title: Rapid recombination screening to test gene essentiality demonstrates that pyrH is essential in Mycobacterium tuberculosis.
abstract: The availability of the complete genome of Mycobacterium tuberculosis affords the possibility of screening genes for essentiality under defined conditions. We tested a rapid recombination method for screening and confirmation of gene essentiality which would be more amenable to higher throughput applications. Non-replicating vectors carrying the internal portion of a gene were used as recombination substrates. Such vectors would lead to inactivation of the target gene in a single recombination step. For non-essential genes, recombinants can be obtained; for essential genes, no recombinants can be obtained, thus providing a rapid screening method to determine essentiality in a targeted manner. The incorporation of a promoter in the vector allowed us to establish the essentiality of a single gene in an operon. We confirmed this method worked with several essential (proC, glnE, mtrB, trpD) and one non-essential (tlyA) gene. In addition, we used the method to demonstrate that the pyrH gene is essential.
authors.affiliation: Centre for Infectious Disease, Institute for Cell and Molecular Science, Barts and the London, Blizard Building, London, UK.
authors.forename: Dina
authors.initials: D
authors.lastname: Robertson
authors.forename: Paul
authors.initials: P
authors.lastname: Carroll
authors.forename: Tanya
authors.initials: T
authors.lastname: Parish
mesh_terms: D005798:Genes, Bacterial / Q000235:genetics*; D020043:Genes, Essential / Q000235:genetics; D005838:Genotype; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011995:Recombination, Genetic / Q000235:genetics*