Mycobacterium Smegmatis Research Articles

A diverse single-stranded DNA–annealing protein library enables efficient genome editing across bacterial phyla

Genome modification is essential for studying and engineering bacteria, yet making efficient modifications to most species remains challenging. Bacteriophage-encoded single-stranded DNA–annealing proteins (SSAPs) can facilitate efficient genome editing by homologous recombination, but their typically narrow host range limits broad application. Here, we demonstrate that a single library of 227 SSAPs enables efficient genome-editing across six diverse bacteria from three divergent classes: Actinomycetia ( Mycobacterium smegmatis and Corynebacterium glutamicum ), Alphaproteobacteria ( Agrobacterium tumefaciens and Caulobacter crescentus ), and Bacilli ( Lactococcus lactis and Staphylococcus aureus ). Surprisingly, the most effective SSAPs frequently originated from phyla distinct from their bacterial hosts, challenging the assumption that phylogenetic relatedness is necessary for recombination efficiency, and supporting the value of a large unbiased library. Across these hosts, the identified SSAPs enable genome modifications requiring efficient homologous recombination, demonstrated through three examples. First, we use SSAPs with Cas9 in C. crescentus to introduce single amino acid mutations with >70% efficiency. Second, we adapt SSAPs for dsDNA editing in C. glutamicum and S. aureus , enabling one-step gene knockouts using PCR products. Finally, we apply SSAPs for multiplexed editing in S. aureus to precisely map the interaction between a conserved protein and a small-molecule inhibitor. Overall, this library-based SSAP screen expands engineering capabilities across diverse, previously recalcitrant microbes, enabling efficient genetic manipulation for both fundamental research and biotechnological applications.

Synthesis, Evaluation of Biological Activity, and Structure–Activity Relationships of New Amidrazone Derivatives Containing Cyclohex-1-ene-1-Carboxylic Acid

In recent years, the incidence of acute and chronic inflammatory diseases has increased significantly worldwide, intensifying the search for new therapeutic agents, especially anti-inflammatory drugs. Therefore, the aim of this work was to synthesize, biologically assess, and explore the structure–activity relationships of new compounds containing the cyclohex-1-ene-1-carboxylic acid moiety. Six new derivatives, 2a–2f, were synthesized through the reaction of amidrazones 1a–1f with 3,4,5,6-tetrahydrophthalic anhydride. Their toxicity was evaluated in cultures of human peripheral blood mononuclear cells (PBMCs). Additionally, their antiproliferative properties and effects on the synthesis of TNF-α, IL-6, IL-10, and IL-1β were assessed in mitogen-stimulated PBMCs. The antimicrobial activity of derivatives 2a–2f was determined by measuring the minimal inhibitory concentration (MIC) values against five bacterial strains—Staphylococcus aureus, Mycobacterium smegmatis, Escherichia coli, Yersinia enterocolitica, and Klebsiella pneumoniae—and the fungal strain Candida albicans. All compounds demonstrated antiproliferative activity, with derivatives 2a, 2d, and 2f at a concentration of 100 µg/mL being more effective than ibuprofen. Compound 2f strongly inhibited the secretion of TNF-α by approximately 66–81% at all studied doses (10, 50, and 100 µg/mL). Derivative 2b significantly reduced the release of cytokines, including TNF-α, IL-6, and IL-10, at a high dose (by approximately 92–99%). Compound 2c exhibited bacteriostatic activity against S. aureus and M. smegmatis, while derivative 2b selectively inhibited the growth of Y. enterocolitica (MIC = 64 µg/mL). Some structure–activity relationships were established for the studied compounds.

Structure and infection dynamics of mycobacteriophage Bxb1.

