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Journal of Infection and Public Health Sep 2020An alternate host for mycobacteria is Mycobacterium smegmatis which is used frequently. It is a directly budding eco-friendly organism not emulated as human infection.... (Review)
Review
An alternate host for mycobacteria is Mycobacterium smegmatis which is used frequently. It is a directly budding eco-friendly organism not emulated as human infection. It is mainly useful for the investigation of various microorganisms in the sort of Mycobacteria in cell culture laboratories. Some Mycobacterium species groups that is normal, unsafe ailments, likely to Mycobacterium leprae, Mycobacterium tuberculosis and Mycobacterium bovis. At present, various laboratories are clean and culture this type of species to make an opinion that fascinating route of harmful Mycobacteria. This publication provides aggregate data on cell shape, genome studies, ecology, pathology and utilization of M. smegmatis.
Topics: Bacterial Proteins; Humans; Liposomes; Models, Biological; Mycobacterium Infections, Nontuberculous; Mycobacterium smegmatis; Mycobacterium tuberculosis
PubMed: 32674978
DOI: 10.1016/j.jiph.2020.06.023 -
Production of recombinant proteins in Mycobacterium smegmatis for structural and functional studies.Protein Science : a Publication of the... Jan 2015Protein production using recombinant DNA technology has a fundamental impact on our understanding of biology through providing proteins for structural and functional... (Review)
Review
Protein production using recombinant DNA technology has a fundamental impact on our understanding of biology through providing proteins for structural and functional studies. Escherichia coli (E. coli) has been traditionally used as the default expression host to over-express and purify proteins from many different organisms. E. coli does, however, have known shortcomings for obtaining soluble, properly folded proteins suitable for downstream studies. These shortcomings are even more pronounced for the mycobacterial pathogen Mycobacterium tuberculosis, the bacterium that causes tuberculosis, with typically only one third of proteins expressed in E. coli produced as soluble proteins. Mycobacterium smegmatis (M. smegmatis) is a closely related and non-pathogenic species that has been successfully used as an expression host for production of proteins from various mycobacterial species. In this review, we describe the early attempts to produce mycobacterial proteins in alternative expression hosts and then focus on available expression systems in M. smegmatis. The advantages of using M. smegmatis as an expression host, its application in structural biology and some practical aspects of protein production are also discussed. M. smegmatis provides an effective expression platform for enhanced understanding of mycobacterial biology and pathogenesis and for developing novel and better therapeutics and diagnostics.
Topics: Bacterial Proteins; Cloning, Molecular; Genetic Vectors; Models, Molecular; Mycobacterium; Mycobacterium smegmatis; Protein Processing, Post-Translational; Recombinant Proteins
PubMed: 25303009
DOI: 10.1002/pro.2584 -
PloS One 2021Reduction of nitrate to nitrite in bacteria is an essential step in the nitrogen cycle, catalysed by a variety of nitrate reductase (NR) enzymes. The soil dweller,...
Reduction of nitrate to nitrite in bacteria is an essential step in the nitrogen cycle, catalysed by a variety of nitrate reductase (NR) enzymes. The soil dweller, Mycobacterium smegmatis is able to assimilate nitrate and herein we set out to confirm the genetic basis for this by probing NR activity in mutants defective for putative nitrate reductase (NR) encoding genes. In addition to the annotated narB and narGHJI, bioinformatics identified three other putative NR-encoding genes: MSMEG_4206, MSMEG_2237 and MSMEG_6816. To assess the relative contribution of each, the corresponding gene loci were deleted using two-step allelic replacement, individually and in combination. The resulting strains were tested for their ability to assimilate nitrate and reduce nitrate under aerobic and anaerobic conditions, using nitrate assimilation and modified Griess assays. We demonstrated that narB, narGHJI, MSMEG_2237 and MSMEG_6816 were individually dispensable for nitrate assimilation and for nitrate reductase activity under aerobic and anaerobic conditions. Only deletion of MSMEG_4206 resulted in significant reduction in nitrate assimilation under aerobic conditions. These data confirm that in M. smegmatis, narB, narGHJI, MSMEG_2237 and MSMEG_6816 are not required for nitrate reduction as MSMEG_4206 serves as the sole assimilatory NR.
