Formation of Streptomyces protoplasts during cultivation in liquid media with lytic enzyme

Many streptomycetes strains are hardly or not at all transformable via protoplasts, or there is a problem with the regeneration of protoplasts. We found that protoplasts are formed directly in cultivation media under submerged conditions in the presence of lytic enzyme. Actinophage μ1/6 endolysin and lysozyme were used in this study. Streptomyces strains were cultivated in several media with glycine and lytic enzyme for 24 and 48h. The highest amounts of protoplasts (about 3 x 107 cfu/ml of cultivation medium) together with the highest regeneration (95%) and transformation frequency (about 2 x 106 – 107 cfu/μg DNA) were obtained reproducibly in YEME medium with high sucrose content. S. aureofaciens B96, as hardly transformable strain because of difficulties with protoplast preparation and their further regeneration, was used in this study. The same procedure was applied to S. lividans 66 TK24 and S. coelicolor A3(2), streptomycetes model strains, to confirm the general use of this method. Moreover, such cultivation process was appropriate for additional quick isolation of either chromosomal as well as plasmid DNA that could be further used in recombinant DNA techniques.

Trichoderma citrinoviride: Anti-Fungal Biosurfactants Production Characteristics

The mechanism of direct impact of Trichoderma fungi on other organisms is a multilayer process. The level of limiting the growth of other microorganisms is determined by the strain and often by the environment. Confirmation of the presence of extracellular biosurfactants in certain strains of Trichoderma considered as biocontrol agents was regarded as a crucial topic complementing the characterization of their interactive mechanisms. Selected strains of T. citrinoviride were cultured in media stimulating biosurfactant biosynthesis, optionally supplemented with lytic enzyme inducers. Results confirmed the anti-fungal properties of surface-active compounds in the tested culture fluids. Preparations that displayed high fungal growth inhibition presented marginal enzymatic activities of both chitinases and laminarinases, implying the inhibitory role of biosurfactants. Fractions from the foam of the culture fluid of the C1 strain, cultured on Saunders medium, and HL strain on MGP medium, without an additional carbon source, exhibited the most prominent ability to inhibit the growth of phytopathogens. Filamentous fungi capable of producing fungicidal compounds, including surfactants, may find applications in protecting the plants against agri-food pathogenic molds.

Isolation and Screening of Plant Growth Promoting Rhizobacteria from the Rhizospheric Soil of Wheat (Triticum aestivum) from Lower Western Himalayan Zone of Himachal Pradesh

The bacteria that colonize the plant's rhizosphere are known as PGPR. The rhizospheric region is the area under the ground surface that is linked with plant roots. PGPR bacteria are free-living bacteria that colonize plant roots and have positive impacts on plant growth. The objectives of this paper were to isolate and identify the most powerful PGPR, as well as to assess their efficacy in terms of P-solubilization, HCN generation, and lytic enzyme activity (protease). A total of 11 bacterial isolates were identified in the Hamirpur district of Himachal Pradesh. All isolates were tested for a variety of plant growth-promoting characteristics, including phosphate solubility, HCN production and protease production. On PVK agar, 8 of the 11 isolates tested positive for P-solubilization in the 5-20 mm zone. One bacterial isolate exhibited positive hydrogen cyanide activity in the event of HCN generation. In the case of lytic enzyme activity, 7 bacterial isolates were positive for protease production.

Influence of Ripening on Polyphenolic Content, Degradative, and Browning Enzymes in Cantaloupe Varieties (C. Melo, L.)

The biochemical changes that occur during the growth and ripening of fruit and vegetable tissues, especially for color and firmness, are the most important factors affecting the quality of fresh products. Cantaloupe (Cucumis melo, L.) is one of the main economically important fruits in the world and its quality parameters, e.g., sweetness, nutritional factors, and texture, influence consumer preferences. Hence, these two features, appearance and texture changes, were investigated in three different genotypes of netted melon, all characterized by an extended shelf life but with different ripening phases. In particular, in all melon cultivars, the cell wall-modifying enzymatic activities and indicators of softening as well as total polyphenols, ortho-diphenols, flavonoids, and tannins, and antioxidant activity were studied. One variety with excellent shelf-life displayed the best nutritional and healthy qualities, in the early stages of ripening, and the lowest degree of browning. The lytic enzyme activities were reduced in the initial stages and after they increased gradually until the overripe stage, with the same trend for all varieties under investigation. The antioxidant activities declined with increasing time of ripeness in all genotypes. The outcomes confirm that the activities of both classes examined, antioxidant and cell wall-modifying enzymes, may vary significantly during ripeness depending on the genotype, suggesting the involvement in determining the postharvest behavior of these fruits.

