Efficient and simple generation of unmarked gene deletions in Mycobacterium smegmatis

Shenkerman, Yael; Elharar, Yifat; Vishkautzan, Marina; Gur, Eyal

doi:10.1016/j.gene.2013.09.082

Cited by 25 publications

(20 citation statements)

References 17 publications

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“…M. smegmatis strain MC 2 155 and its derivative PPS mutants (29) were grown at 30°C in minimal medium (30) containing 40 mM K 2 HPO 4 , 22 mM KH 2 PO 4 , 15 mM (NH 4 ) 2 SO 4 , 1.7 mM sodium citrate, 0.4 mM MgSO 4 , 0.4% glycerol (vol/vol), and 0.05% Tween-80 (vol/vol). To induce nitrogen starvation, exponentially growing cultures were harvested, centrifuged, washed thrice, and resuspended in similar media lacking (NH 4 ) 2 SO 4 .…”

Section: Methodsmentioning

confidence: 99%

“…The same system was used for generation of the Δdop-Δprc-Δpaf triple mutant via consecutive deletion of the respective genes. Generation of the Δprc mutant was previously described (29). For generation of complementation strains, the complementing genes were cloned into the integrative mycobacterial plasmid pMV306 (32) before transformation into M. smegmatis.…”

Section: Methodsmentioning

confidence: 99%

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Posttranslational regulation of coordinated enzyme activities in the Pup-proteasome system

Elharar

Roth

Hecht

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The proper functioning of any biological system depends on the coordinated activity of its components. Regulation at the genetic level is, in many cases, effective in determining the cellular levels of system components. However, in cases where regulation at the genetic level is insufficient for attaining harmonic system function, posttranslational regulatory mechanisms are often used. Here, we uncover posttranslational regulatory mechanisms in the prokaryotic ubiquitin-like protein (Pup)-proteasome system (PPS), the bacterial equivalent of the eukaryotic ubiquitin-proteasome system. Pup, a ubiquitin analog, is conjugated to proteins through the activities of two enzymes, Dop (deamidase of Pup) and PafA (proteasome accessory factor A), the Pup ligase. As Dop also catalyzes depupylation, it was unclear how PPS function could be maintained without Dop and PafA canceling the activity of the other, and how the two activities of Dop are balanced. We report that tight Pup binding and the limited degree of Dop interaction with high-molecular-weight pupylated proteins results in preferred Pup deamidation over protein depupylation by this enzyme. Under starvation conditions, when accelerated protein pupylation is required, this bias is intensified by depletion of free Dop molecules, thereby minimizing the chance of depupylation. We also find that, in contrast to Dop, PafA presents a distinct preference for highmolecular-weight protein substrates. As such, PafA and Dop act in concert, rather than canceling each other's activity, to generate a high-molecular-weight pupylome. This bias in pupylome molecular weight distribution is consistent with the proposed nutritional role of the PPS under starvation conditions. Dop | mycobacteria | PafA | proteasome | Pup

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Posttranslational regulation of coordinated enzyme activities in the Pup-proteasome system

Elharar

Roth

Hecht

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Compared with traditional tools, this system is faster. The total time using one‐plasmid‐based system for N rounds of genome editing is 7 N +2 days (Table S4), while it takes 14–15 days for single‐gene editing using the previous reported homologous recombination method . This is very important for the slow growth strain.…”

Section: Discussionmentioning

confidence: 99%

A CRISPR‐Cpf1‐Assisted Non‐Homologous End Joining Genome Editing System of Mycobacterium smegmatis

Sun

Yang

et al. 2018

Biotechnology Journal

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Mycobacterium smegmatis is an important model strain of Mycobacterium for scientific study because it is non-pathogenic and grows rapidly. However, research is limited by the low efficiency and time-consuming nature of existing genome editing tools. Although the Streptococcus pyogenes CRISPR-Cas9 system is widely used in bacterial genome editing, it cannot be introduced into M. smegmatis because of its toxicity. The authors test 14 different Cas effector proteins in M. smegmatis. Cas9 (TdCas9_m) from Treponema denticola, Cas9 (NmCas9) from Neisseria meningitidis, and Corynebacterium glutamicum codon-optimized Cpf1 (FnCpf1_cg) from Francisella tularensis do not affect cell growth. The numbers of transformant plasmids expressing TdCas9_m, NmCas9, or FnCpf1_cg, and guide RNAs (gRNA) targeting ku(MSMEG_5580), ligD(MSMEG_6301), pta(MSMEG_0783), or ackA(MSMEG_0784) decreases by about 10-, 10-, or 100-fold, respectively, compared with plasmids expressing only the Cas effector proteins. Non-homologous end joining (NHEJ) is detected only in the CRISPR-FnCpf1_cg system. The one-plasmid-based, CRISPR-FnCpf1-assisted NHEJ system enables N iterative rounds of genome editing in 7N + 2 days, with an editing efficiency up to 70%; thus, this system should greatly reduce the necessary genome manipulation time for M. smegmatis.

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“…In M. tb , they observed an increase in HR that was at least an order of magnitude higher compared to control strains not expressing the recombineering proteins. An update of the system used a temperature‐sensitive recombineering plasmid containing a SacB counterselection marker for straightforward curing of the plasmid afterward, a visual screening tool (GFP) to allow easy discrimination of the recombined transformants and site‐specific recombination sites to allow subsequent removal of introduced resistance markers .…”

Section: Targeted Mutagenesismentioning

confidence: 99%

A guide to Mycobacterium mutagenesis

et al. 2019

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The genus Mycobacterium includes several pathogens that cause severe disease in humans, like Mycobacterium tuberculosis (M. tb), the infectious agent causing tuberculosis. Genetic tools to engineer mycobacterial genomes, in a targeted or random fashion, have provided opportunities to investigate M. tb infection and pathogenesis. Furthermore, they have allowed the identification and validation of potential targets for the diagnosis, prevention, and treatment of tuberculosis. This review describes the various methods that are available for the generation of mutants in Mycobacterium species, focusing specifically on tools for altering slow‐growing mycobacteria from the M. tb complex. Among others, it incorporates the recent new molecular biological technologies (e.g. ORBIT) to rapidly and/or genome‐wide comprehensively obtain targeted mutants in mycobacteria. As such, this review can be used as a guide to select the appropriate genetic tools to generate mycobacterial mutants of interest, which can be used as tools to aid understanding of M. tb infection or to help developing TB intervention strategies.

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Efficient and simple generation of unmarked gene deletions in Mycobacterium smegmatis

Cited by 25 publications

References 17 publications

Posttranslational regulation of coordinated enzyme activities in the Pup-proteasome system

Posttranslational regulation of coordinated enzyme activities in the Pup-proteasome system

A CRISPR‐Cpf1‐Assisted Non‐Homologous End Joining Genome Editing System of Mycobacterium smegmatis

A guide to Mycobacterium mutagenesis

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