Regulated ectopic expression and allelic-replacement mutagenesis as a method for gene essentiality testing in Staphylococcus aureus

Conditional expression systems were utilized for the ectopic induction of e ssential genes in Staphylococcus aureus, Resulting strains were then subjec ted to allelic-replacement mutagenesis of the native allele under inducing conditions for expression of the ectopic copy of the gene. This strategy pr oduced test strains whereby cellular viability was uniquely dependent on th e presence of inducer and provided a direct and absolute confirmation of ge netic essentiality for each locus. The procedure is particularly useful for genes that are difficult to analyze by conventional inactivation strategie s due to either small size or complex genomic organization. (C) 2001 Academ ic Press.