Bottromycin A2 Research Articles

Microparticles enhance the formation of seven major classes of natural products in native and metabolically engineered actinobacteria through accelerated morphological development.

Actinobacteria provide a rich spectrum of bioactive natural products and therefore display an invaluable source towards commercially valuable pharmaceuticals and agrochemicals. Here, we studied the use of inorganic talc microparticles (hydrous magnesium silicate, 3MgO·4SiO2 ·H2 O, 10 µm) as a general supplement to enhance natural product formation in this important class of bacteria. Added to cultures of recombinant Streptomyces lividans, talc enhanced production of the macrocyclic peptide antibiotic bottromycin A2 and its methylated derivative Met-bottromycin A2 up to 109 mg L-1 , the highest titer reported so far. Hereby, the microparticles fundamentally affected metabolism. With 10 g L-1 talc, S. lividans grew to 40% smaller pellets and, using RNA sequencing, revealed accelerated morphogenesis and aging, indicated by early upregulation of developmental regulator genes such as ssgA, ssgB, wblA, sigN, and bldN. Furthermore, the microparticles re-balanced the expression of individual bottromycin cluster genes, resulting in a higher macrocyclization efficiency at the level of BotAH and correspondingly lower levels of non-cyclized shunt by-products, driving the production of mature bottromycin. Testing a variety of Streptomyces species, talc addition resulted in up to 13-fold higher titers for the RiPPs bottromycin and cinnamycin, the alkaloid undecylprodigiosin, the polyketide pamamycin, the tetracycline-type oxytetracycline, and the anthramycin-analogs usabamycins. Moreover, talc addition boosted production in other actinobacteria, outside of the genus of Streptomyces: vancomycin (Amycolatopsis japonicum DSM 44213), teicoplanin (Actinoplanes teichomyceticus ATCC 31121), and the angucyclinone-type antibiotic simocyclinone (Kitasatospora sp.). For teicoplanin, the microparticles were even crucial to activate production. Taken together, the use of talc was beneficial in 75% of all tested cases and optimized natural and heterologous hosts forming the substance of interest with clusters under native and synthetic control. Given its simplicity and broad benefits, microparticle-supplementation appears as an enabling technology in natural product research of these most important microbes.

Isolation and Identification of Actinomycetes Strains from Switzerland and their Biotechnological Potential.

Actinomycetes strains isolated from different habitats in Switzerland were investigated for production of antibacterial and antitumoral compounds. Based on partial 16S rRNA gene sequences, the isolated strains were identified to genus level. Streptomyces as the largest genus of Actinobacteriawas isolated the most frequently. A screening assay using the OmniLog instrument was established to facilitate the detection of active compounds from actinomycetes. Extracts prepared from the cultivated strains able to inhibit Staphylococcus aureusand Escherichia coliwere further analysed by HPLC and MALDI-TOF MS to identify the produced antibiotics. In this study, the bioactive compound echinomycin was identified from two isolated Streptomycesstrains. Natural compounds similar to TPU-0037-C, azalomycin F4a 2-ethylpentyl ester, a derivative of bafilomycin A1, milbemycin-α8 and dihydropicromycin were detected from different isolated Streptomyces strains. Milbemycin-α8 showed cytotoxic activity against HT-29 colon cancer cells. The rare actinomycete,Micromonospora sp. Stup16_C148 produced a compound that matches with the antibiotic bottromycin A2. The draft genome sequence from Actinokineospora strain B136.1 was determined using Illumina and nanopore-based technologies. The isolated strain was not able to produce antibacterial compounds under standard cultivation conditions. The antiSMASH bioinformatics analyses of the genome from strain B136.1 identified biosynthetic gene clusters with identity values between 4% to 90% to known gene clusters encoding antibiotics. The combinations of cultivation conditions, screening assays, analytical methods and genome mining are important tools to characterize strains of actinomycetes for the identification of their potential to produce natural compounds with antimicrobial activity.

