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The review concerns discussion of certain aspects of growth and development of streptomycetes, that have an adaptation meaning for their existence under natural conditions and reflect our perception of them as procaryotes which have a range of qualities typical of multicellular organisms. At present, the concept of multicellularity is the key idea in investigation of growth processes, differentiation and physiology of streptomycetes. Streptomyces olivocinereus is presented as an effective model that gives the unique opportunities for investigation of different aspects of biology of streptomycetes within laboratory environment as well as in natural environment in suli. S.olivocinereus produces luminescent antibiotic geliomycin (resistomycin). In this review we summarized the results of the many years of investigation of growth, differentiation and behavior of this streptomycete. The investigations were undertaken by a group of scientists of the Moscow State University. The results can be employed as arguments for the multicellular nature of streptomycetes.

Marine-derived fungi are of great interest as a new promising source of biologically active products such as anticancer compounds, antibiotics, inhibitors of biochemical processes. Since marine organisms inhabit biologically competitive environment with unique conditions, the chemical diversity of the secondary metabolites from marine fungi is considerably high. Recent genomic studies demonstrated that fungi can carry gene clasters encoding production of previously unknown secondary metabolites. Activation of the attenuated or silent genes would be useful either for improving activities of the known compounds or for discovery of new products.

Streptomycetes or mycelial microorganisms are able to form biofilms under the natural, industrial and clinical conditions. The controlled use of biofilms in various industrial processes is much more efficient vs. the cultivation of plankton suspended cells. Optimization of biotechnological processes with the use of streptomycete biofilms is advisable in production of lactic acid and detoxication of the liquor in pyrolysis of plant biomass. Streptomycete biofilms are used in water purification systems. It is recommended to use biofilms for detoxication of wastes and bioremediation of soils contaminated with hard metals. The use of biofilms of streptomycetes producing biologically active substances is of special interest. High yields of.antibiotics and actinomycin D in particular was observed with. cultivation of antibioc-producing streptomycetes as biofilms in bioreactors of unique design.

Streptomycetes, soil-dwelling mycelial bacteria, can form biofilms as indigenous components of the environment. The biofilms formed by streptomycetes exist in different ecological niches, in natural, medical, industrial environments. The biofilm-forming streptomycetes affect water quality, human health, associate with deterioration of artworks and historical monuments. The review should be of interest for researchers of the biofilm mode of streptomycetes growth.

Review represents data on new active metabolites isolated from marine actinomycetes published in 2007 to 2014. Marine actinomycetes are an unlimited source of novel secondary metabolites with various biological activities. Among them there are antibiotics, anticancer compounds, inhibitors of biochemical processes.

The major characteristics of new bioactive microbial secondary metabolites are summarized in the review. A wide range of new molecular targets are implicated in discovery of new nonantibiotic compounds with some other pharmacological activities (noninfectious diseases). Microorganisms represent fascinating resources due to their production of novel products with broad spectra of bioactivities.

It was thought that antibiotics should be produced by soil microorganisms to inhibit the growth of competitors in natural habitats. Yet it has been shown that antibiotics at subinhibitory concentrations may have a role as signalling molecules providing cell-to-cell communication in bacteria in the environment. Antibiotics modulate gene transcription and regulate gene expression in microbial populations. Subinhibitory concentrations of antibiotics may cause a number of phenotypic and genotypic changes in microorganisms. These transcription changes are dependent on the interaction of antibiotics with macromolecular receptors such as ribosome or RNA-polymerase. Antibiotic signalling and quorum-sensing system are important regulatory mechanisms in bacteria. It was demonstrated that antibiotics interfered with quorum-sensing system.

The known conceptions of resistome as a complex of all the antibiotic resistance genes in the genomes of all the microorganisms, pathogenic and nonpathogenic ones, in nature are considered. The data on the origin, evolution and distribution of antibiotic resistance genes and possible approaches to the resistance distribution control are presented.

The third part of the review is concerned with investigation of nonribosomal peptides.

The data on novel polypeptide antibiotics described within the last 10-15 years, as well as new researches on the known antibiotics with respect to their mechanisms of action and microbial resistance are presented.

