Modern technologies can satisfy human needs only with the use of large quantities of fertilizers and pesticides that are harmful to the environment. For this reason, it is possible to develop new technologies for sustainable agriculture. The process could be carried out by using endophytic microorganisms with a (possible) positive effect on plant vitality. Bacterial endophytes have been reported as plant growth promoters in several kinds of plants under normal and stressful conditions. In this study, isolates of bacterial endophytes from the roots and leaves of Miscanthus giganteus plants were tested for the presence of plant growth-promoting properties and their ability to inhibit pathogens of fungal origin. Selected bacterial isolates were able to solubilize inorganic phosphorus, fix nitrogen, and produce phytohormones, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, and siderophore. Leaf bacterial isolate Pantoea ananat is 50 OL 2 had high production of siderophores (zone ≥ 5 mm), and limited phytohormone production, and was the only one to show ACC deaminase activity. The root bacterial isolate of Pseudomonas libanensis 5 OK 7A showed the best results in phytohormone production (N6-(Δ2-isopentenyl)adenine and indole-3-acetic acid, 11.7 and 12.6 ng·mL-1, respectively). Four fungal cultures-Fusarium sporotrichioides DBM 4330, Sclerotinia sclerotiorum SS-1, Botrytis cinerea DS 90 and Sphaerodes fimicola DS 93-were used to test the antifungal activity of selected bacterial isolates. These fungal cultures represent pathogenic families, especially for crops. All selected root endophyte isolates inhibited the pathogenic growth of all tested fungi with inhibition percentages ranging from 30 to 60%. Antifungal activity was also tested in two forms of immobilization of selected bacterial isolates: one in agar and the other on dextrin-coated cellulose carriers. These results demonstrated that the endophytic Pseudomonas sp. could be used as biofertilizers for crops.

• Publication type
• Journal Article MeSH

The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) of pathogenic Bordetellae delivers its adenylyl cyclase (AC) enzyme domain into the cytosol of host cells and catalyzes uncontrolled conversion of cellular ATP to cAMP. In parallel, the toxin forms small cation-selective pores that permeabilize target cell membrane and account for the hemolytic activity of CyaA on erythrocytes. The pore-forming domain of CyaA is predicted to consist of five transmembrane α-helices, of which the helices I, III, IV and V have previously been characterized. We examined here the α-helix II that is predicted to form between residues 529 to 549. Substitution of the glycine 531 residue by a proline selectively reduced the hemolytic capacity but did not affect the AC translocating activity of the CyaA-G531P toxin. In contrast, CyaA toxins with alanine 538 or 546 replaced by diverse residues were selectively impaired in the capacity to translocate the AC domain across cell membrane but remained fully hemolytic. Such toxins, however, formed pores in planar asolectin bilayer membranes with a very low frequency and with at least two different conducting states. The helix-breaking substitution of alanine 538 by a proline residue abolished the voltage-activated increase of membrane activity of CyaA in asolectin bilayers. These results reveal that the predicted α-helix comprising the residues 529 to 549 plays a key role in CyaA penetration into the target plasma membrane and pore-forming activity of the toxin.

• MeSH
• Adenylate Cyclase Toxin chemistry   genetics   toxicity   MeSH
• Bordetella enzymology   MeSH
• Cell Membrane drug effects   MeSH
• Erythrocytes drug effects   MeSH
• Hemolysis MeSH
• Protein Conformation, alpha-Helical MeSH
• Cells, Cultured MeSH
• Mice MeSH
• Sheep MeSH
• Amino Acid Substitution MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Kingella kingae is a member of the commensal oropharyngeal flora of young children. Improvements in detection methods have led to the recognition of K. kingae as an emerging pathogen that frequently causes osteoarticular infections in children and a severe form of infective endocarditis in children and adults. Kingella kingae secretes a membrane-damaging RTX (Repeat in ToXin) toxin, RtxA, which is implicated in the development of clinical infections. However, the mechanism by which RtxA recognizes and kills host cells is largely unexplored. To facilitate structure-function studies of RtxA, we have developed a procedure for the overproduction and purification of milligram amounts of biologically active recombinant RtxA. Mass spectrometry analysis revealed the activation of RtxA by post-translational fatty acyl modification on the lysine residues 558 and/or 689 by the fatty-acyltransferase RtxC. Acylated RtxA was toxic to various human cells in a calcium-dependent manner and possessed pore-forming activity in planar lipid bilayers. Using various biochemical and biophysical approaches, we demonstrated that cholesterol facilitates the interaction of RtxA with artificial and cell membranes. The results of analyses using RtxA mutant variants suggested that the interaction between the toxin and cholesterol occurs via two cholesterol recognition/interaction amino acid consensus motifs located in the C-terminal portion of the pore-forming domain of the toxin. Based on our observations, we conclude that the cytotoxic activity of RtxA depends on post-translational acylation of the K558 and/or K689 residues and on the toxin binding to cholesterol in the membrane.

• MeSH
• Acylation MeSH
• Bacterial Toxins genetics   metabolism   MeSH
• Cell Membrane metabolism   MeSH
• Cell Line MeSH
• Cholesterol metabolism   MeSH
• Kingella kingae enzymology   genetics   MeSH
• Humans MeSH
• Lysine chemistry   MeSH
• Protein Processing, Post-Translational * MeSH
• Recombinant Proteins metabolism   MeSH
• Transaminases genetics   metabolism   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) translocates its adenylate cyclase (AC) enzyme domain into target cells in a step that depends on membrane cholesterol content. We thus examined what role in toxin activities is played by the five putative cholesterol recognition amino acid consensus (CRAC) motifs predicted in CyaA hemolysin moiety. CRAC-disrupting phenylalanine substitutions had no impact on toxin activities and these were not inhibited by free cholesterol, showing that the putative CRAC motifs are not involved in cholesterol binding. However, helix-breaking proline substitutions in these segments uncovered a structural role of the Y632, Y658, Y725 and Y738 residues in AC domain delivery and pore formation by CyaA. Substitutions of Y940 of the fifth motif, conserved in the acylated domains of related RTX toxins, did not impact on fatty-acylation of CyaA by CyaC and the CyaA-Y940F mutant was intact for toxin activities on erythrocytes and myeloid cells. However, the Y940A or Y940P substitutions disrupted the capacity of CyaA to insert into artificial lipid bilayers or target cell membranes. The aromatic ring of tyrosine 940 side chain thus appears to play a key structural role in molecular interactions that initiate CyaA penetration into target membranes.

• MeSH
• Adenylate Cyclase Toxin genetics   metabolism   MeSH
• Amino Acid Motifs MeSH
• Cell Membrane metabolism   MeSH
• Cell Line MeSH
• Cholesterol metabolism   MeSH
• Erythrocytes metabolism   MeSH
• Macrophages metabolism   MeSH
• DNA Mutational Analysis MeSH
• Mice MeSH
• Amino Acid Substitution MeSH
• Protein Transport MeSH
• Tyrosine genetics   metabolism   MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH