CP-78,545, a new monocarboxylic acid ionophore antibiotic related to zincophorin and produced by a Streptomyces.

A new monocarboxylic acid ionophore antibiotic related to zincophorin, CP-78,545 (1), was found in the culture broth of Streptomyces sp. N731-45. CP-78,545 was extracted with organic solvents and purified by column chromatography. The metabolite, which is active in vitro against certain Gram-positive bacteria, as well as the anaerobe Treponema hyodysenteriae, and a coccidium Eimeria tenella, was isolated as a water insoluble magnesium salt (2) in 2:1 (ligand/metal) stoichiometry. The structure of CP-78,545 was elucidated by spectroscopic (NMR and MS) methods, and the relative stereochemistry was determined by single-crystal X-ray analysis of the cadmium salt (3). CP-78,545, i.e., 24-dehydrozincophorin, is unique since its molecular backbone contains a terminal double bond previously not found in other polyether ionophores.

A new channel-forming antibiotic from Streptomyces coelicolor A3(2) which requires calcium for its activity.

A recently discovered antibiotic (CDA; calcium-dependent antibiotic) of Streptomyces coelicolor A3(2) was found to be effective against a wide range of Gram-positive bacteria only in the presence of calcium ions. Producer and non-producer strains were identified and several media tested for their ability to support antibiotic production. The action of calcium was not simulated by any of the other cations tested. The antibiotic was found to induce discrete conductance fluctuations in planar lipid bilayer consistent with a channel-forming action. The electrical potential difference caused by a concentration difference of various salts across the CDA-containing bilayer, showed the channel to be cation-selective but of a size that discriminated against tetramethyl ammonium and choline ions. The data indicate that the antibiotic activity of CDA is due to its action as a calcium-dependent ionophore.

CDA is a new chromosomally-determined antibiotic from Streptomyces coelicolor A3(2).

Mutations (cda) leading to non-production of the new calcium-dependent antibiotic (CDA) of Streptomyces coelicolor A3(2) were closely linked on the chromosome. One representative mutation (cda-1) was mapped precisely between nicA and adeC. No cosynthesis of CDA was found in any pairwise combinations of 14 cda mutants. Mutations lacking aerial mycelium (bald mutations), mapping to the four previously described loci (bldA-D), were pleiotropically defective in production of CDA.

Siderophore production by Vibrio vulnificus.

Previous studies in our laboratory, as well as clinical evidence, have suggested that increased iron levels in the host may be important in infections caused by the halophilic pathogen Vibrio vulnificus. To study iron acquisition, we induced siderophore production by growth in a low-iron medium, and biochemical testing indicated the production of both hydroxamate- and phenolate-type siderophores. The siderophores were extracted from growth filtrates with ethyl acetate (for phenolates) and phenol-chloroform-ether (for hydroxamates). These extracts enhanced the growth of V. vulnificus when the bacterium was grown in iron-limited medium. The ability of these siderophores to stimulate the growth of Salmonella typhimurium LT-2 enb-7 (a mutant deficient in the biosynthesis of enterochelin) and Arthrobacter flavescens JG-9 (a hydroxamate auxotroph) supported the conclusion that V. vulnificus produces both hydroxamate- and phenolate-type siderophores.

Siderophore production by Enterobacter cloacae and a common receptor protein for the uptake of aerobactin and cloacin DF13.

Iron-starved cultures of Enterobacter cloacae produced two siderophores, identified as enterochelin and aerobactin. The aerobactin was excreted in larger amounts than was enterochelin, and it was synthesized preferentially in the late logarithmic and stationary growth phases under iron-deficient conditions. Enterochelin was synthesized by cultures in the logarithmic phase of growth and preferentially in medium with 1 microM ferric chloride. Both siderophores appeared to be excreted immediately after their synthesis, since no intracellular aerobactin or enterochelin could be detected. The killing activity of the bacteriocin cloacin DF13 was inhibited by aerobactin. It was shown that aerobactin and cloacin DF13 bound to the same receptor sites located in the outer membrane. The synthesis of these receptor sites was induced by iron limitation. We conclude that the receptor for the uptake of aerobactin also functions as receptor for cloacin DF13.

Effect of iron limitation on growth, siderophore production, and expression of outer membrane proteins of Vibrio cholerae.