The impact of glycine, added to the cultivation medium, on resistance of Staphylococcus aureus strains to actinomycin D and gramicidin S was studied. The antibiotic resistant strains were isolated after cultivation of the susceptible S. aureus strain 209P on media with increasing concentrations of actinomycin or gramicidin. When the strains were grown on the glycine-containing medium. i. e. under the conditions providing replacement of D-alanine by glycine in the C-end dipeptides of peptidoglycanes, the resistance of the staphylococci to actinomycin markedly decreased. However, in the resistant cells, characterized by significant thickening of the cell walls, the peptidoglycane quantity per a biomass unit did not lower, that was evident of preservation of the wall thickness. At the same time, with addition of glycine to the medium there was observed increased ability of the cells to bind actinomycin. When the gramicidin-adapted strains were grown on the glycine-containing medium, their resistance to the antibiotic did not change. The modification of the peptidoglycane C-end dipeptides probably lowered the protective role of the thicker walls of the cells on their contact with actinomycin but not gramicidin.

Cell walls of Staphylococcus aureus R9/80 resistant to gramicidin S and actinomycin D were investigated. The strain was isolated after passages of a previously isolated strain of S. aureus with resistance to gramicidin and definite changes in the cell walls, a medium with increasing concentrations of actinomycin being used for the passages. The data on the study of the cell walls of the strain with the double resistance were compared with the results of the investigation of the cell walls of the strain susceptible to gramicidin, the gramicidin resistant strain (initial for strain R9/80) and the actinomycin adapted strain that also showed changes in the cell walls. The cell walls of the resistant strains had no significant changes in the peptidoglycane and glucosamine levels, as well as in the peptidoglycane amino acid composition. Teichoic acids of all the strains had different levels of substitution of ribite by D-alanine (a factor influencing the negative charge of teichoic acids and the wall at large). It was noted that all the strains resistant to the tested antibiotics had lower levels of teichoic acids in the cell walls. The resistant cells showed some increase of the lipid component in the walls: from 1.6% in the susceptible strain to 2.1-2.9% in the resistant cells. The main trend of the changes in the resistance development was revealed to be the thickening of the cell wall and its consolidation. The development of resistance to gramicidin, actinomycin and to both the antibiotics provoked respectively a 2.4-, 4- and 5.4-fold increase of the content of the main cell component. i.e. peptidoglycane in the cell biomass. The barrier role of the cell walls in the resistant strains and their ability to bind the antibiotic is discussed.

Cell walls in 2 strains of Staphylococcus aureus 209P, i.e. actinomycin D susceptible and resistant ones were comparatively investigated. The resistant cells contained much more wall material per a unit of the biomass weight vs the susceptible strain cells, that conformed to thickening of the resistant cell walls detected by electron microscopy and a sharp increase of their electron density. Investigation of peptidoglycans and teichoic acids did not reveal any significant alterations in the structure of the wall components in the actinomycin D resistant cells. Only some increase of glucosamine in the peptidoglycan fraction of the resistant cells vs the susceptible ones was observed. It was shown that preparations of the resistant cell walls and peptidoglycan isolated from the resistant cells were able to bind somewhat lower quantities of actinomycin D vs the analogous preparations of the susceptible cells. The significant decrease of the antibiotic binding by live cells of the resistant strain probably slightly depended on the structure characteristics of the main wall components. The barrier properties of the walls in resistant staphylococci are most likely defined by the wall thickening and consolidation while adapting to actinomycin D.

Comparative study of two staphylococcus aureus 209P strains--resistant and susceptible to gramicidin S demonstrated that peptidoglycanes of two strains differ by ratio glycine/serine at peptide bridges. Besides peptidoglycanes significantly differ by amidation of alfa-carboxyles of glutamic acid in muropeptide. This peptidoglycane modification of resistant cells along with enhanced content of etherized D-alanine in teichoic acid provides lower negative charge of cell wall components. It may influence the cell wall ability to react with positively charged gramicidin molecules. It was shown that isolated cell walls and peptidoglycane of resistant cells binds significantly less gramicidin than cell walls and peptodoglyce of susceptable cells. Simultaneous determination of gramicidin binding by intact S. aureus cells and their killing revealed that lower ability of resistant cells to bind gramicidin is significant but not critical factor of gramicidin resistance.

