Georges Michel

The Antifungal Activity of Essential Oils as Determined by Different Screening Methods

Abstract The existing methods for studying the antimicrobial activity of essential oils (e.g. the dilution of the test substances in broth or agar medium) are not adequate to evaluate the effects of the volatile components. Growth inhibition of fungi by various essential oils was determined by direct contact in broth and agar media and compared with the fungistatic action of their vapors using the micro-atmosphere method. Tests were performed on eight cellulolytic mold strains that often contaminate archive and museum reserves. Thirty-seven essential oils were screened to find the most antifungal ones with potential to be employed as atmospheric preservatives. Chenopodium ambrosioides L. var. anthelminticum, Cymbopogon martinii (Roxb.) W. Watson var. martinii, Cymbopogon nardus (L.) W. Watson var. nardus, Syzygium aromaticum (L.) Merr. et L. M. Perry and Pimenta racemosa (Mill.) J.W. Moore were the most active essential oils on the eight strains tested according to direct contact methods. C. martinii was inhibitory even after 12 days of incubation. Using the micro-atmosphere method, C. nardus and C. martinii volatiles were the most fungistatic, but the vapors of Ch. ambrosioides, S. aromaticum and P. racemosa gave moderate results with a specific short effect of P. racemosa.

Pore-forming properties of iturin A, a lipopeptide antibiotic

The addition of iturin A, a lipopeptide antibiotic extracted from Bacillus subtilis, to a bimolecular lipid membrane (BLM) increases dramatically its electrical conductance. For very low concentration of iturin A, discrete conductance steps are observed which are assigned to the formation of conducting pores. The characteristics of these pores depend on the lipid content of the BLM and they change with time. Cholesterol considerably increases the lifetimes of open states. The pores are slightly anion versus cation selective. These first observations unable us to briefly discuss the pore-forming properties of lipopeptides.

Action of peptidolipidic antibiotics of the iturin group on erythrocytes. Effect of some lipids on hemolysis.

Iturin A, bacillomycin L and bacillomycin L dimethyl ester have a strong lytic activity upon human erythrocytes while iturin C is totally inactive. The hemolytic action of the antibiotics is inhibited by free cholesterol as well as by cholesterol included in mixed liposomes of phosphatidylcholine-cholesterol and to a lesser extent by phosphatidylcholine liposomes. This inhibition is the result of an interaction between the antibiotic and added lipids which diminishes the concentration of free antibiotic available to lyse erythrocytes. The inhibitory effect of liposomes on hemolysis demonstrated the affinity of the antibiotic for artificial membrane, especially those containing cholesterol.

Biosynthesis of iturin and surfactin byBacillus subtilis: Evidence for amino acid activating enzymes

SummaryEnzymes catalyzing ATP-PPi exchange reactions mediated by specific amino acids were found inB. subtilis lysates partially purified by gel permeation. The nature of these amino acids varied according to the stage of cell growth: activation of surfactin amino acids was observed during surfactin synthesis; activation of iturin amino acids was not detected at the begining of surfactin synthesis but appeared during iturin synthesis.

Controlled biosynthesis of Val7- and Leu7-surfactins

SummarySurfactin was found to consist of a mixture of two groups of homologous lipopeptides differing by their peptide sequence; Val7-surfactin was recently characterized as a minor companion of the previously described Leu7-surfactin. The addition of various α-amino acids to the culture media led to variations in the production ratios of the two congeners. The supplementation of l-valine or l-isoleucine to the culture medium resulted in a selective enhancement of the production of the Val7-surfactin whereas this production was very low when l-leucine was the nitrogen source in the culture medium.

