Mode of antagonism of Brevibacillus brevis against Botrytis cinerea in vitro

AIMS: To assess the activity of Brevibacillus brevis (formerly Bacillus brevis) Nagano and the antibiotic it produces, gramicidin S, against the plant pathogen Botrytis cinerea. METHODS AND RESULTS: Germination and growth of Bot. cinerea were assessed in the presence of B. brevis or gramicidin S in liquid media, on solid media and on leaf sections of Chinese cabbage. Germination was 10-fold more sensitive to gramicidin S than growth. Inhibition of Bot. cinerea was greater in liquid media compared with on solid media. Activity of gramicidin S against Bot. cinerea on leaf sections was much lower than in vitro. In vitro inhibition of Bot. cinerea by B. brevis Nagano was similar to equivalent levels of gramicidin. CONCLUSIONS: Antibiosis, via gramicidin S, is the mode of antagonism exhibited by B. brevis Nagano against Bot. cinerea in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: The mode of antagonism of B. brevis against Bot. cinerea was elucidated. The differing activity of gramicidin S against Bot. Cinerea in vitro and on leaf sections indicates one mechanism by which biocontrol activity may differ between laboratory and field conditions.     

The biosynthesis of gramicidin S in a cell-free system

1. A cell-free system prepared from Bacillus brevis cells, harvested in the late phase of growth and consisting of the 11000g supernatant, has been shown to incorporate into gramicidin S the five constituent amino acids added in labelled form. The results are consistent with complete synthesis and not merely a completion of pre-existing intermediate peptides. 2. The incorporation of ¹⁴C-labelled amino acids by the 11000g supernatant into gramicidin S requires an energy source. Omission of phosphoenolpyruvate and pyruvate kinase from the incubation mixture prevents incorporation into gramicidin S. The cell-free system incorporates [¹⁴C]-leucine, -proline and -phenylalanine over a period of 4hr. With [¹⁴C]leucine, incorporation into gramicidin S takes place in the range pH6–9 with maximum incorporation at pH7·0. High concentrations of chloramphenicol or puromycin decreased the incorporation into gramicidin S by only about 20%. 3. The 50000g supernatant exhibited no decrease in ability of incorporating [¹⁴C]valine into gramicidin S as compared with the 11000g supernatant. About 40% of the incorporating ability remained in the 105000g supernatant after 3hr. centrifugation. When recombining the 105000g sediment with the 105000g supernatant, some increase in incorporation over that obtained with the supernatant alone was obtained. The findings tend to support the view that gramicidin S is synthesized in a different manner from that of proteins.     

Role of gramicidin S in the sporulation of the R+ and R- variants of Bacillus brevis var. G.-B.]

The effect of gramicidin S added to the cultivation medium on sporulation of the gramicidin S-producing P+ variant and gramicidin S-nonproducing P- variant of Bacillus brevis var. G.-B. was studied. Gramicidin S added to the synthetic medium with glucose in an amount of 30 and 100 microgram/ml 4 and 7 hours after inoculation with the vegetative cells of R- variant had no effect on the growth of the culture but retarded its sporulation. When gramicidin S was added in an amount of 100 microgram/ml 4 hours after inoculation, the sporulation rate of R- variant strongly decreased, rohile sporulation was not suppressed as it was noted before with respect to R+ variant. Active stimulation of Bacillus brevis var. G.-B. sporulation was observed after addition of gramicidin S 13 hours after development of R+ and R- variants without the antibiotic biosynthesis. Synthesis of gramicidin S by R+ strain was suppressed by the specific inhibitor beta-phenyl-beta-alanine. The amount of gramicidin S added to the medium during the sporulation process of R+ and R- variants decreased. On addition of 30 microgram/ml of the antibiotic it was practically not detectable when the culture showed the greatest number of the spores. Therefore, gramicidin S added to the medium is probably adsorbed by the cells of Bac. brevis var. G.-B. and affects sporulation of R- and R+ variants thus accelerating or retarding this process depending on the cultivation conditions.     

