Construction and Use of Transposon MycoTetOP 2 for Isolation of Conditional Mycobacteria Mutants.

Sarah D Riggs-Shute,Zhaomin Yang,Joseph O Falkinham

doi:10.3389/fmicb.2019.03091

Sarah D Riggs-Shute, Zhaomin Yang + Show 1 more

Open Access

https://doi.org/10.3389/fmicb.2019.03091

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Abstract

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.

Highlights

The genus Mycobacterium contains Mycobacterium tuberculosis, the causative agent of tuberculosis (TB)
The remaining 10 mutants belong to the last group with the strongest ATc-dependent growth. They showed no or little growth in the absence of ATc but grew as the wild type in the presence of ATc in this assay. These results indicated that the MycoTetOP2 transposon fulfilled its designed purpose for the isolation of conditional mutants of mycobacteria for the identification of possible essential genes by direct transposon mutagenesis
We screened ∼5,200 MycoTetOP2 transposon insertions and identified 13 mutants with ATcdependent growth phenotypes. These mutants led to nine genetic loci, each with candidate genes with functions in the essential processes of translation and the synthesis of mycobacterial cell wall (Figure 2 and Table 2)

Summary

Introduction

The genus Mycobacterium contains Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). Many aspects of the unique biology of mycobacteria remain to be understood. One approach to study mycobacteria is to decipher the functions of core genes essential for their growth, and these genes may prove valuable for the understanding of mycobacterial biology as well as the research and development of antimycobacterial agents (Borsari et al, 2017; Singh and Mizrahi, 2017; Campanico et al, 2018; Evans and Mizrahi, 2018). Experimental identification and validation of such genes are laborious because their null mutations lead to lethality. Essential genes in mycobacteria are usually inferred statistically from the rarity of their mutants following high density transposon mutagenesis and deep sequencing of mutant pools

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