Composition and properties of the outer cell wall layer in Bacillus brevis--the producer of gramicidin S

Two membrane antigens were found by cross immunoelectrophoresis in the cell walls of Bacillus brevis var. G.-B., R form, which started to synthesize gramicidin S (20 mg per 1 ml of cultural broth). The cell wall contained no membrane components in cells at the beginning of the logarithmic growth phase. The protein with a molecular mass of 100 kDa is a component of the cell wall outer layer. The protein is not digested by trypsin or pronase when it comprises the cell walls of cells synthesizing gramicidin S. In the preparation of isolated cell walls, this protein becomes susceptible to the action of the above proteases only when the peptidoglycan layer is broken down by lysozyme. Electron microscopy of cells treated with proteases and shadowed with a metal revealed that many cells lacked the cytoplasm. Therefore, the outer layer of B. brevis R cell wall contains small regions susceptible to the action of protease along with regions composed of the 100 kDa protein and resistant to these enzymes. It is possible that the small regions contain membrane components.     

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.     

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.     

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.     

Reaction mechanism of gramicidin S synthetase 1, phenylalanine racemase, of Bacillus brevis

We have demonstrated that gramicidin S synthetase 1 (GS 1), phenylalanine racemase [EC 5.1.1.11], of Bacillus brevis catalyzes the exchange between a proton in the medium and alpha-hydrogen of phenylalanine in the course of the racemase reaction by using tritiated water or L-phenyl[2,3-3H]alanine. GS 1 from some gramicidin S non-producing mutants of B. brevis lacking phenylalanine racemase activity did not catalyze the tritium exchange reaction. The proton exchange between phenylalanine bound as thioester on the GS 1-phenylalanine complex and water in the medium was detected, but 5,5'-dithiobis(2-nitrobenzoic acid)-modified complex lacked both the proton exchange and phenylalanine racemase activity. It is suggested that a base group, probably a sulfhydryl group, on the enzyme functions as proton donor and acceptor during the phenylalanine racemase reaction.     

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.     

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.     

Organization of the biosynthesis genes for the peptide antibiotic gramicidin S.

A recombinant bacteriophage containing the intact Bacillus brevis gene for gramicidin S synthetase 1, grsA, and the 5' end of the gramicidin S synthetase 2 gene, grsB, was identified by screening an EMBL3 library with anti-GrsA antibodies. This clone, EMBL315, has a 14-kilobase (kb) insert that hybridizes to the previously isolated 3.9-kb fragment of the grsB gene, which encodes the 155-kilodalton ornithine-activating domain of gramicidin S synthetase 2. Deletion and subcloning experiments with the 14-kb insert located the grsA structural gene and its putative promoter on a 4.5-kb PvuII fragment which encoded the full-length 120-kilodalton protein in Escherichia coli. In addition, hybridization analysis revealed that the 5' end of the grsB gene is located approximately 3 kb from the grsA structural gene. Furthermore, these studies indicated that grsA and grsB are transcribed in opposite orientations.     

Organization and chemical composition of the cell wall of gramicidin S-producing Bacillus brevis

The morphology of cells and cell walls was studied in the Bacillus brevis G.-B. R form during its growth and gramicidin S accumulation in it. The membrane apparatus became more complicated and certain other morphological changes were detected in the cells with aging. The cell wall was rather complex even in young cells and consisted of three electron-dense layers where the external and internal layers had an ordered structure. Only the external layer underwent some modifications in the course of growth and these coincided in time with the beginning of intensive gramicidine S biosynthesis. However, the three-layer structure of the cell wall and the ordered organization of the external and internal layers remained unchanged. A preparation of cell walls and preparations of their external and internal layers were isolated from cells synthesizing gramicidine S in the amount of 20 micrograms/ml of the cultural broth. An acid protein having the molecular mass of 100 kD was shown to be the major component of the external layer according to the data of electrophoresis in PAAG with SDS. The middle layer was sensitive to lysozyme, did not have a ordered structure on electron micrographs, and consisted mainly of peptidoglycan.     

Biosynthesis of the antibiotic gramicidin S by a Bacillus brevis culture associated with a change in growth kinetics

The effect of changes in the kinetics of growth on gramicidin S biosynthesis by Bacillus brevis var. G.-B. was studied. The synthesis of gramicidin S is induced when the specific growth rate decreases, whatever is the factor causing this decrease. As was found experimentally, only cells of a certain age can synthesize 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.     