Mycobacteriophage Bxb1 is a well-characterized virus of Mycobacterium smegmatis with double-stranded DNA and a long, flexible tail. Mycobacteriophages show considerable potential as therapies for Mycobacterium infections, but little is known about the structural details of these phages or how they bind to and traverse the complex Mycobacterium cell wall. Here, we report the complete structure and atomic model of phage Bxb1, including the arrangement of immunodominant domains of both the capsid and tail tube subunits, as well as the assembly of the protein subunits in the tail-tip complex. The structure contains protein assemblies with 3-, 5-, 6-, and 12-fold symmetries, which interact to satisfy several symmetry mismatches. Cryoelectron tomography of phage particles bound to M.smegmatis reveals the structural transitions that occur for free phage particles to bind to the cell surface and navigate through the cell wall to enable DNA transfer into the cytoplasm.

Sequence analysis of two F1 mycobacteriophages, Deb65 and DocMcStuffins.

Isolated from wetland soil, Deb65 and DocMcStuffins are bacteriophages with a siphoviral morphology that infect Mycobacterium smegmatis. Deb65 and DocMcStuffins encode 97 and 91 putative genes, 41 of which are shared. Based on gene content similarity to actinobacteriophages more broadly, both phages are assigned to subcluster F1.

Antimycobacterial activity of new aryloxyethoxy-dialkylaminopropanol derivatives against reference strains of nontuberculous mycobacteria

Objective. To evaluate the in vitro antimycobacterial activity of novel quaternary ammonium salts, derived from alkyl(aryloxyethoxy)dialkylaminopropanol against reference strains of nontuberculous mycobacteria (NTM). Materials and Methods. A total of 52 synthesized compounds, obtained via phase-transfer catalysis, were studied,. The activity was assessed in two stages: initial screening using the agar diffusion method (well method), followed by minimum inhibitory concentration (MIC) determination through serial dilution. Primary testing was performed on Mycobacterium smegmatis, followed by evaluation on M. terrae, M. avium, and M. B5 using the proportion method on Löwenstein–Jensen medium. Compounds were tested at three concentrations: K-I (MIC for M. smegmatis), K-II (10×MIC), and K-III (100×MIC). Results. Ten compounds with the highest activity against M. smegmatis were identified during the screening stage. The most active was Kc12 (MIC – 0.22±0.02 µg/mL). Other active compounds included: Kc16, Kc13, Kc15, Kp15, Kp20, Kp18, Kc22, Kc14, and Kp16. Among the NTM strains, M. terrae and M. B5 were the most sensitive. At K-II, compounds Kc12, Kc13, Kc15, Kc16, Kc22, and Kp18 inhibited bacterial growth by more than 95 %. Kc15 demonstrated the efficacy comparable to rifampicin, while Kp18 and Kc12 surpassed streptomycin. M. avium exhibited resistance, with growth suppression below 5 %, observed only at K-III in a few compounds. The least active was Kp20. Conclusions. Ten promising compounds were identified, notably Kc12, Kc15, Kp18, Kc16, and Kc22, whose activity exceeded that of streptomycin and approached rifampicin in some cases. The findings highlight the potential of these compounds for further preclinical development in the treatment of NTM infections

The emergence of resistance to the antiparasitic selamectin in Mycobacterium smegmatis is improbable and contingent on cell wall integrity.

