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Effect of surfactin on the lipid moiety of Bacillus subtilis cytoplasmic membrane
Sklenářová, Petra ; Seydlová, Gabriela (advisor) ; Lichá, Irena (referee)
Surfactin, a secondary metabolite produced by Bacillus subtilis, is a surface active compound and antibiotic permeabilizing membrane bilayer. The aim of this study was to reveal the self-resistance strategy at the level of the lipid moiety of cytoplasmic membrane, which B. subtilis employs to combat surfactin in concentrations that are lethal for other bacterial species. Non-producing strain B. subtilis 168 was cultivated in the presence of two different sublethal concentrations of surfactin (350 a 650 µg/ml), which was isolated from the culture broth of B. subtilis ATCC 21332. Presence of surfactin in the medium resulted in a concentration dependent lag phase, which took 40 min (350 µg/ml) and 3 h (650 µg/ml), respectively. Afterwards, the culture grew with the altered doubling time of 44 min (350 µg/ml) and 126 min (650 µg/ml), respectively. Surfactin induced substantial changes in the phospholipid composition of the cytoplasmic membrane. The proportion of the major phospholipid component phosphatidylglycerol decreased and inversely, the level of phosphatidylethanolamine increased. Interestingly, the content of phosphatidic acid rose considerably in the presence of surfactin concentration causing stimulation of B. subtilis growth (350 µg/ml). Liposome leakage assay using phospholipids mimicking...
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Effect of membrane lipid composition on resistance against surfactin
Pinkas, Dominik ; Seydlová, Gabriela (advisor) ; Sýkora, Jan (referee)
Surfactin is an antibiotic produced by several strains of B. subtilis. Its broad range of biological activities is interesting from perspective of medicine, food industry and bioremediation and is based on its surface-active properties and interaction with biological membranes. The latter means mainly forming ion channels, conductive pores and with increasing concentration eventually disrupting membrane structure in detergent-like manner. Mechanism of resistance of producing strain against its own toxic product is not yet fully understood. This work shows that it could be based on surfactin target modification - which means altering membrane lipid composition. We were able to recognize surfactin-formed ion channels or pores with a broad range of conductivities spanning from 2 pS to 2 nS using BLM method. Liposome leakage assay with carboxyfluorescein revealed few distinct mechanisms of lysis, differing in amplitude, rate of lysis and cooperativity. Increased content of anionic lipids with conical shape, namely cardiolipin and phosphatidic acid led to substantial increased membrane resistance to surfactin-induced permeabilization. Key words: membrane, surfactin, Bacillus subtilis, cardiolipin, black lipid membranes, liposomes
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Inhibitory effects of B. velezensis strain on plant pathogens of the genus Xanthomonas
Švecová, Magdaléna ; Palyzová, Andrea (advisor) ; Jelínková, Markéta (referee)
Biological control is a method of applied ecology that uses one organism, or its product, to inhibit another pathogenic organism. This method in plant protection should be more environmentally friendly than commonly used pesticides. The rhizobacterium Bacillus velezensis FZB42, which can suppress the growth of other microorganisms, can be used as a biological control agent. Xanthomonas campestris pv. campestris is a phytopathogen that causes significant losses in agricultural production. This diploma thesis proved an antagonistic effect of the bacterial strain B. velezensis FZB42 on the phytopathogenic bacteria Xanthomonas campestris pv. campestris SU in mixed culture. The study confirmed that the antibiotic effect of B. velezensis strain FZB42 against X. campestris pv. campestris SU in mixed culture depends on the inoculation ratios of both strains. The interaction of the two bacterial strains was characterized by metabolomics analysis, it was confirmed that B. velezensis FZB42 produces four dominant secondary metabolites: lipopeptides surfactin, fengycin, and bacillomycin and siderophore bacillibactin, which show antifungal and antibiotic activity. The inhibitory activity of B. velezensis strain FZB42 was further monitored by SEM analysis, which showed damage to X. campesris pv. campestris SU...
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Effect of surfactin on the lipid moiety of Bacillus subtilis cytoplasmic membrane
Sklenářová, Petra ; Seydlová, Gabriela (advisor) ; Lichá, Irena (referee)
Surfactin, a secondary metabolite produced by Bacillus subtilis, is a surface active compound and antibiotic permeabilizing membrane bilayer. The aim of this study was to reveal the self-resistance strategy at the level of the lipid moiety of cytoplasmic membrane, which B. subtilis employs to combat surfactin in concentrations that are lethal for other bacterial species. Non-producing strain B. subtilis 168 was cultivated in the presence of two different sublethal concentrations of surfactin (350 a 650 µg/ml), which was isolated from the culture broth of B. subtilis ATCC 21332. Presence of surfactin in the medium resulted in a concentration dependent lag phase, which took 40 min (350 µg/ml) and 3 h (650 µg/ml), respectively. Afterwards, the culture grew with the altered doubling time of 44 min (350 µg/ml) and 126 min (650 µg/ml), respectively. Surfactin induced substantial changes in the phospholipid composition of the cytoplasmic membrane. The proportion of the major phospholipid component phosphatidylglycerol decreased and inversely, the level of phosphatidylethanolamine increased. Interestingly, the content of phosphatidic acid rose considerably in the presence of surfactin concentration causing stimulation of B. subtilis growth (350 µg/ml). Liposome leakage assay using phospholipids mimicking...
