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Abstract:  The Zygomycete Cunninghamella elegans produces the polycarboxylate siderophore rhizoferrin. Production depends mainly on iron concentration in the medium. With an optimized production medium the yield of rhizoferrin in a bioreactor could be increased to more than 4 g/l. Supplementation of the basic production medium with different precursors led to the formation of nine new rhizoferrins. Both the diaminobutane backbone and the citric acid side-chains of rhizoferrin could be substituted by appropriate analogues. These substitutions led to new siderophores either with a variable length of diamine bridge or with fewer or different functional groups. The proportion of the new diamine analogues relative to the total rhizoferrin could be markedly increased by the use of α-difluoromethylornithine, an inhibitor of the ornithine decarboxylase.

Streptomyces griseus TÜ 6 produces the sideromycin antibiotic albomycin d2 in concentrations of approximately 1 mg/l. The production depends on the phosphate, iron, and ornithine concentrations in the medium. In optimized conditions, the production of albomycin could be increased to 25 mg/l in a fedbatch fermentation. Isolation and purification could be achieved by reversed-phase and size-exclusion chromatography and preparative high-performance liquid chromatography (HPLC). The detection limit in quantitative determination of albomycin by HPLC was reached at a concentration of 1 µg/ml, which was 100 times less sensitive than biological testing, but this method, although time-consuming, was more selective.

At low iron(III)-concentrations (<10-5 M) the fungus Aspergillus viridi-nutans Ducker & Thrower excretes desferri-ferricrocin as the main sideramine into the culture medium. While this compound accounts for 95% of the sideramines produced, small amounts of additional sidermines may also be detected. In a search for an inexpensive nutrient medium for optimum production of desferri-ferricrocin, experiments using shake flasks with good aeration were undertaken initially. The best medium conditions were then employed in a fermentor system.

The uptake of iron from 55Fe-labeled transferrin, ferric citrate, fusigen, coprogen, ferrichrome, ferricrocin, ferrichrysin, ferrirubin and ferrioxamine B was studied on in vitro cultures of bone marrow cells (Detroit-98). From all supplied iron complexes of the sideramine type, fusigen and ferricrocin were especially well accumulated. Transferrin, ferrioxamine B and ferricrocin showed a saturation kinetic within the investigated concentration range (0.5–8.0 µM). For all other iron complexes a linear relationship in the V/S-plot could be observed. After 1 hr uptake the iron content of the cells was approximately the 60-fold of the outer concentration for ferric citrate, the 20-fold for fusigen and ferricrocin and the 6-fold for transferrin and the other sideramines. Uptake of ferric citrate and fusigen was temperature dependent. A pH-dependence could not be observed. Efflux measurements revealed that the largest part of the accumulated iron was retained inside the cell and could not be released by osmotic lysis.

Stenothricin is a new antibiotic, which inhibits the cell wall synthesis of certain bacteria. It was isolated by partition chromatography, ion exchange chromatography, and gel filtration. The structural studies show, that stenothricin is a peptide antibiotic containing several uncommon amino acids, which could be identified by gas chromatography and mass spectrometry.

The production of desferricoprogen in Neurospora crassa, arg-5, ota, aga is suppressed by addition of Fe3+, Ga3+, Al3+, and V3+ to the medium. In the presence of Cr3+ and Co3+, the mutant behaves as under iron-deficient conditions. Once started, the biosynthesis of coprogen is suppressed within 24 h by Fe-Coprogen (10 µM), Ga-Coprogen (10 µM), and Al-Coprogen (>100 µM). The uptake of Coprogen corresponds to a saturation kinetic, whereas the uptake of desferricoprogen and Fe3+ is in accordance with a diffusion line. The different metal analogues of coprogen exhibit competitive behavior during the uptake, and are concentrated by the cells in the following order of affinity: Ga>Fe>Al>V>Cr>Co, which seems to be the same sequence in the regulation of the desferricoprogen biosynthesis. A model for uptake, based on the stability of the metal coprogens, is proposed.

Summary Rhizoferrin is a novel carboxylate-type siderophore which has recently been isolated fromRhizopus microsporus and other fungi of the Mucorales (Zygomycetes). The present investigation shows that a variety of rhizoferrin analogs can be produced by directed fermentation. Thus both the diaminobutane backbone and the citric acid side chains of rhizoferrin have been substituted by diamine and citric acid analogs added to the culture medium. The new ligands as well as their iron complexes have been characterized by physicochemical methods. Conditions of precursor incorporation and implications for the biosynthesis of the new siderophores are discussed.

