Systematic mutagenesis of the model organism Streptomyces coelicolor: completion of an essential resource for the research community

Streptomyces are fascinating soil bacteria of major economic importance as they produce 70% of antibiotics known to man. Unusually for bacteria, they exhibit a complex differentiating life-cycle that has implications for antibiotic production. Due to their importance, the BBSRC and Wellcome Trust funded the complete genome sequencing of the model species S. coelicolor. As a consequence, we know this bacterium possesses about 8000 genes. However, as yet we only understand the biological function of a small proportion of these genes. Understanding gene function is important as it enables us to learn more about the biology of Streptomyces and, in particular, the complexities of the regulation of antibiotic production and differentiation. In addition, S. coelicolor is a genetically tractable model to understand conserved processes it shares with important human pathogens such as Mycobacterium tuberculosis. A critical aspect of understanding gene function is the creation of mutants in which a specific gene is no longer active. By comparing characteristics of the mutant with those of a non-mutant, it is possible to learn about the function of the gene in question. The proposed research intends to complete the construction of a versatile resource that is currently being used by researchers worldwide to create specific gene mutations in S. coelicolor. This resource is made by insertion of a 'jumping gene' or transposon into the DNA sequence of each S. coelicolor gene. These insertions are made in gene copies cloned in a cosmid library: this library was used to obtain the S. coelicolor genome sequence. So for each original cosmid carrying approximately 30 contiguous genes, we construct a sub-library consisting of individual cosmids each carrying an individual insertion in each gene. With access to these mutant cosmids, researchers can easily construct a S. coelicolor mutant defective in a gene of interest. Information on the systematic cosmid mutagenesis programme is accessible to any researcher via an online database, allowing them to select and order specific mutant cosmids for their own use. In addition, we will ourselves use the resource to investigate in more detail how Streptomyces adapt to the varying moisture content of their natural environment. This itself impacts on the life-cycle and antibiotic production.

Streptomyces coelicolor is an important and complex model prokaryote. Creating specific mutants is critical to understanding its differentiating life-cycle, antibiotic production, and processes conserved in other less experimentally tractable actinomycetes such as Mycobacterium tuberculosis. Any procedure for mutagenesis must be sufficiently robust to meet challenges specific to an organism. For Streptomyces, these challenges include a high rate of spontaneous genetic instability and the lack of a reliable means of generalised transduction to prove linkage between genotype and phenotype. In addition, procedures must be efficient with respect to time and cost. We have developed and applied a versatile technology that fulfils these requirements, based on in vitro transposon mutagenesis of the S. coelicolor cosmid library. To date, we have generated over 30,000 fully characterised insertions in more than 4,300 genes, with the data publically accessible at http://streptomyces.org/. Mutated transposon-tagged cosmids are supplied to researchers wordwide who then mobilise them from an E.coli host into S. coelicolor and select for gene replacement to isolate the mutant. We have also constructed various cassettes to tailor individual insertions, for example to remove an antibiotic resistance marker from an insertion in S. coelicolor to permit subsequent construction of multiple mutations in one genetic background. We aim to complete this programme of systematic mutagenesis. Cosmids equivalent to one third of the genome remain to be processed in the 2 years of the proposed funding. In addition, the complete transposon-tagged mutant comids library will be transferred to FTA cards to ensure long-term viability of this critical resource. Lastly, we will use specific cosmids to characterise genes involved in developmental osmoadaptation identified in recent microarray experiments.