Tuberculosis remains the deadliest infectious disease of the 21st century. New antimicrobials are needed to improve treatment outcomes and enable therapy shortening. Drug repurposing is an alternative to the traditional drug discovery process. The avermectins are a family of macrocyclic lactones with anthelmintic activity active against Mycobacterium tuberculosis. However, their mode of action in mycobacteria remains unknown. In this study, we employed traditional mutant isolation approaches using Mycobacterium smegmatis, a non-pathogenic M. tuberculosis surrogate. We were only able to isolate mutants with decreased susceptibility to selamectin using the ∆nucS mutator M. smegmatis strain. This phenotype was caused by mutations in mps1 and mmpL11. Two of these mutants were used for a second experiment in which high-level selamectin-resistant mutants were isolated; however, specific mutations driving the phenotypic change to high-level resistance could not be identified. The susceptibility to selamectin in these mutants was restored to the basal level by subinhibitory concentrations of ethambutol. The selection of ethambutol resistance in a high-level selamectin-resistant mutant also resulted in multiple colonies becoming susceptible to selamectin again. These colonies carried mutations in embB, suggesting that the integrity of the cell envelope is a prerequisite for selamectin resistance. The absence of increased susceptibility to selamectin in an embB deletion strain demonstrated that the target of selamectin is not cytosolic. Our data show that the concurrence of specific multiple mutations and complete integrity of the mycobacterial envelope are necessary for selamectin resistance. Our studies provide first-time insights into the antimycobacterial mode of action of the antiparasitic avermectins.IMPORTANCETuberculosis is the deadliest infectious disease of the 21st century. New antibiotics are needed to improve treatment. However, developing new drugs is costly and lengthy. Drug repurposing is an alternative to the traditional drug discovery process. The avermectins are a family of drugs used to treat parasitic infections that are active against Mycobacterium tuberculosis, the bacterium that causes tuberculosis. However, their mode of action in mycobacteria remains unknown. Understanding how avermectins kill mycobacteria can facilitate its development as an anti-mycobacterial drug, including against M. tuberculosis.In this study, we used Mycobacterium smegmatis, a non-pathogenic M. tuberculosis surrogate model to understand the molecular mechanisms of how selamectin (a drug of the avermectin family selected for this study as a model) acts against mycobacteria. Our data show that the generation of resistance to selamectin is unlikely and that complete integrity of the mycobacterial envelope is necessary for selamectin resistance, providing first-time insights into the antimycobacterial mode of action of the avermectins.

Mycobactin and clofazimine activity are negatively correlated in mycobacteria.

Clofazimine (CFZ) is an anti-leprosy drug shown to improve outcomes in treatment of multidrug-resistant tuberculosis (TB) and nontuberculous mycobacterial infections. Studies in Mycobacterium tuberculosis and Mycobacterium avium identified CFZ resistance mutations in the gene that encodes the MmpR5/MmpT5 regulator, which increase expression of the mycobactin (MBT) transporter, MmpS5/L5. We found exposure of M. tuberculosis to CFZ induced a pattern of gene expression that mirrored low iron conditions, including strong induction of genes that encode MBT synthesis and transport. We identified a corresponding increase in MBT levels indicating a role in iron homeostasis in CFZ activity. CFZ bactericidal activity against both Mycobacterium smegmatis and M. tuberculosis was increased in high iron conditions in which MTB synthesis and transport was limited. We show the presence of MBT correlated with decreased CFZ killing activity while inhibition of MBT synthesis increased killing. Considerable iron efflux was observed during CFZ treatment indicating iron loss may be a feature of CFZ anti-mycobacterial activity. CFZ solubility studies and CFZ-mediated reduction of free iron indicate a potential redox interaction between CFZ and iron. MBT or MBT flux across the cell envelope appears to block CFZ killing in M. smegmatis and potentially M. tuberculosis. The specific mechanism by which MBT inhibits CFZ lethality remains unclear but may involve, increased iron acquisition, the MmpS5/L5 MBT efflux pump, or the CFZ subcellular localization altered by the redox state and solubility of CFZ. CFZ has thus far been proven most effective against Mycobacterium leprae, which lacks MBT, indicating an understanding of the complex interaction of CFZ with iron acquisition systems may suggest more effective therapeutic applications.

Unravelling the genetic contributors to linezolid resistance in Mycobacterium smegmatis: insights from a transposon library.