This paper describes the chemical characterization of staphyloferrin B, a new complexone type siderophore isolated from low iron cultures of Staphylococcus hyicusDSM 20459. Purification of the very hydrophilic metabolite was achieved by anion exchange high performance liquid chromatography HPLC. Mass spectrometry showed a molecular mass of 448 amu. Hydrolysis with 8 mHCl revealed the presence of l-2,3-diaminopropionic acid, citrate, ethylenediamine and succinic semialdehyde. The connections between the four building blocks were determined by two-dimensional nuclear magnetic resonance measurements. UV/Vis and circular dichroism spectra are consistent with the proposed structure, which could also be confirmed by precursor feeding. The siderophore activity of staphyloferrin B was demonstrated by iron transport measurements.

Mutants of Escherichia coli K-12, Staphylococcus aureus, and Bacillus subtilis defective in the general components (enzyme I, or HPr, or both) of the phosphoenolpyruvate:sugar phosphotransferase system are shown to be resistant to the antibiotic streptozotocin. It is shown here, employing 32P-labeled phosphoenolpyruvate, that wild-type cells of E. coli phosphorylate streptozotocin, whereas with a phosphotransferase system-defective mutant of E. coli the drug is recovered in an unaltered, free form. The internal accumulation of streptozotocin at the steady-state level was about 70 times that of the concentration in the external medium. The antibacterial action of streptozotocin, as well as the uptake of the drug, was inhibited by N-acetyl-D-glucosamine. The uptake of the antibiotic was extremely sensitive to p-chloromercuribenzoate. It is concluded that streptozotocin is taken up by E. coli via the phosphoenolpyruvate:sugar phosphotransferase system and consequently accumulates in the cell at first as streptozotocin-phosphate.

The clavams valclavam and hydroxyethylclavam were both bacteriostatic and fungistatic. The molecular basis for growth inhibition of Escherichia coli was a non-competitive inhibition of homoserine-O-succinyltransferase (EC 2.3.1.46), thus blocking methionine biosynthesis. Eucaryotes such as Saccharomyces cerevisiae were inhibited by a different mode of action. Instead of interfering with methionine biosynthesis, the clavams inhibited the formation of RNA in living cells, although the RNA-polymerases of isolated yeast nuclei were not inhibited. The action of valclavam on E. coli was dependent on functional peptide transport systems.

Streptomyces flaveolus, strain Tü 1240 produces besides Tirandamycin A, a hitherto unknown antibiotic, which is closely related to Tirandamycin A. The new antibiotic Tirandamycin B contains one additional hydroxylgroup. Both antibiotics exhibit a similar antimicrobial spectrum and they seem to have the same mechanism of action. According to the data obtained from mass spectrometry, 13C-and 1H-NMR spectra formula II could be deduced for Tirandamycin B.

Candida lipolytica synthesizes the antibiotic tryptanthrin from 1 mole tryptophan and 1 mole anthranilic acid. When feeding tryptophan and substituted anthranilic acids, or substituted tryptophans and anthranilic acid, we could isolate and identify the expected derivatives of tryptanthrin. The enzymes of the biosynthesis of tryptanthrin, with the exception of bromotryptophan, had no specifity for these substrates. In addition to these experiments substituted tryptanthrines were chemically synthesized. We checked them for antibiotic action; the halogen compounds turned out to be especially effective.

From the fermentation broth of Streptomyces tendae Tü 901 a substance was isolated, which inhibits the growth of several fungi. The new antibiotic affects the chitchin biosynthesis. Its structure was identified by mass spectrometry of the products obtained after chemical degradation. Nikkomycin is a nucleoside-peptide antibiotic consisting of uracil, an amino hexuronic acid and a new amino acid containing a pyridin ring.

Rinamycin interfered with DNA-polymerase I of Escherichia coli in cell-free systems more effectively than actinomycin C. Its inhibitory action could be relieved by increasing the concentration of polymerase. The rinamycin concentrations that caused effective inhibition of the RNA-polymerase reaction in vitro were approximately 10 times higher than those required to inhibit the DNA-polymerase reaction in vitro.

From the cultures of Streptomyces violaceoniger, strain Tü 96, two new lipophilic antibioties, Lysolipin I and Lysolipin X were isolated. The latter one is chemically unstable and is easily transformed to Lysolipin I.