Topics: Bacterial Proteins; Gene Deletion; Mycobacterium smegmatis; Nitrate Reductase; Nitrates
PubMed: 33471823
DOI: 10.1371/journal.pone.0245745 -
Proceedings of the National Academy of... Apr 2021Encapsulins containing dye-decolorizing peroxidase (DyP)-type peroxidases are ubiquitous among prokaryotes, protecting cells against oxidative stress. However, little is...
Encapsulins containing dye-decolorizing peroxidase (DyP)-type peroxidases are ubiquitous among prokaryotes, protecting cells against oxidative stress. However, little is known about how they interact and function. Here, we have isolated a native cargo-packaging encapsulin from and determined its complete high-resolution structure by cryogenic electron microscopy (cryo-EM). This encapsulin comprises an icosahedral shell and a dodecameric DyP cargo. The dodecameric DyP consists of two hexamers with a twofold axis of symmetry and stretches across the interior of the encapsulin. Our results reveal that the encapsulin shell plays a role in stabilizing the dodecameric DyP. Furthermore, we have proposed a potential mechanism for removing the hydrogen peroxide based on the structural features. Our study also suggests that the DyP is the primary cargo protein of mycobacterial encapsulins and is a potential target for antituberculosis drug discovery.
Topics: Bacterial Proteins; Cryoelectron Microscopy; Mycobacterium smegmatis; Organelles; Peroxidases
PubMed: 33853951
DOI: 10.1073/pnas.2025658118 -
Hypoxanthine-Guanine Phosphoribosyltransferase Is Dispensable for Mycobacterium smegmatis Viability.Journal of Bacteriology Feb 2020Purine metabolism plays a ubiquitous role in the physiology of and other mycobacteria. The purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT)...
Purine metabolism plays a ubiquitous role in the physiology of and other mycobacteria. The purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is essential for growth ; however, its precise role in physiology is unclear. Membrane-permeable prodrugs of specifically designed HGPRT inhibitors arrest the growth of and represent potential new antituberculosis compounds. Here, we investigated the purine salvage pathway in the model organism Using genomic deletion analysis, we confirmed that HGPRT is the only guanine and hypoxanthine salvage enzyme in but is not required for growth of this mycobacterium or survival under long-term stationary-phase conditions. We also found that prodrugs of HGPRT inhibitors displayed an unexpected antimicrobial activity against that is independent of HGPRT. Our data point to a different mode of mechanism of action for these inhibitors than was originally proposed. Purine bases, released by the hydrolytic and phosphorolytic degradation of nucleic acids and nucleotides, can be salvaged and recycled. The hypoxanthine-guanine phosphoribosyltransferase (HGPRT), which catalyzes the formation of guanosine-5'-monophosphate from guanine and inosine-5'-monophosphate from hypoxanthine, represents a potential target for specific inhibitor development. Deletion of the HGPRT gene () in the model organism confirmed that this enzyme is not essential for growth. Prodrugs of acyclic nucleoside phosphonates (ANPs), originally designed against HGPRT from , displayed anti- activities comparable to those obtained for but also inhibited the strain. These results confirmed that ANPs act in by a mechanism independent of HGPRT.
Topics: Antitubercular Agents; Catalysis; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hypoxanthine Phosphoribosyltransferase; Metabolic Networks and Pathways; Microbial Viability; Mycobacterium smegmatis; Plasmids; Purines
PubMed: 31818925
DOI: 10.1128/JB.00710-19 -
International Journal of Molecular... Oct 2021Regulatory small non-coding RNAs play a significant role in bacterial adaptation to changing environmental conditions. Various stresses such as hypoxia and nutrient...