Localization of gramicidin S on the cytoplasmic membrane of Bacillus brevis and its effect on the activity of membrane enzymes

It was shown that malate dehydrogenase of isolated membranes of the gramicidin S producer Bacillus brevis var. G.-B. (R.-form) is completely inhibited by the antibiotic (approximately 200 mkg/mg of protein). Succinate and NADH dehydrogenases at concentration up to 1 mg per mg of protein are insensitive to it, while corresponding oxidases are inhibited by the antibiotic not more than by 65 -- 75% apparently due to partial damage of the terminal parts of the respiratory chain. The respiration of the producer intact cells is inhibited by exogenous gramicidin S by not more than 55 -- 60%, while the respiration of antibiotic-sensitive cells of M.lysodeikticus is inhibited completely. It was shown that phosphatidyl ethanolamine (50%), phosphatidyl glycerol (15% and diphosphatidyl glycerol (25%) are the major phospholipid components of the membranes of the given strain of Bac. brevis. It was assumed that the resistance of Bac. brevis cells to gramicidin S is partly due to the constant ratio of the charged and amphoteric phospholipids. Using 31P-NMR spectroscopy, the kinetics of free phosphoric compounds in the cells and cell extracts of Bac. brevis during culture growth and gramicidin S synthesis were studied. The content of carbohydrate monophosphate, remained unaffected, while that of nucleoside di- and triphosphates and dinucleotides was low and at definite density and gramicidin S content (above 100 mkg/ml) fell down below the resolution capacity of the method employed. Evidence for gramicidin S localization of the Bac. brevis membrane and possible causes for the manifestation of the NADH dehydrogenase activity at a certain stage of culture growth are discussed.     

The influence of gramicidin S on hydrophobicity of germinating Bacillus brevis spores

Gramicidin S is known to prolong the outgrowth stage of spore germination in the producing culture. Bacillus brevis strain Nagano and its gramicidin S-negative mutant, BI-7, were compared with respect to cell-surface hydrophobicity and germination of their spores. Parental spores were hydrophobic as determined by adhesion to hexadecane, whereas mutant spores showed no affinity to hexadecane. Addition of gramicidin S to mutant spores resulted in a high cell surface hydrophobicity and a delay in germination outgrowth. The hydrophobicity of parental spores was retained throughout most of the germination period. Hydrophobicity was lost as outgrowing spores entered into the stage of vegetative growth. The data indicate that gramicidin S is responsible for the hydrophobicity of B. brevis spores. It is suggested that in making spores hydrophobic, the antibiotic plays a role in concentrating the spores at interfaces where there is a higher probability of finding nutrients for germination and growth.Abbreviation GS Gramicidin S     

Further studies on the biosynthesis of gramicidin S and proteins in a cell-free system from Bacillus brevis

1. An improved 11000g cell-free system for the incorporation of [(14)C]valine into gramicidin S has been obtained. The cell-free extract used was the supernatant obtained by treating Bacillus brevis with ultrasonics for 1min. followed by centrifugation at 11000g. The optimum pH for the incorporation was 8.2-8.4 and the optimum Mg(2+) concentration 0.05m. The presence of ammonium sulphate (0.1m) and K(+) (0.01m) increased the incorporation. 2. Cell-free extracts prepared from cells harvested in the early phase of growth (extinction value 0.1) incorporated negligible amounts of [(14)C]valine into gramicidin S compared with that incorporated by cell-free extracts prepared from cells harvested in the late phase of growth (extinction value 0.5). This was not due to the presence of inhibitors in the cell-free extracts prepared from cells harvested early, since there was no marked decrease in gramicidin S synthesis in a mixture of extracts prepared from cells harvested early and late in the growth phase. 3. The small incorporation of [(14)C]valine into protein, which took place in cell-free extracts from cells harvested in the late growth phase, was not inhibited by puromycin, chloramphenicol and ribonuclease. However, the substantial incorporation that took place in cell-free extracts prepared from cells harvested in the early phase of growth was completely inhibited by puromycin, chloramphenicol and ribonuclease. On mixing cell-free extracts prepared from cells harvested early and late in the growth phase, it appeared that the small incorporation that occurs in extracts from cells harvested in the late phase of growth was not due to cellular inhibitors.     

