Promoter mutagenesis for fine‐tuning expression of essential genes in <i>Mycobacterium tuberculosis</i>

Boldrin, Francesca; Degiacomi, Giulia; Serafini, Agnese; Kolly, Gaëlle S.; Ventura, Marcello; Sala, Claudia; Provvedi, Roberta; Palù, Giorgio; Cole, Stewart T.; Manganelli, Riccardo

doi:10.1111/1751-7915.12875

Cited by 12 publications

(15 citation statements)

References 50 publications

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“…It is expected that single-gene knockout projects for the model organisms, such as mice ( 52 , 53 ) and Arabidopsis thaliana ( 54 ), will soon be completed. Multiple ways to manipulate gene expression are available, such as those based on TetR/Pip-OFF repressible promoter system ( 55 ) and RNA interference ( 56 ). From the aspect of technology, this is a golden era for essential-gene research, because of the availability of Tn-seq and CRISPR/Cas9.…”

Section: Future Perspectivementioning

confidence: 99%

DEG 15, an update of the Database of Essential Genes that includes built-in analysis tools

Luo

Lin

Liu

et al. 2020

Nucleic Acids Research

170

126

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Essential genes refer to genes that are required by an organism to survive under specific conditions. Studies of the minimal-gene-set for bacteria have elucidated fundamental cellular processes that sustain life. The past five years have seen a significant progress in identifying human essential genes, primarily due to the successful use of CRISPR/Cas9 in various types of human cells. DEG 15, a new release of the Database of Essential Genes (www.essentialgene.org), has provided major advancements, compared to DEG 10. Specifically, the number of eukaryotic essential genes has increased by more than fourfold, and that of prokaryotic ones has more than doubled. Of note, the human essential-gene number has increased by more than tenfold. Moreover, we have developed built-in analysis modules by which users can perform various analyses, such as essential-gene distributions between bacterial leading and lagging strands, sub-cellular localization distribution, enrichment analysis of gene ontology and KEGG pathways, and generation of Venn diagrams to compare and contrast gene sets between experiments. Additionally, the database offers customizable BLAST tools for performing species- and experiment-specific BLAST searches. Therefore, DEG comprehensively harbors updated human-curated essential-gene records among prokaryotes and eukaryotes with built-in tools to enhance essential-gene analysis.

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Section: Future Perspectivementioning

confidence: 99%

DEG 15, an update of the Database of Essential Genes that includes built-in analysis tools

Luo

Lin

Liu

et al. 2020

Nucleic Acids Research

170

126

View full text Add to dashboard Cite

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“…Such an elevation in gene expression can easily occur in natural mutants e.g. by point mutations in the promoter region [69,70]. Additionally, plasmas have been shown to be mutagenic [10,13,71], thus they may increase the frequency at which such mutants occur.…”

Section: Implications Of Inherent Plasma Resistance For Medical Applimentioning

confidence: 99%

Plasma-sensitiveEscherichia colimutants reveal plasma resistance mechanisms

Krewing

Jarzina

Dirks

et al. 2019

J. R. Soc. Interface.

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Non-thermal atmospheric pressure plasmas are investigated as augmenting therapy to combat bacterial infections. The strong antibacterial effects of plasmas are attributed to the complex mixture of reactive species, (V)UV radiation and electric fields. The experience with antibiotics is that upon their introduction as medicines, resistance occurs in pathogens and spreads. To assess the possibility of bacterial resistance developing against plasma, we investigated intrinsic protective mechanisms that allow Escherichia coli to survive plasma stress. We performed a genome-wide screening of single-gene knockout mutants of E. coli and identified 87 mutants that are hypersensitive to the effluent of a microscale atmospheric pressure plasma jet. For selected genes ( cysB , mntH , rep and iscS ) we showed in complementation studies that plasma resistance can be restored and increased above wild-type levels upon over-expression. To identify plasma-derived components that the 87 genes confer resistance against, mutants were tested for hypersensitivity against individual stressors (hydrogen peroxide, superoxide, hydroxyl radicals, ozone, HOCl, peroxynitrite, NO•, nitrite, nitrate, HNO 3 , acid stress, diamide, heat stress and detergents). k-means++ clustering revealed that most genes protect from hydrogen peroxide, superoxide and/or nitric oxide. In conclusion, individual bacterial genes confer resistance against plasma providing insights into the antibacterial mechanisms of plasma.

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“…It is noted that the genes identified by Forti et al (2011) and those here have no overlaps. We suggest that it is advantageous to have different and new regulatory schemes (Schnappinger and Ehrt, 2014;Leotta et al, 2015;Boldrin et al, 2018Boldrin et al, , 2019 for the design of transposons for the investigation of mycobacterial biology in the future.…”

Section: Discussionmentioning

confidence: 99%

“…The significant road block here is that mutants in these genes are not readily available for follow-up studies. Rather, their investigation requires reverse genetics to construct conditional mutants using regulated promoters to deplete a gene product (Schnappinger and Ehrt, 2014;Leotta et al, 2015;Boldrin et al, 2018Boldrin et al, , 2019. A tetracycline regulatable system has been successfully adapted for use in mycobacteria for the construction of conditional mutations (Ehrt et al, 2005;Ehrt and Schnappinger, 2006).…”

Section: Introductionmentioning

confidence: 99%

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

2020

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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, MycoTetOP 2 , 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. MycoTetOP 2 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 MycoTetOP 2 and its delivery vector provide valuable tools for the studies of mycobacteria by forward genetics.

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Promoter mutagenesis for fine‐tuning expression of essential genes in Mycobacterium tuberculosis

Cited by 12 publications

References 50 publications

DEG 15, an update of the Database of Essential Genes that includes built-in analysis tools

DEG 15, an update of the Database of Essential Genes that includes built-in analysis tools

Plasma-sensitiveEscherichia colimutants reveal plasma resistance mechanisms

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

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