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

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Abstract
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 σ A - or σ 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.