Genetic manipulation of Fonsecaea pedrosoi using particles bombardment and Agrobacterium mediated transformation.

Fonsecaea pedrosoi, a melanized fungal pathogen that causes Chromoblastomycosis, a human disease with a worldwide distribution. Biolistic is a widely used technique for direct delivery of genetic material into intact cells by particles bombardment. Another well-established transformation method is Agrobacterium-mediated transformation (ATMT), which involves the transfer of a T-DNA from the bacterium to the target cells. In F. pedrosoi there are no reports of established protocols for genetic transformation, which require optimization of physical and biological parameters. In this work, intact conidia of F. pedrosoi were particle bombarded and subjected to ATMT. In addition, we proposed hygromycin B, nourseothricin and neomycin as dominant selective markers for F. pedrosoi and vectors were constructed. We tested two parameters for biolistic: the distance of the particles to the target cells and time of cells recovery in nonselective medium. The biolistic efficiency was 37 transformants/µg of pFpHYG, and 45 transformants/µg of pAN7.1. Transformants expressing GFP were successfully obtained by biolistic. A co-culture ratio of 10: 1 (bacterium: conidia) and co-incubation time of 72 h yielded the largest number of transformants after ATMT. Southern blot analysis showed the number of foreign DNA insertion into the genome is dependent upon the plasmid used to generate the mutants. This work describes for the first time two efficient methods for genetic modification of Fonsecaea and these results open new avenues to better understand the biology and pathogenicity of the main causal agent of this neglected disease.

Identification of streptothricin class antibiotics in the early-stage of antibiotics screening by electrospray ionization mass spectrometry.

Several streptothricin antibiotics have been studied by tandem mass spectrometry. The dominant product ions were derived from the C(7)-N bond cleavage which lead to lose streptolidine from the [M+H](+). The fragmentation pathways of key ions were described and certained by parent scan. According to the generalized principles, streptothricin isomers could be distinguished easily by the difference of CID spectra. A facile method based on ion-pair RP-HPLC coupled with electrospray ionization tandem mass spectrometry has been established for the analysis of streptothricins in the fermentation broth of Streptomyces qinlingensis. A total of 19 streptothricin-like compounds were identified or tentatively characterized based on their mass spectral data, and in which 11 were the first reported compounds. This could be used as an important de-replication method in the programs of screening novel antibiotics.

[A new HPLC method for determination of main constituents of the streptothricin complex. Analysis of nourseothricin, grisin and kormogrisin].

A simple and reliable HPLC method for quantitative analysis of complex antibiotics consisting of a mixture of streptothricins F, E, D and C in a biological matrix was developed. The method is based on ion-pair separation of streptothricins on the reversed-phase C18 analytical column with UV detector (210 nm). Aqueous solution of acetonitrile containing trifluoroacetic acid and octane-1-sulfonic acid sodium salt was used as eluent. Retention times of streptothricins became longer as the molecular weight increased, i.e. the component F was eluted first, then followed components E, D and C. The total time of the analysis was ca. 22 min. Composition of the standard samples of nourseothricin and grisin, as well as the streptothricin content of the commercial grisin-based kormogrisin were determined. Components F and D were found to be dominant in the streptothricin complex comprising totally 70-90%, with streptothricin F prevailing in nourseothricin (56%) and streptothricin D being the major constituent in grisin (51%), while in the kormogrisin the concentrations of components D and F were approximately the same. The portion of E varied from 5 to 20% and the concentration of streptothricin C changed within the range of 3-11%. The peaks of the admixtures present in kormogrisin did not interfere with determination of the streptothricin components. It is suggested that the method described can be applied to determination of the streptothricins in biological objects without a complex preliminary sample preparation.

[Early identification of streptothricin group antibiotics]. / Ranniaia identifikatsiia antibiotikov gruppy streptotritsinov.

Methodical approaches to detecting cultures producing streptothricins at the early stages of screening new antibiotics were developed. The approaches are based on chromatographic and electrophoretic mobility of streptothricins and the products of their hydrolysis in the extracts from agar cultures of actinomycetes. Application of the method for screening new antibiotics is illustrated with an example. Nine strains of actinomyces with broad antibacterial spectra isolated from soil samples were studied and 6 of them belonging to 4 species were shown to produce streptothricins in agar cultures. The new streptothricin-producing culture S. roseolilacinus was isolated.

[Formation of a streptothricin-group antibiotic by a Streptomyces glaucus 1136 culture]. / Obazovanie antibiotika iz gruppy streptotritsinov kul'turoi Streptomyces glaucus 1136.

In screening of new antibiotics a streptomycete (strain 1136) was isolated from a soil sample of Armenia. It showed no antagonistic properties in streek cultures on agarized media. When grown under submerged conditions strain 1136 produced an antibiotic active against grampositive and gramnegative bacteria. By its cultural and morphological properties strain 1136 was classified as Streptomyces glaucus Agre et Preobrazhenskaya, 1983. Microbiological and chemical investigation of the antibiotic produced by strain provided its identification at the early stages of the investigation as an antibiotic of the streptothricin group. Up to now no organisms producing streptothricin antibiotics were detected among streptomycetes of the Azureus section including strain 1136.

A new fluorimetric assay for streptothricins.

In strongly acidic medium (70% HClO4) streptothricins form a fluorophore (lambda ex = 312 nm; lambda em = 381 nm) with unknown structure. A fluorimetric determination of pure or crude products and cultures, respectively, was worked out based on this reaction. Concentrations for fluorescence measurements were in the range of 10(-8) - 2 X 10(-7) moles. Interferences of the assay are discussed, a statistical evaluation of results and a comparison between microbiological and fluorimetric findings are given.