Staphylococcus aureus strains, resistant to actinomycin D (AMD) and to gramicidin S (GS) were selected by S. aureus 209P passing on the media containing the above mentioned drugs. Strain R80 resistant to AMD and strain R9 resistant to GS and AMD and described before didn't perform enzyme inactivation of AMD. Cells of both strains had diminished ability to bind exogenous AMD. Electron microscopy investigation revealed that cells of R80 strain had thickened cell walls and they are characterized by more electron density then cells of R9 strain and of parent strain. Adaption to AMD and GS influenced also on functions of some staphylococcal surface proteins--the activity of endogenous coagulase (clumping factor) was found only in R9 strain. Exogenous coagulase was present in all the strains, but development of resistant to AMD and GS diminished this enzyme activity. It is concluded that development of resistance to AMD and GS causes substantial changes in staphylococcal cell wall, but the type of these changes differ.

Streptomyces chrysomallus is known as an organism producing macrotetrolides (MTL) and actinomycin C. The dynamics of the MTL biosynthesis by some variants of S. chrysomallus in the process of their growth in liquid media was studied. In parallel the ability of the culture mycelium (washed or suspended in physiological solution) to bind exogenous actinomycin D (AMD) was estimated. An inverse correlation between the dynamics of MTL biosynthesis and the rate of the AMD binding by the washed mycelium during the whole period of the culture development was observed: a decrease in the culture ability to bind AMD corresponded to active biosynthesis of MTL and an increase in the culture ability to bind AMD corresponded to lower biosynthesis of MTL. It was suggested that the active biosynthesis of MTL correlated not only with a decrease in the ability of the suspended mycelium to bind AMD but also with a decrease in binding of actinomycin synthesized and excreted to the medium by the culture. A decrease in the reflux of the synthesized antibiotic to the cells was likely one of the components of the system of the S. chrysomallus insensitivity to its own antibiotic.

Binding of exogenous actinomycin D (AMD) by washed mycelium of streptomycetes i.e. variants of Streptomyces chrysomallus producing and not producing actinomycins and Streptomyces lividans not synthesizing the antibiotics was studied. Dependence of the bound quantity of AMD on its concentration, incubation time and temperature, energy source availability, influence of respiration inhibitors and the membranotropic antibiotic gramicidin S was shown. The intracellularly localized portion of the bound AMD likely penetrated to the cells by diffusion and was strongly bound presumably to DNA in the AMD sensitive S.lividans and to the specific intracellular actinomycin-binding proteins in the AMD resistant variants of S.chrysomallus. The ratio of AMD strongly bound by the mycelium and AMD easily washed with physiological solution and probably localized on the surface was determined. The ratio depended on sensitivity of the culture to AMD and for the variants of S.chrysomallus on the age of the culture and its ability to synthesize actinomycins.

The affect of preliminary irradiation of staphylococcus culture by electromagnetic radiation of extremely high frequency (42, 54, 66 + 78 GHz) of nonthermal intensity on the bacteria growth on the media containing various antibiotics is studied. The reliable change in bacteria sensitivity toward 5 antibiotics, mainly having membranotropic properties is observed in the experiments using 14 antibiotics with various mechanisms of action. It has been established that in the presence of subbactericide concentrations of active antibiotics the irradiation could result in both further suppression of bacteria growth and its stimulation. As shown, the development of these effects takes place even in a matter of minutes of preliminary irradiation, and weak changes are observed at further increase of this period up to 60 min.

The absorption of actinomycin D by the cell suspension of Staphylococcus aureus via diffusion linearly depended on the antibiotic concentration in the suspension within the ranges of 2 to 15 micrograms/ml. The absorption of active actinomycins C2, C3 and Au6 was the same as that of actinomycin D. The Staphylococcus intact membranes limited the inlet of the actinomycins to the cells since the membranotropic substances such as gramicidin S and its derivatives and thyrocidin increased their absorption by 30-70 per cent. The absorption of a low active actinomycin D0 and inactive actinomycinic acid even after the exposure to the membranotropic substances was not detectable. These compounds did not form any complexes with DNA. The level of the absorption of the actinomycins by the cells was likely defined by their ability to complex with DNA.

