Isolation and physicochemical study of the components of the new antibacterial antibiotic 2562

An antibacterial antibiotic complex consisting of 2 components designated as 2562 A and 2562 B is produced by Streptomyces griseovarabilis. The antibiotic was isolated from the mycelium and purified chromatographically on a column with aqueous silicic acid. The study of the components showed that component 2562 A was chlorbiocin, while component 2562 B differed from the known antibiotics of this group. Physicochemical assays demonstrated that component 2562 B differed from chlorbiocin by the absence of the methyl group in pyrrol, which is probably attached to sugar at beta-position. It was found that component 2562 B is a new representative of the antibiotic cumero-glycoside group.     

Investigation of the antibiotic complex produced by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> 194, Variant K

Phase variation in the culture of the environmental strain Lactococcus lactis subsp. lactis 194 resulted in the formation of two types of colonies differing by 15% in antibiotic activity. The active variant 194-K produced an antibiotic complex with a broad spectrum of antibacterial and antifungal activity. Five components (194-A, B, C, D, and E) were isolated from the complex by solid-phase extraction and thin-layer chromatography. Components 194-A and 194-B were hydrophobic neutral compounds soluble in organic solvents. Component 194-A possessed fungicidal activity, whereas component 194-B exhibited only bactericidal activity. Physicochemical studies of the isolated components 194-A and 194-B revealed that they had no analogs in the Berdy database of biologically active substances (BNPD) and appeared to be novel antibiotics. Component 194-C was a hydrophilic polar compound inhibiting growth of gram-positive and gramnegative bacteria. Component 194-D belonged to peptide antibiotics; it inhibited growth of only gram-positive bacteria and was similar to nisin A in biological properties but differed in electrophoretic mobility and molecular mass.     

Studies on the production of actinomycin-D by Streptomyces griseoruber– a novel source

Aims:  To isolate and characterize bioactive metabolites produced by a micro-organism isolated from a soil sample associated with the roots of a medicinal plant, Azadirachta indica .
Methods and Results:  Morphological, cultural, physiological and 16S rRNA homology studies revealed that the organism showed 99% similarity with Streptomyces griseoruber NBRC 12873. One bioactive metabolite (Py2) isolated from the fermented broth was characterized as actinomycin-D (act-D). It showed high activity against various Gram-positive and Gram-negative bacterial cultures, Mycobacterium tuberculosis H37Rv and human neoplastic cells in vitro using standard protocols.
Conclusions:  The isolated strain S. griseoruber produced act-D predominantly (210 mg l⁻¹, c. 88% of the crude) under nonoptimized growth conditions.
Significance and Impact of the Study:  Streptomyces griseoruber may be exploited as a potential source for the commercial production of act-D, as this strain is not reported to produce act-D. Further investigations on the strain for commercial application will be of immense pharmaceutical importance.     

Changes in the Extracellular Accumulation of Antibiotics during Growth and Sporulation of Bacillus subtilis in Liquid Culture

Ten antimicrobial metabolites, produced by Bacillus subtilis NCIB 8872, have been categorized into 3 groups on the basis of their antimicrobial spectra, chromatographic mobility and solvent solubility. The maximum concentration of the different groups occurred at different times during fermentation. The accumulation of one antibiotic group was characteristic of the logarithmic growth phase. Groups also differed in their persistence. The intracellular antibiotics contributed little to the total antibiotic activity of the culture. The onset of production and the maintenance of the extracellular accumulation of the 3 antibiotic groups were linked to sporulation and associated specific changes in the intracellular and extracellular protein complement. Production of the antibiotics was not controlled by glucose catabolite repression, since the presence or absence of glucose in the medium did not affect the pattern of antibiotic accumulation.     

Effects of AMO-1618 and B-995 on Growth and Endogenous Gibberellin Content of Cupressus arizonica seedlings

Soil drenches of 250, 500 or 1000 mg/l of the growth retardants AMO-1618 or B-995 effectively reduced dry matter production and stem elongation in young seedlings of Cupressus arizonica Greene. In seedlings treated with AMO-1618, the acidic, ethyl acetate-soluble gibberellin-like substances (GAs), as detected. by bioassay, were reduced to almost undetectable levels. However, the endogenous GA content in seedlings treated with B-995 were at least 11-fold greater than in control seedlings and differed as well in chromatographic characteristics, being of a more polar nature than the endogenous GAs of control seedlings. It was concluded that while AMO-1618 probably acts through interference with GA biosynthesis, B-995 may act through the interconversion of GAs.     

An antibiotic complex produced by Streptomyces werraensis 1365T strain

An antibiotic complex comprising four components (A, B, C, and X) was extracted from a native solution and mycelium of Streptomyces werraensis 1365T. The components were purified by column and thin-layer (TLC) chromatographic procedures to study their physicochemical and biological properties. The results were used to identify the substances isolated. The preliminary data allowed us to identify the components X, A, and B as the previously described compounds undecylprodigiosin, anisomycin, and copiamycin, respectively, whereas component C is a natural compound, which probably has never been described.     