Streptomyces griseus strain Tü 10, which is producing macrotetrolide antibiotics, was compared with a mutant which is not producing any detectable amount of these substances. Following differences were found:1.An increase of Na+-concentration in the medium effects a delay of growth start and growth rate. The effect is more distinct in the case of the mutant than in that of the wild type.2.Growth of the mutant is inhibited at Na+-concentrations higher than 500 mMol/l and K+-concentrations at minimum. The wild type is still growing at 1000 mMol Na/l.3.The mutant is enabled to concentrate K+ intracellular as well as the wild type using a medium without Na+.4.K+-uptake of wild type and mutant is inhibited by ouabain, but there is a difference in sensitivity and grade of inhibition between both organisms.

d-N-hydroxy-L-arginine was isolated from culture filtrates of Nannizzia gypsea, a dermatophyte of the family Gymnoascaceae, order Eurotiales, class Ascomycetes. Several bacteria and fungi are inhibited by the antibiotic. The inhibition is compensated by L-arginine and L-citrulline.

With an ornithine-deficient mutant of Neurospora crassa, arg-5, ota, aga, whose sideramine production is completely blocked, the uptake of various sideramines and iron citrate was studied. Ferrioxamine B, as well as N-acetyl-ferrioxamine B (ferrioxamine D1) and N-benzoyl-ferrioxamine B were not accumulated by the cells of Neurospora. The kinetic data of transport indicate, that under iron-deficient conditions Neurospora evolves two distinguishable iron-transport systems: the coprogen system and the iron citrate system.

Boromycin, at a concentration of 0.05 µg/ml inhibits the synthesis of protein, RNA and DNA in whole cells of Bacillus subtilis. It is being antagonised by surface active compounds and is being bound to lipoprotein. Binding of the boromycin within the cell especially takes place at the cytoplasmic membrane.

A low-density-lipoprotein was isolated from hens egg yolk. This lipoprotein antagonizes ion-transporting antibiotics through extracellular binding. It also binds lipid-dyes, but not the synthetic potassium-complexing compound Dicyclohexyl-18-crown-6. The binding of antibiotics to the lipoprotein is being discussed.

Naphthomycin, a new yellow antibiotic of the quinoid type, has been isolated from cultures of strain Tü 105 of Streptomyces collinus Lindenbeim. It has been characterized by analytical, spectroscopic, and microbiological data. SH compounds prevent the inhibitory effect of the acid antibiotic. Naphthomycin has been found to antagonize vitamin K.

Mutants of E. coli K 12, which are resistant to 1 mM 5-methyltryptophan, excrete 1.5–3 mg/l tryptophan into the medium; in these strains the anthranilate-synthetase is probably insensitive to the feedback-inhibition. A second mutation for resistance to 5-fluoro-tryptophan gives strains, which are derepressed in the synthesis of the enzymes of the tryptophan pathway. The feedback-resistant and derepressed mutant FMT1 produces 50 mg/l tryptophan.

L-2,5-Dihydrophenylalanine inhibits the growth of E. coli K 12 in synthetic liquid media when added during the lag phase at a concentration of 5×10-5 M (8.35 ?/ml). The inhibition is reversed uncompetetively by phenylalanine and competetively by tyrosine.

l-2,5-Dihydrophenylalanine has been isolated from cultures of two strains of Streptomyces. The antibiotic activity of this compound is antagonized by Phenylalanine and Tyrosine.

Aus Kulturen des Actinomyceten-Stammes ETH 28 832 (Streptomyces aureofaciens) wurden die neuen Antibiotica Scopamycin A und B isoliert und charakterisiert. Die Scopamycine sind lipophile Neutralstoffe mit gleichen, charakteristischen UV-Spektren. Scopamycin A und B unterscheiden sich nur im Rf-Wert im Dünnschichtchromatogramm. Die Scopamycine rufen in geringen Konzentrationen starke, Hexenbesen-artige Veränderungen an den Hyphen verschiedener Pilze hervor. Gegen Bakterien zeigen die Scopamycine keine Wirkung.

1.Sideromycine sind Antibiotica, die gekreuzte Resistenz mit Grisein aufweisen und die durch Ferrioxamine in ihrer Wirkung gegen Grampositive Bakterien kompetitiv enthemmt werden.2.Der qualitative und quantitative Nachweis, die Herstellung, Anreicherung und Auftrennung von Ferrioxaminen aus Streptomyceten werden beschrieben.3.Als Ferrioxamine wirkende Stoffe lassen sich in biologischem Material verschiedenen Ursprungs nachweisen (Bakterien, Actinomyceten, Pilze, Algen, Phanerogamen, Leber).4.Die Ferrioxamine fördern das Wachstum verschiedener Mikroorganismen.5.Die Wirkung des Ferrimycins als Beispiel eines Sideromycins wird auf die eines kompetitiven Inhibitors eines Enzymsystems mit Ferrioxaminen oder analogen Verbindungen als Substrat zurückgeführt.6.Die Ferrioxamine aus Streptomyceten sind in bezug auf die Wirkung und im Chemismus mit den Wachstumsfaktoren Ferrichrom, Terregens-Faktor und Coprogen verwandt. Diese eisenhaltigen resp. eisenbindenden Wuchsstoffe werden als Sideramine bezeichnet.7.zwei Hypothesen für die Sideromycinwirkung werden einander gegenübergestellt: — Sideromycine als Konkurrenten eines Atmungsfermentes und — die wahrscheinlichere — Sideromycine als Inhibitoren von “Coenzymen” des Eisentransportes im mikrobiellen Stoffwechsel.

