A transposon insertion single-gene knockout library and new ordered cosmid library for the model organism Streptomyces coelicolor A3(2).

A simple and high-throughput transposon mediated mutagenesis system employing in vitro shuttle transposon mutagenesis has been used to systematically mutagenise the Streptomyces coelicolor genome. To achieve the highest coverage, a new ordered cosmid library was also constructed. Individual cosmids from both the existing and new libraries were disrupted using the Tn5-based mini-transposon Tn5062. A total of 35,358 insertions were sequenced resulting in the disruption of 6,482 genes (83% of the predicted open reading frames). Complete information for both the newly generated cosmids as well as all the insertions has been uploaded onto a central database, StrepDB ( http://strepdb.streptomyces.org.uk/ ). All insertions, new cosmids and a range of transposon exchange cassettes are available for study of individual gene function.

Gene transfer between streptomycetes in soil.

The growth and activity of Streptomyces violaceolatus and Streptomyces lividans was studied in soil under controlled conditions. The life cycle was followed under differing nutrient regimes and the fate of plasmid- and phage-borne genes determined by direct and indirect techniques. Methods were developed for the direct monitoring of plasmid DNA extracted from soil which allowed differentiation of the cellular location of plasmid DNA between mycelium and spores. In a dynamic, nutrient-fed soil microcosm, inoculants survived poorly, but a specific stage was defined by direct and indirect methods when the inoculants were most active and this correlated with the detection of gene transfer events. Plasmid transfer, phage infection and lysogeny only occurred to a significant extent within this stage at days 15-17 during a 60-day incubation. Estimates based on plasmid DNA recovery indicated that viable counts underestimated spore and mycelial propagules by a factor of greater than 100.

Low target site specificity of an IS6100-based mini-transposon, Tn1792, developed for transposon mutagenesis of antibiotic-producing Streptomyces.

To improve transposon mutagenesis of antibiotic-producing Streptomyces, a mini-transposon, Tn1792, was constructed, based on IS6100, originally isolated from Mycobacterium fortuitum. Easily manageable transposition assays were developed to demonstrate inducible transposition of Tn1792 into the Streptomyces genome from a temperature-sensitive delivery plasmid. Introduction of the selectable aac1 gene between the inverted repeats in Tn1792 allowed for both reliable identification of transposition events in Streptomyces, and also subsequent cloning of transposon-tagged sequences in Escherichia coli. This enabled the target site specificity of Tn1792 to be determined at nucleotide resolution, revealing no significant shared homology between different target sites. Consequently, Tn1792 is well suited for random mutagenesis of Streptomyces.

New method for extraction of streptomycete spores from soil and application to the study of lysogeny in sterile amended and nonsterile soil.

A new method for the isolation and enumeration of streptomycete spores from soil was developed. This method makes use of a cation-exchange resin to disperse soil particles. It allowed the detection of 10 spores in 100 g of sterile soil, while ca. 10 could be accurately enumerated in 100 g. This method was applied to studying the fate of a marked actinophage in soil. In sterile amended and nonsterile soil, relatively high numbers of actinophages were only found during the first few days of the experiment when the host streptomycete was in the mycelial form. Later, after sporulation, lysogens could be detected in sterile amended soil and could still be found 60 days after inoculation. Although no lysogens were found in nonsterile soil, the introduced phage could still be detected in the free state after 60 days, albeit at a low titer.