Regulatory small non-coding RNAs play a significant role in bacterial adaptation to changing environmental conditions. Various stresses such as hypoxia and nutrient starvation cause a reduction in the metabolic activity of , leading to entry into dormancy. We investigated the functional role of F6, a small RNA of , and constructed an F6 deletion strain of . Using the RNA-seq approach, we demonstrated that gene expression changes that accompany F6 deletion contributed to bacterial resistance against oxidative stress. We also found that F6 directly interacted with 5'-UTR of mRNA encoding RpfE2, a resuscitation-promoting factor, which led to the downregulation of RpfE2 expression. The F6 deletion strain was characterized by the reduced ability to enter into dormancy (non-culturability) in the potassium deficiency model compared to the wild-type strain, indicating that F6 significantly contributes to bacterial adaptation to non-optimal growth conditions.
Topics: 5' Untranslated Regions; Adaptation, Physiological; Gene Expression Regulation, Bacterial; Genes, Bacterial; Multigene Family; Mycobacterium smegmatis; RNA, Bacterial; RNA, Small Untranslated; RNA-Seq; Sequence Deletion; Stress, Physiological
PubMed: 34768965
DOI: 10.3390/ijms222111536 -
Microbiology (Reading, England) Oct 2018The PrrAB two-component system is conserved across all sequenced mycobacterial species and is essential for viability in Mycobacterium tuberculosis, thus making it a...
The PrrAB two-component system is conserved across all sequenced mycobacterial species and is essential for viability in Mycobacterium tuberculosis, thus making it a promising drug target. The prrAB operon was successfully deleted in nonpathogenic Mycobacterium smegmatis, and the ∆prrAB mutant strain exhibited clumping in ammonium-limited medium and significantly reduced growth during ammonium and hypoxic stress. To assess the influence of M. tuberculosis PrrA overexpression, we constructed a recombinant M. smegmatis ∆prrAB mutant strain which overexpresses M. tuberculosis prrA. M. smegmatis prrAB and M. tuberculosis prrA complemented the M. smegmatis ∆prrAB deletion mutant in Middlebrook M7H9 and ammonium-limited media and during hypoxic and ammonium stress. Based on quantitative untargeted mass spectrometry-based lipidomics, triacylglycerol lipid species were significantly upregulated in the ∆prrAB mutant strain compared to the wild-type when cultured in ammonium-limited medium, revealing that M. smegmatis PrrAB influences triacylglycerol levels during ammonium stress. These results were qualitatively corroborated by thin-layer chromatography. Furthermore, the ∆prrAB mutant significantly upregulated expression of several genes (glpK, GPAT, WS/DGAT, accA3, accD4, accD6 and Ag85C) that participate in triacylglycerol and lipid biosynthetic pathways, thus corroborating the lipidomics analyses.
Topics: Ammonium Compounds; Bacterial Proteins; Culture Media; Gene Deletion; Gene Expression; Gene Expression Regulation, Bacterial; Genetic Complementation Test; Hypoxia; Lipid Metabolism; Mycobacterium smegmatis; Mycobacterium tuberculosis; Operon; Recombinant Proteins; Signal Transduction; Stress, Physiological; Triglycerides
PubMed: 30084767
DOI: 10.1099/mic.0.000705 -
Biotechnology and Applied Biochemistry Jun 2022Tuberculosis, caused by mycobacteria, continues to pose a substantial public health threat. Mycobacteria typically use cholesterol from the membranes of host macrophages...
Tuberculosis, caused by mycobacteria, continues to pose a substantial public health threat. Mycobacteria typically use cholesterol from the membranes of host macrophages as a carbon and energy source. Most genes that control cholesterol degradation are regulated by KstR, which is highly conserved in Mycobacterium tuberculosis and Mycobacterium smegmatis. Through bioinformatic analysis, we found a typical global nitrogen regulator (GlnR)-binding motif (CCGAC-AACAGT-GACAC) in the promoter region of kstR of M. smegmatis, and we determined its binding activity in vitro using electrophoretic mobility shift assays. Using RT-qPCR, we found that nine genes involved in side-chain or sterol-ring oxidation were upregulated in a ΔglnR M. smegmatis strain compared to the WT strain and glnR-complemented strains under nitrogen limitation. ATP assays in macrophages revealed that coordinated GlnR-KstR regulation significantly reduced the viability of M. smegmatis in macrophages. Thus, we found that various genes involved in cholesterol catabolism are regulated by GlnR via KstR in response to environmental nitrogen, and that they further affect the invasive ability of M. smegmatis. These findings revealed a novel regulatory mechanism of cholesterol catabolism, which may be useful in the development of new strategies for controlling tuberculosis.