Unter den 18 von Pridham u. Gottlieb (1948) vorgeschlagenen C-Quellen sind l-Rhamnose, Raffinose, l-Xylose, d-Fructose, l-Arabinose und d-Mannit für die Charakterisierung von Streptomyces-Arten am besten geeignet.

1.29 eigene Isolierungen und 5 Vergleichsstämme lieferten Actinomycine, die sich alle in die Brockmannschen Typen C, I und X einordnen ließen. Es wird die Meinung vertreten, daß Actinomycin A und B mit X und Actinomycin D mit I identisch sind.2.Die untersuchten Actinomycinbildner wurden nach taxonomischen Gesichtspunkten in 4 Gruppen eingeteilt, von denen 3 durch die bekannten Arten Str. antibioticus, Str. parvulus und Str. chrysomallus repräsentiert werden; für die vierte wurde eine neue Art, Str. michiganensis beschrieben.3.Eine Gegenüberstellung von Actinomycintyp und taxonomischer Gruppierung der Stämme ergab wenig Parallelen; immerhin stellten die Actinomycin C-Bildner eine geschlossene Gruppe dar, und die chromogenen Stämme produzierten alle Actinomycin X.4.Einige Stämme von Str. chrysomallus enthielten im Kulturfiltrat neben Actinomycin C zugleich noch das Antibioticum Actidion (Cycloheximid); dies wird als Hinweis auf eine Verwandtschaft mit anderen Actidionbildnern gedeutet.

Von 14 aus Material verschiedener Herkunft isolierten Streptomyceten mit rhodomycinartigen Pigmenten produzierten 13 Stämme Rhodomycin und 1 Stamm Cinerubin. Einer der 13 Rhodomycinbildner ließ sich mit Streptomyces purpurascens Lindenbein identifizieren, die restlichen 12 werden als neue Species Streptomyces cinereoruber beschrieben. Der Cinerubinbildner, der außer im Antibioticum auch im C-Quellenspektrum von S. cinereoruber abweicht, wird als Streptomyces cinereoruber var. fructofermentans nov. var. beschrieben. Cinerubine produzieren ferner authentische Kulturen von Streptomyces bobiliae (Waksman et Curtis) Waksman et Henrici, die im übrigen größte Ähnlichkeit mit S. purpurascens aufweisen.

The transport of potassium ions in a potential gradient across a bulk membrane containing macrotetrolide antibiotics is studied using 14C-labelled macrotetrolide. It is shown that this transport of potassium ions across the membrane is accompanied by an equal transport of macrotetrolide within the membrane indicating that potassium ions are transported as the 1:1 macrotetrolide complex. Since exchange of ligands is shown to occur during the transport, a carrier relay mechanism accounting for all experimental results is proposed.

A strain ofStreptomyces viridochromogenesproduced a new crystalline antibiotic, Avilamycin, related to but not identical with Curamycin and Exfoliatin. Avilamycin, C63H94O35Cl2, gave on solvolytic degradation the following products: dichloroisoeverninic acid, 2-deoxy-d-rhamnose, 2,6-di-O-methylmannose, 4-O-methylfucose,l-lyxose and 3,5-diacetoxy-γ-caprolactone.

a-Lipomycin is a new antibiotic isolated from Streptomyces aureofaciens. —The orange-red compound is a glycoside of the aglycone C26H35NO6 (ß-lipomycin) and the 2.6-dideoxy-ribo-hexopyranose D-digitoxose.—The chromophore of the lipomycins and their methyl esters shows indicator properties. Similar to erythroskyrine a carbonyl group links a pentaene chain with a derivative of N-methyl-tetramic acid.—The polyenic optically active lipomycins are inhibitory against gram-positive bacteria and have no effect upon the growth of fungi and yeasts. The antibiotic activity is probably due to alterations of the cell membrane and is antagonized by lecithin and some sterols.