Thiazoline-Specific Amidohydrolase PurAH Is the Gatekeeper of Bottromycin Biosynthesis.

The ribosomally synthesized and post-translationally modified peptide (RiPP) bottromycin A2 possesses potent antimicrobial activity. Its biosynthesis involves the enzymatic formation of a macroamidine, a process previously suggested to require the concerted efforts of a YcaO enzyme (PurCD) and an amidohydrolase (PurAH) in vivo. In vitro, PurCD alone is sufficient to catalyze formation of the macroamidine, but the process is reversible. We set out to probe the role of PurAH in macroamidine formation in vitro. We demonstrate that PurAH is highly selective for macroamidine-containing precursor peptides and cleaves C-terminal of a thiazoline, thus removing the follower peptide. After follower cleavage, macroamidine formation is irreversible, indicating PurAH as the gatekeeper of bottromycin biosynthesis. The structure of PurAH suggests residues involved in catalysis, which were probed through mutagenesis.

Synthesis and Evaluation of Antibacterial Activity of Bottromycins.

Total synthesis of bottromycin A2 can be accomplished through a diastereoselective Mannich reaction of a chiral sulfinamide, mercury-mediated intermolecular amidination, and cyclization of a constrained tetracyclic peptide. Exploitation of this process allowed the synthesis of several novel bottromycin analogs. The antimicrobial activity of these analogs was evaluated in vitro against Gram-positive bacteria, such as methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE). Structure-activity relationships were explored taking into consideration the unique three-dimensional structure of the compounds. Notably, one of the new analogs devoid of a methyl ester, which is known to lower the in vivo efficacy of bottromycin, exhibited antibacterial bioactivity comparable to that of vancomycin.

The complete genome sequence of Streptomyces autolyticus CGMCC 0516, the producer of geldanamycin, autolytimycin, reblastatin and elaiophylin

Streptomyces autolyticus CGMCC 0516 produces the anti-tumor benzoquinone ansamycins geldanamycin, autolytimycin, and reblastatin and the 16-membered macrodiolide elaiophylin. Here, we report the complete genome sequence of S. autolyticus CGMCC 0516, which consists of a 10,029,028bp linear chromosome and seven circular plasmids. Fifty-seven putative biosynthetic gene clusters for secondary metabolites were found. The geldanamycin, autolytimycin, and reblastatin biosynthetic gene clusters were located on the left arm (2.06–2.15Mb) of the chromosome, and the elaiophylin gene cluster was located on the right arm (9.45–9.53Mb). Twenty-one putative gene clusters with high or moderate similarity to important antibiotic biosynthetic gene clusters were found, including the antitumor agents echoside, bafilomycin, hygrocin, and toxoflavin; the antibacterial/antifungal agents nigericin, skyllamycin, kanamycin, naphthomycin, eco-02301, and bottromycin A2; the immunosuppressants meridamycin and brasilicardin A; the anti-inflammatory agent cyclooctatin; and the acute iron poisoning medication desferrioxamine B. The genome sequence reported here will enable us to study the biosynthetic mechanism of these important antibiotics and will facilitate the discovery of novel secondary metabolites with potential applications to human health.

Dissecting Bottromycin Biosynthesis Using Comparative Untargeted Metabolomics

AbstractBottromycin A2 is a structurally unique ribosomally synthesized and post‐translationally modified peptide (RiPP) that possesses potent antibacterial activity towards multidrug‐resistant bacteria. The structural novelty of bottromycin stems from its unprecedented macrocyclic amidine and rare β‐methylated amino acid residues. The N‐terminus of a precursor peptide (BtmD) is converted into bottromycin A2 by tailoring enzymes encoded in the btm gene cluster. However, little was known about key transformations in this pathway, including the unprecedented macrocyclization. To understand the pathway in detail, an untargeted metabolomic approach that harnesses mass spectral networking was used to assess the metabolomes of a series of pathway mutants. This analysis has yielded key information on the function of a variety of previously uncharacterized biosynthetic enzymes, including a YcaO domain protein and a partner protein that together catalyze the macrocyclization.

