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Applied and Environmental Microbiology, January 2006, p. 327-333, Vol. 72, No. 1
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.1.327-333.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

In Vivo Random Mutagenesis of Bacillus subtilis by Use of TnYLB-1, a mariner-Based Transposon

Yoann Le Breton,^* Nrusingh Prasad Mohapatra, and W. G. Haldenwang

Department of Microbiology and Immunology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900

Received 14 July 2005/ Accepted 19 September 2005

This report describes the construction and characterization of a mariner-based transposon system designed to be used in Bacillus subtilis, but potentially applicable to other gram-positive bacteria. Two pUC19-derived plasmids were created that contain the mariner-Himar1 transposase gene, modified for expression in B. subtilis, under the control of either ^A- or ^B-dependent promoters. Both plasmids also contain a transposable element (TnYLB-1) consisting of a Kan^r cassette bracketed by the Himar1-recognized inverse terminal repeats, as well as the temperature-sensitive replicon and Erm^r gene of pE194ts. TnYLB-1 transposes into the B. subtilis chromosome with high frequency (10^–2) from either plasmid. Southern hybridization analyses of 15 transposants and sequence analyses of the insertion sites of 10 of these are consistent with random transposition, requiring only a "TA" dinucleotide as the essential target in the recipient DNA. Two hundred transposants screened for sporulation proficiency and auxotrophy yielded five Spo^– clones, three with insertions in known sporulation genes (kinA, spoVT, and yqfD) and two in genes (ybaN and yubB) with unknown functions. Two auxotrophic mutants were identified among the 200 transposants, one with an insertion in lysA and another in a gene (yjzB) whose function is unknown.

Present address: Department of Molecular Virology, Immunology and Medical Genetics, Department of Medicine, Division of Infectious Diseases, and The Centre for Microbial Interface Biology, Ohio State University, Columbus, OH 43210.

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