Construction and Use of Transposon MycoTetOP2 for Isolation of Conditional Mycobacteria Mutants

Mycobacteria are unique in many aspects of their biology. The development of genetic tools to identify genes critical for their growth by forward genetic analysis holds great promises to advance our understanding of their cellular, physiological and biochemical processes. Here we report the development of a novel transposon, MycoTetOP2, to aid the identification of such genes by direct transposon mutagenesis. This mariner-based transposon contains nested anhydrotetracycline (ATc)-inducible promoters to drive transcription outward from both of its ends. In addition, it includes the Escherichia coli R6Kγ origin to facilitate the identification of insertion sites. MycoTetOP2 was placed in a shuttle plasmid with a temperature-sensitive DNA replication origin in mycobacteria. This allows propagation of mycobacteria harboring the plasmid at a permissive temperature. The resulting population of cells can then be subjected to a temperature shift to select for transposon mutants. This transposon and its delivery system, once constructed, were tested in the fast-growing model Mycobacterium smegmatis and 13 mutants with ATc-dependent growth were isolated. The identification of the insertion sites in these mutants led to nine unique genetic loci with genes critical for essential processes in both M. smegmatis and Mycobacterium tuberculosis. These results demonstrate that MycoTetOP2 and its delivery vector provide valuable tools for the studies of mycobacteria by forward genetics.
Source: Frontiers in Microbiology - Category: Microbiology Source Type: research

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Tuberculosis remains one of the leading causes of death from a single pathogen globally. It is estimated that 1/4 of the world’s population harbors latent tuberculosis, but only a 5–10% of patients will develop active disease. During latent infection, Mycobacterium tuberculosis can persist unaffected by drugs for years in a non-replicating state with low metabolic activity. The rate of the successful tuberculosis treatment is curbed by the presence of these non-replicating bacilli that can resuscitate after decades and also by the spread of M. tuberculosis drug-resistant strains. International agencies, includi...
Source: Frontiers in Microbiology - Category: Microbiology Source Type: research
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Source: Journal of Biological Chemistry - Category: Chemistry Authors: Tags: Enzymology Source Type: research
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Source: International Journal of Biological Macromolecules - Category: Biochemistry Authors: Tags: Int J Biol Macromol Source Type: research
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Source: Biochimica et Biophysica Acta (BBA) Bioenergetics - Category: Biochemistry Source Type: research
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Source: Journal of Molecular Structure - Category: Molecular Biology Source Type: research
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Source: International Journal of Biological Macromolecules - Category: Biochemistry Authors: Tags: Int J Biol Macromol Source Type: research
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Source: Fight Aging! - Category: Research Authors: Tags: Newsletters Source Type: blogs
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Source: The American Journal of Tropical Medicine and Hygiene - Category: Tropical Medicine Authors: Tags: Am J Trop Med Hyg Source Type: research
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Source: Phytochemistry Letters - Category: Chemistry Source Type: research
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Source: Protein Science - Category: Biochemistry Authors: Tags: Article Source Type: research
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