This study aimed to identify and characterize genes associated with linezolid resistance in Mycobacterium smegmatis using a transposon mutagenesis approach. This research conducted three replicative experiments where 16 isolates displaying pronounced resistance to linezolid were examined, revealing two distinct morphologies. WGS was employed to investigate these isolates, uncovering mutations in specific genes. The binding affinity of linezolid to relevant proteins was assessed through molecular docking studies and validated by drug affinity responsive target stability (DARTS) assays. Complementation of the mspA gene in mutant strains restored linezolid susceptibility, but the Ala127Gln substitution in MSMEG_0965 did not, suggesting a critical role of this residue in resistance. Further investigations revealed that the resistance mechanism in the △MSMEG_0965 mutant involves impaired linezolid uptake. The research successfully identified two genes, MSMEG_4189 and MSMEG_0965, associated with linezolid resistance in M. smegmatis. It also elucidated the role of MSMEG_0965 in the resistance mechanism, providing significant targets and reference points for future studies on clinical strains.

Comparative Chemical Composition and Antimicrobial Activities of The Essential Oils and Solvent Extracts of The Flower, Leaf, And Stem of Epilobium angustifolium Growing in Türkiye

Volatile organic compounds (VOCs) of essential oils (EOs) and solid phase microextract (SPME) obtained from the flower, leaf, and stem of Epilobium angustifolium L. were analyzed by GC-FID/MS. The EOs and SPMEs consist mainly of monoterpenes and aldehydes, which are major classes of compounds. Limonene was found to be a major compound in flower (HD: 42.9% vs. SPME: 95.5%), in leaf (HD: 60.3% vs. SPME: 4.7%), and in stem (HD: 49.06% vs. SPME: 93.6%). The antimicrobial activity of EOs and the solvent extracts (n-hexane, acetonitrile, and methanol) of E. angustifolium were screened in vitro against nine microorganisms. The EO of the leaf showed the best activity (10.2 µg/mL MIC) against Mycobacterium smegmatis. All the EOs and the solvent extracts gave moderate activity against the Staphylococcus aureus, Bacillus cereus, and M. smegmatis within the range of 10.2-1300.0 µg/mL MIC values. The best antibacterial activity was observed against S. aureus and B. cereus in the n-hexane extract of stem and methanol extract of flower samples.

Genome sequences of four A1 subcluster Mycobacterium smegmatis bacteriophages.

Payneful, Marchy, Hami1, and Sorpresa are A1 subcluster tailed bacteriophages belonging to the Caudoviricetes class that infect Mycobacterium smegmatis strain mc2155. They are consistent with other A1 subcluster phages based on their genome length and guanine-cytosine content. Their genomes contain six novel open reading frames.

Structure of Mycobacterial NDH-2 Bound to a 2-Mercapto-Quinazolinone Inhibitor.

Mycobacterial type II NADH dehydrogenase (NDH-2) is a promising drug target because of its central role in energy metabolism in Mycobacterium tuberculosis and other pathogens, and because it lacks a known mammalian homologue. To facilitate optimization of lead compounds, we used electron cryomicroscopy (cryo-EM) to determine the structure of NDH-2 from Mycobacterium smegmatis, a fast-growing nonpathogenic model for respiration in M. tuberculosis. The structure shows that active mycobacterial NDH-2 is dimeric, with an arrangement of monomers in the dimer that differs from the arrangement described for other prokaryotic NDH-2 dimers, instead resembling dimers formed by NDH-2 in the eukaryotes Saccharomyces cerevisiae and Plasmodium falciparum. A structure of the enzyme bound to a 2-mercapto-quinazolinone inhibitor shows that the compound interacts directly with the flavin adenine dinucleotide cofactor, blocking the menaquinone-reducing site. These results reveal structural elements of NDH-2 that could be used to design specific inhibitors of the mycobacterial enzyme.

Mechanism of deamination by mycobacterial deaminase selectively targeting mutagenic bases.