BB0259 Encompasses a Peptidoglycan Lytic Enzyme Function for Proper Assembly of Periplasmic Flagella in Borrelia burgdorferi

Assembly of the bacterial flagellar rod, hook, and filament requires penetration through the peptidoglycan (PG) sacculus and outer membrane. In most β- and γ-proteobacteria, the protein FlgJ has two functional domains that enable PG hydrolyzing activity to create pores, facilitating proper assembly of the flagellar rod. However, two distinct proteins performing the same functions as the dual-domain FlgJ are proposed in δ- and ε-proteobacteria as well as spirochetes. The Lyme disease spirochete Borrelia burgdorferi genome possesses a FlgJ and a PG lytic SLT enzyme protein homolog (BB0259). FlgJ in B. burgdorferi is crucial for flagellar hook and filament assembly but not for the proper rod assembly reported in other bacteria. However, BB0259 has never been characterized. Here, we use cryo-electron tomography to visualize periplasmic flagella in different bb0259 mutant strains and provide evidence that the E580 residue of BB0259 is essential for PG-hydrolyzing activity. Without the enzyme activity, the flagellar hook fails to penetrate through the pores in the cell wall to complete assembly of an intact periplasmic flagellum. Given that FlgJ and BB0259 interact with each other, they likely coordinate the penetration through the PG sacculus and assembly of a functional flagellum in B. burgdorferi and other spirochetes. Because of its role, we renamed BB0259 as flagellar-specific lytic transglycosylase or LTaseBb.

Lytic Enzymes of Aspergillus piperis as a Tool for Attacking Some Phytopathogenic Fungi In vitro with Special Reference to its Cytotoxicity

The antagonistic activity of Aspergillus piperis against Fusarium oxysporum f. sp. fabae (FOF) and Sclerotinia sclerotiorum were examined and showed multiple signs of hyphal interactions. Microscopic examination of contact regions among A. piperis and each pathogen revealed distinct enzymatic lysis of pathogenic hyphal cell walls. Therefore, it is important to estimate the lytic enzyme activity of A. piperis. Extracellular lytic enzymes are important offensive forces for A. piperis as a biological control agent. Chitinase, phospholipase, and protease recorded relatively high activity from a culture age of 10 days (82.3, 42.4, and 6.2 U/ml, respectively). Enzymatic persistence was measured at room temperature, recording relatively long periods, saving 54%, 46%, and 21% of their activity, respectively. The cytotoxicity of the crude culture filtrate of A. piperis was examined in MCF7 and WI38 human cell lines. The cell viability (IC50) value of the fungal filtrate was estimated after 24 h and 48 h. The results revealed that IC50 values against the MCF7 cell line were inoperative after 24 h and were recorded 80 μg/ml after 48 h. In contrast, IC50 values against the WI38 cell line were 85.69 and 69.8 μg/ml after 24 and 48 h, respectively.

Novel Lytic Enzyme of Prophage Origin from Clostridium botulinum E3 Strain Alaska E43 with Bactericidal Activity against Clostridial Cells

Clostridium botulinum is a Gram-positive, anaerobic, spore-forming bacterium capable of producing botulinum toxin and responsible for botulism of humans and animals. Phage-encoded enzymes called endolysins, which can lyse bacteria when exposed externally, have potential as agents to combat bacteria of the genus Clostridium. Bioinformatics analysis revealed in the genomes of several Clostridium species genes encoding putative N-acetylmuramoyl-l-alanine amidases with anti-clostridial potential. One such enzyme, designated as LysB (224-aa), from the prophage of C. botulinum E3 strain Alaska E43 was chosen for further analysis. The recombinant 27,726 Da protein was expressed and purified from E. coli Tuner(DE3) with a yield of 37.5 mg per 1 L of cell culture. Size-exclusion chromatography and analytical ultracentrifugation experiments showed that the protein is dimeric in solution. Bioinformatics analysis and results of site-directed mutagenesis studies imply that five residues, namely H25, Y54, H126, S132, and C134, form the catalytic center of the enzyme. Twelve other residues, namely M13, H43, N47, G48, W49, A50, L73, A75, H76, Q78, N81, and Y182, were predicted to be involved in anchoring the protein to the lipoteichoic acid, a significant component of the Gram-positive bacterial cell wall. The LysB enzyme demonstrated lytic activity against bacteria belonging to the genera Clostridium, Bacillus, Staphylococcus, and Deinococcus, but did not lyse Gram-negative bacteria. Optimal lytic activity of LysB occurred between pH 4.0 and 7.5 in the absence of NaCl. This work presents the first characterization of an endolysin derived from a C. botulinum Group II prophage, which can potentially be used to control this important pathogen.

