title: Gene replacement method for determining conditions in which Bacillus subtilis genes are essential or dispensable for cell viability.
abstract: We describe a method for determining conditions in which Bacillus subtilis genes are essential or dispensable for cell viability. This method utilizes a chloramphenicol-resistant plasmid containing a temperature-sensitive (ts) replication origin. In this method, the target gene is first cloned into the ts vector and the recombinant plasmid is used to transform wild-type B. subtilis. The second step involves transformation of the resulting strain with a linear DNA fragment containing a second antibiotic resistance marker (tet) that disrupts the gene of interest. Selection for tetracycline resistance forces a double crossover between the chromosomal and fragment-borne copies of the gene, thereby replacing the wild-type gene in the chromosome with the disrupted allele. Cells survive even if the gene is essential by virtue of the complementing plasmid. Transformants are then grown at the non-permissive temperature for plasmid replication under various growth conditions. Isolation of chloramphenicol-sensitive colonies indicates that the gene is dispensable, whereas the inability to isolate chloramphenicol-sensitive colonies indicates that the gene is essential. The general utility of this method is demonstrated by allowing disruption of mtrA and trpE under conditions that render each gene non-essential, but not under growth conditions in which each gene is essential.
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: H
authors.initials: H
authors.lastname: Yakhnin
authors.forename: P
authors.initials: P
authors.lastname: Babitzke
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000502:physiology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001412:Bacillus subtilis / Q000235:genetics* / Q000254:growth & development*; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology; D002701:Chloramphenicol / Q000494:pharmacology; D003001:Cloning, Molecular; D004261:DNA Replication; D024881:Drug Resistance, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D020043:Genes, Essential; D016254:Mutagenesis, Insertional; D010957:Plasmids; D011995:Recombination, Genetic; D012641:Selection, Genetic; D013696:Temperature; D013752:Tetracycline / Q000494:pharmacology; D014169:Transformation, Bacterial
chemical_list: D018528:ATP-Binding Cassette Transporters; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; C100938:MtrA protein, Bacteria; D002701:Chloramphenicol; D013752:Tetracycline