The pacidamycins are a new complex of nucleosidyl-peptide antibiotics with highly specific activity against Pseudomonas aeruginosa. They are produced by Streptomyces coeruleorubidus AB 1 183F-64 which was isolated from a soil sample collected at Offenburg in the FRG. The mature spore masses of the producing organism are greenish gray to blue, and the spore chains are arranged in spirals. After the structures of the pacidamycins were determined, the fermentation medium was supplemented with component amino acids. This resulted in the directed biosynthesis of several members of the complex. The overall antibiotic recovered was increased from 1~2 mg/liter to more than 100 mg/liter through a combination of strain selection, mediummanipulation and amino acid feeding experiments.
A 1.3-kb segment of Escherichia coli DNA containing the regulatory gene, araC, and the promoter of the araBAD operon was amplified by the polymerase chain reaction (PCR) and cloned into pUC18, resulting in plasmid pKB130 that produced the alpha fragment of beta-galactosidase upon addition of L-arabinose (L-ara). A synthetic gene for human immunodeficiency virus (HIV)-1 preprotease was placed downstream of the ara-BAD promoter in pKB130 to create a translational fusion inducible by addition of L-ara. The fusion protein correctly autoprocessed in vivo to yield a mature 99-amino-acid HIV-1 protease, which was found predominantly in inclusion bodies. This material could be refolded to an active form, which was purified to homogeneity. A small fraction of the protease was expressed in vivo as a soluble active form, which allowed the monitoring of expression during fermentation by a rapid and simple whole cell assay employing an HIV-1 protease-specific fluorogenic substrate.
In micro-arrayed compound screening (µARCS), an agarose gel is used as a reaction vessel that maintains humidity and compound location as well as being a handling system for reagent addition. Two or more agarose gels may be used to bring test compounds, targets, and reagents together, relying on the pore size of the gel matrix to regulate diffusion of reactants. It is in the microenvironment of the agarose matrix that all the components of an enzymatic reaction interact and result in inhibitable catalytic activity. In an effort to increase the throughput of µARCS-based screens, reduce the effort involved in manipulating agarose gels, and reduce costs, blotter paper was used rather than a second agarose gel to introduce a substrate to a gel containing a target enzyme. In this assay, the matrix of the blotter paper did not prevent the substrate from diffusing into the enzyme gel. The compound density of the µARCS format, the ease of manipulating sheets of paper for reagent addition, and a scheduled protocol for running multiple gels allowed for a throughput capacity of more than 200,000 tests per hour. A protease assay was developed and run in the µARCS format at a rate of 200,000 tests per hour using blotter paper to introduce the substrate. Picks in the primary screen were retested in the µARCS format at a density of 384 compounds per sheet. IC 50 values were confirmed in a 96-well plate format. The screen identified several small molecule inhibitors of the enzyme. The details of the screening format and the analysis of the hits from the screen are presented. (Journal of Biomolecular Screening 2003: 668-675)
Phenelfamycins A, B, C, E, F and unphenelfamycin have been discovered in the fermentation broth of two soil isolates, designated AB 999F-80 and AB1047T-33. These isolates were identified as strains of Streptomyces violaceoniger. The antibiotics were selected for their activity against anaerobic bacteria.Phenelfamycins, a complex of antibiotics related to the elfamycin group, have been found in the fermentation broths of two soil isolates.These isolates have been identified as Streptomyces violaceoniger AB 999F-80 and S. violaceoniger AB 1047T-33. This paper describes the discovery of the antibiotics, the producing organisms and the production of phenelfamycin A by fermentation of Streptomyces violaceoniger AB 999F-80. The isolation and structure determination of the individual membersof the phenelfamycin complex and their biological properties are reported in companion papers1»2). Materials and Methods MicroorganismsStrain AB999F-80 was isolated from a soil collected at Mount Angel, Oregon and strain AB 1047T-33 from a soil collected near Niotaze, Kansas. Both cultures were isolated using media containing species restricting substrates3;. For strain AB 999F-80 the carbon source in the isolation mediumwas inulin and for strain AB1047T-33 it was arabitol. Cultures employed in the screen were from the stock culture collection in our laboratory or from the American Type Culture Collection (ATCC).The Screen For the discovery test, agar plugs were cut from cultures grown for 6 days on ATCCmedium 172* (20 ml/petri dish). The plugs were placed on the surface of seeded agar plates, incubated overnight and scored for inhibition of bacterial growth surrounding the plug. Bacteroides fragilis was grown in Wilkins Chalgren agar and incubated anaerobically at 37°C. The other strains were grown in streptomycin assay agar with yeast extract (BBL) and incubated aerobically at 32°C.Taxonomic Studies Methods and media described by the International Streptomyces Project (ISP)4), Waksman5) and Gordon et alP were used to determine most of the morphological and physiological characteristics. ATCCmedium172 and starch -yeast extract -salts agar7) were also employed for the cultural characteristics. Color names were assigned to the mycelial and diffusible pigments on the American Type Culture Collection. ATCCMedia Handbook. First Ed. American Type Culture Collection, Rockville, 1984.
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