Antibiotics blocking bacterial cell wall assembly (beta-lactams and glycopeptides) are facing a challenge from the progressive spread of resistant pathogens. Lantibiotics are promising candidates to alleviate this problem. Microbisporicin, the most potent antibacterial among known comparable lantibiotics, was discovered during a screening applied to uncommon actinomycetes. It is produced by Microbispora sp. as two similarly active and structurally related polypeptides (A1, 2246-Da and A2, 2230-Da) of 24 amino acids linked by 5 intramolecular thioether bridges. Microbisporicin contains two posttranslational modifications that have never been reported previously in lantibiotics: 5-chloro-trypthopan and mono- (in A2) or bis-hydroxylated (in A1) proline. Consistent with screening criteria, microbisporicin selectively blocks peptidoglycan biosynthesis, causing cytoplasmic UDP-linked precursor accumulation. Considering its spectrum of activity and its efficacy in vivo, microbisporicin represents a promising antibiotic to treat emerging infections.

Anovel antibiotic, GE2270A, was isolated from the fermentation broth of a strain of Planobispora rosea. The product was found to inhibit bacterial protein synthesis. Structural characteristics showed similarities between GE2270 A and thiazolyl peptides such as micrococcin which is known to inhibit protein synthesis by acting directly on the ribosome. Despite this similarity GE2270A showed functional analogy to kirromycin-like antibiotics and pulvomycin, as its molecular target was found to be elongation factor Tu (EF-Tu). GE2270A is active against Gram-positive microorganism and anaerobes and differs from the other EF-Tu inhibitors in its spectrum of antimicrobial activity. 693 GE2270A, a novel peptide antibiotic, emerged from a screening program designed to detect inhibitors of protein synthesis. The present paper deals with the discovery, isolation, initial physico-chemical and biological characterization of this antibiotic. Materials and Methods Cultural and Growth Characteristics of the Producing Strain Colonial and morphological characters were determined with standard methods1'2*. Color determination was madeaccording to Maerz and Paul3). Growthon sole sources of carbon was determined after incubation at 28°C for 2 weeks1*. Chemotaxonomic Characteristics of the Producing Strain Freeze-dried biomass was examined to determine the major chemotaxonomiccharacteristics. Cell wall diamino acids were determined by TLCby a modification of the method of Becker et a/.4)5). Wholecell sugars were hydrolyzed, reduced and derivatized. The resultant alditol acetates were analyzed by GC6). Fatty acid methyl esters were similarly analyzed by GC7). Menaquinones and polar lipids were extracted and analyzed by HPLCand 2D TLC, respectively8*. Fermentation of the Producing Strain A 500-ml Erlenmeyer flask containing 100 ml of seed medium (Pdlypeptone 0.5%, yeast extract 0.3%, beef extract 0.2%, soybean meal 0.2%, starch 2%, calcium carbonate 1%, pH 7.0) was inoculated from an oatmeal slant of the producing strain. After incubation at 28°C for 96 hours on a rotary shaker (200 rpm), the biomass was transferred to a 10-liter jar fermenter containing 4 liters of the seed medium. This culture was grown for 72 hours at 28°C with 2 liters/minute air flow and stirring at 900rpm, prior to inoculating a jar fermenter containing 50 liters of production medium(starch 2%, peptone 0.25%,

In the course of a microbial product screening aimed at the discovery of novel antibiotics acting on bacterial protein synthesis, a complex of three structurally related tetrapeptides, namely, GE81112 factors A, B, and B1, was isolated from a Streptomyces sp. The screening was based on a cell-free assay of bacterial protein synthesis driven by a model mRNA containing natural initiation signals. In this study we report the production, isolation, and structure determination of these novel, potent and selective inhibitors of cell-free bacterial protein synthesis, which stably bind the 30S ribosomal subunit and inhibit the formation of fMet-puromycin. They did not inhibit translation by yeast ribosomes in vitro. Spectroscopic analyses revealed that they are tetrapeptides constituted by uncommon amino acids. While GE81112 factors A, B, and B1 were effective in inhibiting bacterial protein synthesis in vitro, they were less active against Gram-positive and Gram-negative bacterial cells. Cells grown in minimal medium were more susceptible to the compounds than those grown in rich medium, and this is most likely due to competition or regulation by medium components during peptide uptake. The novelty of the chemical structure and of the specific mode of action on the initiation phase of bacterial protein synthesis makes GE81112 a unique scaffold for designing new drugs.