Nucleobase deaminases are important players in maintaining a stringent nucleobase pool and enhancing genetic diversity via judicious base editing. Here, we delineate the mechanism of Mycobacterium smegmatis deaminase, Msd, found predominantly in Mycobacterium species, that selectively catalyzes the deamination of mutagenic bases. Molecular dynamic studies reveal the dynamic nature of unique structural insertions that cycle between 'closed' and 'open' states, enabling zinc-assisted deamination by occluding the solvent. Corroborating X-ray crystallographic and biochemical studies assert that the appropriate length of the two gating loops and proper positioning of the di-proline motif they harbor are paramount to effective closure. Analysis reveals that although both natural base deaminases, guanine and cytosine deaminase, operate via a similar gating mechanism to Msd, they employ topologically differentially placed structural elements to achieve the 'closed' form. The comparison shows that Mycobacterium deaminases lack the dual-proton shuttle, which renders them ineffective for the deamination of natural bases but allows them to selectively target mutagenic s-triazine scaffolds, thereby imparting innate immunity against these drugs. The study highlights how topologically unique insertions in the cytidine deaminase fold play a critical role in harnessing evolutionary versatility, responsible in imparting fidelity for a nucleobase deamination reaction.

In vivo nucleotide excision repair by mycobacterial UvrD1 requires ATP hydrolysis but does not depend on cysteine disulfide-mediated dimerization and DNA unwinding.

Mycobacterial UvrD1 is an SF1-type ATPase that participates in nucleotide excision repair (NER). UvrD1 consists of N-terminal ATPase and C-terminal Tudor domains. The monomeric UvrD1 characterized originally displays vigorous DNA-dependent ATPase activity but only feeble helicase activity. A recent study demonstrated that: (i) cysteine disulfide-mediated homodimerization of UvrD1 generates a highly active helicase; and (ii) an obligate monomeric UvrD1 (by virtue of mutating the domain 2B cysteine) is active as an ATP-dependent 3'-to-5' single-stranded DNA translocase but not as a double-stranded DNA-unwinding helicase. Here we test genetically which physical and functional states of UvrD1 are relevant for its functions in DNA repair, by complementation of an NER-defective Mycobacterium smegmatis ΔuvrD1 strain with a series of biochemically-defined UvrD1 mutants. By assaying complemented strains for sensitivity to UVC, MMC, cisplatin, and psoralen-UVA, we conclude that monomeric UvrD1 ATPase activity suffices for the NER functions of UvrD1 in vivo. Decoupling ATP hydrolysis from duplex unwinding does not affect the repair activity of UvrD1, nor does interdiction of domain 2B cysteine disulfide-mediated dimerization or deletion of the Tudor domain. Our results militate against a proposed model in which UvrD1's repair function is governed by the redox state of the bacterium via its impact on UvrD1 dimerization and helicase activity.

Genome sequence of cluster F1 Mycobacterium smegmatis phage Fastidio.

Fastidio is an F1 subcluster bacteriophage in the Caudoviricetes class, infecting Mycobacterium smegmatis strain mc²155. The genome is 55,839 base pairs in length and contains several putative novel open reading frames. The isolation, annotation, and analysis of Fastidio, together with other bacteriophages, improve our knowledge of phage diversity.

Chemokine CXCL14 Inhibits the Survival of Mycobacterium smegmatis inside Macrophages by Upregulating A20 to Promote ROS Production.

