Rapid recombination screening to test gene essentiality demonstrates that pyrH is essential in Mycobacterium tuberculosis.

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.