Identification and Antimicrobial Activity of Phenylacetic Acid Produced by <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> Isolated from Fermented Soybean,Chungkook-Jang

A bacterial strain, B65-1, which showed strong antimicrobial activity, was isolated from Chungkook-Jang, a traditional Korean fermented-soybean food with antimicrobial properties. Based on carbon utilization pattern and partial 16S rRNA sequence analysis, the B65-1 strain was identified as Bacillus licheniformis. An antibiotic compound, active against bacteria and yeast such as Staphylococcus aureus, Escherichia coli, and Candida albicans, was isolated by various chromatographic procedures from culture filtrates of B. licheniformis B65-1. The purified antibiotic was identified to be phenylacetic acid, with the molecular formula C₈H₈O₂ by analyses of EI-MS and NMR. The phenylacetic acid was detected in fermented soybean made with the strain B65-1 as a starter, but was not present in extracts of nonfermented soybean. Our results indicated that the phenylacetic acid produced by B. licheniformis during fermentation of soybean is one of the main compounds of antimicrobial activity of Chungkook-Jang.     

Conversion of Bacillus subtilis 168 to a subtilin producer by competence transformation.

Subtilin is a ribosomally synthesized peptide antibiotic produced by Bacillus subtilis ATCC 6633. B. subtilis 168 was converted to a subtilin producer by competence transformation with chromosomal DNA from B. subtilis ATCC 6633. A chloramphenicol acetyltransferase gene was inserted next to the subtilin structural gene as a selectable marker. The genes that conferred subtilin production were derived from a 40-kb region of the B. subtilis ATCC 6633 chromosome that had flanking homologies to the B. subtilis 168 chromosome. The subtilin produced by the mutant was identical to natural subtilin in its biological activity, chromatographic behavior, amino acid composition, and N-terminal amino acid sequence.     

Streptomyces species producing the streptovaricin complex

Morphological, cultural and physiological-biochemical properties ofStreptomyces sp. strain 1000 and its antibiotic production were investigated. Antibiotics 1011 (identical with the streptovaricin complex) and 1012 (with antibacterial action) were isolated from the cultural broth of this strain. The overproducing natural variant 1011 was isolated from the population of a strain producing antibiotic 1011 at a concentration of 1000 mg/L (activity of the parent strain represents 41 mg/L only). Comparative taxonomical characteristic ofStreptomyces sp. strain 1000 with strains fromS. spectabilis showed that the strain 1000 differed in some properties and antibiotic production being considered as a new variant ofS. spectabilis. The strain shows an expressed antibiotic activity against G+ as well as G− bacterial and yeasts.     

Heterologous production of daptomycin in Streptomyces lividans

Daptomycin and the A21978C antibiotic complex are lipopeptides produced by Streptomyces roseosporus and also in recombinant Streptomyces lividans TK23 and TK64 strains, when a 128 kbp region of cloned S. roseosporus DNA containing the daptomycin gene cluster is inserted site-specifically in the ϕC31 attB site. A21978C fermentation yields were initially much lower in S. lividans than in S. roseosporus, and detection was complicated by the production of host metabolites. However A21978C production in S. lividans was improved by deletion of genes encoding the production of actinorhodin and by medium optimization to control the chemical form of the calcium dependent antibiotic (CDA). This latter compound has not previously been chemically characterized as a S. lividans product. Adding phosphate to a defined fermentation medium resulted in formation of only the phosphorylated forms of CDA, which were well separated from A21978C on chromatographic analysis. Adjusting the level of phosphate in the medium led to an improvement in A21978C yield from 20 to 55 mg/l.     

Pleiotropic functions of a Streptomyces pristinaespiralis autoregulator receptor in development, antibiotic biosynthesis, and expression of a superoxide dismutase

In Streptomyces, a family of related butyrolactones and their corresponding receptor proteins serve as quorum-sensing systems that can activate morphological development and antibiotic biosynthesis. Streptomyces pristinaespiralis contains a gene cluster encoding enzymes and regulatory proteins for the biosynthesis of pristinamycin, a clinically important streptogramin antibiotic complex. One of these proteins, PapR1, belongs to a well known family of Streptomyces antibiotic regulatory proteins. Gel shift assays using crude cytoplasmic extracts detected SpbR, a developmentally regulated protein that bound to the papR1 promoter. SpbR was purified, and its gene was cloned using reverse genetics. spbR encoded a 25-kDa protein similar to Streptomyces autoregulatory proteins of the butyrolactone receptor family, including scbR from Streptomyces coelicolor. In Escherichia coli, purified SpbR and ScbR produced bound sequences immediately upstream of papR1, spbR, and scbR. SpbR DNA-binding activity was inhibited by an extracellular metabolite with chromatographic properties similar to those of the well known gamma-butyrolactone signaling compounds. DNase I protection assays mapped the SpbR-binding site in the papR1 promoter to a sequence homologous to other known butyrolactone autoregulatory elements. A nucleotide data base search showed that these binding motifs were primarily located upstream of genes encoding Streptomyces antibiotic regulatory proteins and butyrolactone receptors in various Streptomyces species. Disruption of the spbR gene in S. pristinaespiralis resulted in severe defects in growth, morphological differentiation, pristinamycin biosynthesis, and expression of a secreted superoxide dismutase.     