Vibrio cholerae strains secrete a phenolate-type siderophore when grown in low-iron medium. The siderophore was detected as early as 3.5 h after downshift to iron-poor medium, and it continued to accumulate in the medium as the cells entered stationary phase. Two clinical isolates and an environmental isolate were examined for the amount of siderophore produced. The environmental isolate produced more siderophore and continued to secrete it at concentrations of iron that repressed synthesis in the clinical isolates. Concomitant with production of siderophore, at least six new proteins were seen in the outer membranes of iron starved cells. One of the proteins was large (200,000 Mr [220K]) and appeared to be loosely associated with the outer membrane. The other five proteins had approximate Mr values of 77K, 76K, 75K, 73K, and 62K. The 62K protein, like the 40K major outer membrane protein, was heat modifiable. One or more of these proteins may be a component of the receptor for the iron-siderophore complex.

ColV plasmid-specific aerobactin synthesis by invasive strains of Escherichia coli.

Certain strains of Escherichia coli associated with bacteremia of humans and domestic animals harbor plasmids that promote efficient iron uptake. The mechanism, which is an important component of the virulence of invasive strains, is independent of the enterobactin system for iron uptake. Plasmid-specified siderophore was assayed by its ability to support the growth of a chelator-deficient mutant in conditions of iron deprivation. The chelator, which was chemically determined to be a hydroxamate compound, was identical on the basis of field desorption mass spectrometry with aerobactin, a siderophore synthesized by Aerobacter aerogenes. In conditions of iron stress, aerobactin is secreted into the culture medium of plasmid-bearing E. coli strains. Reconstruction experiments involving a chelator-deficient mutant growing with exogenous chelator suggested that association of a small fraction of the total siderophore synthesized with cellular material is due to transient binding of aerobactin to membrane receptors during active bacterial growth.

Siderophore production by pathogenic Neisseria spp.

Previous studies have established the importance of iron acquisition to the growth and virulence of Neisseria meningitidis and Neisseria gonorrhoeae. Although preliminary evidence that the Neisseria spp. produce siderophores has been presented, the exact mechanism of iron acquisition has remained obscure. Siderophore production by N. gonorrhoeae and N. meningitidis was induced in two different low-iron media. The iron-reactive siderophores, "gonobactin" and "meningobactin," were partially purified by ion exchange chromatography followed by extraction with phenol-chloroform-ether or by gel filtration. The compounds were of low molecular weight, their synthesis was repressed by iron in the medium, and they appeared to be hydroxamic acids since they were stimulatory for Arthrobacter flavescens JG-9 (a hydroxamate auxotroph) and gave a positive Csáky reaction for bound hydroxylamine. In the iron form, the compounds had an absorption maximum of approximately 420 nm. Although meningobactin stimulated growth of the gonococcus in low-iron media and vice versa, the homologous activity was more marked, indicating that the compounds, though similar, were probably not identical. As determined by A. flavescens assay the meningococcus produced three to five times more siderophore than did the gonococcus; however, the amount of siderophore present in the culture fluids of even the meningococcus was 100- to 1,000-fold lower than the concentration of hydroxamate siderophores reported to be produced by Bacillus megaterium or Aerobacter aerogenes. Virulent, colony type 1 N. gonorrhoeae produced significantly more gonobactin than did the avirulent colony type 3 gonococci.

Studies on the ionophorous antibiotics. XXIV. Leuseramycin, a new polyether antibiotic produced by Streptomyces hygroscopicus.

A new antibiotic leuseramycin has been isolated from the cultured mycelium of the strain of Streptomyces hygroscopicus TM-531. Physicochemical data, in particular those of 1H- and 13C-NMR spectra, revealed that leuseramycin is closely related to dianemycin in its structure, the former having a methyl group in place of the hydroxymethyl group at C-30. It is active against Gram-positive bacteria, some phytopathogenic fungi and some protozoa.

Studies on the ionophorous antibiotics. XXV. The assignments of the 13C-NMR spectra of dianemycin and lenoremycin.

All the resonances observed in the 13C-NMR spectra of polyether antibiotics, dianemycin and lenoremycin (Ro 21-6150) have been assigned by the aid of selective proton decoupling experiments, T1 value measurements and biosynthetic methods as well as comparison to model compounds such as monensin, nigericin, etheromycin and carriomycin.

X-14547A, a new ionophorous antibiotic produced by Streptomyces antibioticus NRRL 8167. Discovery, fermentation, biological properties and taxonomy of the producing culture.

X-14547A is a novel antibiotic produced by a new strain of Streptomyces antibioticus (NRRL 8167). The antibiotic is active in vitro against Gram-positive bacteria and is capable of complexing and transporting divalent as well as monovalent metal cations.

Emericid, a new polyether antibiotic from Streptomyces hygroscopicus (DS 24 367).

Emericid is a new polyether polycyclic ionophore antibiotic excreted by Streptomyces hygroscopicus (DS 24 367). Active in vitro against Gram-positive bacteria, it is ineffective in vivo. At a 0.006-0.02% level in the diet it protects chickens and rabbits against coccidiosis.