Tuberculosis remains a major global health threat, with traditional antibiotic treatments facing challenges such as drug resistance. Host-directed therapy (HDT) has emerged as a promising approach to combat tuberculosis by enhancing the host immune response. CXCL14, a chemokine family member, plays a crucial role in regulating host antipathogenic immune responses. To elucidate the role of CXCL14 and its key regulatory molecules in mycobacterial infections, we identified new targets for host-directed therapy. RAW264.7 macrophages were pretreated with CXCL14 and infected with Mycobacterium smegmatis. CFU, ROS levels, and apoptosis were assessed. Cell RNA was extracted for high-throughput sequencing, and significantly differentially expressed genes were screened and identified. The effects of candidate genes were verified using knockdown and overexpression techniques. A mouse model of mycobacterial infection was established to validate the role of CXCL14 in vivo. CXCL14 pretreatment significantly reduced intracellular mycobacteria and increased ROS levels in macrophages without affecting apoptosis. Transcriptome analysis identified A20 as a key differentially expressed gene. A20 overexpression promoted ROS production and decreased intracellular mycobacteria, while A20 knockdown reversed these effects. The combination of CXCL14 and A20 overexpression effectively inhibited mycobacterial survival in macrophages. CXCL14 significantly inhibited mycobacterial survival in mice and reduced organ damage in vivo. CXCL14 promoted ROS production in macrophages by upregulating A20 expression, thereby inhibiting mycobacterial survival. In the mouse model, CXCL14 alleviated inflammatory responses and histopathological damage caused by mycobacterial infection. These findings suggest that CXCL14 is a promising new HDT molecule for the treatment of mycobacterial infections.

Conformational landscape of the mycobacterial inosine 5'-monophosphate dehydrogenase octamerization interface.

Conformational landscape of the mycobacterial inosine 5'-monophosphate dehydrogenase octamerization interface.

Genome sequence of cluster A6 bacteriophage Lilbunny, isolated using Mycobacterium smegmatis mc2155.

Bacteriophage Lilbunny is a siphovirus infecting Mycobacterium smegmatis strain mc2155. It was isolated from compost of rabbit fecal matter. The genome of Lilbunny belongs to the A6 subcluster and is 50,789 bp, containing 95 open reading frames, 52.6% of which encode proteins with predicted functions, and three tRNA genes.

Enhancement of GTP hydrolysis and inhibition of polymerization of the cell division protein FtsZ by an N-heterocyclic imine derivative impede growth and biofilm formation in Streptococcus pneumoniae.

Enhancement of GTP hydrolysis and inhibition of polymerization of the cell division protein FtsZ by an N-heterocyclic imine derivative impede growth and biofilm formation in Streptococcus pneumoniae.

Truncation of the C-terminal domain from CgtA of Mycobacterium smegmatis reduces its ribosome binding property but increases its GTPase activity

Truncation of the C-terminal domain from CgtA of Mycobacterium smegmatis reduces its ribosome binding property but increases its GTPase activity

CRISPR/Cas12a-mediated gene silencing across diverse functional genes demonstrates single gene-specific spacer efficacy in Mycobacterium smegmatis

BackgroundTuberculosis, a persistent global health threat, necessitates a comprehensive understanding of the genes and pathways crucial for the survival and virulence of the causative pathogen, Mycobacterium tuberculosis. Working with M. tuberculosis (M.tb) presents significant challenges; therefore, the use of M. smegmatis as a surrogate system for conducting genetic studies of M.tb has proven to be highly valuable. Development of novel genetic tools to probe cellular processes accelerates the progress in the field of drug development and also helps in understanding the basic physiology of the bacterium.ResultsThis study reports the successful implementation and evaluation of the CRISPR-Cas12a system for gene repression in Mycobacterium smegmatis, a surrogate for M. tuberculosis. We engineered a Cas12a-based CRISPR interference (CRISPRi) system and assessed its functionality. Targeting 45 genes with a single sgRNA per gene, we achieved efficient gene repression, leading to marked phenotypic changes. Each knockdown strain was evaluated individually for growth phenotypes, and a comparison of the results with the reported essential gene library probed with dCas9 demonstrated congruous results across diverse gene categories. The study shows that CRISPR/Cas12a system can be effectively utilised with a single gene specific target for efficient silencing of the gene and highlights the importance of subsequent growth assays required to evaluate the vulnerability of targeted gene silencing.ConclusionOur findings reveal the robustness and versatility of the dCas12a-CRISPRi system in M. smegmatis, providing a valuable tool for functional genomics research. This work showcases the potential of the dCas12a-CRISPRi system in investigating essential genes, enabling a deeper understanding of the biology and potential therapeutic targets in mycobacterium species.