Inhibition of fungal spore germination by gramicidin S and its potential use as a biocontrol against fungal plant pathogens

Gramicidin S, as well as being sporicidal to Bacillus spores, also inhibits germination and emergence of fungal-like spores of Dictyostelium discoideum . The fungal plant pathogen Fusarium nivale is also inhibited and gramicidin S, therefore, is a sporicidal and antifungal antibiotic. Considering these findings the potential use of this antibiotic and its producer organism Bacillus brevis as a biocontrol is discussed.     

Lyophilization of the spores and vegetative cells of Bacillus brevis var. G-B.--producer of gramicidin S

Viability, antibiotic properties and variation of 4 variants of Bac. brevis var. G.-B. were studied after lyophilization and storage for a year in the lyophilized state. It was shown that the spores and vegetative cells of S and P- variants not synthesizing gramicidin S were somewhat more stable than the spores and cells of R and P+ variants producing the antibiotic. The latter dissociated by 10 per cent towards the cells producing and not producing gramicidin. The developmental rate of the lyophilized vegetative cells was higher than that of the lyophilized spores. Under analogous cultivation conditions they produced higher amounts of the biomass and antibiotic. The lyophilization method described may be recommended for the maintenance of viability and stability of the spores and vegetative cells of Bacillus brevis var. G.-B. producing gramicidin S.     

Biological properties of a Bacillus brevis 101 mutant

Some features of the Bacillus brevis 101 mutant producing the antibiotic gramicidin S are described. The mutant is very close to the initial P+-variant of Bacillus brevis var. G-B by cultural, physiological and biochemical characteristics. The most typical features of Bacillus brevis 101 are high antibiotic activity (up to 2 g/l) and the specific phenotype of the colonies. The phenotypical features of Bacillus brevis 101 are dependent on the conditions of its cultivation. On minimal media rich in organics a change of the culture correlated with a lower antibiotic activity.     

Germination-initiated spores of Bacillus brevis Nagano retain their resistance properties.

Initiated spores and vegetative cells of the gramicidin S-producing Bacillus brevis Nagano were compared with respect to their resistance to various forms of stress (osmotic shock-starvation, exposure to ethanol, sonic oscillation, and heat). The resistance of initiated spores to all of these stress situations was considerably greater than that of vegetative cells and approached that of dormant spores. The period during which the initiated spores remained resistant to heat was extended by addition of gramicidin S. The antibiotic may therefore be of survival value to the species in nature by slowing down the development of initiated spores in the outgrowth phase of germination, thereby extending the period during which the cells are resistant to environmental stress.     

Sporulation and spore properties of Bacillus brevis and its gramicidin S-negative mutant

The function(s) of the peptide antibiotic, gramicidin S, in its producer, Bacillus brevis Nagano, was investigated. Particular attention was paid to the possible role of gramicidin S in sporulation and spore properties. Sporulation was similar in both the gramicidin S-producing parental strain and a gramicidin S-negative mutant of this strain. Mature parental and mutant spores were equally resistant to UV irradiation, solvents (reported previously) and heat. Thus, the lack of gramicidin S synthesis impairs none of these properties. Contrary to results reported by others, we also found no difference in heat resistance between spores of B. brevis ATCC 8185 and its linear gramicidin-negative mutant, Ml.     

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.     

Selective medium based on tyrosine metabolism for the isolation and enumeration of Brevibacillus brevis (Bacillus brevis)

AIMS: To develop a selective medium for the enumeration of Brevibacillus brevis Nagano spores from soil and plant material. METHODS AND RESULTS: Tyrosine agar was developed as a selective medium and compared with nutrient agar for the enumeration of B. brevis Nagano spores from sterile and non-sterile plant and soil extracts. Brevibacillus brevis Nagano colonies could be easily identified only on tyrosine agar due to their clear halo and distinct colony morphology. Identification was confirmed by thin layer chromatography of the antibiotic, gramicidin S, produced by this strain. CONCLUSIONS: Tyrosine agar was shown to be a suitable selective medium for the enumeration of B. brevis Nagano. SIGNIFICANCE AND IMPACT OF THE STUDY: The medium developed, tyrosine agar, can be used to monitor the population of the biological control agent, B. brevis Nagano, and will allow detailed studies within the crop environment.     

Suppression of tyrocidine production by purine nucleotides and related substances in Bacillus brevis

Bacillus brevis (ATCC 8185) produces an antibiotic peptide, tyrocidine. We found that adenosine or 5'-AMP suppressed the production of tyrocidine with half-maximum inhibition at 100-300 microM. This inhibition was specific to the production of tyrocidine since neither adenosine nor 5'-AMP showed any effect on bacterial growth. Cyclic nucleotides had no effect. These results suggest that adenosine, 5'-AMP or its metabolite was specifically involved in the regulation of tyrocidine production.     