Screening and Identification of Antimicrobial Compounds from Streptomyces bottropensis Suppressing Rice Bacterial Blight

Xanthomonas oryzae pv. oryzae (Xoo) is the most devastating pathogen to Oryza sativa and has been shown to cause bacterial blight. Two bioactive compounds showing antimicrobial activities against Xoo strain KACC 10331 were isolated from a Streptomyces bottropensis strain. The ethyl acetate extract was fractionated on a Sephadex LH-20 column, and then purified by preparative HPLC. The purified compounds were identified as bottromycin A2 and dunaimycin D3S by HR/MS and 1H NMR analyses. The MIC value against Xoo and the lowest concentration still capable of suppressing rice bacterial blight were 2 microgram/ml and 16 microgram/ml for bottromycin A2, and 64 microgram/ml and 0.06 microgram/ml for dunaimycin D3S, respectively. These two compounds were shown to exert different bioactivities in vitro and in rice leaf explants.

Studies on bottromycins. I. 1H and 13C NMR assignments of bottromycin A2, the main component of the complex.

Studies on bottromycins. I. 1H and 13C NMR assignments of bottromycin A2, the main component of the complex.

Production, purification, and characterization of chandramycin, a polypeptide antibiotic from Streptomyces lydicus.

A plant pathogenic actinomycete identified as Streptomyces lydicus was isolated from the deep-pitted scab lesions of potato tubers. This strain produces a new polypeptide antibiotic named chandramycin. The antibiotic was isolated from culture broth by extraction with organic solvents and purified by chromatography. The purified antibiotic is a light-yellow crystalline compound soluble in water and in most organic solvents. Amino acid analysis of the acid hydrolysates of chandramycin revealed the presence of glycine, cis-methyl proline, valine, beta, beta-dimethylaminobutyric acid, beta-methyl-phenylalanine, and beta-2-thioazolyl-beta-alanine. The amino acid composition of chandramycin is qualitatively similar to that of a known antibiotic, bottromycin A2. Chandramycin showed activity against several gram-positive and a few gram-negative species of bacteria. It showed a strong activity against anaerobic microorganisms. Oral doses of antibiotic when administered up to 466 mg/kg of body weight failed to produce any observable toxic effect in mice.

The revised structure of bottromycin A2.

The revised structure of bottromycin A2.

Mode of action of bottromycin A2. Release of aminoacyl- or peptidyl-tRNA from ribosomes.

Bottromycin A2 inhibited MS2 phage RNA-dependent protein synthesis as well as polyuridylic acid-(poly(U))- or polyadenylic acid (poly(A))-dependent polypeptide synthesis. When the ribosomal complex with N-acetyl-[14C]phenylalanyl-tRNA (N-acetyl-[14C]Phe-tRNA) at the A site was subjected to bottromycin A2, the release of N-acetyl-[14C]Phe-tRNA was observed while no release of N-acetyl-[14C]Phe-tRNA from the donor site was observed, indicating that the action of bottromycin A2 is specific to the A site of ribosomes. Due to bottromycin's capacity to release [14C]Phe-tRNA or N-acetyl-[14C]Phe-tRNA from the ribosomal acceptor site (A site), bottromycin A2 inhibited the nonenzymatic binding of N-acetyl-[14C]Phe-tRNA and elongation factor T (EF-T)-dependent binding if the concentration of EF-Tu-GTP-[14C]Phe-tRNA ternary complex was low. Our data are consistent with the possibility that the inhibition of overall polypeptide synthesis by bottromycin A2 is at least partly due to bottromycin A2's activity to release aminoacyl- or oligopeptidyl-tRNA from ribosomes. Among 10 antibiotics tested, bottromycin A2 and lincomycin released aminoacyl-tRNA from ribosomes.

The revised structure of bottromycin A2.

The revised structure of bottromycin A2.