Ferric iron reductase of Rhodopseudomonas sphaeroides.

Partially digested chromosomal DNA of Bacillus brevis ATCC 9999, a producer of the cyclic peptide antibiotic gramicidin S, was ligated into the BamHI site of the Escherichia coli expression vector pUR2-Bam. The ligated molecules were used to transfer E. coli to ampicillin resistance. Of 5 X 10(3) colonies tested by in situ immunoassay for a cross-reaction with antibodies against the gramicidin S synthetase 2, 6 colonies were found to be immunoreactive. A clone designated MK2, which had a 3.9-kilobase insert of B. brevis DNA, directed in E. coli under the lac promoter control the synthesis of polypeptides that were cross-reactive with the antibody to the gramicidin S synthetase 2. Partial purification of the gene products by gel filtration revealed a major fraction with an approximate molecular weight of 140,000 and with specific ornithine-dependent ATP-32PPi and 2'-dATP-32PPi exchange activities. These unique activities of the gramicidin S synthetase 2 were not detected in the E. coli strain harboring the vector.     

Interaction of gramicidins with the cellular phospholipids of the producer Bacillus brevis

Phospholipid fractions were isolated from the cells of Bacillus brevis var. G.-B. variants, some, of which produced gramicidin S and some did not. As was found by thin layer chromatography, phosphatidyl ethanolamine predominated in the fraction of phospholipids. Interaction of the isolated phospholipids with gramicidin S in vitro resulted in a loss of the antibiotic activity. Presumably, formation of a complex between the polypeptide and phospholipids may decrease the actual concentration of gramicidin S in cells producing the antibiotic.     

Identification of two Saccharomyces cerevisiae cell wall mannan chemotypes

We have obtained evidence for two structurally and antigenically different Saccharomyces cerevisiae cell wall mannans. One, which occurs widely and is found in S. cerevisiae strain 238C, is already known to be a neutral mannan which yields mannose, mannobiose, mannotriose, and mannotetraose on acetolysis of the (1 --> 6)-linked backbone. The other, which was found in S. cerevisiae brewer's strains, is a phosphomannan with a structure very similar to that of Kloeckera brevis mannan. S. cerevisiae (brewer's yeast strain) was agglutinated by antiserum prepared against Kloeckera brevis cells. The mannan, isolated from a proteolytic digest of the cell wall of the former, did not react with S. cerevisiae 238C antiserum, whereas it cross-reacted strongly with K. brevis antiserum. Controlled acetolysis cleaved the (1 --> 6)-linkages in the polysaccharide backbone and released mannose, mannobiose, mannotriose, and mannotriose phosphate. Mild acid treatment of the phosphomannan hydrolyzed the phosphodiester linkage, yielding phosphomonoester mannan and mannose. The resulting phosphomonoester mannan reacted with antiserum prepared against K. brevis possessing monoester phosphate groups on the cell surface. alpha-d-Mannose-1-phosphate completely inhibited the precipitin reaction between brewer's yeast mannan and the homologous antiserum. Flocculent and nonflocculent strains of this yeast were shown to have similar structural and immunological properties.     

Effect of different factors on the germination of spores in S and P- variants of Bacillus brevis]

The effect of temperature, duration of heating and the presence of L-alanine and L-glutamine in the medium on the spore germination was studied with the S and P- variants of Bacillus brevis which did not contain gramicidin S and with the R and P+ varants obtained on a defined medium with beta-phenyl-beta-alanine, an inhibitor of the biosynthesis of gramicidin S. The experiments were carried out according to the scheme of complete factor experiment. Germination of the spores was found upon their incubation in a defined medium with L-alanine within two hours after their preliminary heating at 80 degrees C during 45 minutes (S variant), at 60 degrees C during 45 minutes (R variant+trace amounts of gramicidin S), at 80 degrees C during 15 minutes (P+ variant/trace amounts of gramicidin S). Germination of the spores of the P- variant was best upon heating to 60 degrees C during 45 minutes. Gramicidin S is presumed to inhibit, to a certain extent, germination of the spores of its producing culture.     

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.