Production, isolation and characterization of (Leu4) - and (Ile4)surfactins from Bacillus subtilis

Bacillus subtilis coproduces several surfactin variants that are powerful biosurfactants and have potential applications in biology and industry. A single amino acid substitution in the heptapeptide moiety of surfactins strongly modifies their properties. To better establish structure-activity relationships and to search new variants with enhanced properties, Bacillus subtilis was grown into two modified culture media. Two new variants were isolated by chromatographic methods and studied by NMR spectroscopy. As planned, modifications consisted in the substitution of the l-valine residue at the fourth position by a more hydrophobic residue, i.e., leucine or isoleucine. These [Leu4]- and [Ile4]surfactins have a higher affinity for hydrophobic solvents and a twice improved surfactant power. Structure-property correlations were confirmed by analysis of the hydrophobic residue distribution in the three-dimensional model of the structure of surfactin in solution.

Ionophorous and sequestering properties of surfactin, a biosurfactant from Bacillus subtilis

Abstract Surfactin is a bacterial lipopeptide containing two carboxylic groups. Its sequestering properties (surfactin binds calcium and magnesium ions) were determined in aqueous solution at pH 9.5. The calculated association constants were K = 1.5 × 105 M−1 for Ca2− and 1.9 × 104 M−1 for Mg2+. Its complexing properties were determined by the two-phase distribution system; the aqueous phase contained Ca2+ or Rb+ ions and the lipidic phase contained surfactin. At pH 9, surfactin forms a 1:1 complex with Ca2+ and a 2:1 complex with Rb+ and it shows a higher affinity for divalent cations than for monovalent cations. Surfactin is non-selective in binding monovalent cations but is slightly more effective in binding Ca2+ than Mg2+ or Ba2+. Surfactin is also a mobile carrier to transport monovalent and divalent cations across a solvent barrier and mediates the exchange between H− and inorganic cation.

Two high affinity estrogen binding proteins of different specificity in the immature rat uterus cytosol

Abstract The presence of two high affinity estrogen binding proteins in the uterine cytosol of the immature rat has been observed. Besides the 8 S cytosol estrogen receptor , there is a 4–5 S fraction binding estradiol and estrone with a large capacity. In fact, the two binding systems have a different affinity for estradiol and estrone, the receptor binding more the former and the 4–5 S fraction more the latter. Exposure of the cytosol to specific anti-α1-Fetoprotein antibodies suppresses a large part of the 4–5 S binder, if not the totality. Moreover the estrogen binding 4–5 S fraction decreases with increasing age until puberty, while the receptor increases. These results suggest therefore that the estradiol-estrone binding 4–5 S peak of the uterine cytosol is mainly made up of Estradiol Binding Plasma Protein-α1-Fetoprotein (EBP-AFP). Also they confirm that “cytosol” should be taken as an operational fraction which may include extracellular components. During the course of these experiments, it has been observed that the increase of the estradiol receptor is more rapid than the other uterine cytosol proteins until the 8th day, and that there is a second period of growth when it follows the development of the uterus and of the animal, as if it had reached a constant number of binding sites per cell.

Mode of action of iturin A, an antibiotic isolated from Bacillus subtilis, on Micrococcus luteus

Abstract Iturin A has an antibacterial activity on M. luteus which is strongly reduced in presence of MgCl 2 . Iturin A lyses M. luteus protoplast, this lysis is enhanced by EDTA and inhibited by MgCl 2 . These results suggest an action of iturin A on cytoplasmic membrane with interactions of both lipophilic and polypeptidic moieties of the antibiotic, respectively with membrane lipids and membrane polar components. Polar interactions involve the participation of mineral ions as magnesium ions have a strong inhibition effect on the activity of iturin A. The effect of iturin A on the incorporation of radio-active thymidine, uracil, isoleucine and alanine seems unspecific and is probably a consequence of the primary action on cytoplasmic membrane.

Lytic enzymes in sporulating Bacillus subtilis.

Abstract Two specific lytic enzymes were found in sporulating B. subtilis cells: a N-acetyl muramyl L-alanine amidase and a γ-D-glutamyl-(L) meso diaminopimelyl endopeptidase. Both enzyme activities were measured using radioactive synthetic substrates. They are low in vegetative cells and increase during sporulation. The highest rates of increase are concomitant with cortex formation. In a mutant with delayed sporulation enzyme synthesis is also delayed. We suggest that both enzymes play a role in the synthesis of the specific cortex peptidoglycan.