Production of a broad spectrum streptothricin like antibiotic from halotolerant Streptomyces fimbriatus isolate G1 associated with marine sediments.

Streptomyces have been reported as a remarkable source for bioactive secondary metabolites with complex structural and functional diversity. In this study, 35 isolates of genus Streptomyces were purified from rhizospheric and marine soils collected from previously unexplored habitats and screened for antimicrobial activities. One of these isolates, G1, when tested in vitro, was found highly active against wide range of microbes including Gram-positive, Gram-negative bacteria, and different fungal pathogens. It was identified as mesophilic, alkaliphilic, and moderately halotolerant as it showed optimum growth at temperature 30 °C, pH 8.0 in casein-starch-peptone-yeast extract-malt extract medium supplemented with 5% NaCl. Sequence analysis of the 16S rRNA gene indicated 100% identity of this isolate to Streptomyces fimbriatus. Moreover, maximum antimicrobial activity was achieved in starch nitrate medium supplemented with 1% glycerol as carbon and 0.03% soy meal as nitrogen source. The antimicrobial compounds produced by this isolate were extracted in methanol. Bioassay-guided fractionation through thin layer chromatography of methanolic extract resulted in the separation of a most active fraction with an Rf value of 0.46. This active fraction was characterized by FTIR and LCMS analysis and found similar to streptothricin D like antibiotic with m/z 758.42.

Separation of Streptothricin Antibiotics from Culture Broth with Colorimetric Determination Using Dipicrylamine.

Our earlier method for the detection and separation of ε-poly-L-lysine using a yellow anionic dye, the dipicrylamine (DPA-) anion, was herein optimized for streptothricin antibiotics (ST), which contains the ß-lysine oligopeptides moiety, H-[NH-(CH2)3-CH(NH2)-CH2-CO]n-. We then applied this method to the detection and separation of ST in a commercially available nourseothricin, a mixture of ST species with n = 1, 2, 3, and 4. The ST species were precipitated with the DPA- anion. The precipitate was found to consist of the salts of the fully protonated ST species, STz+ (z = n + 1), with the DPA- anion. The ST(DPA)z precipitate was re-dissolved in acetonitrile. The solution was yellowish, and gave an absorption maximum at around 420 nm. Thus, the equivalent concentration of the ST species referred to the charge numbers of STz+ can be determined colorimetrically. By the addition of bis(triphenylphosphoranylidene)ammonium chloride, the ST species could be re-precipitated from the acetonitrile solution as hydrochloride salts. All of the ST species were found in the precipitate with high yields. The method was thus successfully applied to the detection and separation of ST species from the culture broth.

Three new 12-carbamoylated streptothricins from Streptomyces sp. I08A 1776.

Two new streptothricins (1 and 2) and a new streptothricin acid derivative (3), all with the carbamoyl group substituted at C-12 of the gulosamine moiety, together with the known N(ß)-acetylstreptothricin D acid (4), have been isolated from the culture broth of Streptomyces sp. I08A 1776. The structures of the new compounds were determined by MS, CD, and 1D and 2D NMR spectroscopic data analysis. The isolated compounds were evaluated for antibacterial and antifungal activities. Streptothricin E (6) showed potent activity against the clinically isolated extensively drug-resistant Mycobacterium tuberculosis with MIC values of 0.25-0.5µg/mL.

Two streptothricins with a cis-streptolidine lactam moiety from Streptomyces sp. I08A 1776.

Two unique cis-fused streptothricins (1 and 2) were isolated from the culture broth of Streptomyces sp. I08A 1776. Their structures were determined by MS, CD, and 1D and 2D NMR spectroscopic data analysis. Compound 2 showed weak antibacterial activities against Bacillus subtilis and Enterococcus faecalis with MIC values of 32 and 64 µg ml(-1), respectively.

Streptothricin derivatives from Streptomyces sp. I08A 1776.

Five new streptothricin derivatives with a carbamoyl group substituted at C-12 (1-5) and three known analogues have been isolated from the culture broth of Streptomyces sp. I08A 1776 by ion exchange and hydrophilic interaction chromatographic techniques. Their structures were determined by spectroscopic and chemical methods. Compound 3 was a streptothricin derivative possessing a cis-streptolidine moiety. Its absolute configuration was defined by comparison of quantum chemical TDDFT calculated and experimental ECD spectra. Compound 5 and streptothricin E (6) displayed antibacterial and antifungal activity with MIC values in the range 1-64 µg/mL.

