Antibiotic Complex Research Articles

Factors influencing cell fatty acid composition and A40926 antibiotic complex production in Nonomuraea sp. ATCC 39727

A40926 is a glycopeptide antibiotic complex consisting of several structurally related factors. It is produced by fermentation of Nonomuraea sp. ATCC 39727 and the complex components differ in the structure of the fatty acid moiety linked to the aminoglucuronic acid unit. In previous work, we observed that the production of single factors in glycopeptide antibiotic complexes could be selectively enhanced by the addition of suitable precursors to the culture medium. In this contribution, we examine the effects of branched amino acid addition to fermentation of Nonomuraea sp. in a chemically defined minimal medium. The changes in the composition of cell fatty acids correlate to the fatty acid distribution within the A40926 complex in diverse cultivation conditions. Nonomuraea sp. prefers isobutyric, butyric and propionic acids as initiators of fatty acid biosynthesis. The relative amount of the produced fatty acids is significantly influenced by the availability of intermediates or final products from the amino acid catabolic pathways. Antibiotic complex composition closely reflects the cell fatty acid pattern, in agreement with the assumption that the antibiotic fatty acid moieties are synthesized by shortening the chain of cell fatty acids.

An Antibiotic Complex from Lysobacter enzymogenes Strain C3: Antimicrobial Activity and Role in Plant Disease Control

Lysobacter enzymogenes C3 is a bacterial biological control agent that exhibits antagonism against multiple fungal pathogens. Its antifungal activity was attributed in part to lytic enzymes. In this study, a heat-stable antifungal factor (HSAF), an antibiotic complex consisting of dihydromaltophilin and structurally related macrocyclic lactams, was found to be responsible for antagonism by C3 against fungi and oomycetes in culture. HSAF in purified form exhibited inhibitory activity against a wide range of fungal and oomycetes species in vitro, inhibiting spore germination, and disrupting hyphal polarity in sensitive fungi. When applied to tall fescue leaves as a partially-purified extract, HSAF at 25 mug/ml and higher inhibited germination of conidia of Bipolaris sorokiniana compared with the control. Although application of HSAF at 12.5 mug/ml did not reduce the incidence of conidial germination, it inhibited appressorium formation and suppressed Bipolaris leaf spot development. Two mutant strains of C3 (K19 and DeltaNRPS) that were disrupted in different domains in the hybrid polyketide synthase-nonribosomal peptide synthetase gene for HSAF biosynthesis and had lost the ability to produce HSAF were compared with the wild-type strain for biological control efficacy against Bipolaris leaf spot on tall fescue and Fusarium head blight, caused by Fusarium graminearum, on wheat. Both mutant strains exhibited decreased capacity to reduce the incidence and severity of Bipolaris leaf spot compared with C3. In contrast, the mutant strains were as efficacious as the wild-type strain in reducing the severity of Fusarium head blight. Thus, HSAF appears to be a mechanism for biological control by strain C3 against some, but not all, plant pathogenic fungi.

Spontaneous White Sectored-Mutants in Streptomyces hygroscopicus 111-81: Characterization and Antibiotic Productivity

Spontaneous white mutants from sectors of Streptomyces hygroscopicus 111-81 were isolated. The comparison of morphological, cultural, and biochemical properties of the mutants and ancestor showed the differences in colors of aerial, substrate mycelia, and sporulation. Changes in resistance to antibiotics and sensitivity to lysozyme indicated alterations in cell walls and cell membranes of the mutants. They showed antifungal activity close to that of the parent strain on fermentation medium FM2, with unchanged component composition of the AK-111-81 antibiotic complex. The cells of spontaneous white mutants are characterized with electron-transparent structures, vacuoles, aggregation of ribosomes, intrahyphal growth, and lack of multiple cell septa, which was established by transmission electron microscopy. The appearance of white sectored-mutants in S. hygroscopicus 111-81 is connected with exhausting of nutrients causing the substrate limitation and is a stress response to starvation.