Factors defining actinomycin D resistance in Staphylococcus aureus resistant to gramicidin S were investigated. The results of the thin layer chromatography, high-voltage electrophoresis and bioautography showed that the resistant cells did not inactivate actinomycin D by the hydrolysis of the lactone bond in the antibiotic molecule. The estimation of the cell ability to bind actinomycin D revealed that the antibiotic binding to the resistant cells was lower by 70-75 per cent as compared to the cells of the susceptible strains. Gramicidin S impaired the intactness of the cytoplasmic membranes and increased the absorption of actinomycin D by the susceptible cells and to a much lesser extent by the cells of the resistant strains. Actinomycin D bound by the susceptible cells could not be washed out with a buffer solution. It could be separated from the cells only by extraction with an organic solvent. Comparative electron microscopy of the susceptible and resistant cells demonstrated that the cell walls in the resistant strains were 1.5-2-fold thicker than the cell walls in the susceptible strains. The actinomycin D resistance of the Staphylococcus strains resistant gramicidin S was likely conditioned by the barrier properties of the morphologically changed cell walls.

Strains of Staphylococcus aureus 209P growing in the presence of 20 micrograms/ml of gramicidin S were isolated after the successive subculture on a liquid medium with increasing concentrations of the antibiotic. The resistance was stable and preserved after the subculture on media not containing the antibiotic. The development of the resistance to gramicidin S did not lower the cell sensitivity to a large number of antibiotics known as inhibitors of the cell wall synthesis, protein synthesis and RNA-polymerase reaction. There was observed the development of moderate resistance to actinomycin D but not to other antibiotics interacting with DNA. The gramicidin resistant strains were also resistant to tyrocidine, a membrane active polypeptide. By the amount of the bound gramicidin S the cells of the sensitive and resistant strains did not practically differ.

Polarographic determination of the rate of endogenic respiration of the cells of Staphylococcus aureus 209P showed that the respiration activity of the cells of the strain resistant to 20 micrograms/ml of gramicidin S was 20 to 30 per cent lower than that of the sensitive strain. The rate of oxygen consumption in oxidation of NADH by the membrane preparations of the resistant cells was also 25 to 30 per cent lower. By comparison with the initial sensitive strain the activity of endogenic DPI-reductases of the intact cells and NADH-dehydrogenases of the membranes of the resistant strain was also lower. The velocity of the valine transport to the resistant cells was much lower than that of the amino acid transport to the cells of the sensitive strain. Development of gramicidin S resistance in the staphylococcal strain was likely accompanied by a decrease in the activity of the energy metabolism in the membranes.

The absence of pyruvate and insignificant levels of alpha-keto-glutarate in the mycelium during the fermentation cycle were characteristic of a highly active erythromycin-producing strain of Saccharopolyspora erythraea. Alpha-keto-glutarate partially excreted to the fermentoffon broth. The activity of pyruvate decarboxylase and alpha-keto-glutarate decarboxylase was detected in the cells during the entire period of the cultivation. The same regularities were observed in the chloramphenicol resistant mutant of the strain. The mycelium of a low productive strain of S.erythraea contained not only alpha-keto-glutarate but also pyruvate and excreted large amounts of keto-acids. By the activity levels of the decarboxylases the low productive strain did not differ from the highly productive one. Propanol did not influence the growth of the low productive strain and the synthesis of erythromycin by it. However, it stimulated accumulation of keto-acids and especially pyruvate in both the mycelium and fermentation broth. Relation between the intensity of keto-acid metabolism and erythromycin biosynthesis is discussed.

The work was aimed at studying the effect of gramicidin S on the intact cells, spheroplasts and membrane specimens of Escherichia coli K12S with the natural resistance to this antibiotic. The resistance was shown to be caused by the barrier properties of the cell wall: the spheroplasts were highly sensitive to the lytic action of gramicidin S. The differences in the sensitivity to gramicidin S of substrate oxidation carried by the membranes of E. coli and Micrococcus luteus, a sensitive organism, were not of crucial significance for the manifestation of the resistance. The resistance was not associated with the decrease of gramicidin S adsorption: the cells were capable of binding large quantities of the antibiotic and remaining viable. Gramicidin S appeared to be attached to the cell walls (most likely, the outer membranes) rather than the cytoplasmic membranes.

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.

The effect of membrane active antibiotics, such as gramicidin S, its derivatives and carbonyl-conjugated pentaens on suspended bacterial protoplasts resulted in lysis of the protoplasts accompanied by a marked decrease in the optical density (OD) of the suspensions. However, when the drug concentrations were lower than those inducing the lysis, an increase in the suspension OD by 10-30 per cent as compared to the control values of the OD was often observed. The increase in the protoplast suspension OD was most pronounced with the use of the drugs with a relatively low lytic activity or under the conditions lowering the lytic activity of the antibiotics. Under such conditions no agglutination of the protoplasts was observed. The comparative estimation of the protoplast diameters by the method of Klenin et al. in the control suspensions of M. lysodeikticus protoplasts and in the suspensions with a stable increase in the OD showed that the OD increase was associated with swelling of the protoplasts: an increase in the suspension OD by approximately 30 per cent corresponded to an increase in the protoplast diameter by approximately 15 per cent. The observed increasing of the suspension OD must be due to the fact that the membrane active antibiotics induced a decrease in the osmotic stability of the protoplasts not sufficient for their lysis.

