The broad objective of this proposal is to elucidate some of the molecular connections between the complex, interrelated processes of antibiotic production and morphological development in the model bacterium Streptomyces coelicolor. The streptomycetes are an incredibly valuable source of bioactive molecules, including the majority of antibiotics clinically used. These mycelial bacteria typically synthesize antibiotics and other secondary metabolites concurrently with broader developmental events that result in the generation of an aerial mycelium on the colony surface whose filamentous cells ultimately sporulate.
The aims of this study concern two particular types of proteins that influence both antibiotic production and development in S. coelicolor. First, the phosphopantetheinyl transferase are enzymes that catalyze a posttranslational modification required for the activity of many antibiotic biosynthetic enzymes. Mutation of the SCO6673 PPTase gene in S. coelicolor results in loss of the ability to produce calcium dependent-antibiotic but also conversely hyperproduction of the potential anti-cancer molecule undecylprodigiosin. Sporulation is also delayed in this mutant. In order to understand the basis for these diverse phenotypic effects, real-time PCR will be used to assess any differences in expression of known antibiotic regulatory and developmental genes in the PPTase mutant as compared to the wild type. This data will inform a subsequent targeted mutational strategy to engineer S. coelicolor for maximum undecylprodigiosin production.
The second aim focuses on understanding how activity of a likely stress response ECF sigma factor, ?U, can block both polyketide antibiotic production and aerial mycelium formation in S. coelicolor. A combination of microarray analysis and proteomic studies comparing wild type S. coelicolor to a rsuA mutant with high, unregulated ?U activity will establish the complete regulon of ?U-dependent genes as well as those indirectly affected by ?U activity. The relevance of the observed elevation of both extracellular protease and pentose phosphate pathway enzyme activity in the rsuA mutant to its antibiotic production and developmental defects will be tested by mutation or overexpression of the relevant genes and analysis of the resulting strains. Mutational analysis will also be used to test the involvement of the likely membrane protein SCO4110 in the regulation of ?U activity. A better understanding of how secondary metabolism and development are mutually influenced may ultimately suggest generalizable strategies for effective pharmaceutical production in the streptomycetes.
The goal of the proposed research is to understand how Streptomyces bacteria make useful compounds, such as antibiotics, as part of their overall life cycle. The better we understand how these beneficial bacteria grow, the more likely it is that we can engineer that growth to produce life-saving drugs for fighting infections and cancers.