A comparison of the properties of bacteriocins formed by Lactococcus lactis subsp. lactis strains of diverse origin

Bacteriocins formed by four strains of Lactococcus lactis subsp. lactis have been studied and compared: 729 (a natural strain isolated from milk), 1605 (a mutant of strain 729), F-116 (a recombinant obtained by fusing of protoplasts of the two related strain 729 and 1605), and a nisin-forming strain obtained by adaptive selection at Moscow State University. Antimicrobial activity studies revealed differences between the strains in the effects on individual groups of microorganisms; the activities of the strains were also distinct from that of Nisaplin (a commercial preparation of the bacteriocin nisin). Methods for isolation and purification of bacteriocins have been developed, making it possible to obtain individual components of antibiotic complexes as chromatographically pure preparations. Bacteriocins formed by the strains of Lactococcus lactis subsp. lactis have been identified and differences in their biological and physicochemical properties, established. A novel potent broad-spectrum antibiotic substance distinct from nisin has been isolated from the recombinant strain F-116.

Changes of conductance and compressibility of bilayer lipid membranes induced by oligonucleotide-cationic polyene antibiotic complexes

The positively charged polyene molecule amphotericin B 3-dimethylaminopropylamide (AMA) is an efficient agent for the delivery of antisense oligodeoxyribonucleotides (ODN) into target cells. In the present study, bilayer lipid membrane (BLM) conductance, elasticity modulus perpendicular to the membrane plane, surface potential and electrical capacitance were measured by conductance and electrostriction methods in the presence of AMA, pure or complexed to 20-mer single stranded ODN at different ratios. Pure AMA did not induce changes in conductance of cholesterol-containing BLM, but did induce an increase in elasticity modulus and surface potential. ODN/AMA complexes changed BLM properties depending on the charge ratio. The most pronounced effect on membrane conductance was observed for positively charged ODN/AMA complexes (charge ratio ρ−/+=0.1), while for negatively charged complexes these changes were less marked/apparent, correlating to substantially lower binding constants. The effect of ODN/AMA complexes on elasticity modulus and charge potential was biphasic. After an increase in both values, a decrease was observed for higher incubation times and ODN/AMA concentrations. These results are interpreted as indicating that the membrane property changes result from the large AMA aggregates induced by the presence of the negatively charged ODN, which condensate on these aggregates. It is suggested that the decrease of elasticity modulus and surface potential in the presence of increasing incubation time and AMA concentration result from desorption of the complexes in the complex-free compartment of the BLM cell, or appearance of a non-linear conductance of the lipid bilayer. The first alternative would explain the AMA-induced transmembrane transfer of ODN.

Biological and Chemical Detections in Adsorbent Layer for Monitoring Microbial Production of Primycin

Primycin is a macrolide antibiotic complex produced in microbiological fermentation processes. The microbial production of primycin requires an in-process analytical method suitable for monitoring the level of the active agents. In this paper, a method fulfilling the described requirement is presented. This method consists of a simple, efficacious extraction step, an instrumental sample application followed by a high-performance thin-layer chromatographic separation in relatively short time and a quantitative chromatogram evaluation. A dipping technique, in a solution containing sulfuric acid followed by heating at 120 degrees C, is used for chromogen formation, resulting in an absorption maximum at 290 nm. A progress diagram of the fermentation obtained by this technique is compared with one obtained by a microbiological agar diffusion method. The bioautographic evaluation of the active spots in the chromatogram are also presented. By our TLC method, the group of the active primycin components in the fermentation broth and by-products formed during the fermentation can be well separated. The relative intensities of the different TLC spots provide some information on the formation of the active components.

Metabolic flux analysis for directing metabolism of Streptomyces olindensis from growth to anti-tumor drug (cosmomycin) production