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.

Neotelomycin induced lysis of the protoplasts of Bac. megaterium and inhibited their succinate dehydrogenase activity. Direct correlation between the lytic activity of the antibiotic and its effect on succinate dehydrogenase was found. Neotelomycin had no effect on the dehydrogenase activity of the protoplast lysates. Possibly, suppression of the protoplast succinate dehydrogenase of Bac. megaterium under the effect of neotelomycin was due to significant structural changes caused by the antibiotic in the protoplast membranes and leading to their lysis and not to the direct effect on the enzyme. Neotelomycin had practically no effect on the spheroplast dehydrogenase activity of E. coli resistant to the antibiotic and did not induce their lysis. Resistance of E. coli to neotelomycin must be associated not with the presence of the antibiotic non-permeable cell wall but the peculiar properties of the membrane cytoplasm.

In concentrations of 0.05-0.10 gamma/ml geliomycin suppresses inclusion of C14-uracyl into the acid-soluble fractions of the staphylococcal cells. Inclusion of C14-leucine at the same concentrations of the antibiotics was suppressed to a lesser extent. The above concentrations of geliomycin had practically no effect on inclusion of C14-thimine. Suppression of C14-uracyl inclusion was observed in 5 minutes of the cell incubation with the antibiotic, a significant decrease in C14-leucine inclusion being observed only in 30 minutes of the incubation. The effect of geliomycin on the staphylococcal cells within the concentrations inhibiting the microbial growth and suppressing inclusion of labeled uracyl and leucine into the cells was of the bacteriostatic nature. It was supposed that the antibacterial effect of geliomycin was based on suppression of RNA synthesis.

The effect of heliomycin and known uncouplers of oxidative phosphorylation on respiration and oxidative phosphorylation was studied comparatively. Heliomycin, as well as 2,4-dinitrophenol, valinomycin and gramicidin S inhibited the mitochondrial synthesis of ATP. This process was inhibited completely by heliomycin at a concentration of 1.5 x 10(-5) M. The synthesis of inorganic pyrophosphate, the other macroergic compound, was also inhibited by heliomycin, ATPase and pyrophosphatase of uncoupled mitochondria being not inhibited by the antibiotic. Like 2,4-dinitrophenol, heliomycin stimulated the synthesis of ATPase and respiration in intact mitochondria. Probably, heliomycin inhibited the synthesis of ATP and pyrophosphate by uncoupling the processes of respiration and oxidative phosphorylation. It was shown earlier that heliomycin, a specific inhibitor of bacterial RNA synthesis, also affected energy metabolism of bacterial cells by inhibiting the synthesis of ATP and active transport.

The effect of cyclic decapeptide of gramicidin S on electrical conductivity of bilayer lipid membranes has been studied. The integral conductivity of bilayer has been shown to increase with the growth of antibiotic concentration. The integral conductivity increase occurs as series of conductivity discrete leaps, differing in amplitude from fluctuations of conductivity caused by linear gramicidins. In the series of selectivity of bilayer membranes for cations of alkaline metals the rubidium ion is before the cesium ion. This is the only difference between this series and the series of relative ionic mobility series of cations of alkaline metals in water solutions.

Heliomycin at a concentration of 100 mcg/ml had almost no effect on the growth of Escherichia coli, which was inhibited only upon the combined action of heliomycin and ethanol that facilitated the penetration of the antibiotic into the cell. Heliomycin inhibited in these conditions the synthesis of RNA and had no effect on the synthesis of DNA in the three studied strains of E. coli. The synthesis of protein either was not inhibited by heliomycin at all or was inhibited only to a small extent.