Studies on the Formation of Transfer Ribonucleic Acid-Ribosome Complexes: XV. EFFECT OF ANTIBIOTICS ON STEPS OF BACTERIAL PROTEIN SYNTHESIS: SOME NEW RIBOSOMAL INHIBITORS OF TRANSLOCATION

Abstract The effect of some antibiotics on several steps of protein biosynthesis was examined. Thiostrepton and siomycin A inhibited translocation through interference with ribosomal function; they also inhibited Phe-tRNA binding to ribosomes and GTP hydrolysis dependent on ribosomes and factor G. Micrococcin inhibited translocation, but did not inhibit either the associated GTP hydrolysis or Phe-tRNA binding to ribosomes. Althiomycin appeared to inhibit transpeptidation, although the inhibition was substrate-dependent: it inhibited peptidyl transfer to C-A-C-C-A(Phe), but did not inhibit either acetyl-phenylalanyl-puromycin formation or GTP hydrolysis dependent on factor G and ribosomes. Bottromycin A2, carbomycin, chloramphenicol, griseoviridin, and lincomycin inhibited acetyl-phenylalanyl-puromycin formation, but not GTP hydrolysis dependent on ribosomes and factor G. It appeared that inhibitors of transpeptidation and some inhibitors of translocation (micrococcin for example) can uncouple GTP hydrolysis from functional translocation.

Mechanism of inhibition of protein synthesis by fusidic acid and related steroidal antibiotics.

Fusidic acid and related steroidal antibiotics were demonstrated to inhibit ribosomc-dependent GTPase activity of G factor in the presence and absence of sRNA, phenyl-alanyl-sRNA and/or poly U. They also inhibited polypeptide synthesis, and the grade of inhibition of both reactions was parallel. The inhibition of polypeptide synthesis by fusidic acid was reversed by excess of G factor, but not by excess of T factor or ribo-somes, Puromycin-dependent release of peptide from the ribosomes was not significantly affected by fusidic acid in the absence of GTP and G factor; but the puromycin reaction, enhanced by GTP and G factor, was inhibited by the antibiotic. The results indicate that fusidic acid and related antibiotics affect polypeptide synthesis by inhibiting GTPase activity of G factor and by inhibiting translocation of peptidyl-sRNA on the ribosomes. The results also seem to support the assumption that GTP split reaction, linked to the ribosomes and G factor, is essential for translocation of peptidyl-sRNA on the ribosomes. The ribosome-associated GTPase activity of G factor was not significantly affected by blasticidin S, mikamycin B, bottromycin A2, chloramphenicol, thiophenicol, and erythromycin.

MECHANISM OF PROTEIN SYNTHESIS INHIBITION BY BOTTROMYCIN A2 : STUDIES WITH PUROMYCIN

Bottromycin A2 inhibits polylysine synthesis with poly A at low concentrations of E. coli ribosomes. Peptide release by puromycin from ribosomes was significantly inhibited by blasticidin S, chloramphenicol and thiophenicol, but not by bottromycin A2 in the absence of GTP and G factor. Puromycindependent release of peptide from ribosomes was stimulated by the presence of GTPand G factor; it was inhibited by bottromycin A2. The results indicate that bottromycin A2 inhibits translocation of peptidyl-tRNA on the ribosomes, but does not affect peptide bond synthesis.

Inhibition of protein synthesis by bottromycin A2 and its hydrazide.

Inhibition of protein synthesis by bottromycin A2 and its hydrazide.

The structure of bottromycin A2, a new component of bottromycins.

Bottromycin A2 is hydrolyzed to tetrapeptide and dipeptide by alkaline hydrolysis. The structures of both peptides are determined to be 3, 3-dimethyl-2-aminobutylylvalyl-3-methylprolylglycine and 3-methyl-3-phenylalanyl-3-(2-thiazolyl)-β-alanine. Hydrochloric acid hydrolysis of bottromycin A2 gives Δ1-isocaproyltetrapeptide and the structure is discussed on the bases of pKa studies. The structure of bottromycin A2 is proposed from the results above described.