Characterization of Streptomyces D3 derived from protoplast fusion between Streptomyces cyaneus 190-1 and Streptomyces griseoruber 42-9

Streptomyces D3, derived from protoplast fusion between Streptomyces cyaneus 190-1 and Streptomyces griseoruber 42-9, has the ability to produce high levels of xylose isomerase when grown on hemicellulosic materials such as xylan as the carbon source. Comparison between the partial nucleotide sequences of the 16S ribosomal RNA genes from S. cyaneus 190-1, S. griseoruber 42-9, and fusant D3 showed that the 16S rRNA gene of fusant D3 was identical to that of S. cyaneus 190-1. Partial sequence analysis of the xylose isomerase genes also indicated that the gene of fusant D3 was identical to that of S. cyaneus 190-1. The partial DNA fragments for the xylanase genes (xlnA and xlnB) of fusant D3 were amplified by PCR, and subjected to Southern hybridization analysis. The results revealed that the xlnB gene of fusant D3 was similar to that of S. cyaneus 190-1, but that the xlnA gene of fusant D3 was similar to that of S. griseoruber 42-9. These results suggest that the majority of the genome of fusant D3 may be derived from S. cyaneus 190-1.     

Comparative characteristics of some actnomycetes, producers of antifungal antibiotics related to chondamycin]

Three actinomycetous strains designated as LIA-0773, LIA-0783 and LIA-0780 were isolated from various soil samples. The cultures actively inhibited the growth of Trichophyton gipseum and produced a non-polyenic antibiotic of the chondamycin type. The strains were identified with Act. griseochromogenes Fukunaga et. al., 1955. The cultures differed within the species by some morphological, cultural, physiological and antibiotic properties, as well as by the component composition of the antibiotic produced. Thus, strain LIA-0773 had larger spiral sporophores, satisfactorily hydrolized starch and inverted sucrose. The strain inhibited the growth of not only the fungi but also grampositive bacteria and mycobacteria and produced an antibiotic composed of 6 components. Strain LIA-0780 had small sporophores with close spirals and low amilolytic activity. It inhibited only the growth of the fungi and produced a monocomponent antibiotic. Strain LIA-0783 was intermediate. By its biological properties it was closer to strain LIA-0780. The antibiotic produced by it consisted of 6 components, while by its physico-chemical properties the antibiotic was close to that produced by strain LIA-0780. All the 3 actinomycetous cultures were considered as different variants of Act. griseochromogenes Fukunaga et al., 1955.     

Penicillin production and its mode of inheritance inAspergillus nidulans

Previous workers have shown that some strains ofAspergillus nidulans produce penicillin-like substances. In the present studies, shake-flask cultures of 101 wild-type strains ofA. nidulans, representatives of 18 different heterokaryon-compatible groups, were examined and filtrates of most found to inhibit the growth of a strain ofBacillus subtilis sensitive to penicillin, although members of two of these groups had no detectable antibiotic activity. Five strains with antibacterial properties were chosen for detailed investigation as well as two genetically labelled derivatives obtained from one of these after ultraviolet light treatments; one derivative had a similar antibiotic yield to its original wild-type parent but the other was selected as having increased antibiotic yield. The antibiotic produced by these seven strains was by all tested criteria, including chromatographic and electrophoretic behaviour, indistinguishable from penicillin. A heterokaryon test between the two mutants indicated that antibiotic productivity was under nuclear control.     

Streptomyces albus G produces an antibiotic complex identical to paulomycins A and B

An antibiotic complex active against multiply resistant strains of staphylococci and other Gram-positive bacteria was isolated from cultures of Streptomyces albus G. Silica gel and Sephadex LH-20 column chromatography gave two congeners with Mr values of 786 and 772, which differed by one -CH2-group. The two homologues contained an isothiocyanate group, and proved to be identical with paulomycins A and B produced by Streptomyces paulus; the FAB mass spectra, in addition, proved the same two congeners to be present in proceomycin obtained from Streptomyces alboniger.     

