Secondary Metabolites from Marine Microorganisms. II. Marine Fungi and Their Habitats.

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.

[Secondary Metabolites from Marine Microorganisms. I. Secondary Metabolites from Marine Actinomycetes].

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.

[Microbial secondary metabolites as potential reserve of pharmaceuticals].

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.

[Action of antibiotics as signalling molecules].

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.

[Biologically active nonribosomal peptides. III. Mechanism of biosynthesis of nonribosomal peptides].

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

[Biologically active nonribosomal peptides. I. Nonribosamal polypeptide antibiotics].

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.

[Impact of exogenous glycine on actinomycin D resistance of Staphylococcus aureus strain adapted to the antibiotic].

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 wall components in Staphylococcus aureus with double resistance to gramicidin S and actinomycin D].

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.

[Investigation of cell wall components in actinomycin D resistant Staphylococcus aureus]. / Izuchenie komponentov kletochnoi stenki Staphylococcus aureus, ustoichivogo k aktinomitsinu D.

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.

[Cell wall components of gramicidin S resistant Staphylococcus aureus]. / Izuchenie komponentov kletochnoi stenki Staphylococcus aureus, ustoichivogo k gramitsidinu S.

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.

[The study of Staphylococcus aureus strain resistant to actinomycin D]. / Izuchenie shtamma Staphylococcus aureus, ustoichivogo k aktinomitsinu D.

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.

[The capacity of the mycelia of Streptomyces chrysomallus variants to bind exogenous actinomycin D and the synthesis by these variants of macrotetralides]. / Sposobnost' mitseliia varinatov Streptomyces chrysomallus sviazyvat' ékzogennyi aktinomitsin D i sintez étimi variantami makrotetrolidov.

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.

[Interaction of actinomycin D with suspended mycelium of streptomyces]. / Vzaimodelstvie aktinomitsina D s suspendirovannym mitseliem streptomitsetov

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 effect of millimeter-band radiation of nonthermal intensity on sensitivity of Staphylococcus to various antibiotics]. / Vliianie millimetrovogo izlucheniia neteplovoi intensivnosti na chuvstvitel'nost' stafilokokka k razlichnym antibiotikam.

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.

[Absorption of various actinomycins by Staphylococcus aureus cells]. / Pogloshchenie kletkami Staphylococcus aureus razlichnykh aktinomitsinov.

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.

[Actinomycin D resistance of gramicidin-resistant strains of Staphylococcus]. / Ustoichivost' k aktinomitsinu D gramitsidinustoichivykh shtammov stafilokokka.

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.

[Biologically active substances formed by a number of strains of the actinomycin C producer--Streptomyces chrysomallus]. / Biologicheski aktivnye veshchestva, obrazuemye riadom shtammov produtsenta aktinomitsina C--Streptomyces chrysomallus.

Biologically active substances produced by some strains of S. chrysomallus and their mutants with different levels of the differentiation were studied. Along with actinomycin C the strains produced macrotetrolides, bacteriocins and A factor-like substances. It was shown that the plasmid status of the strains was different. This suggested that the plasmid presence was a characteristic of the strains and the production of the studied substances was likely typical of S. chrysomallus.

[Macrotetrolide antibiotics from a recombinant strain of Streptococcus chrysomallus]. / Makrotetrolidnye antibiotiki iz rekombinantnogo shtamma Streptococcus chrysomallus.

A mixture of antibiotic substances was isolated from the culture fluid of a recombinant strain of Streptomyces chrysomallus. The substances were identified as macrotetrolides (nonactin, monactin, dinactin and trinactin) by the mass spectrometry, ionoform properties and thin layer chromatography in the antibiotic samples and ethylacetate extracts of the fermentation broth filtrate. The parent strain of Streptomyces chrysomallus also synthesized a mixture of macrotetrolides. However, it contained no nonactin and the total level of the macrotetrolide biosynthesis was 5 to 7 time lower than that in the recombinant strain (determined by a chemical method).