Background Cosmomycin, an aromatic polyketide antibiotic complex produced by Streptomyces olindensis, belongs to anthracycline family of chemotherapy drugs. It is a powerful antitumor drug similar to doxorubicin and daunorubicin. The structure of cosmomycin consists of one tetracycline aglycon (β-rhodomycinone) and six deoxysugars (two dTDPL-2-deoxifucose, two dTDP-L-rodosamine and two dTDPL-rodinose). The tetracycline aglycon, a polyketide structure, is obtained by condensation of one propionyl-CoA with nine acetate molecules derived from malonyl-CoA by the action of enzymes known as minimal Polyketide Synthetases (minimal PKS). Model construction The in silico metabolism was reconstructed involving more than 240 stoichiometrically balanced metabolic reactions in matrix formalism using the information from the literature and databases (top-down and bottom-up). Then, computational metabolic flux balancing method was used in order to obtain fluxes of all the metabolic reactions with linear programming and optimisation in GAMS environment (General Algebraic Modeling System). The objective function of optimisation was either the maximisation of the specific growth rate or the maximisation of the specific cosmomycin production rate. The experimental specific glucose uptake and growth rates were used as the model constraints, as appropriate. The solution of the metabolic model gave the specific growth and/or product formation rates as well as the specific rates of all 242 metabolic reactions.

Crystal structure, conformation, and absolute configuration of kanamycin A

Kanamycin, an antibiotic complex produced by Streptomyces kanamycetius isolated from Japanese soil, was described by Okami and Umezawa as early as 1957 and consists of three components: Kanamycin A (the major component), B, and C. The disulfate salt of kanamycin A [4- O-(6-amino-6-deoxy-α- d-glucopyranosyl)-6- O-(3-amino-3-deoxy-α- d-glucopyranosyl)-2-deoxystreptamine] is a broad-spectrum antibiotic that is used to treat gonorrhea, salmonella, tuberculosis, and many other diseases. Crystals of kanamycin A monosulfate monohydrate obtained from water are triclinic, space group P1, with a = 7.2294(14), b = 12.4922(15), c = 7.1168(9), α = 94.74(1), β = 89.16(1), γ = 91.59(1), V = 640.2(2) Å 3, μ(Cu Kα) = 18.4 cm −1, FW 600.6, D calc = 1.558 g/cm 3, CAD-4 diffractometric data (2693 reflections, 2554 ⩾ 3 σ( I)), structure by shelx-86 and refined by full-matrix least squares to a final R value of 0.038. The wrong conformer had an R value of 0.043. Both of the d-glucose moieties are attached to the deoxystreptamine by α linkages. This absolute configuration agrees with the earlier determination by both chemical and X-ray methods with photographic data. The ( ϕ, ψ) values for the glycosidic linkages are 101.6°, −121.1°, 106.3°, and −140.4°, respectively. Kanamycin interacts with the ribosomal S12 protein to stabilize the codon–anticodon binding between mRNA and the aminoacyl tRNA and inhibits the elongation of peptide chains through a series of reactions resulting in the prevention of ribosomes from moving along mRNA. ▪

Characterization of Moenomycin Antibiotic Complex by Multistage MALDI-IT/RTOF-MS and ESI-IT-MS

Flavomycin is a commercially available antimicrobial growth promoter and an authorized additive for feeding stuffs in the EU and in the USA. As most antibiotically active products biosynthesized by microorganisms, it contains not only a single active compound but is a complex mixture of structurally closely related substances. Multistage matrix-assisted laser desorption/ionization-ion trap/reflectron time-of-flight mass spectrometry (MALDI-IT/RTOF-MS) and liquid chromatography-electrospray ionization-ion trap-mass spectrometry (LC-ESI-IT-MS) were utilized for a detailed analysis of the constituents of the Flavomycin complex based on low-energy collision induced dissociation (CID). An optimal sample preparation for negative ion vacuum MALDI-MS for this compound class was developed. The MALDI-IT/RTOF-MS2 and -MS3 analysis starting with the precursor [M - H]- ions of these interesting phosphoglycolipids, named moenomycins, yielded a large variety of product ions that facilitated the structural characterization of this class of compounds. Based on the derived CID fragmentation pathway of the five known major constituents, namely moenomycin A, moenomycin A12, moenomycin C4, moenomycin C3. and moenomycin C1, four not yet described moenomycin-type constituents could be characterized. They were assigned as 4F-demethyl-6E-O-de-beta-D-glucopyranosyl-moenomycin A, 6B-N-de(2-hydroxy-5-oxo-1-cyclopenten-1-yl)-moenomycin A, 6B-hydroxy-6B-de[N-(2-hydroxy-5-oxo-1-cyclopenten-1-yl)amino]-moenomycin A, and 6C-hydroxy-moenomycin A. In addition, a moenomycin A carrying an oxygen in the moenocinol-group was found, which is most probably a chemical degradation product. These new compounds were verified by LC-ESI-IT-MS.