Variants of Micrococcus lysodeikticus resistant to 100 micrograms/ml of gramicidin S with preserved resistance in subcultures on media without the antibiotic were isolated as a result of prolonged adaptation on a solid medium with increasing concentrations of gramicidin. The sensitive and resistant cells did not differ by their ability to bind gramicidin. Under the antibiotic effect permeability of the cytoplasmic membranes of the intact cells in the sensitive bacteria appeared to be impaired to a greater extent than that of the membranes of the cells in the resistant variant. Comparison of the lytic activity of gramicidin and its derivatives with respect to the protoplasts prepared with the cells of the initial and resistant variants of M. lysodeikticus revealed much higher resistance of the resistant variant protoplasts to the membrane-disorganizing effect of the preparations. Malate dehydrogenase and NADH-oxidase in the membrane preparations of the resistant variant cells differed from analogous enzymes from the membranes of the initial strain by the levels of their activity and sensitivity to gramicidin. It is likely that during adaptation of M. lysodeikticus to gramicidin significant changes in the cell cytoplasmic membranes occurred.

The membrane activity of gramicidin S against intact cells of staphylococci and micrococci determined by the loss of intracellular low-molecular compounds with the adsorption maxima at 260 nm by the bacteria markedly increased in the presence of phosphates. Acetate and hydrochlorides had no effect on the membranotropic action of the antibiotic. Analogous results were obtained for gramicidin S derivatives by free ornithine amino groups possessing basic and antibiotic activity. The increased membrane action of the antibiotic on the cells in the presence of phosphates was probably due to the changes under these conditions in the aggregate state of the substance in solution. With the use of labeled gramicidin S it was shown that centrifugation of the antibiotic solution in a concentration of 50-1000 micrograms/ml at 18000-20000 rpm resulted in sedimentation of a significant part of gramicidin S dissolved in the phosphate buffer. Sedimentation of the drug in aqueous and NaCl solutions was insignificant. The presence of phosphates in the medium had no effect on the quantity of the antibiotic bound to the micrococcal membrane preparations. It is suggested that the increase in the level of cytoplasmic membrane disorganization in the presence of phosphates was due to binding of the antibiotic molecule associations to the membranes and/or additional intermolecular association of gramicidin S bound to the membranes.

Exposure of intact mycelium and protoplasts of Streptomyces sp. 26-115 to gramicidin S resulted in impairment of permeability of the cytoplasmic membranes thus inducing a loss of low molecular compounds by the cells and protoplasts. Aminoacyl and guanyl derivatives of gramicidin S with respect to the delta-amine group of ornithine were somewhat less effective with respect to the streptomycete membranes. The same as the initial antibiotic, these derivatives had positively charged groups. The changes in the membranes of the streptomycete protoplasts induced by gramicidin S and its derivatives did not result in the protoplast lysis accompanied by decreasing of the suspension optical density. The neutral derivative having no positive charge and not capable of complexing with the membrane phospholipids and the carboxyl-containing derivative with a negative charge showed a low membrane activity when applied to the protoplasts and induced no impairment of permeability of the intact mycelium membranes. The use of phosphates as factors increasing the membrane activity of the gramicidin derivatives having no positive charge is not possible since in the presence of phosphates streptomycete protoplasts are readily destroyed.

Heliomycin inhibited formation of the pool or purine and pyrimidine containing compounds in the cells of the growing cultures of Staphylococcus aureus and Escherichia coli both in the lag phase at the maximum rate of formation of the pool of compounds absorbing at 260 nm and in the exponential phase. A decrease in the concentration of purine and pyrimidine containing compounds in the acid-soluble fraction of the cells by the antibiotic was not caused by the disturbed permeability of the membranes. Apparently, heliomycin interfered with the regulation of RNA synthesis, either directly or through its action on the energy metabolism of the cells.

Gramicidin S and its derivatives by the free amino groups of the ornithine moieties were studied with respect to their effect on interface tension of mercury and the polarographic background for comparative estimation of the surface active properties of these compounds. The same way as the compounds preserving their main properties, the neutral and acid derivatives lowered the polarographic maximum. No correlation between the level of the antimicrobial and membrane activity and the surface active properties of the tested compounds was observed. It is likely that the direct cause of the induced biological effect is not connected with the surface active properties responsible for possible interaction of the tested substances with the cell (protoplast).

