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Microorganisms
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Genetic Engineering in Mycobacteria and Modern Microbiological Studies
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Genetic Engineering in Mycobacteria and Modern Microbiological Studies

A special issue of Microorganisms (ISSN 2076-2607). This special issue belongs to the section "Microbial Biotechnology".

Deadline for manuscript submissions: closed (15 November 2023) | Viewed by 6559

Special Issue Editor

Dr. Anna V. Goncharenko

E-Mail Website
Guest Editor
Bach Institute of Biochemistry, Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences, 119071 Moscow, Russia
Interests: mycobacteria; tuberculosis; gene knockout; gene knockdown; gene insertion; overexpression constructs; CRISPR editing; CRISPR interference

Special Issue Information

Dear Colleagues,

Tuberculosis kills 1.5 million people every year. The tuberculosis causative agent (Mycobacterium tuberculosis) has a unique ability to evade host immunity effectively and survive for years in the host organism in acute or dormant forms. The situation is further worsened by the spread of multiresistant strains, while existing TB vaccines do not provide secure protection.

Targeted changes introduced in the bacterial genome make it possible to investigate molecular mechanisms of interactions between mycobacteria and the host, explore the physiology of transition to and from latent tuberculosis infection, and find new targets for antimycobacterial drugs.

We are pleased to invite you to contribute to a Special Issue of Microorganisms (IF=4.926) concerning any aspects of genetic engineering of mycobacteria, both Mycobacterium tuberculosis and closely related species, and their applications in breakthrough mycobacterial studies.

The Special Issue is aimed at the exchange of information among leading scientists dealing with the study of mycobacteria, their physiology, and the development of antituberculosis drugs and vaccines using genetic methods.

I/We look forward to receiving your contributions.

Dr. Anna V. Goncharenko
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Microorganisms is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • mycobacteria
  • tuberculosis
  • gene knockout
  • gene knockdown
  • gene insertion
  • overexpression constructs
  • CRISPR editing
  • CRISPR interference

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Published Papers (3 papers)