Isolation of peptide antibiotic virginiamycin components and selection of their producer Streptomyces virginiae]

A method for chromatographic separation and quantitative determination of individual components of the antibiotic virginiamycin, produced by microbiological synthesis (Streptomyces virginiae strain 147), is described. The components, M1-2 and S1-5, were isolated from fermentation broth and identified by HPTLC and HPLC (the results obtained using the two methods correlate well with each other). Conditions of culturing of the producer and compositions of nutritive media were optimized. Using UV irradiation as a mutagenic factor, the producer was selected for increased level of synthesis of the antibiotic; this was achieved by inducing mutations that impart resistance to virginiamycin and meta-fluorophenylalanine, an analog of phenylalanine.     

Structural basis for hygromycin B inhibition of protein biosynthesis

Aminoglycosides are one of the most widely used and clinically important classes of antibiotics that target the ribosome. Hygromycin B is an atypical aminoglycoside antibiotic with unique structural and functional properties. Here we describe the structure of the intact Escherichia coli 70S ribosome in complex with hygromycin B. The antibiotic binds to the mRNA decoding center in the small (30S) ribosomal subunit of the 70S ribosome and induces a localized conformational change, in contrast to its effects observed in the structure of the isolated 30S ribosomal subunit in complex with the drug. The conformational change in the ribosome caused by hygromycin B binding differs from that induced by other aminoglycosides. Also, in contrast to other aminoglycosides, hygromycin B potently inhibits spontaneous reverse translocation of tRNAs and mRNA on the ribosome in vitro. These structural and biochemical results help to explain the unique mode of translation inhibition by hygromycin B.     

New tetraene antibiotic, abkhazomycin]

The cultures of Act. L10-0740 and L10-0772 were isolated from a soil sample. By their morphological and cultural features they were close to Act. badiocolor and differed from the latter by their antibiotic properties. Because of this they were classified as a new variant of Act. badiocolor var. abhasus var. nov. The cultures produced a new tetraen antibiotic, named abkhazomycin. Its physico-chemical properties are presented.     

A kinetic and structural characterization of adenosine-5'-triphosphate: ribonucleic acid adenylyltransferase from Pseudomonas putida.

A catalytic and structural study of ATP:RNA adenylyltransferase (EC 2.7.7.19) from the particulate fraction of Pseudomonas putida was made. During the large-scale purification of this enzyme, designated adenylyltransferase B, a previously undetected ATP-incorporating activity, designated adenylyltransferase A, was observed. Adenylyltransferases A and B were indistinguishable catalytically; however, they differed in their chromatographic and sedimentation properties. Adenylyltransferases A and B were resolved by phosphocellulose, by poly (U)-Sepharose and by Bio-Gel P-100 chromatographies. Adenylytransferase A was determined to have a sedimentation coefficient (S020,w) of 9.3 S and B of 4.3 S. The molecular weight of adenylyltransferase A was estimated to be 185000 and that of adenylyltransferase B to be 50000-60000. Apparently, adenylyltransferase A was generated from adenylyltransferase B during the purification. The AMP incorporation catalyzed by adenylyltransferases A and B was inhibited by two derivatives of the antibiotic rifamycin, AF/013 (50% at 5 mug/ml) and AF/DNFI (50% at 10 mug/ml). The 5'-triphosphate derivative (3'-dATP) of the drug cordycepin (3'-deoxyadenosine/ was a competitive inhibitor with ATP for both adenylyltransferases. The Ki for 3'-deoxyadenosine 5'-triphosphate was 6 - 10(-4)--10 - 10(-4) M, while the Km for ATP was 1 - 10(-4)--2 - 10(-4) M. Several other anaolgs of ATP, 2'-deoxyadenosine 5' triphosphate, 2'-O-methyl ATP, or the fluorescent 3-beta-D-ribofuranosylimidazo [2,1-i] purien 5'-triphosphate did not affect the activity of adenylyltransferase A or B. Poly(U) and poly(dT) were competitive inhibitors of the ribosomal RNA-primed polymerization reaction. The Ki for poly(U) or poly(dT), in terms of nucleotide phosphate, was 4 - 10-6)--10 - 10(-6) M for adenylyltransferases A and B, compared to 2 - 10(-4)--4 - 10(-4) M for the Km of ribosomal RNA. The inhibition was a result of the competition between the non-priming poly(U), or poly(dT), and ribosomal RNA for the primer binding site on the enzyme.     

Preservation of microorganisms by the method of L-drying

A procedure for L-drying of microorganisms or their drying under vacuum from liquid state is described in detail. The procedure is used in the Culture Collection of the All-Union Research Institute of Antibiotics. Preservation of Acremonium chrysogenum VNIIA 313A, the cephalosporin-producing culture, with the described procedure allowed one not only to maintain its viability for prolonged periods, but also to completely reproduce its initial antibiotic activity. L-Drying of some poorly kept cultures belonging to Acremonium was a success.