High productivity tank fermentation for gramicidin S synthetases

A high productivity tank fermentation for gramicidin S synthetases has been developed to supply biocatalyst for a preparative-scale ATP-driven cell-free enzymatic synthesis employing the polypeptide antibiotic, gramicidin S, as a model product. A rich, complex medium supports rapid and dense growth of the enzyme-producing microorganism, Bacillus brevis ATCC 9999, accompanied by the appearance of excellentenzyme activities. Under conditions used, the two enzyme fractions of the gramicidin S synthesizing system, as well as the total enzymatic activity for synthesis of gramicidin S, all reach their maxima simultaneously at the point where growth enters the stationary phase. Successful batch enzyme fermentations have been performed at the bench (14 liter) and pilot (180 liter)scales.     

Environment-dependent conformation and antimicrobial activity of a gramicidin S analog containing leucine and lysine residues

An analog of gramicidin S, cyclo(-L-Leu-L-Lys-L-Leu-D-Leu-L-Leu-)2, in which four out of five amino acid components of gramicidin S were substituted, has been synthesized. This analog assumes a conformation similar to that of gramicidin S in acidic liposomes and a random conformation in neutral liposomes. The antimicrobial activity of this analog corresponded to one-fourth of that of gramicidin S. A possible mechanism for conformational changes in acidic liposomes is discussed.     

The status and the role of glutathione under disturbed ionic balance and pH homeostasis in Escherichia coli

The study of glutathione status in aerobically grown Escherichia coli cultures showed that the total intracellular glutathione (GSHin + GSSGin) level falls by 63% in response to a rapid downshift in the extracellular pH from 6.5 to 5.5. The incubation of E. coli cells in the presence of 50 mM acetate or 10 micrograms/ml gramicidin S decreased the total intracellular glutathione level by 50 and 25%, respectively. The fall in the total intracellular glutathione level was accompanied by a significant decrease in the (GSHin:GSSGin) ratio. The most profound effect on the extracellular glutathione level was exerted by gramicidin S, which augmented the total glutathione level by 1.8 times and the (GSHout:GSSGout) ratio by 2.1 times. The gramicidin S treatment and acetate stress inhibited the growth of mutant E. coli cells defective in glutathione synthesis 5 and 2 times more severely than the growth of the parent cells. The pH downshift and the exposure of E. coli cells to gramicidin S and 50 mM acetate enhanced the expression of the sodA gene coding for superoxide dismutase SodA.     

Permeability of dormant spores of Bacillus subtilis to gramicidin S

Abstract Gramicidin S, dissolved in ethanol, penetrated into the inside of the dormant spores of Bacillus subtilis , had a partial inhibitory effect on l-alanine-initiated germination and completely inhibited their outgrowth and vegetative growth. The activity of particulate NADH oxidase of the antibiotic-treated dormant spores was also influenced significantly. Abnormal morphological changes were observed in germinated spores from gramicidin S-treated dormant spores. An immunoelectron microscopy method with colloidal gold-IgG complex showed that the penetration site of gramicidin S inside dormant spores was mainly the core region. These facts suggest that gramicidin S induces the damage of not only the outer membrane-spore coat complex but also the inner membrane surrounding the spore protoplast, and is able to penetrate into the core region of B. subtilis dormant spores.     

The proline-activating activity of the multienzyme gramicidin S synthetase 2 can be recovered on a 115-kDa tryptic fragment

The multienzyme gramicidin S synthetase 2 was treated with trypsin to obtain fragments capable of activating proline. Three different active fragments were detected. The course of proteolysis was simulated by using a concentration range of trypsin; the cleavage pattern indicated that one of the fragments was particularly stable. This fragment was purified and shown to have a molecular mass of 115 kDa. It was compared chromatographically, by SDS/PAGE, and enzymatically to a Pro-activating fragment produced by a gramicidin-S-negative mutant. It can be concluded that the proteolytic fragment represents a structure which is contained on a continuous part of the polypeptide chain of gramicidin S synthetase 2 and has a relatively compact structure. This provides evidence that the multienzyme gramicidin S synthetase 2 is, at least in part, constructed from functional domains. An approach towards extending these studies to other parts of the gramicidin S synthetase 2 molecule has also been devised. This work complements recombinant DNA studies in the area, providing stable functional fragments.     