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Research

16 pages, 1267 KiB  
Article
The Benefits of Toxicity: M. smegmatis VapBC TA Module Is Induced by Tetracycline Exposure and Promotes Survival
by Mikhail Zamakhaev, Julia Bespyatykh, Anna Goncharenko and Mikhail Shumkov
Microorganisms 2023, 11(12), 2863; https://doi.org/10.3390/microorganisms11122863 - 26 Nov 2023
Viewed by 1712
Abstract
Toxin–antitoxin (TA) systems are widely present in bacterial genomes. Mycolicibacterium smegmatis, a common model organism for studying Mycobacterium tuberculosis physiology, has eight TA loci, including mazEF and vapBC. This study aims to investigate the physiological significance of these TA systems. Proteomic profiling [...] Read more.
Toxin–antitoxin (TA) systems are widely present in bacterial genomes. Mycolicibacterium smegmatis, a common model organism for studying Mycobacterium tuberculosis physiology, has eight TA loci, including mazEF and vapBC. This study aims to investigate the physiological significance of these TA systems. Proteomic profiling was conducted on a culture overexpressing the VapC toxin, and the involvement of VapC in M. smegmatis stress responses to heat shock and antibiotic treatment was examined. While deciphering the underlying mechanisms of the altered stress resistance, we assessed the antibiotic susceptibility of vapBC, mazEF, and double vapBC-mazEF deletion mutants. Additionally, the mRNA levels of vapC and mazF were measured following tetracycline supplementation. The results reveal changes in the abundance of metabolic enzymes and stress response proteins associated with VapC overexpression. This activation of the general stress response leads to reduced thermosensitivity in M. smegmatis, but does not affect susceptibility to ciprofloxacin and isoniazid. Under tetracycline treatment, both vapC and mazF expression levels are increased, and the fate of the cell depends on the interaction between the corresponding TA systems. Full article
(This article belongs to the Special Issue Genetic Engineering in Mycobacteria and Modern Microbiological Studies)
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7 pages, 2689 KiB  
Communication
The Nocardial aph(2″) Gene Confers Tobramycin and Gentamicin Resistance and Is an Effective Positive Selection Marker in Mycobacteria and Nocardia
by Yizhak Hershko, Amos Adler and Daniel Barkan
Microorganisms 2023, 11(7), 1697; https://doi.org/10.3390/microorganisms11071697 - 29 Jun 2023
Cited by 2 | Viewed by 1628
Abstract
The current study aimed to evaluate the feasibility of using the aminoglycoside 2″-O-phosphotransferase aph(2″) gene as a positive selection marker in N. asteroides, M. smegmatis, M. abscessus and M. tuberculosis. The aph(2″) gene, known to confer resistance to tobramycin, was [...] Read more.
The current study aimed to evaluate the feasibility of using the aminoglycoside 2″-O-phosphotransferase aph(2″) gene as a positive selection marker in N. asteroides, M. smegmatis, M. abscessus and M. tuberculosis. The aph(2″) gene, known to confer resistance to tobramycin, was PCR amplified from N. farcinica and cloned into two plasmid vectors, pMSG383 and pDB151, harboring hygromycin and zeocin selection markers, respectively. The recombinant plasmids were transformed into the target microorganisms, and selectability was assessed against varying concentrations of tobramycin and using an E-test against gentamicin. The results indicated that the aph(2″) gene is a useful selection marker in Mycobacteria and Nocardia against tobramycin, with a good selectability at 2.5–10 µg/mL for M. smegmatis mc2-155 and N. asteroides ATCC 19,247, and 60–160 µg/mL for M. abscessus ATCC 19,977 and M. tuberculosis H37Ra. The minimum inhibitory concentration (MIC) of gentamicin for recombinant N. asteroides, M. smegmatis and M. abscessus was >256 µg/mL, whereas respective MIC in wild-type strains was 0.125 µg/mL, 0.38 µg/mL and 8 µg/mL, respectively. These findings demonstrate the potential of aph(2″) as a positive selection marker for genetic manipulation processes in Mycobacteria and Nocardia, thus facilitating their research and improving the efficiency of biotechnology applications. Conclusions: the aph(2″) gene is a useful, new selection marker for genetic manipulation of Nocardia and various Mycobacteria. Full article
(This article belongs to the Special Issue Genetic Engineering in Mycobacteria and Modern Microbiological Studies)
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14 pages, 1893 KiB  
Article
The Mycobacterium smegmatis HesB Protein, MSMEG_4272, Is Required for In Vitro Growth and Iron Homeostasis
by Nandi Niemand Wolhuter, Lerato Ngakane, Timothy J. de Wet, Robin M. Warren and Monique J. Williams
Microorganisms 2023, 11(6), 1573; https://doi.org/10.3390/microorganisms11061573 - 14 Jun 2023
Viewed by 2072
Abstract
A-type carrier (ATC) proteins are proposed to function in the biogenesis of Fe-S clusters, although their exact role remains controversial. The genome of Mycobacterium smegmatis encodes a single ATC protein, MSMEG_4272, which belongs to the HesB/YadR/YfhF family of proteins. Attempts to generate an [...] Read more.
A-type carrier (ATC) proteins are proposed to function in the biogenesis of Fe-S clusters, although their exact role remains controversial. The genome of Mycobacterium smegmatis encodes a single ATC protein, MSMEG_4272, which belongs to the HesB/YadR/YfhF family of proteins. Attempts to generate an MSMEG_4272 deletion mutant by two-step allelic exchange were unsuccessful, suggesting that the gene is essential for in vitro growth. CRISPRi-mediated transcriptional knock-down of MSMEG_4272 resulted in a growth defect under standard culture conditions, which was exacerbated in mineral-defined media. The knockdown strain displayed reduced intracellular iron levels under iron-replete conditions and increased susceptibility to clofazimine, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), and isoniazid, while the activity of the Fe-S containing enzymes, succinate dehydrogenase, and aconitase were not affected. This study suggests that MSMEG_4272 plays a role in the regulation of intracellular iron levels and is required for in vitro growth of M. smegmatis, particularly during exponential growth. Full article
(This article belongs to the Special Issue Genetic Engineering in Mycobacteria and Modern Microbiological Studies)
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