In the course of a search for glycopeptide antibiotics having novel biological properties, we isolated A40926. Produced by an actinomycete of the genus Actinomadura, A40926 is a complex of four main factors which contain a fatty acid as part of a glycolipid attached to the peptide backbone. Its activity was, in most respects, similar to that of other glycopeptides, such as vancomycin and teicoplanin. However, in addition to inhibiting gram-positive bacteria, A40926 was very active against Neisseria gonorrhoeae. A40926 was rapidly bactericidal for N. gonorrhoeae clinical isolates at concentrations equal to or slightly higher than the MIC. In mice, levels in serum were higher and more prolonged than those of an equivalent subcutaneous dose of teicoplanin. These properties suggest that A40926 may have potential in the therapy of gonorrhea.The increased frequency of isolation of multiply antibioticresistant gram-positive bacteria has led to wider use of antibacterial agents which were originally rarely considered for first-line therapy, such as the glycopeptide vancomycin (18). One of the advantages of this antibiotic is that, thus far, resistance is rare and restricted to certain coagulase-negative staphylococci (25). The success of vancomycin has stimulated the development of new glycopeptides, such as teicoplanin (5,13,32,33). These antibiotics inhibit cell wall biosynthesis by binding to the D-alanyl-D-alanine portion of nascent peptidoglycan (22). Specific binding to an affinity resin containing acyl-D-alanyl-D-alanine groups (9) formed the basis of our search for new glycopeptides.Among the new glycopeptides identified, we focused our attention on those having novel biological properties. A40926 is the most interesting antibiotic which has emerged thus far from our screening campaign. In fermentation broths, we found it as a complex of four factors (PA, PB, A, and B). PA and PB, described here for the first time, predominated in the broth; during purification, these two compounds were largely converted to A and B, whose structures have been described recently (31a). One feature of all four factors, which relates them to teicoplanin and some other glycopeptides (aridicins and kibdellins) (3, 27, 12) while distinguishing them from vancomycin, ristocetin, and others, is the presence of a fatty acid moiety attached to one of the sugars. In terms of biological activity, the most striking difference between the components of the A40926 complex and other glycopeptide antibiotics is their activity against Neisseria gonorrhoeae.MATERIALS AND METHODS Isolation of A40926. We screened specifically for glycopeptide antibiotics by passing fermentation broths over the affinity resin Sepharose-D-alanyl-D-alanine (9) and eluting adsorbed glycopeptides with 1% aqueous ammonia. The strain producing A40926 was found in a soil sample collected in India. Batch adsorption to the same affinity resin described above yielded nearly pure A40926 complex in one step. Quick neutralization of the ammonia eluate yielded primarily the two fac...

Teicoplanin, a new glycopeptide antibiotic belonging to the same family as vancomycin, inhibits cell wall synthesis in Bacillus subtilis; the inhibition is accompanied by an intracellular accumulation of UDP-N-acetylmuramyl-pentapetide. A cell-free system from Bacillus stearothermophilus, capable of synthesizing peptidoglycan, is 50% inhibited by teicoplanin at 40 ,ug/ml and 100,% inhibited at 100 ,ug/ml; suppression of peptidoglycan synthesis is accompanied by parallel accumulation of the lipid intermediate. Teicoplanin binds to cell walls and forms a complex with N,N'-diacetyl-L-lysyl-D-alanyl-D-alanine. The association constant of this complex is 2.56 X 106 liters mol'1, calculated by spectrophotometric titration. The mechanism of action of teicoplanin is discussed in comparison with those of other inhibitors of cell wall biosynthesis, namely, vancomycin, ristocetin, and gardimycin.Teicoplanin is a new antibiotic chemically related to the group of glycopeptides which also includes vancomycin and ristocetin. It was isolated from the fermentation broth of Actinoplanes teichomyceticus nov. sp. (1, 12) and is endowed with outstanding bactericidal activity against grampositive pathogenic bacteria (11). The antibiotic is a mixture of five components of very similar polarity (the T-A2 complex, formerly named teichomycin A2) and a more polar product (T-A3); the components of the T-A2 complex, accounting for 90 to 95% of the product, have the same molecular weight, ca. 1,900 (2).The main features distinguishing teicoplanin from the other glycopeptide antibiotics are the occurrence of glucosamine as the basic sugar and the presence of aliphatic acid residues. Teicoplanin is not active against the L form of Staphylococcus aureus derived from a susceptible strain (12); this is a primary indication that the antibiotic interacts with the bacterial cell wall. We made further investigations on the mechanism of action of teicoplanin, and we present data showing that: (i) teicoplanin inhibits peptidoglycan synthesis in intact cells and in "cell-free" systems; (ii) the inhibition by teicoplanin is reverted by cell wall; (iii) the antibiotic binds to walls of whole cells, rendering them resistant to the action of lysozyme; and (iv) a complex occurs between teicoplanin and the peptide N,N'-diacetyl-L-lysyl-D-alanyl-D-alanine (Ac2-L-lys-D-ala-D-ala), a synthetic analog of the pentapeptide precursor in the peptidoglycan synthesis.Part of this work was presented at the 12th International Congress of Chemotherapy, July 1981, Florence, Italy. MATERIALS AND METHODSBacterial strains and growth conditions. Bacillus subtilis SB25 (His-Try-) and PB556/1 (Thy-), derived from strain W168, were grown in Difco Penassay medium (PY) or in Davis minimal medium (4) with the appropriate supplements in flasks at 37°C with reciprocating shaking. Bacillus stearothermophilus (calidolactis) ATCC 10149 was grown at 55°C in 4-liter tanks with forced aeration in a medium containing Incorporation of radioactive macromolecule precursors. B. subtilis 556/1 wa...