Two new members of streptothricin class antibiotics from Streptomyces qinlingensis sp. nov.

Four streptothricin-group antibiotics (1~4) were isolated from the fermentation broth of Streptomyces qinlingensis sp. nov. Along with the known antibiotics streptothricins F (1) and D (3), two new members of this class (2, 4) were identified as 12-carbamoyl derivatives of 1 and 3, respectively, mainly by analysis of the IR, HR-MS and NMR spectral data. The antibacterial activities of 1~4 against Escherichia coli (MICs 3.1, 25.0, 3.1 and 12.5 microg/ml), Bacillus subtilis (MICs 6.3, 25.0, 3.1 and 50 microg/ml), Staphylococcus aureus (MICs 12.5, >100.0, 6.3, >100.0 microg/ml), Bacillus cereus (MICs 25.0, 50.0, 25.0 and 50.0 microg/ml) and Pseudomonas aeruginosa (MICs 50.0, >100.0, 50.0, >100.0 microg/ml) were assayed by micro-broth dilution. The results based on MIC data indicated that 2 and 4 exhibited significantly less potent antibacterial activities when compared to that of 1 and 3.

A-53930A and B, novel N-type Ca2+ channel blockers.

A-53930A, B and C, which inhibit N-type Ca2+ channels, were isolated from the culture broth of Streptomyces vinaceusdrappus SANK 62394. A-53930A and B were new compounds which contained a carbamoyl group on the 6-hydroxyl group of the D-gulosamine part of streptothricin. A-53930C was identical to streptothricin B. A-53930A, B and C inhibited [125I]omega-conotoxin MVIIA binding to N-type Ca2+ channels (IC50= 0.17, 0.091 and 0.071 microM), but did not inhibit [3H]PN200-110 binding to L-type Ca2+ channels (IC50 > 50 microM). These compounds also inhibited [3H]norepinephrine release from chick cerebral cortex synaptosomes (IC50=91.0, 20.6 and 39.5 microM), indicating these compounds selectively block N-type Ca2+ channels which are important for neurotransmitter release. It was also revealed that although A-53930C had antimicrobial activity against gram-negative and -positive bacteria and fungi, A-53930A and B showed weak activity only against gram-negative bacteria.

Streptothricin biosynthesis is catalyzed by enzymes related to nonribosomal peptide bond formation.

In a search for strains producing biocides with a wide spectrum of activity, a new strain was isolated. This strain was taxonomically characterized as Streptomyces rochei F20, and the chemical structure of the bioactive product extracted from its fermentation broth was determined to be a mixture of streptothricins. From a genomic library of the producer strain prepared in the heterologous host Streptomyces lividans, a 7.2-kb DNA fragment which conferred resistance to the antibiotic was isolated. DNA sequencing of 5.2 kb from the cloned fragment revealed five open reading frames (ORFs) such that ORF1, -2, -3, and -4 were transcribed in the same direction while ORF5 was convergently arranged. The deduced product of ORF1 strongly resembled those of genes involved in peptide formation by a nonribosomal mechanism; the ORF2 product strongly resembled that of mphA and mphB isolated from Escherichia coli, which determines resistance to several macrolides by a macrolide 2'-phosphotransferase activity; the ORF3 product had similarities with several hydrolases; and the ORF5 product strongly resembled streptothricin acetyltransferases from different gram-positive and gram-negative bacteria. ORF5 was shown to be responsible for acetyl coenzyme A-dependent streptothricin acetylation. No similarities in the databases for the ORF4 product were found. Unlike other peptide synthases, that for streptothricin biosynthesis was arranged as a multienzymatic system rather than a multifunctional protein. Insertional inactivation of ORF1 and ORF2 (and to a lesser degree, of ORF3) abolishes antibiotic biosynthesis, suggesting their involvement in the streptothricin biosynthetic pathway.

A streptothricin-like antibiotic mixture, A-269A (and A-269A').