Inhibiting mild steel corrosion from sulfate-reducing bacteria using antimicrobial-producing biofilms in Three-Mile-Island process water

Biofilms were used to produce gramicidin S (a cyclic decapeptide) to inhibit corrosion-causing, sulfate-reducing bacteria (SRB). In laboratory studies these biofilms protected mild steel 1010 continuously from corrosion in the aggressive, cooling service water of the AmerGen Three-Mile-Island (TMI) nuclear plant, which was augmented with reference SRB. The growth of both reference SRB (Gram-positive Desulfosporosinus orientis and Gram-negative Desulfovibrio vulgaris) was shown to be inhibited by supernatants of the gramicidin-S-producing bacteria as well as by purified gramicidin S. Electrochemical impedance spectroscopy and mass loss measurements showed that the protective biofilms decreased the corrosion rate of mild steel by 2- to 10-fold when challenged with the natural SRB of the TMI process water supplemented with D. orientis or D. vulgaris. The relative corrosion inhibition efficiency was 50–90% in continuous reactors, compared to a biofilm control which did not produce the antimicrobial gramicidin S. Scanning electron microscope and reactor images also revealed that SRB attack was thwarted by protective biofilms that secrete gramicidin S. A consortium of beneficial bacteria (GGPST consortium, producing gramicidin S and other antimicrobials) also protected the mild steel.     

Production of gramicidin S synthetases by Bacillus brevis in continuous culture.

The effects of different nutrient limitations on the production of the two enzymes of gramicidin S biosynthesis were studied during continuous culture of Bacillus brevis. Gramicidin S synthetases I and II were produced in the chemostat under carbon, nitrogen, phosphorus or sulphur limitation. The growth rate, rather than the nature of the limitation, was the major controlling factor in regulating the level of the gramicidin S synthetases. Synthetase production was low at high dilution rates (0.45 to 0.50 h-1) but increased as the dilution rate was lowered. The highest specific activities occurred at dilution rates that were different for each type of limitation: 0.40 h-1 for nitrogen, 0.32 h-1 for carbon, 0.24 h-1 for sulphur and 0.20 h-1 for phosphorus. Phosphorus limitation gave the highest specific activities. At low dilution rates (0.10 to 0.15 h-1), enzyme activities were again low. Sporulation occurred under carbon limitation, but at a lower dilution rate than that which supported optimal gramicidin S synthetase formation. The specific productivity of the synthetases in the chemostat was higher than the highest productivity obtained in batch growth.     

Dependence of in vivo inactivation kinetics of gramicidin S synthetase on cell physiology

Gramicidin S synthetase, the enzyme complex catalyzing the biosynthesis of the antibiotic gramicidin S in Bacillus brevis, is subject to O(2)-dependent in vivo inactivation during exponential aerobic growth after reaching a peak in specific activity. The five amino acid substrates of the synthetase are capable of stabilizing its activity to varying degrees in whole cells shaken aerobically. Depending on the time of cell harvesting before, during, or after the peak in intracellular gramicidin S synthetase specific activity, the enzyme has a long, medium, or short half-life, respectively. The kinetic profiles of gramicidin S synthetase in B. brevis cells indicate that both the kinetics of synthetase loss and the degree of its amino-acid-mediated stabilization are a strong function of the cells' physiological development.     

Heavy metal cation-induced increase in the antimicrobial activity of gramicidin S. Increased sensitivity of metal-resistant mutants of Escherichia coli B to the antibiotic]

Gramicidin S response of metal resistant mutants of E. coli B and the effect of concentrations of Cu2+, Ag+, Co2+ and Cd2+ on the growth and sensitivity of E. coli B to cationic antibiotics, i.e. gramicidin S2+ and streptomycin2+, were studied. It was shown that the metal-cumulating mutants of E. coli B with two different mechanisms of cross resistance to Cu2+, Cd2+ and Ag+ had higher sensitivity to gramicidin S than the initial wild type strain of E. coli B. It was found that in the threshold or higher doses the salts of Cu, Ag, Co and Cd increased the gramicidin S antimicrobial action on actively metabolizing cells of E. coli B. Analysis of the experimental data as well as the literature ones suggested that the synergic action of gramicidin S and the heavy metals stemmed from an increase in the cationic conductivity of the cytoplasma membrane modified by the metals in the threshold doses which induced an increase in the transport and accumulation of the cations in the bacterial cells by the electric field gradient (with the negative sign inside). Withdrawal of Ca2+ and Mg2+ from the E. coli outer structures into the cytoplasm impaired the barrier properties of the outer membrane and promoted binding of the gramicidin S cations to the liberated anionic groups of the E. coli outer structures and potentiation of the gramicidin S antimicrobial activity as was shown in our experiments.     