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BackgroundTeicoplanin is a glycopeptide antibiotic used clinically in Europe and in Japan for the treatment of multi-resistant Gram-positive infections. It is produced by fermenting Actinoplanes teichomyceticus. The pharmaceutically active principle is teicoplanin A2, a complex of compounds designated T-A2-1-A2-5 differing in the length and branching of the fatty acid moiety linked to the glucosamine residue on the heptapeptide scaffold. According to European and Japanese Pharmacopoeia, components of the drug must be reproduced in fixed amounts to be authorized for clinical use.ResultsWe report our studies on optimizing the fermentation process to produce teicoplanin A2 in A. teichomyceticus ATCC 31121. Robustness of the process was assessed on scales from a miniaturized deep-well microtiter system to flasks and 3-L bioreactor fermenters. The production of individual factors T-A2-1-A2-5 was modulated by adding suitable precursors to the cultivation medium. Specific production of T-A2-1, characterized by a linear C10:1 acyl moiety, is enhanced by adding methyl linoleate, trilinoleate, and crude oils such as corn and cottonseed oils. Accumulation of T-A2-3, characterized by a linear C10:0 acyl chain, is stimulated by adding methyl oleate, trioleate, and oils such as olive and lard oils. Percentages of T-A2-2, T-A2-4, and, T-A2-5 bearing the iso-C10:0, anteiso-C11:0, and iso-C11:0 acyl moieties, respectively, are significantly increased by adding precursor amino acids L-valine, L-isoleucine, and L-leucine. Along with the stimulatory effect on specific complex components, fatty acid esters, oils, and amino acids (with the exception of L-valine) inhibit total antibiotic productivity overall. By adding industrial oils to medium containing L-valine the total production is comparable, giving unusual complex compositions.ConclusionsSince the cost and the quality of teicoplanin production depend mainly on the fermentation process, we developed a robust and scalable fermentation process by using an industrial medium in which a complex composition can be modulated by the combined addition of suitable precursors. This work was performed in the wild-type strain ATCC 31121, which has a clear genetic background. This is important for starting a rational improvement program and also helps to better control teicoplanin production during process and strain development.

GE 23077 factors A1, A2, B1 and B2 are novel antibiotics isolated from fermentation broths of an Actinomadura sp. strain. GE23077 antibiotics are cyclic peptides, which inhibit Escherichia coli RNA polymerase at nM concentrations. Both rifampicin-sensitive and rifampicin-resistant polymerases are inhibited, whereas E. coli DNA polymerase and wheat germ RNA polymerase are substantially not affected. In spite of the potent activity on the enzyme, the antibiotics generally show poor activity against whole cell bacteria. The spectrum of activity is restricted to Moraxella catarrhalis, including clinical isolates, with partial activity against Neisseria gonorrhoeae and Mycobacterium smegmatis.