glnE is an essential gene in Mycobacterium tuberculosis.
Mycobacterium tuberculosis possesses a homologue of glnE, potentially encoding a regulator of glutamine synthetase activity. We attempted to construct glnE-disrupted mutants using a two-step strategy, whereby a single-crossover 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 isolated. When a second copy of the wild-type glnE was integrated into the chromosome, we could isolate both wild-type and mutant double-crossover strains. Thus, the chromosomal gene could only be replaced with a disrupted copy when another functional copy of the gene was provided, demonstrating that this gene is essential under the conditions tested.
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Department of Infectious & Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom. Tanya.Parish@lshtm.ac.uk
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D003434:Crossing Over, Genetic; D004171:Diploidy; D005798:Genes, Bacterial; D020043:Genes, Essential; D005838:Genotype; D016296:Mutagenesis; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D009713:Nucleotidyltransferases / Q000235:genetics*; D011995:Recombination, Genetic; D015183:Restriction Mapping; D013395:Sucrose / Q000494:pharmacology
D013395:Sucrose; D009713:Nucleotidyltransferases; C020658:glutamine-synthetase adenylyltransferase