Heterologous production of daptomycin in Streptomyces lividans

Daptomycin and the A21978C antibiotic complex are lipopeptides produced by Streptomyces roseosporus and also in recombinant Streptomyces lividans TK23 and TK64 strains, when a 128 kbp region of cloned S. roseosporus DNA containing the daptomycin gene cluster is inserted site-specifically in the phiC31 attB site. A21978C fermentation yields were initially much lower in S. lividans than in S. roseosporus, and detection was complicated by the production of host metabolites. However A21978C production in S. lividans was improved by deletion of genes encoding the production of actinorhodin and by medium optimization to control the chemical form of the calcium dependent antibiotic (CDA). This latter compound has not previously been chemically characterized as a S. lividans product. Adding phosphate to a defined fermentation medium resulted in formation of only the phosphorylated forms of CDA, which were well separated from A21978C on chromatographic analysis. Adjusting the level of phosphate in the medium led to an improvement in A21978C yield from 20 to 55 mg/l.

Cover Picture: Cervimycin A–D: A Polyketide Glycoside Complex from a Cave Bacterium Can Defeat Vancomycin Resistance (Chem. Eur. J. 19/2005)

A novel antibiotic complex was purified from a strain of Streptomyces tendae, isolated from the wall of an ancient cave in southern Italy, shown in the cover picture. These polyketide glycosides, cervimycins A–D, demonstrated potent activity against multi‐drug‐resistant staphylococci and vancomycin‐resistant enterococci. Results of NMR and chemical degredation studies revealed the cervimycins to have a common tetracyclic polyketide core substituted with unusual di‐ and tetrasaccharide chains (see picture), but with differences in their acetyl and carbamoyl ring substituents, and in the terminal methylmalonyl and dimethylmalonyl residues. For more details, see the Full Paper by C. Hertweck and co‐workers on page 5523 ff.

ANTIBIOTICS TARGETING RIBOSOMES: Resistance, Selectivity, Synergism, and Cellular Regulation

Antibiotics target ribosomes at distinct locations within functionally relevant sites. They exert their inhibitory action by diverse modes, including competing with substrate binding, interfering with ribosomal dynamics, minimizing ribosomal mobility, facilitating miscoding, hampering the progression of the mRNA chain, and blocking the nascent protein exit tunnel. Although the ribosomes are highly conserved organelles, they possess subtle sequence and/or conformational variations. These enable drug selectivity, thus facilitating clinical usage. The structural implications of these differences were deciphered by comparisons of high-resolution structures of complexes of antibiotics with ribosomal particles from eubacteria resembling pathogens and from an archaeon that shares properties with eukaryotes. The various antibiotic-binding modes detected in these structures demonstrate that members of antibiotic families possessing common chemical elements with minute differences might bind to ribosomal pockets in significantly different modes, governed by their chemical properties. Similarly, the nature of seemingly identical mechanisms of drug resistance is dominated, directly or via cellular effects, by the antibiotics' chemical properties. The observed variability in antibiotic binding and inhibitory modes justifies expectations for structurally based improved properties of existing compounds as well as for the discovery of novel drug classes.

Metabolic network analysis of Streptomyces tenebrarius, a Streptomyces species with an active entner-doudoroff pathway.

Streptomyces tenebrarius is an industrially important microorganism, producing an antibiotic complex that mainly consists of the aminoglycosides apramycin, tobramycin carbamate, and kanamycin B carbamate. When S. tenebrarius is used for industrial tobramycin production, kanamycin B carbamate is an unwanted by-product. The two compounds differ only by one hydroxyl group, which is present in kanamycin carbamate but is reduced during biosynthesis of tobramycin. (13)C metabolic flux analysis was used for elucidating connections between the primary carbon metabolism and the composition of the antibiotic complex. Metabolic flux maps were constructed for the cells grown on minimal medium with glucose or with a glucose-glycerol mixture as the carbon source. The addition of glycerol, which is more reduced than glucose, led to a three-times-greater reduction of the kanamycin portion of the antibiotic complex. The labeling indicated an active Entner-Doudoroff (ED) pathway, which was previously considered to be nonfunctional in Streptomyces. The activity of the pentose phosphate (PP) pathway was low (10 to 20% of the glucose uptake rate). The fluxes through Embden-Meyerhof-Parnas (EMP) and ED pathways were almost evenly distributed during the exponential growth on glucose. During the transition from growth phase to production phase, a metabolic shift was observed, characterized by a decreased flux through the ED pathway and increased fluxes through the EMP and PP pathways. Higher specific NADH and NADPH production rates were calculated in the cultivation on glucose-glycerol, which was associated with a lower percentage of nonreduced antibiotic kanamycin B carbamate.