A streptothricin-like antibiotic, A-269A (referred to as A-269A hereafter) was isolated from the culture broth of Streptomyces sp., strain No. A-269. In this paper, the characterization of the producer, and the production, isolation, physico-chemical properties and biological properties of A-269A are reported. The structure of the antibiotic was also examined by 1H NMR, 13C NMR and fast atom bombardment mass spectra studies. From the spectroscopic data, A-269A was assumed to be a mixture of antibiotic LL-BL 136 and an isomeric compound in which the carbamoyl group is substituted on C-12' hydroxyl instead of C-10 hydroxyl.

Nourseothricin (streptothricin) inactivated by a plasmid pIE636 encoded acetyl transferase: nature of the inactivated nourseothricin.

Nourseothricin, a mixture of several streptothricins, is inactivated by an acetyl transferase produced by Escherichia coli containing the plasmid pIE636. Nourseothricin inactivated in the presence of 14C-acetate was purified and submitted to partial hydrolysis. In the hydrolysate besides others a radioactive and ninhydrin-reactive substance moving only slightly towards the cathode was found. It proved to be [14C]-acetyl beta-lysine.

[Formation of a streptothricin-group antibiotic by a Streptomyces glaucus 1136 culture]. / Obazovanie antibiotika iz gruppy streptotritsinov kul'turoi Streptomyces glaucus 1136.

In screening of new antibiotics a streptomycete (strain 1136) was isolated from a soil sample of Armenia. It showed no antagonistic properties in streek cultures on agarized media. When grown under submerged conditions strain 1136 produced an antibiotic active against grampositive and gramnegative bacteria. By its cultural and morphological properties strain 1136 was classified as Streptomyces glaucus Agre et Preobrazhenskaya, 1983. Microbiological and chemical investigation of the antibiotic produced by strain provided its identification at the early stages of the investigation as an antibiotic of the streptothricin group. Up to now no organisms producing streptothricin antibiotics were detected among streptomycetes of the Azureus section including strain 1136.

New streptothricin-group antibiotics, AN-201 I and II. Screening, fermentation, isolation, structure and biological activity.

Two streptothricin-group antibiotics, AN-201 I and II, were newly discovered and isolated from the culture broth of Streptomyces nojiriensis C-13. These antibiotics were purified by IRC-50 (H+) and CM-Sephadex C-50 chromatography, and paper electrophoresis. Structural analysis of AN-201 I and II showed that they were N beta-acetylated derivatives of streptothricin E and D, respectively. They had antibacterial activities against several strains of Escherichia coli, Bacillus subtilis, Micrococcus luteus and Staphylococcus aureus, and showed a strong selective cytotoxic effect on 3T3 cells transformed with SV-40 as compared with their normal cells in a test system in vitro as well as in vivo.

Separation and biologic activities of individual components of S15-1, a streptothricin class antibiotic.

A method is described for isolation of gram quantities of the components of the streptothricin complex S15-1 utilizing CM Sephadex column chromatography eluted with 10% acetic acid as an eluant followed by gradient elution with 10% acetic acid containing 0.02 N approximately 0.03 N HCI. Streptothricins F and E, as well as an unidentified component C1, have been isolated and their comparative biological activities determined. Streptothricins F and E were comparable in taeniacidal activity in mice infected with Hymenolepis nana ia feeding either one at 0.05% in the diet removed 92 approximately 100% of the adult tapeworms. The unidentified component C1 was inactive at the levels tested. In contrast, component C1 was the most active in antimicrobial activity against Bacillus subtilis and in inhibiting the urease activity of proteus mirabilis. In the former test, the ratios of activity were; 1:7:30 for F:E:C1 and in the latter; 1:2:4 for F:E:C1.

Glycinothricin, a new streptothricin-class antibiotic from Streptomyces griseus.

Glycinothricin is a streptothricin-class antibiotic obtained for the first time from the culture broth of a strain of Streptomyces griseus. Glycinothricin, the deformimino derivative of antibiotic LL-AB664, gives N-methylstreptolidine, N-methyl-D-glucosamine and glycine on acid hydrolysis. In comparison with LL-AB664, glycinothricin is less active against gram-positive and gram-negative bacteria and less toxic to mice.