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Mode of action of antimicrobial lipopeptides produced by Bacillus subtilis
Pinkas, Dominik ; Seydlová, Gabriela (advisor) ; Žíla, Vojtěch (referee)
Increasing bacterial resistance to classical antibiotics and emergence of multi-resistant strains impose a constant threat. Antimicrobial compounds of bacterial origin are an important source of new antibacterial therapeutic agents needed to answer this issue. Three families of lipopeptides produced by Bacillus subtilis - surfactins, fengycins and iturins represent an interesting class of such compounds. They exert a wide range of biological activities and possess a good potential for modifications and improvement of their structure and function. Lipopeptides produced by B. subtilis are surface active compounds capable of reducing surface/interface tension. The variety of their biological activities stems from their ability to insert into lipid membranes leading to disruption and permeabilization of the membrane. Specific mode of action differs between the three families but the common feature is that it is concentration dependent. First, lipopeptides induce ion leakage, pore formation and then the increasing concentration eventually causes complete solubilisation of the membrane in a detergent-like manner. In addition, surfactin can inhibit some enzymes by chelating divalent cations required for their activity. These properties make the B. subtilis lipopeptides promising compounds for commercial applications.
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Mechanism of surfactin self-resistance in the Bacillus subtilis cytoplasmatic membrane
Seydlová, Gabriela ; Svobodová, Jaroslava (advisor) ; Spížek, Jaroslav (referee) ; Julák, Alois (referee)
Surfactin, a lipopeptide surfactant and antibiotic produced by Bacillus subtilis, exhibits a strong membrane perturbation. One of the drawbacks hindering its commercial applications is the unknown mechanism of surfactin self-resistance in the producer. Therefore, the aim of this study was to examine the self-protective mechanisms of the cytoplasmic membrane of B. subtilis against the deleterious effect of surfactin. In order to reach this task, two isogenic pairs of strains differing only in surfactin production were constructed. It was found out that the early response of the producer is realized by increasing the amount of total lipid content in the membrane already with the onset of surfactin synthesis. This process leads to lowering the surfactin-lipid ratio in the membrane. In parallel with the growing surfactin concentration the content of anionic phospholipids with cardiolipin as the major representative rises up to 24 % of the total. Together with the fall of phosphatidylethanolamine these changes promote the membrane stabilization and protect it against the interaction with surfactin. These alterations result in higher rigidity both of the polar head and hydrophobic chain region of the membrane as the steady state anisotropy of DPH and TMA-DPH showed. After 24 h of cultivation induction of...
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Cytoplasmic membrane adaptation to surfactin in Bacillus subtilis non-producing strain
Stružinská, Olga ; Svobodová, Jaroslava (advisor) ; Fišer, Radovan (referee)
Cytoplasmic membrane adaptation to surfactin in Bacillus subtilis 168 non-producing strain Surfactin, the most potent surface active compound and antibiotic is produced by bacteria of the genus Bacillus. Surfactin interacts with membrane bilayers, that results in destabilization and permeabilization of this structure. However mechanism of surfactin self-resistance in the producer's membrane is not understood. The aim of this study was to characterize the adaptive processes occurring at the level of cytoplasmic membrane of surfactin non-producing strain B. subtilis 168, which was exposed to exogenously added surfactin during the exponential phase of growth. The cultivation protocol of B. subtilis growth on agar media plates supplemented with surfactin was developed. Two surfactin concentrations that inhibit (400 g/ml) and even stimulate (300 g/ml) the growth of B. subtilis 168 strain were assessed. Surfactin brought about the growth arrest for 3 hours and the restored growth rate decreased in the case of inhibitory concentration, whereas the stimulatory concentration increased the growth rate and resulted in higher final density of the population. TLC was performed to analyze the polar head groups of membrane phospholipids. The portion of phosphatidylserine was found to increase at both surfactin...
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Effect of membrane lipid composition on resistance against surfactin
Pinkas, Dominik ; Seydlová, Gabriela (advisor) ; Sýkora, Jan (referee)
Surfactin is an antibiotic produced by several strains of B. subtilis. Its broad range of biological activities is interesting from perspective of medicine, food industry and bioremediation and is based on its surface-active properties and interaction with biological membranes. The latter means mainly forming ion channels, conductive pores and with increasing concentration eventually disrupting membrane structure in detergent-like manner. Mechanism of resistance of producing strain against its own toxic product is not yet fully understood. This work shows that it could be based on surfactin target modification - which means altering membrane lipid composition. We were able to recognize surfactin-formed ion channels or pores with a broad range of conductivities spanning from 2 pS to 2 nS using BLM method. Liposome leakage assay with carboxyfluorescein revealed few distinct mechanisms of lysis, differing in amplitude, rate of lysis and cooperativity. Increased content of anionic lipids with conical shape, namely cardiolipin and phosphatidic acid led to substantial increased membrane resistance to surfactin-induced permeabilization. Key words: membrane, surfactin, Bacillus subtilis, cardiolipin, black lipid membranes, liposomes
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