Arenaemycin E (epoxide), a new, colorless, lipophilic antibiotic has been isolated from cultures of strain Tü 469 of Streptomyces arenae. The antibiotic inhibits the growth of microorganisms in synthetic, but not in complex media. After adjusting the culture filtrate with hydrochloric acid to pH 2.5 Arenaemycin C (chlorohydrin) was isolated. However, when the pH of the original culture filtrate was adjusted to pH 2.5 with sulphuric acid, Arenaemycin D (diol) was isolated.

Kirromycin, a new yellow antibiotic, has been isolated from cultures of strain Tü 365 of Streptomyces collinus. It has been characterized by analytical, spectroscopic, and microbiological data.

All compounds tested (ferrioxamines, sideramines of the ferrichrome type except ferrichrome A, fusigen, coprogens, dihydroxamates, monohydroxamates except fusarinine, iron citrate, iron-EDTA, hemin and hemoproteins) promote the growth of Microbacterium lacticum. The first four groups mentioned are of high and nearly equal activity. As in other cases the sideramine requirement of Microbacterium lacticum is inoculum dependent.

Chlorothricin, a new macrolide-type antibiotic, inhibits growth of the gram-positive organism, Bacillus subtilis, on chemically defined media with glucose or pyruvate as sole carbon source. Acetate has an antagonizing effect on the action of this antibiotic, but the inhibition is not completely overcome. On a medium with asparagine or aspartate, respectively, chlorothricin affects growth only at higher concentrations. In vivo 14CO2 incorporation experiments show that chlorothricin inhibits the anaplerotic CO2 fixation catalyzed by pyruvate carboxylase (EC 6.4.1.1.).

After feeding tryptophan, Candida lipolytica produces reddish-brown pigments and the antibiotic ‘Tryptanthrin’. The pigments are—probably spontaneous arising-derivatives of indolepyruvic acid. Indolepyruvic acid and one of its derivatives-ß-3H-indolydenopyruvate acid-could be demonstrated in the culture filtrate. According to feeding experiments, the synthesis of ‘Tryptanthrin’ starts from anthranilic acid-anthranilic acid could also be demonstrated in the culture filtrate- and indolepyruvic acid. The structure-6,12-dihydro-6,12-dioxoindolo-(2,1-b)-quinazoline-is in agreement with this assumption.

From culture filtrates of the Streptomyces strain Tü 99 the antibiotic ketomycin, in addition to chlorothricin, was isolated by solvent extraction, counter-current distribution and crystallization of the sodium salt. Spectroscopic investigations and chemical transformation showed, that ketomycin is identical with 3-cyclohexeneglyoxylic acid. Degradation of ketomycin by means of ozone yielded the dextrorotatory (R)-ß-carboxyadipic acid. Ketomycin is therefore (R)-3-cyclohexeneglyoxylic acid.

Besides iron deficiency a suitable carbon source is the condition for the production of sideramines by Aspergillus melleus. If no other carbon sources are added, glucose, fructose and the tested intermediary products of glucose metabolism bring about growth but no sideramine production. Optimal amount of sideramines could be obtained by using combinations of glucose, acetate, asparagine and ornithine (up to 3 g/l).

A pure form of sideramine was extracted from cultures of Arthrobacter simplex and was identified as ferrioxamine B. Chromobacterium violaceum produces ferrioxamine E.

The mode of action of borrelidin has been studied and compared with two macrolide antibiotics. Borrelidin specifically inhibits the attachment of threonine to sRNA at a concentration of 1,4·10-6m in a cell-free system of Bacillus subtilis. However, the macrolide antibiotics lankamycin (neutral) and erythromycin (basic), at concentrations which are ten times higher than those required to inhibit growth, still do not inhibit the binding of threonine or other amino acids to sRNA.

In iron free cultures Pseudomonas fluorescens secretes a polypeptide which reacts with ferric ions to form a stable complex. This iron complex called Ferribactin is a dihydroxamate and belongs to the Siderochromes. Ferribactin contains at least the following components: A mole each of glycine, L-serine, d-serine, L-glutamic acid, d-N-hydroxy-L-ornithine, d-N-hydroxy-d-ornithine, d-tyrosine, 3 moles of L-lysine and 2 moles of acetic acid.

The macrolide-antibiotics are discussed in respect to their chemistry and biology. The single substances are listed. An investigation and classification of the streptomycetes producing macrolides, is added; the single species are critically revised. Relations are established between the producers and their products.

Die Resistenz von Mikroorganismen genenüber Ferrimycin wird untersucht. Es können drei Formen von Resistenz aufgezeigt werden: