glnE is an essential gene in Mycobacterium tuberculosis

Mycobacterium tuberculosis possesses a homologue of glnE, potentially encod ing a regulator of glutamine synthetase activity, We attempted to construct glnE-disrupted mutants using a two-step strategy, whereby a single-crossov er strain was first isolated, followed by sacB counterselection to isolate the double-crossover strain. Of 192 sucrose-resistant colonies tested, none were mutants, although the wild-type double crossover could be easily isol ated. When a second copy of the wild-type glnE was integrated into the chro mosome, we could isolate both wild-type and mutant double-crossover strains . Thus, the chromosomal gene could only be replaced with a disrupted copy w hen another functional copy of the gene was provided, demonstrating that th is gene is essential under the conditions tested.