Rhizocticin A, the main component of the antifungal, hydrophilic phosphono-oligopeptides of Bacillus subtilis ATCC 6633, was used for sensitivity testing and experiments into the molecular mechanism of the antibiotic action. Budding and filamentous fungi as well as the cultivated nematode Caenorhabditis elegans were found to be sensitive, whereas bacteria and the protozoon Paramecium caudatum were insensitive. Rhizoctonia solani was inhibited in agar dilution tests but not in diffusion tests. The antifungal effect of rhizocticin A was neutralized by a variety of amino acids and oligopeptides. Oligopeptide influence was mainly understood as transport antagonism, and it was concluded that the antibiotic enters the recipient cell via the peptide transport system. L- and D-cystine were also identified as potent, general antagonists of the oligopeptide transport. The rhizocticin-antagonism of four other amino acids was taken as a clue to the site of action. Provided that rhizocticin A is split by peptidases of the target cell into inactive L-arginine and toxic L-2-amino-5-phosphono-3-cis-pentenoic acid (L-APPA), the latter may interfere with the threonine or threonine-related metabolism.
The widely uscd and wcll-known bacterial strain Bucillrcs suh/i/is ATCC 6633 was found to produce two novel. antifungal hydrophilic pcptidc anlibiotics, 1.-arginyl-~-2-amino-5-phosphono-3-cis-pcnlenoic acid (I.-A~~-L-APPA, rhizocticin A) and r-valyl-L~irgit~yI-~-2-amino-5-phosphono-3-ci.~-pentenoic acid (L-Val-rArp-L-APPA. rhizocticin B). Besides rhizocticin A and B. the main components. small amounts of related tripeptidcs were detcctcd. Instead of the t-Val of rhizocticin B thcy contain r-Ile or I.-LCU and are rcfcrred to as rhizocticins C and D, respectively. The C-tcrminal rcsiduc was idcntificd by NMR spectroscopy as the unsaturated phosphono amino acid L-APPA. known till now only as D cnantiomcr. Enzymatic cleavages of rhizocticin B yielded both I.-APPA and rhizocticin A.In cultures of Bacillus subtilis ATCC 6633, known to produce the antibacterial antibiotic subtilin'), Michener and Snell" demonstrated two antifungal activities referred to as "aspergillus factor" and "rhizoctonia factor". No chemical structures were then implicated, but during a preliminary study on hydrophilic antibiotics from strains of Bacillus subtilis it became obvious that s u b~p o r i n s~~ and aspergillus factor are based on the same activity and that the bacterial strain used in both investigation^^,^) was the same, although the latter authors3) had designated it as "PCI 219".In the investigation described here, the structures of the antibiotics representing the rhizoctonia factor have been elucidated, and the name "rhizocticins" applied to them. Chro- Rbizocticine -ncue Phosphono-Oligopeptide mit antifungi.wher matographic and spectroscopic analyses of the highly hydrophilic metabolites revealed them to be formerly unknown antifungal di-and tripeptides, possessing the unusual Cterminal amino acid ~-2-amino-5-phosphono-3-cis-pentenoic acid (L-APPA, Scheme 1). APPA has been described as a part of the molecule of the plumbemycins, threonine antagonists from Streptomyces plumbeus4). However, D configuration of APPA was assumed, whereas rhizocticins contain L-APPA. Plumbemycins are active against bacteria, whereas rhizocticins show antifungal effects. Fermentation and Isolation of RhizocticinsRhizocticins can be detected in complex culture media (yeast extract, soy bean flour) of Bacillus subtilis ATCC 6633. During optimization experiments the complex medium was replaced by a chemically defined production medium. The change of the nutrient medium resulted in advantages for the detection and purification of the antibiotics. The fermentation broth was harvested after 72 h of incubation in 5-1 and 25-1 fermenter vessels at 27 "C. Preliminary examination had indicated that two naturally occurring components of the antibiotic, rhizocticins A and B, exist. They are of low molecular mass, hydrophilic, amphoteric, highly charged, and could not be extracted from the culture broth with organic solvents.After acidification, the culture broth was centrifuged to remove the bacterial growth together with precipitated components (Scheme 2). Other ...