Effect of cyclopeptide antibiotic gramicidin S on some enzymes and physical state of isolated Micrococcus lysodeikticus membranes is studied. Malate and lactate dehydrogenases were monotonously inhibited under the increase of gramicidin S concentration, while the activity of NADH-dehydrogenase firstly decreased and then reversed to the initial level under further increase of gramicidin S concentration. The oxygen uptake under oxidation of NADH and malate with membranes almost completely inhibited by the antibiotic, while the activity of ascorbate-TMPD-oxidase activity slightly inhibited by the same concentration of gramicidin. The addition of Triton X-100 completely eliminated the inhibitory effect of gramicidin on malate dehydrogenase. The introduction into the membrane of spine probes (2,2,6,6-tetramethyl-4-palmitoylamidopiperidine-1-oxile and 2(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxyazolidinyloxile) revealed that gramicidin caused the condensation of membrane lipid component. It is suggested that ionic interaction of gramicidin S with membrane phospholipids brings to "a freezing" of lipids which is a direct cause of impairing the activity of membrane respiration enzymes and the change of their position in the lipid matrix, thus inhibiting energy-producing processes in cell.

Heliomycin inhibited synthesis of RNA in Staph. aureua which was clearly shown in the study of the antibiotic effect on RNA synthesis in the lag phase of the culture development: heliomycin markedly lowered the maximum RNA level in the biomass observed in the culture at the beginning of the exponential growth. On further growth of the culture heliomycin induced a significant retardness of the process of the natural decrease in the RNA biomass level resulting in increased content of RNA in the cells growing in the presence of heliomycin as compared to the control culture. Retarded natural decrease in the RNA biomass level in the presence of heliomycin was observed also on the antibiotic addition just at the beginning of the exponential growth, during the period of maximum RNA accumulation in the cells. Heliomycin had no effect on synthesis and biomass levels of DNA. Heliomycin inhibited the protein synthesis and was close to chloramphenicol by the level of inhibition of the summation protein synthesis in the biomass. However, comparison of the effect of the above antibiotics on synthesis of beta-galactosidase, an individual enzyme protein showed that heliomycin was much less active as an inhibitor of protein synthesis in comparison to chloramphenicol.

The gramicidin S-producing organisms Bacillus brevis was grown under submerged conditions in fermenters at a temperature of 32 degrees C which is the lower limit of the temperature ranges providing the culture growth. The culture was also grown under the optimal temperature conditions, i.e. 40 degrees C. At the lower temperature the biomass synthesis at the early stage of the culture development was decreased, the intensity of the cell respiration was low and solubility of oxygen in the liquid was increased. Under such conditions the decrease in the levels of dissolved oxygen in the medium was less pronounced than that under the control conditions. At the lower temperature limitation of the culture growth by the deficit of dissolved oxygen lowered and the period of the antibiotic synthesis increased which brought about a rise of the gramicidin S yield by 30-40 per cent.

When used in concentrations of 10-15 micrograms per 10(8) cells, flavofungin and nigrofungin, carbonyl-conjugated pentaenes lowered the rate of oxygen absorption by thymocyte suspensions in the presence of glucose. Flavopentin inhibited glucose oxidation in higher concentrations. ADP and succinate did not penetrate through the thymocyte membranes and were not oxidized with the cell suspensions. In concentrations of 30 micrograms per 10(8) cells the pentaenes did not change the membrane permeability for ADP. Still, they induced oxidation of succinate by the thymocytes (in the presence of amital). Flavofungin and nigrofungin were more effective with respect to increasing the membrane permeability for succinate as compared to flavopentin. The differences in the membrane permeability for various substrates induced by the antibiotics indicated a definite specific nature of the impairment of the thymocyte cytoplasmic membranes by carbonyl-conjugated pentaenes. The antibiotics tested are very similar as to their chemical structure and physicochemical properties. It is suggested that the differences in the biological activity of these substances are not associated with the presence of definite functional groups but are due to the general configuration of the antibiotic molecules, determining their hydrophilic-lipophilic balance and capacity for interaction with the biological membranes.