Synthesis and structure investigation of the antibiotic amoxicillin complexes of d-block elements

The study of some transition metals (M) and amoxicillin trihydrate (ACT) ligand complexes (M–ACT) that formed in solution involved the spectrophotometric determination of stoichiometric ratios and their stability constants and these ratios were found to be M:ACT = 1:1, 1:2 and 2:1 in some instances. The calculated stability constants of these chelates, under selected optimum conditions, using molar ratio method have values ranging from K f = 10 7 to 10 14. These data were confirmed by calculations of their free energy of formation Δ G, which corresponded to their high stabilities. The separated solid complexes were studied using elemental analyses, IR, reflectance spectra, magnetic measurements, mass spectra and thermal analyses (TGA and DTA). The proposed general formulae of these complexes were found to be ML ( H 2 O ) w ( H 2 O ) x ( OH ) y ( Cl ) z , where M = Fe(II), Co(III), w = 0 , x = 2, y = 1, z = 0; M = Co(II), w = 0 , x = 1, y = 0, z = 1; M = Fe(III), w = 0 , x = 1, y = 2, z = 0; M = Ni(II), Cu(II) and Zn(II), w = 2 , x = 0, y = 1, z = 0, where w = water of crystallization, x = coordinated water, y = coordinated OH − and z = Cl − in the outer sphere of the complex. The IR spectra show a shift of ν(NH) (2968 cm −1) to 2984–2999 cm −1of imino group of the ligand ACT and the absence of ν(CO) (β-lactame) band at 1774 cm −1 and the appearance of the band at 1605–1523 cm −1 in all complexes suggest that 6,7-enolization takes place before coordination of the ligand to the metal ions. The bands of M N (at 625–520 cm −1) and of M O (at 889–7550 cm −1) proved the bond of N (of amino and imino groups) and O of C O group of the ligand to the metal ions. The reflectance spectra and room temperature magnetic measurements refer to octahedral complexes of Fe(II) and Fe(III); square planner form of Co(II), reduced Co(III), Ni(II) and Cu(II)–ACT complexes but tetrahedral form of Zn–ACT complex. The thermal degradation of these complexes is confirmed by their mass spectral fragmentation. These data confirmed the proposed structural and general formulae of these complexes.

Application of liquid chromatography-ion trap mass spectrometry to the characterization of the 16-membered ring macrolide josamycin propionate.

Coupled liquid chromatography and ion trap mass spectrometry (LC/MS) was used for the characterization of the semi-synthetic 16-membered ring macrolide josamycin propionate. On-line identification of impurities in this antibiotic complex was performed with an ion trap mass spectrometer without recourse to time-consuming isolation and purification procedures. Ion trap mass spectrometry is ideally suited to identification of impurities because it provides MSn capability, enabling multiple stages of mass spectrometry to obtain the maximum amount of structural information for a given molecule. The ion trap was used with an electrospray ionization source operated in the positive ion mode or with an atmospheric pressure chemical ionization source operated in the negative ion mode. The identity of the unknown compounds was deduced using the MS/MS and MSn collision-induced dissociation spectra of reference substances or structural analogs as interpretative templates, combined with knowledge about the nature of functional group fragmentation behavior. Given the importance attached to the identification of impurities of unknown identity in pharmaceutical substances, this study is useful for companies producing josamycin propionate. The knowledge of the fragmentation behavior is also of importance in further research on other 16-membered macrolides.

