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3 results on '"Msm, Mycobacterium smegmatis"'

1. Biochemical Properties of Single-Stranded DNA-Binding Protein from Mycobacterium smegmatis, a Fast-growing Mycobacterium and Its Physical and Functional Interaction with Uracil DNA Glycosylases

Author

Acharya, Narottam and Varshney, Umesh

Subjects
*DNA-binding proteins, *MYCOBACTERIUM
Abstract

The single-stranded DNA-binding proteins (SSBs) are vital to virtually all DNA functions. Here, we report on the biochemical properties of SSB from a fast-growing mycobacteria, Mycobacterium smegmatis, and the interaction of the homotetrameric SSBs with uracil DNA glycosylases (UDGs) from M. smegmatis (Msm), Mycobacterium tuberculosis (Mtu) and Escherichia coli (Eco). UDG is a crucial DNA repair enzyme, which removes the promutagenic uracil residues. MsmSSB stimulates activity of the homologous MsmUDG and of the heterologous Mtu-, and Eco-UDGs. On the contrary, while the MtuSSB stimulates the MtuUDG, it inhibits the other two UDGs. Although the MsmSSB shares 84% identity with MtuSSB, the two are strikingly different, in that MsmSSB contains a glycine-rich segment (11 out of 13 residues) in the spacer connecting the N-terminal DNA-binding domain with the C-terminal acidic tail. While the DNA-binding properties of MsmSSB, such as its affinity to oligomeric DNA, requirement of minimum size DNA and the modes of interaction are indistinguishable from those of Eco-, and Mtu-SSBs, it is unclear if the glycine-rich segment confers structural advantage to MsmSSB, responsible for its stimulatory effect on all UDGs tested. More importantly, by using a small polypeptide inhibitor of UDGs, and the deletion mutants of SSBs, we suggest that the C-terminal acidic tail of the SSBs interacts within the DNA-binding groove of the UDGs, and propose a role for SSBs in the recruitment of UDGs to the damaged DNA. [Copyright &y& Elsevier]

Published
2002
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2. A Cas12a-based CRISPR interference system for multigene regulation in mycobacteria

Author

Christoph Grundner and Neil Fleck

Subjects
mycobacteria, CRISPR-Associated Proteins, Gene Expression, Computational biology, Biochemistry, Mycobacterium tuberculosis, Bacterial Proteins, Humans, CRISPR, CRISPR, clustered regularly interspaced short palindromic repeat, Molecular Biology, Gene, Subgenomic mRNA, Gene Editing, Regulation of gene expression, CRISPR interference, Endodeoxyribonucleases, Cas12a, biology, Cas9, Mycobacterium smegmatis, sgRNA, single guide RNA, CRISPRi, Gene Expression Regulation, Bacterial, Cell Biology, biology.organism_classification, Cas, CRISPR-associated protein, CRISPRi, CRISPR interference, Gene Knockdown Techniques, Msm, Mycobacterium smegmatis, CRISPR-Cas Systems, gene regulation, Research Article
Abstract

Mycobacteria are responsible for a heavy global disease burden, but their relative genetic intractability has long frustrated research efforts. The introduction of clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) has made gene repression in mycobacteria much more efficient, but limitations of the prototypical Cas9-based platform, for example, in multigene regulation, remain. Here, we introduce an alternative CRISPRi platform for mycobacteria that is based on the minimal type V Cas12a enzyme in combination with synthetic CRISPR arrays. This system is simple, tunable, reversible, can efficiently regulate essential genes and multiple genes simultaneously, and works as efficiently in infected macrophages as it does in vitro. Together, Cas12a-based CRISPRi provides a facile tool to probe higher-order genetic interactions in mycobacteria including Mycobacterium tuberculosis (Mtb), which will enable the development of synthetically lethal drug targets and the study of genes conditionally essential during infection.

Published
2021
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3. Mycobacterium tuberculosis has diminished capacity to counteract redox stress induced by elevated levels of endogenous superoxide

Author

Amit Singh, Ashima Bhaskar, Harinath Chakrapani, Priyanka Tyagi, and Allimuthu T. Dharmaraja

Subjects
MABA, microplate Alamar blue assay, Mycobacterium smegmatis, Antitubercular Agents, Mtb, Mycobacterium tuberculosis, Gene Expression, Endogeny, Oxidative phosphorylation, Microbial Sensitivity Tests, medicine.disease_cause, Redox, Biochemistry, Clofazimine, chemistry.chemical_compound, Bacterial Proteins, Superoxides, Lipid biosynthesis, Physiology (medical), Drug Resistance, Multiple, Bacterial, Drug Resistance, Bacterial, medicine, XDR, extensively drug-resistant, chemistry.chemical_classification, Microbiology & Cell Biology, Reactive oxygen species, biology, Superoxide, Reactive oxygen species (ROS), Gene Expression Regulation, Bacterial, Mycobacterium tuberculosis, Original Contribution, MDR, multidrug-resistant, biology.organism_classification, CFZ, clofazimine, Oxidative Stress, DNA Repair Enzymes, chemistry, Genes, Bacterial, Redox regulation, Drug resistance, Msm, Mycobacterium smegmatis, Oxidation-Reduction, DHE, dihydroethidium, Oxidative stress
Abstract

Mycobacterium tuberculosis (Mtb) has evolved protective and detoxification mechanisms to maintain cytoplasmic redox balance in response to exogenous oxidative stress encountered inside host phagocytes. In contrast, little is known about the dynamic response of this pathogen to endogenous oxidative stress generated within Mtb. Using a noninvasive and specific biosensor of cytoplasmic redox state of Mtb, we for first time discovered a surprisingly high sensitivity of this pathogen to perturbation in redox homeostasis induced by elevated endogenous reactive oxygen species (ROS). We synthesized a series of hydroquinone-based small molecule ROS generators and found that ATD-3169 permeated mycobacteria to reliably enhance endogenous ROS including superoxide radicals. When Mtb strains including multidrug-resistant (MDR) and extensively drug-resistant (XDR) patient isolates were exposed to this compound, a dose-dependent, long-lasting, and irreversible oxidative shift in intramycobacterial redox potential was detected. Dynamic redox potential measurements revealed that Mtb had diminished capacity to restore cytoplasmic redox balance in comparison with Mycobacterium smegmatis (Msm), a fast growing nonpathogenic mycobacterial species. Accordingly, Mtb strains were extremely susceptible to inhibition by ATD-3169 but not Msm, suggesting a functional linkage between dynamic redox changes and survival. Microarray analysis showed major realignment of pathways involved in redox homeostasis, central metabolism, DNA repair, and cell wall lipid biosynthesis in response to ATD-3169, all consistent with enhanced endogenous ROS contributing to lethality induced by this compound. This work provides empirical evidence that the cytoplasmic redox poise of Mtb is uniquely sensitive to manipulation in steady-state endogenous ROS levels, thus revealing the importance of targeting intramycobacterial redox metabolism for controlling TB infection., Graphical abstract, Highlights • Dynamic response of Mycobacterium tuberculosis (Mtb) to endogenous ROS is poorly characterized. • Biochemically and genetically validated ROS generators were used to enhance ROS in Mtb. • Application of a cytoplasmic redox biosensor revealed dynamic changes in redox potential of Mtb on elevated endogenous ROS. • Mtb has diminished capacity to restore redox balance in response to endogenous ROS. • Maintenance of cytoplasmic redox potential in drug-resistant Mtb is crucial for its survival.

Published
2014

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