RESUMO
To further explore possible avenues for accessing microbial biodiversity for drug discovery from natural products, we constructed and screened a 5,000-clone "shotgun" environmental DNA library by using an Escherichia coli-Streptomyces lividans shuttle cosmid vector and DNA inserts from microbes derived directly (without cultivation) from soil. The library was analyzed by several means to assess diversity, genetic content, and expression of heterologous genes in both expression hosts. We found that the phylogenetic content of the DNA library was extremely diverse, representing mostly microorganisms that have not been described previously. The library was screened by PCR for sequences similar to parts of type I polyketide synthase genes and tested for the expression of new molecules by screening of live colonies and cell extracts. The results revealed new polyketide synthase genes in at least eight clones. In addition, at least five additional clones were confirmed by high-pressure liquid chromatography analysis and/or biological activity to produce heterologous molecules. These data reinforce the idea that exploiting previously unknown or uncultivated microorganisms for the discovery of novel natural products has potential value and, most importantly, suggest a strategy for developing this technology into a realistic and effective drug discovery tool.
Assuntos
Antibacterianos/metabolismo , Bactérias/classificação , Produtos Biológicos/metabolismo , Biblioteca Gênica , Variação Genética , Recombinação Genética , Microbiologia do Solo , Sequência de Aminoácidos , Bactérias/genética , Bactérias/metabolismo , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Cosmídeos , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/genética , Vetores Genéticos , Dados de Sequência Molecular , Complexos Multienzimáticos/genética , Reação em Cadeia da Polimerase , Streptomyces/genética , Transformação BacterianaRESUMO
The antibiotic kirromycin (Kr) inhibits bacterial protein synthesis by binding to elongation factor Tu (EF-Tu). Streptomyces cinnamoneus and Nocardia lactamdurans, producers of antibiotics of the Kr class, are known to possess an EF-Tu resistant to Kr. Both micro-organisms appear to possess a single tuf gene and we have characterized the one from S. cinnamoneus, which belongs to the tuf1 family. To assess the molecular determinants of Kr resistance, the S. cinnamoneus tuf gene was expressed in Escherichia coli as a translational fusion to malE, which enabled the recovery by affinity chromatography of the recombinant protein uncontaminated by the host factor. The recombinant EF-Tu was able to catalyse polyU-directed polyPhe synthesis in two heterologous cell-free systems, even as an uncleaved fusion. When tested for antibiotic sensitivity it behaved like the natural S. cinnamoneus protein, showing equivalent resistance to Kr but sensitivity to the antibiotic GE2270, indicating that all the determinants for Kr resistance are intrinsic to the EF-Tu sequence. Multiple sequence analysis of EF-Tu proteins, together with knowledge of mutations conferring Kr resistance, allowed the identification of key residues as likely candidates for the natural Kr resistance of the S. cinnamoneus EF-Tu. One of these, Thr378, was mutated to the consensus Ala and the resulting mutant protein was sensitive to Kr. Interestingly, it retained some activity (30% of the control) even at high Kr concentrations.