Thermal and photochemical behaviour of zn(II) complexes of some cephalosporins

Zn(II) complexes of some cephalosporin antibiotics namely cephalexin, cephapirin, cefamandole, cefuroxime, cefotaxime and ceftazidime were synthesised and characterized. The stoichiometrics and the mode of bonding of the complexes were deduced from their elemental analysis, IR and electronic spectroscopies. Thermal stabilities and the photochemical behaviour of the complexes were studied. The Zn(II) complex of cephalexin exhibited a high light sensitivity. The remaining Zn(II) complexes behaved similarly to their free antibiotics, upon irradiation.

Gene cluster in Micromonospora echinospora ATCC15835 for the biosynthesis of the gentamicin C complex.

Gentamicin is a 4,6-disubstituted aminocyclitol antibiotic complex synthesised by some members of the actinomycete genus Micromonospora. In a search for the gentamicin biosynthetic gene cluster we identified, using a cosmid library approach, a region of the M. echinospora ATCC15835 chromosome that encodes homologues of aminoglycoside biosynthesis genes including gntB-a close homologue of the 2-deoxy-scyllo-inosose synthase gene (btrC) from butirosin-producing Bacillus circulans. Insertional inactivation was achieved by homologous recombination with an internal gntB fragment-containing suicide plasmid, delivered by conjugal transfer from Escherichia coli. gntB disruptants were gentamicin nonproducing mutants as assayed by an ELISA antibiotic detection system, proving the association of gntB (or a downstream region) with gentamicin biosynthesis. The function of some open reading frames within the cluster, predicted by nucleotide database homology searching, is discussed with regards to their potential roles in gentamicin biosynthesis. The discovery of this genetic region represents the first report of a gene cluster involved in the biosynthesis of a 4,6-disubstituted aminocyclitol antibiotic.

Biosynthesis of cervimycin C, an aromatic polyketide antibiotic bearing an unusual dimethylmalonyl moiety.

Cervimycin C is the major component of an antibiotic complex produced by Streptomyces tendae HKI-179 consisting of a tetracycline-type aglycon, six tridesoxysugars and a rare dimethylmalonyl moiety. The biosynthetic origin of cervimycin was studied by molecular studies and feeding experiments, which reveal that the dimethylmalonate unit is not derived from malonate, but from valine.

Hyphenation of liquid chromatography to ion trap mass spectrometry to identify minor components in polypeptide antibiotics.

The application of liquid chromatography-ion trap mass spectrometry for the characterization of linear and cyclic polypeptide antibiotics was investigated. The aim was on-line identification of impurities in those antibiotic complexes without recourse to time-consuming isolation and purification procedures. Hyphenated techniques, such as liquid chromatography coupled to mass spectrometry, are ideally suited for this purpose. Characterization was performed with an ion trap mass spectrometer offering MS(n) capability; this enables more structural information to be obtained. Liquid chromatography in combination with ion trap mass spectrometry was successfully applied for the characterization of impurities in gramicidin, polymyxin B, polymyxin E, and bacitracin and the study of the degradation products of polymyxins B and E.

AN INVESTIGATION OF THE Cu(II) COMPLEXES OF CERTAIN CEPHALOSPORIN ANTIBIOTICS: SPECTRAL, THERMAL, AND PHOTOCHEMICAL STUDIES

ABSTRACT The following Cu(II) complexes of some cephalosporin antibiotics were synthesized and characterized: [Cu(ATB)Cl]ċ-xH2O, [Cu(ATB)2]ċxH2O and [Cu(ATB)2]ċxH2, where HATB=cephalexin, cephapirin, cefamandole, cefuroxime or cefotaxime, H2ATB=ceftazidime and x=1–4. The stoichiometries and structures of the complexes were deduced from their elemental analyses, IR and electronic spectra and thermal decomposition. Thermal stabilities of the complexes were discussed and kinetic parameters were calculated using the Coats-Redfern and Horowitz-Metzger equations. The photochemical behaviour of the complexes was investigated. Upon irradiation, the Cu(II) complex of cefamandole underwent two competitive processes, one is photodegradation, the other is intramolecular hydrogen abstraction, as indicated from the spectral changes. The remaining studied Cu(II) complexes behave similar to their corresponding free antibiotics.