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Generation Of Dominant Selectable Markers For Resistance To Pseudomonic Acid By Cloning And Mutagenesis Of The IleS Gene From The Archaeon Methanosarcina Barkeri Fusaro

Paolo Boccazzi, Jun Kai Zhang, William W. Metcalf

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ABSTRACT Currently, only one selectable marker is available for genetic studies in the archaeal genus Methanosarcina . Here we report the generation of selectable markers that encode resistance to pseudomonic acid (PA r ) in Methanosarcina species by mutagenesis of the isoleucyl-tRNA synthetase gene ( ileS ) from Methanosarcina barkeri Fusaro. The M. barkeri ileS gene was obtained by screening of a genomic library for hybridization to a PCR fragment. The complete 3,787-bp DNA sequence surrounding and including the ileS gene was determined. As expected, M. barkeri IleS is phylogenetically related to other archaeal IleS proteins. The ileS gene was cloned into a Methanosarcina-Escherichia coli shuttle vector and mutagenized with hydroxylamine. Nine independent PA r clones were isolated after transformation of Methanosarcina acetivorans C2A with the mutagenized plasmids. Seven of these clones carry multiple changes from the wild-type sequence. Most mutations that confer PA r were shown to alter amino acid residues near the KMSKS consensus sequence of class I aminoacyl-tRNA synthetases. One particular mutation (G594E) was present in all but one of the PA r clones. The MIC of pseudomonic acid for M. acetivorans transformed with a plasmid carrying this single mutation is 70 μg/ml of medium (for the wild type, the MIC is 12 μg/ml). The highest MICs (560 μg/ml) were observed with two triple mutants, A440V/A482T/G594E and A440V/G593D/G594E. Plasmid shuttle vectors and insertion cassettes that encode PA r based on the mutant ileS alleles are described. Finally, the implications of the specific mutations we isolated with respect to binding of pseudomonic acid by IleS are discussed.