Purification and characterization of a new metal protease which hydrolyzes the cyclic decapeptide,gramicidin S

We screened a strain which can produce a new protease. The strain, Lactobacillus sp. no. 1, was isolated from a natural environment as an organism which could utilize gramicidin S as a sole nitrogen source. This strain was proved to produce much protease because it formed a large halo on a plate containing casein, and the protease was purified using ion exchange column chromatography. The amino-terminal amino acid sequence of the hydrolyzed products by the cleavage of gramicidin S was determined by a protein sequencer, and sizes of those products were analyzed by a mass spectrometer. The protease could cleave two peptide bonds between l-Orn-l-Leu in gramicidin S. These cleavage sites were different from other reported cleavage sites of gramicidin S by protease. The molecular weight of the protease was 42,000 by SDS-polyacrylamide gel electrophoresis. The optimum pH and temperature for the enzyme activity were 5.5 and 45°C, respectively. The enzyme activity was inhibited by EDTA, but not by phenylmethylsulfonyl fluoride (PMSF). Because the reported protease that can hydrolyze gramicidin S was a serine protease and the cleavage site was different from that of this protease from Lactobacillus sp. no. 1, we concluded that this enzyme was a new type of metal protease which can cleave both linear and cyclic peptide substrates with a unique substrate specificity.     

The effect of ionophores on growth and glycosyltransferase production of Streptococcus sanguis

Abstract Batch cultures of Streptococcus sanguis NCTC7865 were grown in complex medium in the presence and absence of the ionophores gramicidin, valinomycin and nigericin, to study their effects on growth and glycosyltransferase production. Growth of S. sanguis was markedly inhibited by nigericin or gramicidin, whereas valinomycin had no significant effect. The presence of ionophores caused only slight decreases in glucosyltransferase activity. Fructosyltransferase activity was however reduced by at least 90%. The results indicate that ΔpH rather than ΔΨ is essential for maintaining normal growth in S. sanguis . However, both ΔpH and ΔΨ are necessary for fructosyltransferase synthesis and secretion, but are not apparently involved in the synthesis and secretion of glucosyltransferase.     

Synthesis and biological evaluation of backbone-aminated analogues of gramicidin S

We report the parallel synthesis of gramicidin S derivatives featuring backbone N-amino substituents. Analogues were prepared by incorporation of N-amino dipeptide subunits on solid support. Nine backbone-aminated macrocycles were evaluated for growth inhibitory activity against ESKAPE pathogens and hemolytic activity against human red blood cells. Diamination of the Orn residues in the β-strand region of gramicidin S was found to enhance broad-spectrum antimicrobial activity without a corresponding increase in hemolytic activity.     

The Status and the Role of Glutathione under Disturbed Ionic Balance and pH Homeostasis in Escherichia coli

The study of glutathione status in aerobically grown Escherichia coli cultures showed that the total intracellular glutathione (GSH_in + GSSG_in) level falls by 63% in response to a rapid downshift in the extracellular pH from 6.5 to 5.5. The incubation of E. coli cells in the presence of 50 mM acetate or 10 g/ml gramicidin S decreased the total intracellular glutathione level by 50 and 25%, respectively. The fall in the total intracellular glutathione level was accompanied by a significant decrease in the (GSH_in : GSSG_in) ratio. The most profound effect on the extracellular glutathione level was exerted by gramicidin S, which augmented the total glutathione level by 1.8 times and the (GSH_out : GSSG_out) ratio by 2.1 times. The gramicidin S treatment and acetate stress inhibited the growth of mutant E. coli cells defective in glutathione synthesis 5 and 2 times more severely than the growth of the parent cells. The pH downshift and the exposure of E. coli cells to gramicidin S and 50 mM acetate enhanced the expression of the sodA gene coding for superoxide dismutase SodA.