Abstract
Non-tuberculosis mycobacteria (NTMs) comprise a large group of organisms that are phenotypically diverse. Analysis of the growing number of completed NTM genomes has revealed both significant intra-genus genetic diversity, and a high percentage of predicted genes that appear to be unique to this group. Most NTMs have not been studied, however, the rise in NTM infections in several countries has prompted increasing interest in these organisms. Mycobacterial research has recently benefitted from the development of new genetic tools and a growing number of studies describing the genetic manipulation of NTMs have now been reported. In this review, we discuss the use of both site-specific and random mutagenesis tools in NTMs, highlighting the challenges that exist in applying these techniques to this diverse group of organisms.
Highlights
The genus Mycobacterium is a diverse group of organisms that includes more than 180 species
A comparison of Tn5367 and the MycoMarT7 mariner transposon in M. avium subsp. paratuberculosis revealed a >3-fold higher number of insertions for MycoMarT7, and a higher percentage of insertions occurring within genes (83% vs. 74%) (Rathnaiah et al, 2016)
Expression of the M. marinum non-homologous end joining (NHEJ) (MmNHEJ) locus with the CRISPRFnCpf1 system in M. smegmatis resulted mutations in 0.75% of transformants. This efficiency was increased to 90% when the experiment was performed in a recA null mutant, suggesting that inhibiting homologous recombination (HR) is required for the NHEJ system to repair double strand break (DSB) in M. smegmatis
Summary
The genus Mycobacterium is a diverse group of organisms that includes more than 180 species. The first step of the process uses positive selection (usually antibiotic resistance) to isolate clones that have undergone a single homologous recombination event (single cross-over) This results in integration of the AES, creating a strain that carries both the wild-type and mutant alleles. Spontaneous resistance to antibiotics used for selection arises at a similar frequency to homologous recombination (Parish et al, 1999; Bardarov et al, 2002; Viljoen et al, 2018) This can be problematic during allelic exchange mutagenesis as it increases the number of colonies that need to be screened following selection. Twostep allelic exchange is more efficient, the process is slow and TABLE 1 | Examples of one-step and two-step allelic exchange mutagenesis in non-tuberculosis mycobacteria
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