S = 112 excited state, the three fits are each quite different. The first gives an S = 7 / 2 ground state with an S = 9 / 2 excited state only 9 cm-' higher in energy. The second fit has an S = 9 / 2 ground state and an S = 7 / 2 excited state, separated by only 0.5 cm-' with seven other spin states lying within 50 cm-' of the ground state; whereas, the third fit has an S = 9 / 2 ground state with the closest excited state being S = 7 / 2 at 226 cm-' above the ground state. This third fit was used for the calculation of the unbroken curve in Figure 2a.To our surprise, magnetization data for complex 2, collected over the temperature range 1.8 to 40 K at field values from 2.5 to 4.5 T, conclusively show an isolated S=9/2 ground state (Fig. 2b). This is in agreement with the third set of parameters obtained from the susceptibility data. Additional support for the isolation of this S = 9 / 2 level is found in the fact that, although there are some changes and loss of resolution at elevated temperatures, the EPR spectrum of 2 (powder or glass) is readily seen at both liquid-helium and liquid-nitrogen temperatures. Such behavior would not be expected from the manifold of spin states found in the second fit.It is likely that all the features in the glass EPR spectrum of 2 at g z 2, 6, and 9 (similar to those seen for 1 ,@I) are attributable to transitions between components of the S = 9/2 ground state, which, as a detailed analysis of the M versus H/T data (Fig. 2b) shows, experiences an axial zero-field splitting (0s:) with D = 0.25-0.35 cm-'. Whether the S, state of PS I1 contains a cluster similar to that of 2 is still unclear, but the described work does suggest that the possibility that the &-state EPR spectral features might also arise from components of a large-spin ground state should be seriously considered. Interestingly, we have just characterized"" a dinuclear Mn"Mn"' complex which is also ferromagnetically coupled and has an S = 912 ground state (verified by M versus H/T data). The EPR spectrum for this Mn"Mn"' complex is similar to those for 1 and 2.
A lipophilic antibiotic was isolated horn the culture filtrate 01 S i r q m n t y e s o/it~aceus TU 3010. Its structure was elucidated spectroscopically using 2D-'H-and "C-NMR experiments (e.g C'OLOC. NOESY). FD-and GC-MS, and by chemical dcgradaiion and dcrivatixation. The antibiotic Tij 3010 was idenlitled as (1S)-4e~hyI-2.5-dihydro-3-hydroxy-2-[( 1 E)-bmethyl-I ,3-butadienyl]-S-oh;o-2-ihienylacet-amide (I), therefore it is a new member of natural compounds with thiotetronic acid structure. The metabolite shows antibacterial activity cspccially against StrepronipThe strain Streptomyces olivaceus TU 3010 was found to produce a new antibacterial metabolite, which was named TU 3010. This metabolite was discovered for the first time in a chemical screening using detection with blue tetrazolium chloride on thin-layer plates and bioautography using the sensitive test organism Streptomyces viridochromogenes TU 57.In the following we describe the isolation and structure elucidation of the new thiolactone antibiotic 1, which can (1) exists only as a single tautomer, a so-called thiotetronic acid2), and this parent compound is completely substituted. Fermentation, Isolation, and Antimicrobial ActivityThe antibiotic Tii 3010 was produced by fermentation of Streptomyces olivaceus Tii 3010 at 27°C for 96 h in a 100-1 fermenter containing complex medium with 2% oatmeal. During fermentation and isolation the antibiotic content was determined quantitatively by HPLC analysis.The antibacterial agent was isolated from the culture broth by adsorption on Amberlite XAD-4. After elution with 50% aqueous methanol the acidic antibiotic was extracted with ethyl acetate from the aqueous concentrate at pH 4 (Scheme 2). Flash chromatography on silica gel followed by gel chromatography on Sephadex LH-20 yielded a colorless pure product.The antibiotic TU 3010 was found to be an acidic compound of low molecular mass [m/z(M+) = 267, FD-MS], which is soluble in alkaline water and most organic solvents. High solution mass spectra revealed the molecular formula All Gram-negative bacterial test strains were nonsensitive against the antibiotic Tii 3010. From the Gram-positive strains in particular Streptomycetes were inhibited (Table 1). No inhibition was observed on the tested yeasts and fungi, C13H17N03S.
Biodegradation ofH. brasiliensis wood by R. lignosus 159 NDS, D. ; NICOLE, M. ; GEIGER, J. P., 1983: Infections artificielles de jeunes plantules d'Hevea hrasiliensis par Rigidoporus lignosus (Kl.) Imaz. et Phellinus noxius (Corner) G. H. Cunn. Eur. J. For. Path. 13, 65-76. NICOLE, M.; NANDRIS, D.; GEIGER, J. P.; Rio, B., 1985: Variability among African populations of Rigidoporus lignosus and Phellinus noxius. Eur. J. For. Path. 15, 293-300. NICOLE, M.; GEIGER, J. P.; NANDRIS, D., 1986: Root rot disease of Hevea brasiliensis. II. Some host reactions. Eur. J. For. Path. 37-55. PICHEL, R. J., 1956: Les pourridies de l'Hevea dans la cuvette congolaise. INEAC, Ser. Techn. 49, 480 pp. REYNOLDS, E. S., 1963: The use of lead citrate at high pH as an electron opaque stain in electron microscopy. AbstractOn the anatomical features of well functioning and "damaged" rootlets of spruce: meristem and differentiation of root apices and mycorrhizae. The root apices of various developmental stages oi Picea abies (L.) Karst. are described in detail and the differences between long-and short roots discussed.
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