The effect of neotelomycin derivatives on the cells of Bac. megaterium was studied. Derivatives with modification of one of the two active centers of the antibiotic molecule, i.e. the free alpha-amine group of the residue of asparaginic acid or hydrophobic triptophanic structure were studied. The derivative with modified indol rings of the residues of beta-methyl-and dehydrotriptophane induced the same though lower damages as the natural antibiotic: increased permeability of the cytoplasmic membranes, protoplast lysis, suppression of the dehydrogenase activity. The activity of this derivative was due to the free amino group and amounted approximately to 3 per cent of the activity of neotelomycin. The derivative with the free amino group of the asparaginic acid residue replaced by the benzoylic group showed a high antibacterial activity but had almost no effect on the membrane permeability and a very low lytic effect. The capacity of this derivative to inhibit the bacterial dehydrogenase activity remained relatively high. Possibly the free amino group of the asparaginic acid residue provided neotelomycin with the capacity for damaging the structure of the bacterial cytoplasmic membranes. No detectable damages in the membrane state after exposure to the benzoylic derivative, as well as its high antibacterial activity are evident of the fact that the mechanism of action of the benzoylic derivative on the cells was in principal different from that of the derivative preserving the free amino group. The triptophane structure was probably not only the center actively affecting the cell but also the factor that provided the antibiotic molecule with conformation most favourable for the action of the free amino group on the membrane structures.

The work was concerned with studying the effect of gramicidin S derivatives with modified free amino groups of ornithine residues on bacterial cells and protoplasts. The substitution of the amino groups with neutral or carboxyl-containing groups eliminated or sharply decreased the antibacterial activity of gramicidin S, its binding to the cells, and the ability to change the permeability of the cytoplasmic membranes of the intact cells. However, the neutral derivatives and the derivative with acidic properties showed a considerable lytic activity when they were incubated with the protoplasts of Micrococcus lysodeikticus, Bacillus megaterium and Bacillus subtilis. Hence, these compounds preserved a certain membranotropic level. Those gramicidin S derivatives with modified ornithine amino groups which possessed basic properties were similar to gramicidin S in the antibiotic activity, the modified permeability of the membranes, the ability to bind with the cells, and the lytic action on the protoplasts.

The lysis of Bacillus subtilis protoplasts by gramicidin S, a membrane active antibiotic, and its derivatives was studied according to free amino groups of the ornithine residue. The initial antibiotic and guanylgramicidin , a positive charge-preserving derivative, had a high lytic activity. Succinylgramicidin , a gramicidin S derivative with acid properties, and carbomoylgramicidin , a neutral derivative, actively lysed B. subtilis protoplasts suspended in 1/15 M phosphate buffer solution with sucrose . No lytic activity of succinylgramicidin was observed with respect to B. subtilis protoplasts suspended in an aqueous solution of sucrose. Comparative study on the sensitivity of the protoplasts of Micrococcus lysodeikticus, B. megaterium and B. subtilis to the lytic action of gramicidin S and its derivatives showed in the main a similar character of their interaction with the membranes of the protoplasts of the taxonomically close species (B. megaterium and B. subtilis). It is likely that the specificity of the action of the above substances on the protoplasts of M. lysodeikticus, i. e. a complicated character of the dependence of the lytic action of gramicidin S on its concentration, manifestation of the lytic activity of the neutral and acid derivatives in the presence of phosphates or other salts and in sucrose aqueous solution was mainly defined by the properties of the micrococcal membranes.

Influence of gramicidin S on electric parameters of nitrocellulose ultrafilter as a biomembrane model was studied, the ultrafilters being impregnated with fatty acids or their ethers. It was shown that addition of the antibiotic to the solution over one side of the model membrane resulted in generation of electric potential. With increasing of the drug concentration by one order there was observed more than a 10-fold drop in the membrane resistance while the electric capacitance actually remained unchanged. It was suggested that gramicidin S was localized in thin water layers covering the surface of the ultrafilter pores and separating the polymer matrix and impregnating liquid filling the pores. Such incorporation led to changes in the state of water and water channel surfaces which defined the increase in the model membrane electric conductivity.

The activity of superoxide dismutase (SOD) in the cells of the microorganisms producing gramicidin S, B. brevis var. G.-B., ranged from 7 to 22 units per mg protein. When the culture was grown in a fermenter, the decrease in the concentration of dissolved oxygen in the medium to the minimum values corresponded to the decrease in the enzyme activity. By the end of the fermentation process, when consumption of oxygen by the culture lowered and its level in the medium increased, the enzyme activity increased. When the culture was grown in flasks, no effect of the medium aeration level on the enzyme activity was noted. This might be associated with rather high levels of dissolved oxygen in the medium in the flasks under all aeration conditions. Addition of manganese ions to the cultivation medium activated SOD in the cells of B. brevis. Synthesis of gramicidin S by the bacterial culture had no effect on the activity of SOD in the cells.

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.