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 essential genes in Staphylococcus aureus. Resulting strains were then subjected to allelic-replacement mutagenesis of the native allele under inducing conditions for expression of the ectopic copy of the gene. This strategy produced test strains whereby cellular viability was uniquely dependent on the presence of inducer and provided a direct and absolute confirmation of genetic essentiality for each locus. The procedure is particularly useful for genes that are difficult to analyze by conventional inactivation strategies due to either small size or complex genomic organization.
Fan
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F
Antimicrobials and Host Defense CEDD, Collegeville, Pennsylvania 19426, USA. Frank_K_Fan@sbphrd.com
Lunsford
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RD
Sylvester
D
D
Fan
J
J
Celesnik
H
H
Iordanescu
S
S
Rosenberg
M
M
McDevitt
D
D
D000483:Alleles; D017920:Antiporters / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics*; D003598:Cytoskeletal Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008565:Membrane Proteins; D016296:Mutagenesis; D012697:Serine Endopeptidases / Q000235:genetics*; D013211:Staphylococcus aureus / Q000235:genetics*
D017920:Antiporters; D001426:Bacterial Proteins; D003598:Cytoskeletal Proteins; C071525:FtsZ protein, Bacteria; D008565:Membrane Proteins; C079051:tetA protein, Bacteria; D012697:Serine Endopeptidases; C026515:type I signal peptidase