Novel Amycolatopsis balhimycina biochemical abilities unveiled by proteomics.

Amycolatopsis balhimycina DSM5908 is an actinomycete producer of balhimycin, an analogue of vancomycin, the antibiotic of 'last resort' against multidrug-resistant Gram-positive pathogens. Most knowledge on glycopeptide biosynthetic pathways comes from studies on A. balhimycina as this strain, among glycopeptide producers, is genetically more amenable. The recent availability of its genome sequence allowed to perform differential proteomic analyses elucidating key metabolic pathways leading to antibiotic production in different growth conditions. To implement proteomic data on A. balhimycina derived from 2-DE approaches and to identify novel components, a combined approach based on protein extraction with different detergents, SDS-PAGE resolution of intact proteins and nanoLC-ESI-LIT-MS/MS analysis of their tryptic digests was carried out. With this procedure, 206 additional new proteins such as very basic, hydrophobic or large species were identified. This analysis revealed either components whose expression was previously only inferred by growth conditions, that is, those involved in glutamate metabolism or in resistance, or proteins that allow the strain to metabolize alkanes. These findings will give additional insight into metabolic pathways that could really contribute to A. balhimycina growth and antibiotic production and metabolic enzymes that could be manipulated to generate a model producing strain to use for synthetic biology.

Two heterologously expressed Planobispora rosea proteins cooperatively induce Streptomyces lividans thiostrepton uptake and storage from the extracellular medium.

BACKGROUND: A bacterial artificial chromosomal library of Planobispora rosea, a genetically intractable actinomycete strain, was constructed using Escherichia coli-Streptomyces artificial chromosome (ESAC) and screened for the presence of genes known to be involved in the biosynthesis of antibiotics. RESULTS: One clone with a 40 kb insert showed antimicrobial activity against Gram positive bacteria. Insert sequence analysis and subcloning experiments revealed that the bioactivity was due to a 3.5 kb DNA fragment containing two open reading frames. These orfs encode two proteins with high similarity to a putative membrane protein of Streptomyces coelicolor and to the nogalamycin resistance protein SnorO of Streptomyces nogalater, respectively. The role of these two Orfs is unknown in Planobispora. Disruption and complementation experiments revealed that both proteins are necessary for the antibacterial activity and chemical analysis demonstrated that the antibiotic activity was due to thiostrepton, antibiotic used as recombinant clone selection marker. CONCLUSION: Two Planobispora rosea orfs are responsible for increasing intracellular amounts and storage of thiostrepton in Streptomyces lividans.

Artificial chromosome libraries of Streptomyces coelicolor A3(2) and Planobispora rosea.

Using an Escherichia coli-Streptomyces shuttle vector derived from a bacterial artificial chromosome (BAC), we developed methodologies for the construction of BAC libraries of filamentous actinomycetes. Libraries of Streptomyces coelicolor, the model actinomycete, and Planobispora rosea, a genetically intractable strain, were constructed. Both libraries have an average insert size of 60 kb, with maximal insert larger than 150 kb. The S. coelicolor library was evaluated by selected hybridisations to DraI fragments and by end sequencing of a few clones. Hybridisation of the P. rosea library to selected probes indicates a good representation of the P. rosea genome and that the library can be used to facilitate the genomic analysis of this actinomycete.