Optimization of Mariner Transposon for Bacillus

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DESCRIPTION (provided by applicant): Transposable elements are among the most useful tools available for genetic analysis in bacteria. Not only can transposons disrupt operons or tag interesting alleles with selectable markers, but specialized versions of these elements, carrying origins of replication or reporter genes, can be used to directly clone or analyze the expression of the operons that they disrupt. Studies of Gram-positive bacteria, including Bacillus anthracis have benefited from a transposon system (Tn917) developed in B. subtilis. Although the Tn917 system has been successfully used in a number of instances, it is not without its limitations. Most notably, 99% of all Tn917 transpositions in B. subtilis target "hot spots" on the B. subtilis chromosome. Recently, the eukaryotic transposable element Himar1 has been modified for use in a number of bacterial systems, where random transposition occurs into a dinucleotide sequence (TA). The current application seeks to develop Himar1 for use in Bacillus species. Antibiotic resistance cassettes, selectable in Bacillus will be bracketed by the transposon's inverted terminal repeats (ITR) and placed on a plasmid with a temperature-sensitive Bacillus origin of replication. The plasmid will also include a hyperactive variant of the Himar1 transposase gene with expression elements appropriate for Bacillus. The system will be evaluated in the more genetically tractable B. subtilis, and once optimized, embellished with additional elements (e.g., a reporter gene system, origin of replication for cloning) and tested for effectiveness in B. anthracis. During development, Himar1 transposase variants, hyperactive in Bacillus will be sought using a modification of a papillation assay developed for use in Escherichia coli and a sporulation selection protocol unique to Bacillus. The transposons created in this study should be of use both in Bacillus species and Gram-positive bacteria in general. Additionally, the hyperactive Himar1 transposase variants isolated in this study could be incorporated into existing Himar1 systems to enhance their efficiency, and provide insights into elements of the transposase that are important for its activity or stability.