glnE is an essential gene in Mycobacterium tuberculosis

Abstract

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.

FIG. 1

Plasmid constructs. (A) Arrangement of genes in the M. tuberculosis chromosome, showing the 6.5-kb restriction fragment used in the construction of pGLN13 and pGLINT1. (B) pGLN13 delivery vector, showing the M. tuberculosis fragment used to construct the single-crossover strain. The internal 1.7-kb NotI fragment of glnE was replaced by the hyg gene. (C) pGLINT1 integrating vector, showing the M. tuberculosis fragment cloned into the integrating vector.

FIG. 2

Construction of single-crossover and double-crossover strains of wt M. tuberculosis. (A) The nonreplicating delivery vector pGLN13 was transformed into M. tuberculosis. A single recombination event gives rise to one of two possible single-crossover strains (B). (C) The second crossover can result in either restoration of the wt gene or a mutant double-crossover strain.

FIG. 3

Construction of a merodiploid strain and isolation of double-crossover strains. (A) The integrating vector was transformed into the single-crossover strain (single crossover 2 in Fig. 2B), providing an extra copy of the glnE gene elsewhere on the chromosome. A second crossover event results in either of two double-crossover strains (B) being isolated. Both double-crossover strains will still retain a functional glnE gene in the integrated vector.