Topics: Bacterial Proteins; Cholesterol; Gene Expression Regulation, Bacterial; Humans; Mycobacterium smegmatis; Nitrogen; Tuberculosis
PubMed: 34008246
DOI: 10.1002/bab.2197 -
Applied Microbiology and Biotechnology Dec 2019Mycobacterium tuberculosis is able to transition into a dormant state, causing a latent state of tuberculosis. Dormant mycobacteria acquire phenotypic resistance to all...
Mycobacterium tuberculosis is able to transition into a dormant state, causing a latent state of tuberculosis. Dormant mycobacteria acquire phenotypic resistance to all known antibacterial drugs; they are also able to maintain vitality in the host for decades and become active, causing the active form of the disease. In order to cure latent tuberculosis, new approaches should be developed. Earlier, we discovered accumulation in significant concentrations of porphyrins in dormant Mycobacterium smegmatis, which is a close, fast-growing relative of the causative agent of tuberculosis. In this study, we explore a new possibility to kill dormant mycobacteria by photodynamic inactivation (PDI) using accumulated porphyrins as endogenous photosensitisers. The dormant M. smegmatis were obtained under gradual acidification in Sauton's medium, for 14 days. Cells were exposed to light with different wavelengths emitted by three Spectra X light-emitting diodes (395/25, 470/24, 575/25 nm) and one separated 634-nm LED for 15 min. An increase in the concentration of coproporphyrin in M. smegmatis after 6 days of growth correlated with the beginning of a decrease in metabolic activity and formation of ovoid dormant forms. Dormant bacteria were sensitive to PDI and killed after 15-30 min of illumination, in contrast to active cells. The greatest inactivation of dormant mycobacteria occurred at 395 and 575 nm, which coincides with the main maximum of the absorption spectrum of extracted porphyrins. We, for the first time, demonstrate a successful application of PDI for inactivation of dormant mycobacteria, due to significant accumulation of endogenous photosensitisers-porphyrins.
Topics: Culture Media; Light; Microbial Sensitivity Tests; Microbial Viability; Mycobacterium smegmatis; Photosensitizing Agents; Porphyrins
PubMed: 31713670
DOI: 10.1007/s00253-019-10197-3 -
Canadian Journal of Microbiology Jul 2015Fast-growing mycobacteria such as Mycobacterium sp. and Mycobacterium smegmatis degrade natural sterols. They are a model to study tuberculosis. Interestingly, M....
Fast-growing mycobacteria such as Mycobacterium sp. and Mycobacterium smegmatis degrade natural sterols. They are a model to study tuberculosis. Interestingly, M. smegmatis has been found in river effluents derived from paper production, and therefore, it would be important to gain further insight into its capacity to synthesize steroids that are potential endocrine disruptors affecting the development and reproduction of fishes. To our knowledge, the capacity of M. smegmatis to synthesize estrogens and even testosterone has not been previously reported. Therefore, the objective of this study was to investigate the capacity of M. smegmatis to synthesize in vitro testosterone and estrogens from tritiated precursors and to investigate the metabolic pathways involved. Results obtained by thin-layer chromatography showed that (3)H-progesterone was transformed to 17OH-progesterone, androstenedione, testosterone, estrone, and estradiol after 6, 12, or 24 h of incubation. (3)H-androstenedione was transformed into testosterone and estrogens, mainly estrone, and (3)H-testosterone was transformed to estrone and androstenedione. Incubation with (3)H-dehydroepiandrosterone rendered androstenediol, testosterone, and estrogens. This ability to transform less potent sex steroids like androstenedione and estrone into other more active steroids like testosterone and estradiol or vice versa suggests that M. smegmatis can influence the amount of self-synthesized strong androgens and estrogens and can transform those found in the environment.
Topics: Androgens; Chromatography, Thin Layer; Dehydroepiandrosterone; Estrogens; Female; Humans; Mycobacterium smegmatis; Steroids
PubMed: 25994226
DOI: 10.1139/cjm-2015-0025