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 radiobiological characteristics of the P+ variant of Bacillus brevis var. G.--B. in relation to gramicidin S synthesis]

The radiosensitivity of P(+) variant Bacillus brevis var. G.-B. cells cultured under condition of normal and inhibited gramicidin S synthesis, antibiotically high-active strain and high radioresistant cells has been studied. It has been shown that the radioresistance of bacterial cells correlates, in general, with their antibiotic activity: the antibiotic superproduced is more radioresistant than P(+) variant, the inhibition of antibiotic synthesis by beta-phenil-beta-alanin rises a little the sensitivity of P(+) variant cells. But the radioresistant fraction of P(+) variant contains the lower antibiotic amount than the whole population. It has been concluded that the radioprotective action of gramicidin S can not be the only reason of the above-mentioned differences in radiosensitivity.     

Structuro-functional properties of the bacteria Bacillus brevis in relation to the accumulation of gramicidin S in cells

The culture of Bacillus brevis var. G-B R-form was grown in the presence of beta-phenyl-beta-alanine, the inhibitor of gramicidin S synthesis, is characterized by enhanced endogenous respiration and the DPI-reductase activity as compared to the culture synthezising antibiotic. The increased synthesis of the antibiotic in the region of the culture transition from the logarithmic growth phase to the linear one is associated with a decrease in the number of viable cells despite the fact that the culture on the whole does not die but continues to grow. The membranes prepared from young gramicidin S-free cells and from the cells enriched with the antibiotic possess identical electron micrograph images, IR spectra and protein sets as determined by polyacrylamide gel electrophoresis in a Na-DS system. However, in young cell membranes NADH and succinate dehydrogenase are insensitive to gramicidin S and only malate dehydrogenase is inhibited by this antibiotic. In aged cell membranes the activities of all mentioned dehydrogenases are suppressed. Malate dehydrogenase from young cells is weakly inhibited by thyrotrycin obtained from Bac. brevis ATCC 10068; succinate dehydrogenase is entirely insensitive to this antibiotic, while NADH-dehydrogenase is almost completely inhibited by it. The specificity of action on the respiratory chain of peptide antibiotics synthesized by the cells of one strain of Bac. brevis is suggestive of a possible regulatory role of these peptides in the metabolism of the producent. Hence the accumulation of gramicidin S which is adsorbed on the membrane and destroys the respiratory chain function to the cause of the low rate of oxygen uptake by the culture of Bac. brevis var. G-B R-form and of the low activities of DPI-reductases.     

Effect of polymyxins on Bacillus polymyxa sporogenesis

Bacillus polymyxa var. Ross. producing polymyxin M and Bacillus polymyxa 153 producing polymyxin B form spores during submerged cultivation when the rate of biosynthesis of antibiotic peptides is low and when the production of antibiotics is over. However, sporogenesis is stimulated if polymyxins are added at the early stage of cultural growth. Inhibition of the synthesis of antibiotics suppresses the formation of spores. Substances other than polymyxins do not exhibit such a specific effect on sporogenesis. The fact that the culture requires endogenous polymyxins which are most effective in the period prior to the appearance of spores in the culture suggests the regulatory action of these peptides at the stage between vegetative growth and spore formation in Bacillus polymyxa.     

Selective inhibition of sporulation of Bacillus subtilis by netropsin

At low concentrations, the basic-polypeptide antibiotic, netropsin, did not inhibit growth, over-all RNA synthesis, replication of phage Øe, or synthesis of some catabolite-repressed enzymes in $\text{Bacillus subtilis}$ 168. Cells developed normally until t₂ of sporulation, but no refractile spores were formed in the presence of the antibiotic. The selective inhibition of sporulation by netropsin may be related to the base composition or sequence of some sporulation specific genes.     

Induction of sporulation in Bacillus brevis. 2. Dependence on the presence of the peptide antibiotics tyrocidine and linear gramicidin

This paper presents evidence that the two peptide antibiotics tyrocidine and linear gramicidin, produced by Bacillus brevis ATCC 8185, are required for the induction of sporulation in the producer organism. When tyrocidine synthesis was specifically blocked with 2-amino-3-hydroxy-3-phenylpropanoic acid [Mach, B., Reich, E., and Tatum, E. L. (1963) Proc. Natl Acad. Sci. USA, 50, 175-181], sporulation and gramicidin synthesis were inhibited, but both processes could be restored by the addition of tyrocidine. Certain other amino acids such as L-tyrosine inhibited both sporulation and peptide antibiotic synthesis in nitrogen-limited cultures. When either tyrocidine or linear gramicidin was added together with L-tyrosine, neither sporulation nor peptide antibiotic synthesis was restored. On the other hand, the addition of both tyrocidine and linear gramicidin effectively reversed the inhibition of sporulation by L-tyrosine. These experiments demonstrate that sporulation of B. brevis depends on either the endogenous synthesis or the addition of both tyrocidine and linear gramicidin. The fact that endogenous as well as exogenous peptides could effect sporulation argues against the involvement of artifacts, such as the depletion of intracellular nucleotide pools caused by the surfactant properties of added peptide antibiotics.     

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.