Systematic mutagenesis of the model organism Streptomyces coelicolor: completion of an essential resource for the research community
Lead Research Organisation:
Swansea University
Department Name: Institute of Life Science Medical School
Abstract
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 A critical aspect of understanding gene function is the creation of mutants in which a specific gene is no longer active.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.
Technical Summary
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.
Organisations
Publications
Akpe San Roman S
(2010)
A heterodimer of EsxA and EsxB is involved in sporulation and is secreted by a type VII secretion system in Streptomyces coelicolor.
in Microbiology (Reading, England)
Aldridge M
(2013)
A novel bifunctional histone protein in Streptomyces: a candidate for structural coupling between DNA conformation and transcription during development and stress?
in Nucleic acids research
Hitchings MD
(2014)
A tale of tails: deciphering the contribution of terminal tails to the biochemical properties of two Dps proteins from Streptomyces coelicolor.
in Cellular and molecular life sciences : CMLS
Fernández-Martínez LT
(2011)
A transposon insertion single-gene knockout library and new ordered cosmid library for the model organism Streptomyces coelicolor A3(2).
in Antonie van Leeuwenhoek
Fuchino K
(2017)
Cell-Biological Studies of Osmotic Shock Response in Streptomyces spp.
in Journal of bacteriology
Schaechter, Moselio
(2009)
Encyclopedia of Microbiology
Jones G
(2011)
Forkhead-associated proteins genetically linked to the serine/threonine kinase PknB regulate carbon flux towards antibiotic biosynthesis in Streptomyces coelicolor.
in Microbial biotechnology
Mistry BV
(2008)
FtsW is a dispensable cell division protein required for Z-ring stabilization during sporulation septation in Streptomyces coelicolor.
in Journal of bacteriology
Fernández Martínez L
(2009)
Osmoregulation in Streptomyces coelicolor: modulation of SigB activity by OsaC.
in Molecular microbiology
Dyson P
(2014)
Reference Module in Biomedical Sciences
Lamb DC
(2010)
Streptomyces coelicolor A3(2) CYP102 protein, a novel fatty acid hydroxylase encoded as a heme domain without an N-terminal redox partner.
in Applied and environmental microbiology
Godinez O
(2015)
Targeting the Osmotic Stress Response for Strain Improvement of an Industrial Producer of Secondary Metabolites.
in Journal of microbiology and biotechnology
Facey PD
(2013)
The evolution of an osmotically inducible dps in the genus Streptomyces.
in PloS one
Description | A comprehensive library of gene knockouts was generated for the model streptomycete, Streptomyces coelicolor. This resource has been used extensively by researchers the world over, investigating the functions of genes in this important antibiotic-producing organism. |
Exploitation Route | This resource is being used extensively by researchers the world over. |
Sectors | Pharmaceuticals and Medical Biotechnology |
URL | http://strepdb.streptomyces.org.uk/ |