Alexander Fleming (to the 140th anniversary of his birth)

Alexander Fleming is a prominent Scottish scientist, Nobel laureate, and bacteriologist who discovered penicillin. He was born near the small town Darwell in Scotland. He studied at St. Mary’s Hospital Medical School and received his master’s and bachelor’s degrees from London University. The research of the young scientist began with the study of the etiology, diagnosis and vaccine therapy of infectious diseases, he soon began working on finding treatment options for patients with bacterial infections with a talented military doctor Almroth Wright. Working in the Royal Military Medical Service during the First World War, A. Wright and O. Fleming showed that the use of antiseptics after injury does not destroy bacterial pathogens effectively. After the war, O. Fleming made an extremely important discovery of a lytic enzyme with a pronounced antibacterial effect - lysozyme. This discovery prompted him to search for antibacterial drugs actively. In 1928, Alexander Fleming discovered the first antibiotic – penicillin. He found that penicillin is an effective antibacterial substance that can affect pyogenic cocci and diphtheria bacilli. The widespread introduction of penicillin into clinical practice has saved hundreds millions of lives. Keywords: penicillin, lysozyme, antibiotics, bacterial infections.

Cold Atmospheric Pressure Plasma Treatment of Maize Grains—Induction of Growth, Enzyme Activities and Heat Shock Proteins

Zea mays L. is one of the most produced crops, and there are still parts of the world where maize is the basic staple food. To improve agriculture, mankind always looks for new, better methods of growing crops, especially in the current changing climatic conditions. Cold atmospheric pressure plasma (CAPP) has already showed its potential to enhance the culturing of crops, but it still needs more research for safe implementation into agriculture. In this work, it was shown that short CAPP treatment of maize grains had a positive effect on the vitality of grains and young seedlings, which may be connected to stimulation of antioxidant and lytic enzyme activities by short CAPP treatment. However, the prolonged treatment had a negative impact on the germination, growth, and production indexes. CAPP treatment caused the increased expression of genes for heat shock proteins HSP101 and HSP70 in the first two days after sowing. Using comet assay it was observed that shorter treatment times (30–120 s) did not cause DNA damage. Surface diagnostics of plasma-treated grains showed that plasma increases the hydrophilicity of the surface but does not damage the chemical bonds on the surface.

Modulation of the enzymatic activity of the flagellar lytic transglycosylase SltF by rod components, and the scaffolding protein FlgJ in Rhodobacter sphaeroides .

Macromolecular cell-envelope-spanning structures such as the bacterial flagellum must traverse the cell wall. Lytic transglycosylases enzymes are capable of enlarging gaps in the peptidoglycan meshwork to allow the efficient assembly of supramolecular complexes. In the periplasmic space, the assembly of the flagellar rod requires the scaffold protein FlgJ, which includes a muramidase domain in the canonical models Salmonella enterica and Escherichia coli . In contrast, in Rhodobacter sphaeroides , FlgJ and the dedicated flagellar lytic transglycosylase SltF are separate entities that interact in the periplasm. In this study we show that sltF is expressed along with the genes encoding the early components of the flagellar hierarchy that include the hook-basal body proteins, making SltF available during the rod assembly. Protein-protein interaction experiments demonstrated that SltF interacts with the rod proteins FliE, FlgB, FlgC, FlgF and FlgG through its C-terminal region. A deletion analysis that divides the C-terminus in two halves revealed that the interacting regions for most of the rod proteins are not redundant. Our results also show that the presence of the rod proteins FliE, FlgB, FlgC, and FlgF displace the previously reported SltF-FlgJ interaction. In addition, we observed modulation of the transglycosylase activity of SltF mediated by FlgB and FlgJ that could be relevant to coordinate rod assembly with cell wall remodeling. In summary, different mechanisms regulate the flagellar lytic transglycosylase, SltF ensuring a timely transcription, a proper localization and a controlled enzymatic activity. Importance Several mechanisms participate in the assembly of cell-envelope-spanning macromolecular structures. The sequential expression of substrates to be exported, selective export, and a specific order of incorporation are some of the mechanisms that stand out to drive an efficient assembly process. In this work we analyze how the structural rod proteins, the scaffold protein FlgJ and the flagellar lytic enzyme SltF, interact in an orderly fashion to assemble the flagellar rod into the periplasmic space. A complex arrangement of transient interactions directs a dedicated flagellar muramidase towards the flagellar rod. All these interactions bring this protein to the proximity of the peptidoglycan wall while also modulating its enzymatic activity. This study suggests how a dynamic network of interactions participates in controlling SltF, a prominent component for flagellar formation.