer (EDS). SEM and TEM analyses revealed that CE has a spherical and hexagonal structure ranging from 20 to 80 nm in size, while CS has boulder-like and tubular structures of about 320 nm in size. FTIR and EDS showed that both nanoclay types have different functional groups and contain many vital plant nutrients that are necessary for every stage and process of the plant, including development, productivity, and metabolism. Under greenhouse conditions, a 1% nanoclay foliar application enhanced potato growth, reduced disease symptoms, and reduced PVY accumulation levels compared with non-treated plants. Significant increases in levels of antioxidant enzymes (PPO and POX) and considerable decreases in oxidative stress markers (MDA and H2O2) were also reported. Moreover, a significant increase in the transcriptional levels of defense-related genes (PAL-1, PR-5, and CHI-2) was observed. All experiment and analysis results indicate that the CE type is more effective than the CS type against PVY infection. Based on these results, the foliar applications of nanoclay could be used to manage plant viral infections in a way that is both effective and environmentally friendly. To our knowledge, this is the first report of the antiviral activity of the foliar application of nanoclay against PVY infection.

Mycosynthesis of Hematite (α-Fe2O3) Nanoparticles Using Aspergillus niger and Their Antimicrobial and Photocatalytic Activities.

Nanoparticles (NPs) and nanomaterials (NMs) are now widely used in a variety of applications, including medicine, solar energy, drug delivery, water treatment, and pollution detection. Hematite (α-Fe2O3) nanoparticles (Hem-NPs) were manufactured in this work by utilizing a cost-effective and ecofriendly approach that included a biomass filtrate of A. niger AH1 as a bio-reducer. The structural and optical properties of Hem-NPs were investigated using X-ray diffraction (XRD), transmission electron microscopy (TEM), dynamic light scattering (DLS), and UV-visible and Fourier-transform infrared (FTIR) spectroscopies. The results revealed that all of the studied parameters, as well as their interactions, had a significant impact on the crystallite size. The average diameter size of the biosynthesized Hem-NPs ranged between 60 and 80 nm. The antimicrobial and photocatalytic activities of Hem-NPs were investigated. The antimicrobial results of Hem-NPs revealed that Hem-NPs exhibited antibacterial activity against E. coli, B. subtilis, and S. mutans with MICs of 125, 31.25, and 15.62 µg/mL, respectively. Moreover, Hem-NPs exhibited antifungal activity against C. albicans and A. fumigatus, where the MICs were 2000 and 62.5 µg/mL, respectively. The efficiency of biosynthesized Hem-NPs was determined for the rapid biodegradation of crystal violet (CV) dye, reaching up to 97 percent after 150 min. Furthermore, Hem-NPs were successfully used more than once for biodegradation and that was regarded as its efficacy. In conclusion, Hem-NPs were successfully biosynthesized using A. niger AH1 and demonstrated both antimicrobial activity and photocatalytic activity against CV dye.

Plant Growth-Promoting Fungi as Biocontrol Tool against Fusarium Wilt Disease of Tomato Plant.

Plant growth-promoting fungi (PGPF) improve plant health and resist plant pathogens. The present study was carried out to biocontrol tomato Fusarium wilt using PGPF through antifungal activity and enhance tomato plant immune response. Four PGPF were identified genetically as Aspergillus flavus, Aspergillus niger, Mucor circinelloides and Pencillium oxalicum. In vitro antagonistic activity assay of PGPF against Fusariumoxysporum was evaluated, where it exhibited promising antifungal activity where MIC was in the range 0.25-0.5 mg/mL. Physiological markers of defense in a plant as a response to stimulation of induced systemic resistance (ISR) were recorded. Our results revealed that A. niger, M. circinelloides, A. flavus and P. oxalicum strains significantly reduced percentages of disease severity by 16.60% and 20.83% and 37.50% and 45.83 %, respectively. In addition, they exhibited relatively high protection percentages of 86.35%, 76.87%, 56.87% and 59.06 %, respectively. With concern to the control, it is evident that the percentage of disease severity was about 87.50%. Moreover, the application of M. circinelloides, P. oxalicum, A. niger and A. flavus successfully recovered the damage to morphological traits, photosynthetic pigments' total carbohydrate and total soluble protein of infected plants. Moreover, the application of tested PGPF enhanced the growth of healthy and infected tomato plants.

Antifungal Activity of Biosynthesized Silver Nanoparticles (AgNPs) against Aspergilli Causing Aspergillosis: Ultrastructure Study.

Currently, nanoparticles and nanomaterials are widely used for biomedical applications. In the present study, silver nanoparticles (AgNPs) were successfully biosynthesized using a cell-free extract (CFE) of Bacillus thuringiensis MAE 6 through a green and ecofriendly method. The size of the biosynthesized AgNPs was 32.7 nm, and their crystalline nature was confirmed by XRD, according to characterization results. A surface plasmon resonance spectrum of AgNPs was obtained at 420 nm. Nanoparticles were further characterized using DLS and FTIR analyses, which provided information on their size, stability, and functional groups. AgNPs revealed less cytotoxicity against normal Vero cell line [IC50 = 155 µg/mL]. Moreover, the biosynthesized AgNPs exhibited promising antifungal activity against four most common Aspergillus, including Aspergillus niger, A. terreus, A. flavus, and A. fumigatus at concentrations of 500 µg/mL where inhibition zones were 16, 20, 26, and 19 mm, respectively. In addition, MICs of AgNPs against A. niger, A. terreus, A. flavus, and A. fumigatus were 125, 62.5, 15.62, and 62.5 µg/mL, respectively. Furthermore, the ultrastructural study confirmed the antifungal effect of AgNPs, where the cell wall's integrity and homogeneity were lost; the cell membrane had separated from the cell wall and had intruded into the cytoplasm. In conclusion, the biosynthesized AgNPs using a CFE of B. thuringiensis can be used as a promising antifungal agent against Aspergillus species causing Aspergillosis.

Suppression of Pepper Root Rot and Wilt Diseases Caused by Rhizoctonia solani and Fusarium oxysporum.

Pepper is vulnerable to soil-borne fungal pathogens such as Rhizoctonia solani and Fusarium oxysporum. The potential of beneficial rhizosphere microorganisms to control R. solani and F. oxysporum f.sp. capsici was evaluated in pepper plants. Paenibacillus polymyxa and Trichoderma longibrachiatum were isolated from rhizospheric soil samples of healthy pepper plants. In vitro, both isolates caused clear reductions in the radial growth of root rot and wilt pathogens. Scanning electron microscopy displayed lysis and abnormal shape of the pathogens in dual cultures with P. polymyxa and T. longibrachiatum. The incidence and severity of root rot and wilt diseases were significantly reduced in pepper plants treated with the growth-promoting fungi (PGPF isolates; Fusarium equiseti GF19-1, Fusarium equiseti GF18-3, and Phoma sp. GS8-3), P. polymyxa, or T. longibrachiatum in comparison to the control. Moreover, the induction treatments led to increased pepper growth compared with their control. The defense related gene (CaPR4) expression was shown to be significantly higher in the treated plants than in the control plants. In conclusion, the antagonistic isolates (P. polymyxa and T. longibrachiatum) and PGPF isolates have a clear impact on the prevention of root rot and wilt diseases in pepper plants incited by R. solani and F. oxysporum f.sp. capsici. The expression of the CaPR4 gene added to the evidence that PGPF isolates generate systemic resistance to pathogen infections.